WO2004066945A2 - Enzymes, acides nucleiques codant pour ces enzymes et procedes pour les produire et les utiliser - Google Patents

Enzymes, acides nucleiques codant pour ces enzymes et procedes pour les produire et les utiliser Download PDF

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Publication number
WO2004066945A2
WO2004066945A2 PCT/US2004/002242 US2004002242W WO2004066945A2 WO 2004066945 A2 WO2004066945 A2 WO 2004066945A2 US 2004002242 W US2004002242 W US 2004002242W WO 2004066945 A2 WO2004066945 A2 WO 2004066945A2
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seq
activity
sequence
polypeptide
nucleic acid
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PCT/US2004/002242
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English (en)
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WO2004066945A3 (fr
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Nelson Barton
Dan Robertson
Kristine Chang
James Elkins
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Diversa Corporation
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Publication of WO2004066945A3 publication Critical patent/WO2004066945A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes

Definitions

  • This invention relates generally to medical products and microbiology.
  • the invention provides enzymes having a surE protein activity, a deacetylase, an amidase, a cellulase, an esterase, a glycosidase, a xylanase, an amylase, a transaminase, a laminarinase, a beta-galactosidase, a beta-mannosidase, a pullulanase, and/or a phosphatase activity, and the nucleic acids that encode them, and antibodies that specifically bind to them.
  • biofilm control compositions e.g., enzymes and antibodies, for the control of biofilms, polynucleotides encoding the polypeptides and methods of making and using these polynucleotides and polypeptides.
  • these proteins are biofilm matrix-hydrolyzing enzymes.
  • the invention provides products comprising these biofilm control compositions.
  • the biofilm-control compositions of the invention have an amidase activity, e.g., the ability to hydrolyze amides, including enzymes having secondary amidase activity, including a peptidase, a protease and/or a hydantoinase activity.
  • the biofilm control compositions of the invention have a deacetylase, an amidase, a cellulase, an esterase, a glycosidase, a xylanase, an amylase, a transaminase, a laminarinase, a beta- galactosidase, a beta-mannosidase, a pullulanase, and/or a phosphatase activity.
  • Microbial fouling is a common problem in a variety of industrial, household, personal hygiene and medical settings.
  • the formation of microbial biofilms is an important aspect of microbial colonization and contamination of these environments and chemical biocides are typically applied in an effort to prevent and mitigate microbe growth. This approach is only partially effective due the resistance these films impart to the microbe and macro-organism communities in the films.
  • Weaknesses of current approaches to controlling microbial contamination and growth include 1) ineffective penetration and release of microbe-harboring biofilms, 2) the need for large amounts of toxic and corrosive chemicals, and finally 3) instability of existing biofilm-targeted enzymes.
  • biocidal and bacteriostatic agents have been used with limited success against industrial biofilms (REF). These include common chemical oxidants such as NaOCl, ClO 2 , H 2 O , or other compounds such as peracetic acid, methylene bis- thiocyanate, glutaraldehyde, sodium dimethyldithiocarbamate, and isothiazolones. Lack of efficacy is presumed to be due to poor penetration of the biofilm matrix. Indeed, biocidal killing of sessile microbial forms is estimated to be 1000 to 10,000 times less effective than killing of planktonic organisms (REF). Water processing systems are susceptible to formation of biofilm. Unless countermeasures are taken, microbes may colonize the system.
  • Microbial biofilms can cause both process and health-related problems. Control or elimination of biofilms can control or prevent their adverse effects. The pathogenicity of bacteria in biofilms is a problem, particularly when humans consume water from biofilm-contaminated systems. Natural biofilms can be composed of a single species or mixed populations of gram-negative and gram-positive organisms. Toxic biocides, even at high concentrations, often fail to control problematic biofilms.
  • Biofilms are surface-attached consortia of microorganisms. Such communities are problematic in many settings due to properties such as antibiotic resistance, and a large number of industrial and medically relevant materials harbor such sessile, attached microbial communities. Health risks stemming from biofilms are also prevalent in the medical industry. Medical implants and prosthetic devices may harbor biofilms of potentially pathogenic organisms. In the dental industry, biofilms are implicated in a number of health-associated problems. As an example, biofilms of acidogenic, gram-positive cocci on the tooth surface are known to cause dental decay.
  • Materials used in ballast tanks are typically low-alloy steels, which are protected with a coating, often epoxy coatings. Tar-epoxy coatings were once commonly used but now a pure epoxy or a modified epoxy is more common.
  • coating failures the most common type of corrosion failure being blisters, which is caused by impurities on the metal surface when the coating is apphed.
  • One of the most important steps in the coating process is cleaning. If cleaning of the steel is not performed in a proper way the coating cannot protect the steel. The anodes placed in the ballast tanks will then protect the steel as much as possible. The environment in the ballast tanks is humid and dirty.
  • micro-organisms are one of the main reasons why the ballast tanks corrode.
  • a thin layer can line the bottom of the ballast tanks.
  • the corrosion in the ballast tanks inspected is microbial influenced corrosion, MIC, probably in combination with ordinary corrosion. The corrosion can be decreased if the quantity of micro-organisms is removed.
  • ballast water of ships Vibrio cholerae can invade some species of algae, then enter a dormant state awaiting favorable conditions that facilitate its re-emergence as an infectious agent.
  • Ballast water can carry V. cholerae, multiple viruses, Escherichia coli and other pathogenic forms, including Clostridium perfringins, Salmonella species, and enteroviruses, from port to port around the world.
  • the load of bacteria and viruses in the ballast water of ships, as well as the biofilm that lines the ballast tanks, is substantial. Further, credible scientific evidence exists inferring that ballast water exchange at sea does little to decrease the content and concentration of these pathogens and may actually stimulate and increase the bacterial and viral load.
  • ballast water can also transfer a range of species of micro-algae, including toxic species (dinoflagellates) that may form harmful algae blooms or "red tides.”
  • toxic species dinoflagellates
  • red tides harmful algae blooms or "red tides.”
  • the public health impacts of such outbreaks are well documented and include paralytic shellfish poisoning, which can cause severe illness and death in humans.
  • ballast problem is re-ballasting, an exchange of ballast water in deep waters at sea.
  • the assumption is that organisms found in the deep open ocean are not adapted to live close to shore and that greater levels of salinity may kill ballast organisms.
  • re-ballasting is not highly effective. Recent studies have found that organisms from ports remain inside along with sediment and biofilm.
  • ballast tanks Three factors combine to prevent the elimination of microorganisms from ballast tanks by exchange at sea. First, currently there is no mechanism for totally emptying the ballast tanks on the high seas. A residual amount of ballast water and sediment always remains in the tanks. Second, the greatest source of continuing contamination of the ballast water is the biofilm produced by the microorganisms and macro organisms in the ballast tanks, which adheres to the inner surfaces of ballast tanks.
  • the biofilm is largely unaffected by water exchange at sea and resistant to most proposed methods of removal.
  • the biocide concentrations necessary to inactivate pathogens imbedded in the biofilm matrix are orders of magnitude higher than that necessary to kill pathogens that are suspended in water.
  • the biofilm provides a protective environment for pathogenic bacteria, which is consistent with the notion that the biofilm is causally related to pathogen transmission.
  • Third, following exchange at sea the environmental conditions in the ballast tanks may favor a population expansion and increased biodiversity. In fact, when conditions are favorable the numbers of disease producing organisms can reach levels considerably higher than were recorded before the exchange.
  • the salinity of the exchanged water in the ballast tanks can be higher and the water temperature can be lower, the conditions for expanded growth are more favorable because the flushed sea water will likely contain more oxygen and less nitrogen.
  • ballast water When a ship arrives at a port and discharges its ballast water the possibility of contaminating the local waters with foreign bacteria, viruses, plankton, crustaceans, etc. is initiated. This begins a cycle of pathogen transport that continues as ships enter and leave the port taking on and discharging their ballast water. Even ships that are only involved in coastal trade and never leave the territorial waters of their homeland can transport foreign organisms to each of their ports of call. The ballast water of all ships is a potential source for the dissemination of pathogens as well as macro organisms.
  • nosocomial pneumonia Most patients with nosocomial pneumonia are those with extremes of age, severe underlying disease, immunosuppression, depressed sensorium, and cardiopulmonary disease, and those who have had thoraco-abdominal surgery. Although patients with mechanically assisted ventilation do not comprise a major proportion of patients with nosocomial pneumonia, they have the highest risk of developing the infection. Most bacterial nosocomial pneumonias occur by aspiration of bacteria colonizing the oropharynx or upper gastrointestinal tract of the patient. Intubation and mechanical ventilation greatly increase the risk of nosocomial bacterial pneumonia because they alter first-line patient defenses. Pneumonias due to
  • the invention provides enzymes having a deacetylase, an amidase, a cellulase, an esterase, a glycosidase, a xylanase, an amylase, a fransaminase, a laminarinase, a beta-galactosidase, a beta-mannosidase, a pullulanase, and/or a phosphatase activity, and nucleic acids that encode them, and antibodies that specifically bind to them.
  • the invention provides compositions and methods for enzymatic biofilm removal and for controlling problematic biofilms.
  • the enzymes of the invention are effective in removing biofilms or preventing them from forming.
  • the enzymes of the invention can be used in conjunction with traditional biocides, and, in some aspects, they can resist the harsh effects of frequently used biocides, corrosion inhibitors and surfactants.
  • the enzymes of the invention can be used for industrial applications and in some aspects can function in extreme and fluctuating temperatures and pH.
  • the enzymes of the invention have extreme heat and/or pH stable properties.
  • the enzymes of the invention are biofilm matrix-hydrolyzing enzymes.
  • the invention also provides biofilm micro-assays using matrix-hydrolyzing enzymes of the invention.
  • the invention also provides methods for characterizing enzymes for biofilm control activity and to test enzyme combinations for additive or synergistic biofilm control activities.
  • the enzymes of the invention have activity against biofilms composed of gram-negative (e.g., P. fluorescens) and/or gram-positive (e.g., S. epidermidis) biofilms.
  • the invention provides isolated or recombinant nucleic acids (including nucleic acid probes, e.g., for identifying or isolating nucleic acids) comprising (or consisting of) a nucleic acid sequence having at least about 50%, 51%, 52%, 53%, 54%), 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to an exemplary nucleic acid of the invention, e.g., SEQ ID NO:l; SEQ ID NO:3; SEQ ID NO:
  • the invention also provides nucleic acids encoding polypeptides having a sequence as set forth in SEQ ID NO:2; SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO: 10; SEQ ID NO: 12; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:2; SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO: 10; SEQ ID NO: 12; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:2; SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO: 10;
  • the mvention provides expression cassettes comprising a nucleic acid of the mvention, e.g., a nucleic acid comprising a sequence as set forth in SEQ ID NO:l; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:ll; SEQ ID NO: 13; SEQ ID NO: 15; SEQ ID NO: 17; SEQ ID NO: 19; SEQ ID NO-21; SEQ ID NO:23; SEQ ID NO:25; SEQ ID NO:27; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:33; SEQ ID NO:35; SEQ ID NO:37; SEQ ID NO:39; SEQ ID NO:41; SEQ ID NO:43; SEQ ID NO:45; SEQ ID NO:47; SEQ ID NO:49; SEQ ID NO:51 ; SEQ ID NO:53; SEQ ID NO:55; SEQ ID NO:57; SEQ ID NO:59; S
  • the expression cassettes can comprise a nucleic acid of the invention that is operably linked to a promoter.
  • the promoter can be a viral, bacterial, mammalian or plant promoter.
  • the plant promoter can be a potato, rice, corn, wheat, tobacco or barley promoter.
  • the promoter can be a constitutive promoter.
  • the constitutive promoter can comprise CaMV35S.
  • the promoter can be an inducible promoter.
  • the promoter can be a tissue- specific promoter or an environmentally regulated or a developmentally regulated promoter.
  • the promoter can be, e.g., a seed-specific, a leaf-specific, a root-specific, a stem-specific or an abscission-induced promoter.
  • the expression cassette can further comprise a plant or plant virus expression vector.
  • the invention provides vectors comprising a nucleic acid of the invention.
  • the invention provides cloning vehicles comprising an expression cassette (e.g., a vector) of the invention or a nucleic acid of the invention.
  • the transformed cell can be a bacterial cell, a mammalian cell, a fungal cell, a yeast cell, an insect cell or a plant cell.
  • the plant cell can be a potato, wheat, rice, corn, tobacco or barley cell.
  • the invention provides transgenic non-human animals comprising a nucleic acid of the invention or an expression cassette (e.g., a vector) of the invention.
  • the animal is a mouse.
  • the invention provides transgenic plants comprising a nucleic acid of the invention or an expression cassette (e.g., a vector) of the invention.
  • the transgenic plant can be a corn plant, a potato plant, a tomato plant, a wheat plant, an oilseed plant, a rapeseed plant, a soybean plant, a rice plant, a barley plant or a tobacco plant.
  • the invention provides transgenic seeds comprising a nucleic acid of the invention or an expression cassette (e.g., a vector) of the invention.
  • the transgenic seed can be a corn seed, a wheat kernel, an oilseed, a rapeseed, a soybean seed, a palm kernel, a sunflower seed, a sesame seed, a peanut or a tobacco plant seed.
  • the RNAi is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides in length.
  • the invention provides methods of inhibiting the expression of a polypeptide (e.g., an enzyme) in a cell comprising admimstering to the cell or expressing in the cell a double-stranded inhibitory RNA (iRNA), wherein the RNA comprises a subsequence of a sequence of the invention.
  • a polypeptide e.g., an enzyme
  • iRNA double-stranded inhibitory RNA
  • the mvention provides isolated or recombinant polypeptides (i) having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26
  • the activity is thermostable, e.g., the polypeptide retains activity under conditions comprising a temperature range of between about 1°C to about 5°C, between about 5°C to about 15°C, between about 15°C to about 25°C, between about 25°C to about 37°C, between about 37°C to about 95°C, between about 55°C to about 85°C, between about 70°C to about 95°C, between about 70°C to about 75°C, or between about 90°C to about 95°C.
  • the activity is thermotolerant, e.g., the polypeptide retains activity after exposure to a temperature in the range from between about 1°C to about 5°C, between about 5°C to about 15°C, between about 15°C to about 25°C, between about 25°C to about 37°C, between about 37°C to about 95°C, between about 55°C to about 85°C, between about 70°C to about 75°C, or between about 90°C to about 95°C, or more.
  • the invention provides isolated or recombinant polypeptides comprising a polypeptide of the invention lacking a signal sequence and/or a prepro sequence.
  • the invention provides isolated or recombinant polypeptides comprising a polypeptide of the invention having a heterologous signal sequence or a heterologous prepro sequence.
  • the activity comprises a specific activity at about 37°C in the range from about 100 to about 1000 units per milligram of protein, from about 500 to about 750 units per milligram of protein, from about 500 to about 1200 units per milligram of protein, or from about 750 to about 1000 units per milligram of protein.
  • the polypeptide of the invention retains activity under conditions comprising about pH 6.5, pH 6.0, pH 5.5, 5.0, pH 4.5 or 4.0. In one aspect, the polypeptide of the invention retains activity under conditions comprising about pH 7.5, pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10 or pH 10.5.
  • the invention provides protein preparations comprising a polypeptide of the invention, wherein the protein preparation comprises a liquid, a solid or a gel.
  • the invention provides heterodimers comprising a polypeptide of the invention and a second domain.
  • the second domain can be a polypeptide and the heterodimer can be a fusion protein.
  • the second domain can be an epitope or a tag.
  • the invention provides homodimers comprising a polypeptide of the invention.
  • the invention provides immobilized polypeptides, wherein the polypeptide comprises a sequence of the invention, or a subsequence thereof.
  • the polypeptide can be immobilized on a cell, a metal, a resin, a polymer, a ceramic, a glass, a microelectrode, a graphitic particle, a bead, a gel, a plate, an anay or a capillary tube.
  • the invention provides anays comprising an immobilized polypeptide of the invention.
  • the mvention provides anays comprising an immobilized nucleic acid of the invention.
  • the polypeptide can retain biofilm control, or other activity under conditions comprising a temperature range of between about 37°C to about 95°C, between about 55°C to about 85°C, between about 70°C to about 95°C, or between about 90°C to about 95°C.
  • biofilm control, or other activity can be thermotolerant.
  • the polypeptide can retain an biofilm control, or other activity after exposure to a temperature in the range from greater than 37°C to about 95°C, or in the range from greater than 55°C to about 85°C.
  • the polypeptide can retain biofilm control, or other activity after exposure to a temperature in the range from greater than 90°C to about 95°C at pH 4.5.
  • the isolated or recombinant polypeptide can comprise the polypeptide of the invention that lacks a signal sequence.
  • the isolated or recombinant polypeptide can comprise the polypeptide of the invention comprises a heterologous signal sequence, such as an amylase signal sequence.
  • biofilm control, or other activity comprises a specific activity at about 37°C in the range from about 100 to about 1000 units per milligram of protein.
  • biofilm control, or other activity comprises a specific activity from about 500 to about 750 units per milligram of protein.
  • biofilm control, or other activity comprises a specific activity at 37°C in the range from about 500 to about 1200 units per milligram of protein.
  • biofilm control, or other activity comprises a specific activity at 37°C in the range from about 750 to about 1000 units per milligram of protein.
  • the thermotolerance comprises retention of at least half of the specific activity of the enzyme at 37°C after being heated to the elevated temperature.
  • the thermotolerance can comprise retention of specific activity at 37°C in the range from about 500 to about 1200 units per milligram of protein after being heated to the elevated temperature.
  • the mvention provides heterodimers comprising a polypeptide of the invention and a second domain.
  • the second domain can be a polypeptide and the heterodimer can be a fusion protein.
  • the second domain can be an epitope or a tag.
  • the invention provides immobilized polypeptides having biofilm control, wherein the polypeptide comprises a polypeptide of the invention, a polypeptide encoded by a nucleic acid of the invention, or a polypeptide comprising a polypeptide of the invention and a second domain.
  • the polypeptide can be immobilized on a cell, a metal, a resin, a polymer, a ceramic, a glass, a microelectrode, a graphitic particle, a bead, a gel, a plate, an anay or a capillary tube.
  • the mvention provides anays comprising an immobilized nucleic acid of the mvention.
  • the invention provides anays comprising an antibody of the invention.
  • the invention provides isolated or recombinant antibodies that specifically bind to a polypeptide of the invention or to a polypeptide encoded by a nucleic acid of the invention.
  • the antibody can be a monoclonal or a polyclonal antibody.
  • the invention provides hybridomas comprising an antibody of the invention, e.g., an antibody that specifically binds to a polypeptide of the invention or to a polypeptide encoded by a nucleic acid of the invention.
  • the invention provides methods of making an antibody of the mvention comprising admimstering to a non-human animal a nucleic acid of the invention or a polypeptide of the invention or subsequences thereof in an amount sufficient to generate a humoral immune response, thereby making an antibody.
  • the invention provides methods of making an immune response comprising a ⁇ - ⁇ inistering to a non-human animal a nucleic acid of the mvention or a polypeptide of the invention or subsequences thereof in an amount sufficient to generate an immune response.
  • the invention provides methods of producing a recombinant polypeptide comprising the steps of: (a) providing a nucleic acid of the invention operably linked to a promoter; and (b) expressing the nucleic acid of step (a) under conditions that allow expression of the polypeptide, thereby producing a recombinant polypeptide.
  • the method can further comprise transforming a host cell with the nucleic acid of step (a) followed by expressing the nucleic acid of step (a), thereby producing a recombinant polypeptide in a transformed cell.
  • the sequence comparison algorithm is a BLAST version 2.2.2 algorithm where a filtering setting is set to blastall -p blastp -d "nr pataa" -F F, and all other options are set to default.
  • the invention provides isolated or recombinant nucleic acids comprising a sequence that hybridizes under stringent conditions to a nucleic acid of the mvention.
  • the nucleic acid can be at least about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500 or more residues in length or the full length of the gene or transcript.
  • the stringent conditions include a wash step comprising a wash in 0.2X SSC at a temperature of about 65°C for about 15 minutes.
  • the mvention provides nucleic acid probes for identifying an enzyme having a biofilm control or biofilm modifying activity, or a surE protein activity (e.g., survival protein surE), or a deacetylase, an amidase, a cellulase, an esterase (e.g., hydroxyesterase or lipase activity), a glycosidase, a xylanase, an amylase, a transaminase, a laminarinase, a beta-galactosidase, a beta-mannosidase, a pullulanase, a phosphatase activity, a hydrolase activity, a 6-aminohexanoate-cyclic dimer hydrolase activity, a pyrazinamidase and/or a nicotinamidase activity, and in alternative aspects, probes for identifying nucleic acids encoding a polypeptide having a biofilm or other activity, where
  • the probe can comprise an oligonucleotide comprising at least about 10 to 50, about 20 to 60, about 30 to 70, about 40 to 80, or about 60 to 100 consecutive bases of a sequence comprising a sequence of the invention, or fragments or subsequences thereof.
  • the invention provides a nucleic acid probe for identifying an enzyme having a biofilm control or biofilm modifying activity, or a surE protein activity (e.g., survival protein surE), or a deacetylase, an amidase, a cellulase, an esterase (e.g., hydroxyesterase or lipase activity), a glycosidase, a xylanase, an amylase, a fransaminase, a laminarinase, a beta-galactosidase, a beta-mannosidase, a pullulanase, a phosphatase activity, a hydrolase activity, a 6-aminohexanoate-cyclic dimer hydrolase activity, a pyrazinamidase and/or a nicotinamidase activity, and in alternative aspects, probes for identifying nucleic acids encoding a polypeptide having biofilm control, or other activity,
  • the probe can comprise an oligonucleotide comprising at least about 10 to 50, about 20 to 60, about 30 to 70, about 40 to 80, or about 60 to 100 consecutive bases of a nucleic acid sequence of the invention.
  • the invention provides an amplification primer sequence pair for amplifying a nucleic acid encoding a polypeptide of the invention or a nucleic acid of the invention.
  • One or each member of the amplification primer sequence pair can comprise an oligonucleotide comprising at least about 10 to 50 consecutive bases of the sequence.
  • the invention provides methods of amplifying a nucleic acid of the invention or a nucleic acid encoding a polypeptide of the invention with amplification primer sequence pairs, e.g., with amplification primer sequence pairs capable of amplifying a nucleic acid sequence of the invention or a subsequence thereof, or, a nucleic acid encoding a polypeptide of the invention, or fragments or subsequences thereof.
  • the invention provides methods for identifying a polypeptide having a biofilm control activity comprising the following steps: (a) providing a polypeptide of the invention; or a polypeptide encoded by a nucleic acid of the invention; (b) providing a biofilm; and (c) contacting the polypeptide or a fragment or variant thereof of step (a) with the biofilm of step (b) and detecting a decrease in the amount of biofilm or an increase in the amount of a biofilm breakdown product, wherein a decrease in the amount of biofilm or an increase in the amount of the product detects a polypeptide having an biofilm control activity.
  • the invention provides methods of determining whether a test compound specifically binds to a polypeptide comprising the following steps: (a) expressing a nucleic acid or a vector comprising the nucleic acid under conditions permissive for translation of the nucleic acid to a polypeptide, wherein the nucleic acid comprises a nucleic acid of the invention, or, providing a polypeptide of the invention; (b) providing a test compound; (c) contacting the polypeptide with the test compound; and (d) dete-tmining whether the test compound of step (b) specifically binds to the polypeptide.
  • the invention provides computer systems comprising a processor and a data storage device wherein said data storage device has stored thereon a polypeptide sequence or a nucleic acid sequence of the invention.
  • the computer system can further comprise a sequence comparison algorithm and a data storage device having at least one reference sequence stored thereon.
  • the sequence comparison algorithm comprises a computer program that indicates polymorphisms.
  • the computer system can further comprise an identifier that identifies one or more features in said sequence.
  • the invention provides computer readable media having stored thereon a polypeptide sequence or a nucleic acid sequence of the invention.
  • the invention provides methods for identifying a feature in a sequence comprising the steps of: (a) reading the sequence using a computer program wliich identifies one or more features in a sequence, wherein the sequence comprises a polypeptide sequence or a nucleic acid sequence of the invention; and (b) identifying one or more features in the sequence with the computer program.
  • the invention provides methods for comparing a first sequence to a second sequence comprising the steps of: (a) reading the first sequence and the second sequence through use of a computer program which compares sequences, wherein the first sequence comprises a polypeptide sequence or a nucleic acid sequence of the invention; and (b) determining differences between the first sequence and the second sequence with the computer program.
  • the step of determining differences between the first sequence and the second sequence can further comprise the step of identifying polymorphisms.
  • the method can further comprise an identifier that identifies one or more features in a sequence.
  • the method can comprise reading the first sequence using a computer program and identifying one or more features in the sequence.
  • the invention provides methods for isolating or recovering a nucleic acid encoding a polypeptide having a biofilm control activity from an environmental sample comprising the steps of: (a) providing an amplification primer sequence pair for amplifying a nucleic acid encoding a polypeptide of the invention or a nucleic acid of the invention; (b) isolating a nucleic acid from the environmental sample or treating the environmental sample such that nucleic acid in the sample is accessible for hybridization to the amplification primer pair; and, (c) combining the nucleic acid of step (b) with the amplification primer pair of step (a) and amplifying nucleic acid from the environmental sample, thereby isolating or recovering a nucleic acid encoding a polypeptide having a biofilm control activity from an environmental sample.
  • One or each member of the amplification primer sequence pair can comprise an oligonucleotide comprising at least about 10 to 50 consecutive bases of a nucleic acid encoding a polypeptid
  • the mvention provides methods for isolating or recovering a nucleic acid encoding a polypeptide having a biofilm control activity from an environmental sample comprising the steps of: (a) providing a polynucleotide probe comprising a nucleic acid of the mvention or a subsequence thereof; (b) isolating a nucleic acid from the environmental sample or treating the environmental sample such that nucleic acid in the sample is accessible for hybridization to a polynucleotide probe of step (a); (c) combining the isolated nucleic acid or the treated environmental sample of step (b) with the polynucleotide probe of step (a); and (d) isolating a nucleic acid that specifically hybridizes with the polynucleotide probe of step (a), thereby isolating or recovering a nucleic acid encoding a polypeptide having an biofilm control activity from an environmental sample.
  • the invention provides methods of generating a variant of a nucleic acid encoding a polypeptide having a biofilm control, or other, activity comprising the steps of: (a) providing a template nucleic acid comprising a nucleic acid of the invention or a nucleic acid encoding a polypeptide of the invention; and (b) modifying, deleting or adding one or more nucleotides in the template sequence, or a combination thereof, to generate a variant of the template nucleic acid.
  • the method can further comprise expressing the variant nucleic acid to generate a variant polypeptide.
  • the modifications, additions or deletions can be introduced by a method comprising enor- prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, gene site saturated mutagenesis (GSSM), synthetic ligation reassembly (SLR) or a combination thereof.
  • the modifications, additions or deletions are introduced by a method comprising recombination, recursive sequence recombination, phosphothioate- modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid multimer creation and a combination thereof.
  • the method can be iteratively repeated until a polypeptide having an altered or different activity or an altered or different stability from that of a polypeptide encoded by the template nucleic acid is produced.
  • the variant polypeptide is thermotolerant, and retains some activity after being exposed to an elevated temperature.
  • the variant polypeptide has increased glycosylation as compared to the polypeptide encoded by a template nucleic acid.
  • the variant polypeptide has an activity under a high temperature, wherein the polypeptide encoded by the template nucleic acid is not active under the high temperature.
  • the method can be iteratively repeated until a polypeptide coding sequence having an altered codon usage from that of the template nucleic acid is produced.
  • the method can be iteratively repeated until a gene having higher or lower level of message expression or stability from that of the template nucleic acid is produced.
  • the invention provides methods for modifying codons in a nucleic acid encoding a polypeptide of the invention to increase its expression in a host cell, the method comprising the following steps: (a) providing a nucleic acid of the invention; and, (b) identifying a non-prefened or a less prefened codon in the nucleic acid of step (a) and replacing it with a prefened or neutrally used codon encodmg the same amino acid as the replaced codon, wherein a prefened codon is a codon over-represented in coding sequences in genes in the host cell and a non-prefened or less prefened codon is a codon under-represented in coding sequences in genes in the host cell, thereby modifying the nucleic acid to increase its expression in a host cell.
  • the mvention provides methods for modifying codons in a nucleic acid encoding a polypeptide of the invention to increase its expression in a host cell, the method comprising the following steps: (a) providing a nucleic acid of the invention encoding a polypeptide of the invention; and, (b) identifying a non-prefened or a less prefened codon in the nucleic acid of step (a) and replacing it with a prefened or neutrally used codon encoding the same amino acid as the replaced codon, wherein a prefened codon is a codon over-represented in coding sequences in genes in the host cell and a non-prefened or less prefened codon is a codon under-represented in coding sequences in genes in the host cell, thereby modifying the nucleic acid to increase its expression in a host cell.
  • the invention provides methods for modifying a codon in a nucleic acid encoding a polypeptide of the invention to decrease its expression in a host cell, the method comprising the following steps: (a) providing a nucleic acid of the invention; and (b) identifying at least one prefened codon in the nucleic acid of step (a) and replacing it with a non-prefened or less prefened codon encoding the same amino acid as the replaced codon, wherein a prefened codon is a codon over-represented in coding sequences in genes in a host cell and a non-prefened or less prefened codon is a codon under-represented in coding sequences in genes in the host cell, thereby modifying the nucleic acid to decrease its expression in a host cell.
  • the host cell can be a bacterial cell, a fungal cell, an insect cell, a yeast cell, a plant cell or a mammalian cell.
  • the invention provides methods for producing a library of nucleic acids encoding a plurality of modified active sites or substrate binding sites, wherein the modified active sites or substrate binding sites are derived from a first nucleic acid comprising a sequence encodmg a first active site or a first subsfrate binding site the method comprising the following steps: (a) providing a first nucleic acid encoding a first active site or first substrate binding site, wherein the first nucleic acid sequence comprises a nucleic acid of the invention, and the nucleic acid encodes an active site or a substrate binding site; (b) providing a set of mutagenic oligonucleotides that encode naturally- occurring amino acid variants at a plurality of targeted codons in the first nucleic acid; and, (c) using the set of mutagenic oligonu
  • the method comprises mutagenizing the first nucleic acid of step (a) by a method comprising an optimized directed evolution system, gene site-saturation mutagenesis (GSSM), synthetic ligation reassembly (SLR), enor-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, gene site saturated mutagenesis (GSSM), synthetic ligation reassembly (SLR) and a combination thereof.
  • GSSM gene site-saturation mutagenesis
  • SLR synthetic ligation reassembly
  • the method comprises mutagenizing the first nucleic acid of step (a) or variants by a method comprising recombination, recursive sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid multimer creation and a combination thereof.
  • the invention provides methods for modifying a small molecule comprising the following steps: (a) providing an enzyme of the invention; (b) providing a small molecule; and (c) reacting the enzyme of step (a) with the small molecule of step (b) under conditions that facilitate an enzymatic reaction catalyzed by the enzyme, thereby modifying a small molecule by an enzymatic reaction.
  • the method can comprise a plurality of small molecule substrates for the enzyme of step (a), thereby generating a library of modified small molecules produced by at least one enzymatic reaction catalyzed by an enzyme of the invention.
  • the method can comprise a plurality of additional enzymes under conditions that facilitate a plurality of biocatalytic reactions by the enzymes to form a library of modified small molecules produced by the plurahty of enzymatic reactions.
  • the method can further comprise the step of testing the library to determine if a particular modified small molecule which exhibits a desired activity is present within the library.
  • the step of testing the library can frirther comprise the steps of systematically eliminating all but one of the biocatalytic reactions used to produce a portion of the plurality of the modified small molecules within the library by testing the portion of the modified small molecule for the presence or absence of the particular modified small molecule with a desired activity, and identifying at least one specific biocatalytic reaction that produces the particular modified small molecule of desired activity.
  • the invention provides methods for deten ' ning a functional fragment of an enzyme of the invention comprising the steps of: (a) providing an enzyme of the invention; and (b) deleting a plurality of amino acid residues from the sequence of step (a) and testing the remaining subsequence for a biofilm control activity, thereby determining a functional fragment of the enzyme.
  • the activity is measured by providing a biofilm substrate and detecting a decrease in the amount of the substrate or an increase in the amount of a reaction product.
  • the invention provides methods for whole cell engineering of new or modified phenotypes by using real-time metabolic flux analysis, the method comprising the following steps: (a) making a modified cell by modifying the genetic composition of a cell, wherein the genetic composition is modified by addition to the cell of a nucleic acid of the invention; (b) culturing the modified cell to generate a plurality of modified cells; (c) measuring at least one metabolic parameter of the cell by monitoring the cell culture of step (b) in real time; and, (d) analyzing the data of step (c) to determine if the measured parameter differs from a comparable measurement in an unmodified cell under similar conditions, thereby identifying an engineered phenotype in the cell using real-time metabolic flux analysis.
  • the genetic composition of the cell can be modified by a method comprising deletion of a sequence or modification of a sequence in the cell, or, knocking out the expression of a gene.
  • the method can further comprise selecting a cell comprising a newly engineered phenotype.
  • the method can comprise culturing the selected cell, thereby generating a new cell strain comprising a newly engineered phenotype.
  • compositions of the invention can comprise at least one, several or all polypeptides of the invention, or, in alternative aspects, can comprise a polypeptide having a SurE activity, e.g., a polypeptide having a sequence as set forth in SEQ ID NO: 118; SEQ ID NO: 120; SEQ ID NO: 122; SEQ ID NO: 124; SEQ ID NO: 126 SEQ ID NO:128; SEQ ID NO:130; SEQ ID NO:132; SEQ ID NO:134; SEQ IDNO:136 SEQ ID NO: 138; SEQ ID NO: 140; SEQ ID NO: 142; SEQ ID NO: 144; SEQ ID NO: 146 SEQ ID NO: 148; SEQ ID NO: 150; SEQ ID NO: 152; SEQ ID NO: 154; SEQ ID NO: 156 SEQ ID NO:158; SEQ ID NO: 118; SEQ ID NO: 120; SEQ ID NO: 122; SEQ ID NO: 124; SEQ ID NO:
  • the invention provides biofilm confrol products for preventing or controlling biofilm accumulation on food processing equipment and medical devices.
  • the compositions and methods of the invention can be used to confrol, or prevent, microbial colonization of food processing equipment and medical devices. This can prevent a serious health threat, especially when biofilms harbor pathogenic organisms.
  • compositions e.g., the biofilm control products
  • the compositions of the invention are stable in the presence of chemical biocides and varying adverse reaction conditions.
  • the compositions and methods of the invention are used against mixed-species biofilms.
  • biofilm methods of the invention can be used to identify biofilm matrix-hydrolyzing enzymes, including polypeptides having an amidase, a cellulase, an esterase, a glycosidase, a xylanase, an amylase, a transaminase, a laminarinase, a beta-galactosidase, a beta-mannosidase, a pullulanase, and/or a phosphatase activity.
  • biofilm matrix-hydrolyzing enzymes including polypeptides having an amidase, a cellulase, an esterase, a glycosidase, a xylanase, an amylase, a transaminase, a laminarinase, a beta-galactosidase, a beta-mannosidase, a pullulanase, and/or a phosphatase activity.
  • the invention also provides methods and compositions for treating water processing systems, including regenerative water processing systems, using the enzymes of the invention, or, in alternative aspects, polypeptides having a SurE activity, e.g., a polypeptide having a sequence as set forth in SEQ ID NO: 118; SEQ ID NO: 120; SEQ ID NO:
  • SEQ ID NO:222 SEQ ID NO:222, or any combination thereof.
  • These enzymes can be used to remove or control biofilms in any part of any water processing system. Water processing systems and routing methods for monitoring biofilms in them are described, e.g., in U.S. Patent No. 6,498,862.
  • the methods and compositions of the invention are used in products to target biofilms and "slimes" found on equipment surfaces to prevent film formation and enable facile decontamination of water systems. These products can be used as enhancements to suitable chemical biocide chemicals.
  • the invention also provides methods and compositions for water freatment in the pulp and paper industry using the enzymes of the invention, or, in alternative aspects, polypeptides having a SurE activity, e.g., a polypeptide having a sequence as set forth in SEQ ID NO: 118; SEQ IDNO.120; SEQ IDNO:122; SEQ IDNO:124; SEQ ID NO: 118; SEQ ID NO: 118; SEQ IDNO.120; SEQ IDNO:122; SEQ IDNO:124; SEQ ID
  • These enzymes can be used to remove or confrol biofilms in any aspect of the pulp and paper industry involving water, such as water treatment in pulp and paper processing.
  • the methods and compositions of the invention can be used for the modification of existing paper and pulp mills, to increase stringency limits on effluents, to increase paper recycling, to increase demand for high quality paper, for water loop closure and as biostat enzymes at the research level.
  • the methods and compositions of the invention can be used to decrease pitch deposits, to lower chemical/maintain integrity and usage in retention aids, sizing, pitch control, chemical biocides, wet-end size, drainage aids.
  • the methods and compositions of the invention can enable use of environment-friendly chemicals.
  • the invention also provides methods and compositions for treating or coating cooling systems; food and beverage processing systems; industrial processing systems (e.g., for water); pulp and paper mill systems; brewery pasteurizers; sweetwater systems; air washer systems; oil field drilling fluids and muds; petroleum recovery processes; industrial lubricants; cutting fluids; heat transfer systems; gas scrubber systems; latex systems; clay and pigment systems; decorative fountains; water intake pipes; ballast water tanks; and ship reservoirs, and the like.
  • industrial processing systems e.g., for water
  • pulp and paper mill systems e.g., for water
  • brewery pasteurizers e.g., for water
  • sweetwater systems e.g., air washer systems
  • oil field drilling fluids and muds e.g., oil field drilling fluids and muds
  • petroleum recovery processes e.g., industrial lubricants
  • cutting fluids e.g., heat transfer systems; gas scrubber systems; latex systems; clay and pigment systems; decorative fountains; water intake pipes; bal
  • the invention provides cooling systems; food and beverage processing systems; industrial processing systems (e.g., for water); pulp and paper mill systems; brewery pasteurizers; sweetwater systems; air washer systems; oil field drilling fluids and muds; petroleum recovery processes; industrial lubricants; cutting fluids; heat transfer systems; gas scrubber systems; latex systems; clay and pigment systems; decorative fountains; water intake pipes; ballast water tanks; and ship reservoirs, and the like comprising at least one polypeptide (e.g., enzymes and antibodies) of the invention, or, in alternative aspects, polypeptides having a SurE activity, e.g., a polypeptide having a sequence as set forth in SEQ ID NO:118; SEQ ID NO:120; SEQ ID NO:122; SEQ ID NO:124; SEQ ID NO:126 SEQ ID NO: 128; SEQ ID NO: 130; SEQ ID NO: 132; SEQ ID NO: 134; SEQ ID NO: 136 SEQ ID NO: 138; SEQ ID NO:
  • methods and compositions of the invention can be used in water management engineering to set the stage to utilize multiple and combined technologies and for reduced water usage and disposal.
  • the methods and compositions of the invention can be used to improve heat transfer efficiency, lower biocide chemical usage, decrease/eliminate pitting and conosion in pipes & equipment, reduce risk of human illness (e.g. Legionelld).
  • the invention also provides methods and compositions for treating drugs and pharmaceuticals, including tablets, pills, implants, suppositories, inhalers, sprays, ointments, and the like, using the enzymes of the mvention, and drugs and pharmaceuticals comprising these.
  • the polypeptides (e.g., enzymes and antibodies) of the invention can be used to remove or confrol biofilms from any medical device, drug or pharmaceutical.
  • the invention provides medical devices, drugs and pharmaceuticals comprising an enzyme of the invention, or, in alternative aspects, polypeptides having a SurE activity, e.g., a polypeptide having a sequence as set forth in SEQ ID NO: 118; SEQ ID NO: 118; SEQ ID NO: 118; SEQ ID NO: 118; SEQ ID NO: 118; SEQ ID NO: 118; SEQ ID NO: 118; SEQ ID NO: 118; SEQ ID NO: 118
  • compositions wherein it may be advantageous to prevent or remove a biofilm comprising an enzyme of the invention.
  • compositions can further comprise a antimicrobial agent or a antimicrobial composition e.g., rifamycins (e.g., rifampin), tefracyclines (e.g., minocycline), macrolides (e-g-, erythromycin), penicillins (e.g., nafcillin), cephalosporins (e.g., cefazolin), carbepenems (e.g., imipenem), monobactams (e.g., aztreonam), aminoglycosides (e.g., gentamicin), chloramphenicol, sulfonamides (e.g., sulfamethoxazole), glycopeptides (e.g., vanomycin), metronidazole, clind
  • compositions of the invention may further comprise microbial activity indicators which indicate the presence of microorganisms in or on the surface of the composition.
  • the invention provides medical devices comprising one or more enzymes ' of the invention, or, in alternative aspects, polypeptides having a SurE activity, e.g., a polypeptide having a sequence as set forth in SEQ ID NO: 118; SEQ ID NO: 120; SEQ ID NO:122; SEQ ID NO:124; SEQ ID NO:126; SEQ ID NO:128; SEQ ID NO:130; SEQ ID
  • the invention provides medical devices comprising one or more enzymes of the mvention, these medical devices including any device which may be inserted or implanted into a human being or other animal, or placed at the insertion or implantation site such as the skin near the insertion or implantation site, and which include at least one surface which is susceptible to colonization by biofilm embedded microorganisms.
  • enzymes of the invention can be used in conjunction with (e.g., be coated onto, use to treat) any surface which may be desired or necessary to prevent biofilm embedded microorganisms from growing or proliferating in or on at least one surface of a medical device or a drug or pharmaceutical, or to remove or clean biofilm embedded microorganisms from the at least one surface of a medical device or a drug or pharmaceutical, such as the surfaces of equipment in operating rooms, emergency rooms, hospital rooms, clinics, and bathrooms.
  • the mvention provides adhesives, such as tapes, comprising at least one of these enzymes (e.g., an enzyme of the invention).
  • compositions and solutions including buffer solutions (e.g., phosphate buffered saline), saline, water, polyvinyl, polyethylene, polyurethane, polypropylene, silicone (e.g., silicone elastomers and silicone adhesives), polycarboxylic acids, (e.g., polyacrylic acid, polymethacrylic acid, polymaleic acid, poly- (maleic acid monoester), polyaspartic acid, polyglutamic acid, aginic acid or pectimic acid), polycarboxylic acid anhydrides (e.g., polymaleic anhydride, polymethacrylic anhydride or polyacrylic acid anhydride), polyamines, polyamme ions (e.g., polyethylene imine, polyvinylarnine, polylysine, poly-(dialkylamine
  • compositions and methods of the invention can be used in biofilm reduction and to treat or prevent any surface film formation.
  • the compositions and methods of the invention can be used to treat or prevent biofilms on the surfaces of pipes, tanks, storage containers, and industrial and personal appliances, paints and coatings.
  • the invention also provides means to deliver an enzyme formulation of the invention.
  • the compositions and methods of the invention can be used for industrial water treatment, sanitizers & disinfectants and personal care.
  • compositions and methods of the invention can be used for primary industrial water treatments, including pulp and paper and cooling systems.
  • the compositions and methods of the invention can be used in conjunction with oxidizing biocides such as chlorine, bromine and sodium hypochlorite.
  • oxidizing biocides such as chlorine, bromine and sodium hypochlorite.
  • the compositions and methods of the invention can be used in conjunction with (e.g., for in pulp and paper treatments or cooling systems) organosulfur chemicals (e.g. dazomet, dithiocarbamates, MBT (methylene bis-thiocyanate) and benzothiazoles).
  • organosulfur chemicals e.g. dazomet, dithiocarbamates, MBT (methylene bis-thiocyanate) and benzothiazoles.
  • Other biocides in use include DBNPA (2,2-dibromo-3-nitrilopropionamide), glutaraldehyde and quaternary ammonia compounds.
  • compositions and methods of the invention can be used in conjunction with (e.g., for cooling systems) quaternary ammonium compounds such as cocobenzyl- dimethyl ammonium chloride and other chemicals such as BNPD (2-bromo-2- nitropropane-l,3-diol), DBNPA, glutaraldehyde, active halogens and phenolics.
  • quaternary ammonium compounds such as cocobenzyl- dimethyl ammonium chloride and other chemicals such as BNPD (2-bromo-2- nitropropane-l,3-diol), DBNPA, glutaraldehyde, active halogens and phenolics.
  • BNPD 2-bromo-2- nitropropane-l,3-diol
  • DBNPA glutaraldehyde
  • active halogens phenolics.
  • the compositions and methods of the invention can be used in sanitizers and disinfectants, including janitorial/
  • compositions and methods of the invention can be used to improve cleaning efficiency and decrease mechanical contact requirement and to decrease chemical usage.
  • the compositions and methods of the invention can be used in dairy and food processing products to dislodge and remove microbes, to decrease pitch deposits, to decrease chemical usage and to enable use of equipment and environment-friendly chemicals.
  • compositions and methods of the invention can be to increase usage of low temperature cleaners and to set higher cleanliness standards.
  • the compositions and methods of the invention can be used in conjunction with quaternary ammonium compounds, miscellaneous biocides such as glycine-based amphoterics, glyoxal, biguanides, followed by active halogens, phenolics, organic acids/salts, and organosulfur chemicals, and amine-based chemicals and organic acids/salts.
  • the compositions and methods of the invention can be used as preservatives in food, medicinal (e.g., drug), hygiene and cosmetic products.
  • the compositions and methods of the invention can be used in personal care products such as toothpastes, mouthwashes, dental appliance cleaners, contact lens cleaners.
  • compositions and methods of the invention can be used in any skin and tissue related environment, e.g., products used in the medical fields.
  • anti-biofilm enzymes can be used in products such as surgical implants, bone fixtures and catheters.
  • the compositions and methods of the invention can be used to dislodge and remove plaque from dental and oral surfaces, to prevent tartar formation and to decrease toxicity and skin irritation.
  • the mvention provides methods for treating (including removing, slowing the growth of or preventing the growth of) biofilms comprising contacting a composition (e.g., a water treatment device, a water conduit such as a pipe, a medical device, a drug, etc.) by contacting the composition with at least one polypeptide (e.g., antibody or enzyme) of the invention.
  • a composition e.g., a water treatment device, a water conduit such as a pipe, a medical device, a drug, etc.
  • the methods can comprise soaking, rinsing, flushing, submerging or washing with a composition (e.g., a solution, fluid, gas, spray) comprising at least one polypeptide of the invention.
  • a composition e.g., a solution, fluid, gas, spray
  • the composition can be contacted with a biofilm control composition of the invention for a period of time sufficient to remove some, or, substantially all, of the biofilm, including, e.g., embedded microorganisms.
  • the composition can be submerged in a biofilm confrol composition of the invention for at least 1, 5, 10, 15, 20, 30, 40, 50 or 60 or more minutes.
  • a composition e.g., medical device, water pipe
  • the biofilm control composition of the invention may be poured into the pipe or tubing and both ends of the pipe or tube sealed or clamped such that the biofilm control composition of the invention is retained within the lumen of the pipe or tube.
  • the pipe or tube is then allowed to remained filled with the biofilm control composition of the invention for a period of time sufficient to remove some or, substantially all, of the biofilm embedded microorganisms, e.g., from at least one surface.
  • the treatment can last from at least about 1 minute to about 48 or more hours.
  • Figure 1 is a block diagram of a computer system.
  • Figure 2 is a flow diagram illustrating one aspect of a process for comparing a new nucleotide or protein sequence with a database of sequences in order to determine the homology levels between the new sequence and the sequences in the database.
  • Figure 4 is a flow diagram illustrating one aspect of an identifier process 300 for detecting the presence of a feature in a sequence.
  • Figure 5 illustrates four exemplary biofilm micro-assays, as described in Example 1, below, how enzymes are evaluated in four separate biofilm micro-assays to measure enzymatic control or enzymatic removal of biofilms formed by gram-negative (Pseudomonas fluorescens) and gram-positive (Staphylococcus epidermidis) bacteria.
  • Figure 6 illustrates a summarization of data evaluating well-to-well variation in biofilm growth in a 96-well microtiter plate, as described in Example 2, below; the bar graph shows the relative fluorescence units for each well in the microtiter plate; the numbers along the x-axis designate the column number.
  • Figure 7 illustrates dose response data using a protease in a biofilm confrol micro-assay with Pseudomonas fluorescens, as described in Example 2, below.
  • Figure 8 illustrates data obtained from a biofilm removal micro-assay assay using P. fluorescens biofilm, as described in Example 2, below.
  • Figure 9 is a table summarizing primary and secondary hits identified in biofilm micro-assay screening, as described in Example 2, below.
  • Figure 10 illustrates an exemplary reactor, a drip-flow reactor, as described in Example 2, below.
  • Figure 11 is a chart summary and description of exemplary nucleic acids and polypeptides of the invention, including initial sources from which they were isolated, exemplary activities, and sequence comparisons to known nucleic acids and proteins.
  • Figure 12 shows enzyme activity against biofilm measured as initial rate of reaction at varying biocide concentrations, as discussed in further detail, below.
  • Figure 13 shows enzyme activity against biofilm measured as initial rate of reaction at varying biocide concentrations, as discussed in further detail, below.
  • Figure 14 shows enzyme activity against biofilm measured as initial rate of reaction at varying biocide concentrations, as discussed in further detail, below.
  • Figure 15 shows enzyme activity against biofilm measured as initial rate of reaction at varying biocide concentrations, as discussed in frirther detail, below.
  • Figure 16 shows enzyme activity against biofilm measured as initial rate of reaction at varying biocide concentrations, as discussed in further detail, below.
  • Figure 17 shows enzyme activity against biofilm measured as initial rate of reaction at varying biocide concentrations, as discussed in further detail, below.
  • biofilm confrol compositions e.g., enzymes and antibodies
  • polynucleotides encoding the polypeptides (e.g., enzymes and antibodies) for the control of biofilms
  • polynucleotides and methods of making and using these polynucleotides and polypeptides The invention provides enzymes and methods for biofilm control, including to prevent or slow the growth of biofilm formation and/or to completely or partially remove an established biofihn or to disrupt a biofilm.
  • these proteins are biofilm matrix-hydrolyzmg enzymes.
  • the invention provides products comprising these biofilm control compositions.
  • the biofilm-control compositions of the invention have an amidase activity, e.g., the ability to hydrolyze amides, including enzymes having secondary amidase activity, including a peptidase, a protease and/or a hydantomase activity.
  • an amidase activity e.g., the ability to hydrolyze amides, including enzymes having secondary amidase activity, including a peptidase, a protease and/or a hydantomase activity.
  • the biofilm control compositions of the invention have a deacetylase, an amidase, a cellulase, an esterase, a glycosidase, a xylanase, an amylase, a transaminase, a laminarinase, a beta-galactosidase, a beta-mannosidase, a pullulanase, and/or a phosphatase activity. While the invention is not limited to any particular mechamsm of action, the biofilm-control compositions of the invention can have a biofilm matrix degrading activity.
  • biofilm control includes partial or complete biofilm removal, preventing or slowing the formation of biofilms, disruption of a biofilm (e.g., making it less adherent and thus easier to wash off a surface) or to make a biofilm more susceptible to another anti-biofilm freatment or reagent (such as a drug or toxic compound) or any variation thereof.
  • biofilm control compositions of the invention comprise polypeptides encoded by at least one nucleic acid of the invention (e.g., SEQ ID NO:l, SEQ ID NO:9, SEQ ID NO:l l, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:l, SEQ ID NO:9, SEQ ID NO:l l, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID
  • compositions of the invention can be used in and the methods of the invention can be practiced with "effective amounts” or "effective concentrations” of the nucleic acids or polypeptide of the invention.
  • an "effective amount” or an a “effective concentration” of biofilm confrol composition of the invention is used in the method or composition.
  • An “effective concentration” can be a sufficient amount of the biofilm control composition of the invention to prevent (e.g., substantially prevent) the attachment, viability, growth or proliferation of a microorganism involved in biofilm attachment, growth and/or survival.
  • the biofilm confrol composition of the invention can be on the at least one surface of a composition (e.g., a medical device or water pipe), e.g., it can be a coating.
  • biofilm confrol composition of the invention can be a sufficient amount of biofilm confrol composition of the invention to substantially penetrate, or break-up, the biofilm on or in a composition (e.g., on or in at least one surface of a medical device).
  • the effective concentration facilitates access of the biofilm confrol composition of the invention, and, in some aspect, other antimicrobial agents, and/or antifungal agents.
  • the amount can vary for each use or biofilm confrol composition of the invention. Routine screening can be used to determine an effective amount or concentration for each biofilm confrol composition of the invention for any particular application or method; see, e.g., U.S.
  • 6,326,190 describing a method where bacteria are incubated to form a biofilm on biofilm adherent sites by providing a flow of liquid growth medium across the sites, the direction of the flow of liquid being repeatedly changed, and an assay made of the resulting biofilm. See also U.S. Patent No. 6,405,582, describing o method and apparatus for detennining the deposition of organic and inorganic contaminants, such as biofilm, on surfaces such as water pipes and tanks.
  • the biofilm-control enzymes of the invention can have activity in alkaline pHs as high as pH 9.5, pH 10, pH 10.5, and pH 0 11.
  • the biofilm-control enzymes of the invention are active in the temperature range of between about 40°C to about 70°C under conditions of low water activity (low water content).
  • biofilm-control enzymes generated by the methods of the invention can have altered enzymatic activity, thermal stability, pH/activity profile, pH/stability profile (such as increased stability at low, e.g. pH ⁇ 6 or ⁇ H ⁇ 5, or high, e.g. pH>9, pH values), stability towards oxidation, Ca 2+ dependency, specific activity and the like.
  • the invention provides for altering any property of interest. For instance, the alteration may result in a 0 variant which, as compared to a parent enzyme, has altered enzymatic activity, or, pH or temperature activity profiles.
  • biofilm-control enzyme includes all polypeptides, e.g., enzymes, catalytic antibodies, and the like, having a biofilm-control activity, including disrupting, slowing, preventing or otherwise modifying the growth, structure or compositional nature of a biofilm, including the complete dissolution of a biofilm, or, simply modifying the architecture or chemical (compositional) makeup of a biofilm.
  • the biofilm can be on any surface (e.g., a medical device, a tooth, a utensil, an implant, a surgical device), in a composition (e.g., a food, a sponge, a gel, an animal tissue, a tooth) or suspended in a solution or a gel.
  • the biofilm can be on any surface, natural (e.g. a tooth or a tissue, e.g., an organ or a scar tissue) or a product of manufacture (e.g., a medical device, implant or instrument), a pharmaceutical (e.g., a tablet, a pill, an implant, a suppository) or a food or a food processing equipment.
  • the term "biofilm-control enzyme” includes enzymes that catalyze the hydrolysis of amides.
  • biofilm-control enzyme includes polypeptides having secondary amidase activity, e.g., having activity in the hydrolysis of amides.
  • the term includes enzymes having a peptidase, a protease and/or a hydantoinase activity.
  • the term includes enzymes having a cellulase or an esterase activity.
  • antibody includes antigen-binding portions, i.e., "antigen binding sites,” (e.g., fragments, subsequences, complementarity determining regions (CDRs)) that retain capacity to bind antigen, including (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR).
  • Antigen binding sites e.g., fragments, subs
  • a "coding sequence of or a “sequence encodes” a particular polypeptide or protein is a nucleic acid sequence which is transcribed and translated into a polypeptide or protein when placed under the confrol of appropriate regulatory sequences.
  • a "vector” comprises a nucleic acid which can infect, transfect, transiently or permanently transduce a cell. It will be recognized that a vector can be a naked nucleic acid, or a nucleic acid complexed with protein or lipid.
  • the vector optionally comprises viral or bacterial nucleic acids and/or proteins, and/or membranes (e.g., a cell membrane, a viral lipid envelope, etc.).
  • Vectors include, but are not limited to replicons (e.g., RNA replicons, bacteriophages) to which fragments of DNA may be attached and become replicated.
  • tissue-specific promoters are transcriptional confrol elements that are only active in particular cells or tissues or organs, e.g., in plants or animals. Tissue- specific regulation may be achieved by certain intrinsic factors which ensure that genes encoding proteins specific to a given tissue are expressed. Such factors are known to exist in mammals and plants so as to allow for specific tissues to develop.
  • Plasmids can be commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. Equivalent plasmids to those described herein are known in the art and will be apparent to the ordinarily skilled artisan.
  • gene includes a nucleic acid sequence comprising a segment of DNA involved in producing a transcription product (e.g., a message), which in turn is translated to produce a polypeptide chain, or regulates gene transcription, reproduction or stability.
  • Genes can include regions preceding and following the coding region, such as leader and trailer, promoters and enhancers, as well as, where applicable, intervening sequences (introns) between individual coding segments (exons).
  • nucleic acid or “nucleic acid sequence” includes oligonucleotide, nucleotide, polynucleotide, or to a fragment of any of these, to DNA or RNA (e.g., mRNA, rRNA, tRNA) of genomic or synthetic origin which may be single- stranded or double-stranded and may represent a sense or antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material, natural or synthetic in origin, including, e.g., iRNA, ribonucleoproteins (e.g., iRNPs).
  • DNA or RNA e.g., mRNA, rRNA, tRNA
  • PNA peptide nucleic acid
  • DNA-like or RNA-like material natural or synthetic in origin, including, e.g., iRNA, ribonucleoproteins (e.g., iRNPs).
  • nucleic acids i.e., oligonucleotides, containing known analogues of natural nucleotides.
  • the term also encompasses nucleic-acid-like structures with synthetic backbones, see e.g., Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197; Strauss-Soukup (1997) Biochemistry 36:8692-8698; Straussense Nucleic Acid Drug Dev 6:153- 156.
  • amino acid or amino acid sequence include an oligopeptide, peptide, polypeptide, or protein sequence, or to a fragment, portion, or subunit of any of these, and to naturally occurring or synthetic molecules.
  • a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
  • an isolated material or composition can also be a "purified" composition, i.e., it does not require absolute purity; rather, it is intended as a relative definition.
  • Individual nucleic acids obtained from a library can be conventionally purified to electrophoretic homogeneity.
  • the invention provides nucleic acids which have been purified from genomic DNA or from other sequences in a library or other environment by at least one, two, three, four, five or more orders of magnitude.
  • nucleic acids can include nucleic acids adjacent to a “backbone” nucleic acid to which it is not adjacent in its natural environment.
  • nucleic acids represent 5% or more of the number of nucleic acid inserts in a population of nucleic acid "backbone molecules.”
  • Backbone molecules include nucleic acids such as expression vectors, self-replicating nucleic acids, viruses, integrating nucleic acids, and other vectors or nucleic acids used to maintain or manipulate a nucleic acid insert of interest.
  • the enriched nucleic acids represent 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or more of the number of nucleic acid inserts in the population of recombinant backbone molecules.
  • Recombinanf ' polypeptides or proteins refer to polypeptides or proteins produced by recombinant DNA techniques; e.g., produced from cells transformed by an exogenous DNA construct encoding the desired polypeptide or protein.
  • synthetic polypeptides or protein are those prepared by chemical synthesis, as described in further detail, below.
  • a promoter sequence can be "operably linked to" a coding sequence when RNA polymerase which initiates transcription at the promoter will transcribe the coding sequence into mRNA, as discussed further, below.
  • Oligonucleotide includes either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus will not Ugate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide can ligate to a fragment that has not been dephosphorylated.
  • nucleic acid and polypeptide sequences having substantial identity to an exemplary sequence of the invention over a region of at least about 10, 20, 30, 40, 50,
  • Nucleic acid sequences of the invention can be substantially identical over the entire length of a polypeptide coding region.
  • a "substantially identical" amino acid sequence also can include a sequence that differs from a reference sequence by one or more conservative or non- conservative amino acid substitutions, deletions, or insertions, particularly when such a substitution occurs at a site that is not the active site of the molecule, and provided that the polypeptide essentially retains its functional properties.
  • a conservative amino acid substitution substitutes one amino acid for another of the same class (e.g., substitution of one hydrophobic amino acid, such as isoleucine, valine, leucine, or metl-ionine, for another, or substitution of one polar amino acid for another, such as substitution of argierine for lysine, glutamic acid for aspartic acid or glutamine for asparagine).
  • One or more amino acids can be deleted, for example, from a biofilm- control enzyme, resulting in modification of the structure of the polypeptide, without significantly altering its biological activity. For example, amino- or carboxyl-terminal amino acids that are not required for biofilm-confrol enzyme activity can be removed.
  • Hybridization includes the process by which a nucleic acid strand joins with a complementary strand through base pairing. Hybridization reactions can be sensitive and selective so that a particular sequence of interest can be identified even in samples in which it is present at low concentrations. Stringent conditions can be defined by, for example, the concentrations of salt or formamide in the prehybridization and hybridization solutions, or by the hybridization temperature, and are well known in the art. For example, stringency can be increased by reducing the concentration of salt, increasing the concentration of formamide, or raising the hybridization temperature, altering the time of hybridization, as described in detail, below.
  • optical directed evolution system or “optimized directed evolution” includes a method for reassembling fragments of related nucleic acid sequences, e.g., related genes, and explained in detail, below.
  • SLR synthetic ligation reassembly
  • the invention provides nucleic acids, including expression cassettes such as expression vectors and the like, encoding enzymes, e.g., biofilm-control enzyme polypeptides, and polypeptides having biofilm control or biofilm modifying activity.
  • the invention also includes methods for discovering new biofilm-control enzyme sequences using the nucleic acids of the invention.
  • the invention also includes methods for inhibiting the expression of biofilm-control enzyme genes, transcripts and polypeptides using the nucleic acids of the invention.
  • methods for modifying the nucleic acids of the invention by, e.g., synthetic ligation reassembly, optimized directed evolution system and/or saturation mutagenesis.
  • the invention provides novel nucleic acids and the polypeptides (e.g., enzymes, such as biofilm control enzymes) they encode.
  • the polypeptides and nucleic acids of the invention can be initially isolated from an environmental or a microbial source, e.g., an archaeon or bacterial source.
  • the following table summarizes the initial source of exemplary nucleic acids and polypeptides of the invention (e.g., the nucleic acid having a sequence as set forth in SEQ ID NO: 139 encodes a polypeptide as set forth in SEQ ID NO: 140, and was initially isolated from an archaeon, etc.; the nucleic acid having a sequence as set forth in SEQ ID NO:45 encodes a polypeptide as set forth in SEQ ID NO:46, and was initially isolated from an bacterial source, etc.; and, the nucleic acid having a sequence as set forth in SEQ ID NO:37 encodes a polypeptide as set forth in SEQ ID NO: 38, and was initially isolated from an environmental source.
  • the nucleic acid having a sequence as set forth in SEQ ID NO: 139 encodes a polypeptide as set forth in SEQ ID NO: 140, and was initially isolated from an archaeon, etc.
  • the nucleic acid having a sequence as set forth in SEQ ID NO:45 encode
  • nucleic acids of the invention can be made, isolated and/or manipulated by, e.g., cloning and expression of cDNA libraries, amplification of message or genomic DNA by PCR, and the like.
  • homologous genes can be modified by mampulating a template nucleic acid, as described herein.
  • the invention can be practiced in conjunction with any method or protocol or device known in the art, which are well described in the scientific and patent literature.
  • RNA, iRNA, antisense nucleic acid, cDNA, genomic DNA, vectors, viruses or hybrids thereof may be isolated from a variety of sources, genetically engineered, amplified, and/or expressed/ generated recombinantly. Recombinant polypeptides generated from these nucleic acids can be individually isolated or cloned and tested for a desired activity. Any recombinant expression system can be used, including bacterial, mammalian, yeast, insect or plant cell expression systems.
  • these nucleic acids can be synthesized in vitro by well- ' known chemical synthesis techniques, as described in, e.g., Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Terra. Lett. 22:1859; U.S. Patent No. 4,458,066.
  • nucleic acids such as, e.g., subcloning, labeling probes (e.g., random-primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization and the like are well described in the scientific and patent literature, see, e.g., Sambrook, ed., MOLECULAR CLONING: A
  • Another useful means of obtaining and manipulating nucleic acids used to practice the methods of the invention is to clone from genomic samples, and, if desired, screen and re-clone inserts isolated or amplified from, e.g., genomic clones or cDNA clones.
  • Sources of nucleic acid used in the methods of the invention include genomic or cDNA libraries contained in, e.g., mammalian artificial chromosomes (MACs), see, e.g., U.S. Patent Nos. 5,721,118; 6,025,155; human artificial chromosomes, see, e.g., Rosenfeld (1997) Nat. Genet.
  • MACs mammalian artificial chromosomes
  • Detection and purification facilitating domains include, e.g., metal chelating peptides such as polyhistidine tracts and histidine- tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle WA).
  • metal chelating peptides such as polyhistidine tracts and histidine- tryptophan modules that allow purification on immobilized metals
  • protein A domains that allow purification on immobilized immunoglobulin
  • the domain utilized in the FLAGS extension/affinity purification system Immunex Corp, Seattle WA.
  • the inclusion of a cleavable linker sequences such as Factor Xa or enterokinase (Invitrogen, San Diego CA) between a purification domain and the motif-comprising peptide or polypeptide to facilitate purification.
  • an expression vector can include an epitope-encoding nucleic acid sequence linked to six histidine residues followed by a thioredoxin and an enterokinase cleavage site (see e.g., Williams (1995) Biochemistry 34:1787-1797; Dobeli (1998) Protein Expr. Purif. 12:404-414).
  • the histidine residues facilitate detection and purification while the enterokinase cleavage site provides a means for purifying the epitope from the remainder of the fusion protein.
  • Technology pertaining to vectors encodmg fusion proteins and application of fusion proteins are well described in the scientific and patent literature, see e.g., Kroll (1993) DNA Cell. Biol., 12:441-53.
  • the invention provides nucleic acid (e.g., DNA) sequences of the invention operatively linked to expression (e.g., transcriptional or translational) control sequence(s), e.g., promoters or enhancers, to direct or modulate RNA synthesis/ expression.
  • expression control sequence can be in an expression vector.
  • Exemplary bacterial promoters include lad, lacZ, T3, T7, gpt, lambda PR, PL and tip.
  • Exemplary eukaryotic promoters include CMN immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein I.
  • the mvention provides expression cassettes that can be expressed in a tissue-specific manner, e.g., that can express a biofilm-control enzyme of the invention in a tissue-specific manner.
  • the invention also provides plants or seeds that express a biofilm-control enzyme of the invention in a tissue-specific manner.
  • the tissue- specificity can be seed specific, stem specific, leaf specific, root specific, fruit specific and the like.
  • a constitutive promoter such as the CaMV 35S promoter can be used for expression in specific parts of the plant or seed or throughout the plant.
  • a plant promoter fragment can be employed which will direct expression of a nucleic acid in some or all tissues of a plant, e.g., a regenerated plant.
  • constitutive promoters are refened to herein as “constitutive” promoters and are active under most environmental conditions and states of development or cell differentiation.
  • constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the 1'- or 2'- promoter derived from T-D ⁇ A of Agrobacterium tumefaciens, and other transcription initiation regions from various plant genes known to those of skill.
  • Such genes include, e.g., ACTll from Arabidopsis (Huang (1996) Plant Mol. Biol. 33: 125-139); Cat3 from Arabidopsis (GenBank No. U43147, Zhong (1996) Mol. Gen. Genet.
  • the invention uses tissue-specific or constitutive promoters derived from viruses which can include, e.g., the tobamovirus subgenomic promoter (Kumagai (1995) Proc. Natl. Acad. Sci. USA 92: 1679-1683; the rice tungro bacilliform virus (RTBV), which replicates only in phloem cells in infected rice plants, with its promoter which drives strong phloem-specific reporter gene expression; the cassava vein mosaic virus (CVMV) promoter, with highest activity in vascular elements, in leaf mesophyll cells, and in root tips (Verdaguer (1996) Plant Mol. Biol. 31 : 1129-1139).
  • viruses which can include, e.g., the tobamovirus subgenomic promoter (Kumagai (1995) Proc. Natl. Acad. Sci. USA 92: 1679-1683; the rice tungro bacilliform virus (RTBV), which replicates only in phl
  • the plant promoter may direct expression of biofilm-confrol enzyme-expressing nucleic acid in a specific tissue, organ or cell type (i.e. tissue-specific promoters) or may be otherwise under more precise environmental or developmental control or under the control of an inducible promoter.
  • tissue-specific promoters examples include anaerobic conditions, elevated temperature, the presence of light, or sprayed with chemicals/hormones.
  • the invention incorporates the drought-inducible promoter of maize (Busk (1997) supra); the cold, drought, and high salt inducible promoter from potato (Kirch (1997) Plant Mol. Biol. 33:897 909).
  • nucleic acids of the invention are operably linked to a promoter active primarily only in cotton fiber cells.
  • nucleic acids of the invention are operably linked to a promoter active primarily during the stages of cotton fiber cell elongation, e.g., as described by Rinehart (1996) supra.
  • the nucleic acids can be operably linked to the
  • a leaf-specific • promoter see, e.g., Busk (1997) Plant J. 11 : 1285 1295, describing a leaf-specific promoter in maize
  • the ORF 13 promoter from Agrobacterium rhizogenes which exhibits high activity in roots
  • a tomato promoter active during fruit ripening, senescence and abscission of leaves and, to a lesser extent, of flowers can be used (see, e.g., Blume (1997) Plant J. 12:731 746); a pistil-specific promoter from the potato SK2 gene (see, e.g., Ficker (1997) Plant Mol. Biol.
  • plant promoters which are inducible upon exposure to plant hormones, such as auxins, are used to express the nucleic acids of the invention.
  • the invention can use the auxin-response elements El promoter fragment (AuxREs) in the soybean (Glycine max L.) (Liu (1997) Plant Physiol. 115:397-407); the auxin-responsive Arabidopsis GST6 promoter (also responsive to salicylic acid and hydrogen peroxide) (Chen (1996) Plant J. 10: 955-966); the auxin-inducible parC promoter from tobacco (Sakai (1996) 37:906-913); a plant biotin response element (Sokot (1997) Mol. Plant Microbe Interact. 10:933-937); and, the promoter responsive to the stress hormone abscisic acid (Sheen (1996) Science 274:1900-1902).
  • auxin-response elements El promoter fragment AuxREs
  • the invention can use the auxin-response elements El
  • the nucleic acids of the invention can also be operably linked to plant promoters which are inducible upon exposure to chemicals reagents which can be applied to the plant, such as herbicides or antibiotics.
  • plant promoters which are inducible upon exposure to chemicals reagents which can be applied to the plant, such as herbicides or antibiotics.
  • the maize In2-2 promoter activated by benzenesulfonamide herbicide safeners, can be used (De Veylder (1997) Plant Cell Physiol. 38:568-577); application of different herbicide safeners induces distinct gene expression patterns, including expression in the root, hydathodes, and the shoot apical meristem.
  • Coding sequence can be under the control of, e.g., a tetracycline-inducible promoter, e.g., as described with transgenic tobacco plants containing t Avena sativa L. (oat) arginine decarboxylase gene (Masgrau (1997) Plant J. 11:465-473); or, a salicylic acid-responsive element (Stange (1997) Plant J. 11:1315-1324).
  • a tetracycline-inducible promoter e.g., as described with transgenic tobacco plants containing t Avena sativa L. (oat) arginine decarboxylase gene (Masgrau (1997) Plant J. 11:465-473); or, a salicylic acid-responsive element (Stange (1997) Plant J. 11:1315-1324).
  • chemically- (e.g., hormone- or pesticide-) induced promoters i.e., promoter responsive to a chemical which
  • the invention also provides for transgenic plants containing an inducible gene encoding for polypeptides of the invention whose host range is limited to target plant species, such as corn, rice, barley, wheat, potato or other crops, inducible at any stage of development of the crop.
  • tissue-specific plant promoter may drive expression of operably linked sequences in tissues other than the target tissue.
  • a tissue-specific promoter is one that drives expression preferentially in the target tissue or cell type, but may also lead to some expression in other tissues as well.
  • the nucleic acids of the invention can also be operably linked to plant promoters which are inducible upon exposure to chemicals reagents.
  • These reagents include, e.g., herbicides, synthetic auxins, or antibiotics which can be applied, e.g., sprayed, onto transgenic plants.
  • Inducible expression of the biofilm-confrol enzyme- producing nucleic acids of the invention will allow the grower to select plants with desired biofilm-control activity. The development of plant parts can thus controlled. In this way the invention provides the means to facilitate the harvesting of plants and plant parts.
  • polyadenylation region at the 3 '-end of the coding region should be included.
  • the polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from genes in the Agrobacterial T-DNA. Expression vectors and cloning vehicles
  • the invention provides expression vectors and cloning vehicles comprising nucleic acids of the invention, e.g., sequences encoding the biofilm-control enzymes and antibodies of the invention.
  • Expression vectors and cloning vehicles of the invention can comprise viral particles, baculovirus, phage, plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes, viral DNA (e.g., vaccinia, adenovirus, foul pox virus, pseudorabies and derivatives of SV40), PI -based artificial chromosomes, yeast plasmids, yeast artificial chromosomes, and any other vectors specific for specific hosts of interest (such as bacillus, Aspergillus and yeast).
  • Vectors of the invention can include chromosomal, non-chromosomal and synthetic DNA sequences. Large numbers of suitable vectors are known to those of skill in the art, and are commercially available. Exemplary vectors are include: bacterial: pQE vectors (Qiagen), pBluescript plasmids, pNH vectors, (lambda-ZAP vectors (Sfratagene); ⁇ trc99a, pKK223-3, pDR540, pRIT2T (Pharmacia); Eukaryotic: pXTl, pSG5 (Sfratagene), pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia). However, any other plasmid or other vector may be used so long as they are replicable and viable in the host. Low copy number or high copy number vectors may be employed with the present invention.
  • the expression vector can comprise a promoter, a ribosome binding site for translation initiation and a transcription terminator.
  • the vector may also include appropriate sequences for amplifying expression.
  • Mammalian expression vectors can comprise an origin of replication, any necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking non-transcribed sequences.
  • DNA sequences derived from the SV40 splice and polyadenylation sites may be used to provide the required non-transcribed genetic elements.
  • the expression vectors contain one or more selectable marker genes to permit selection of host cells containing the vector.
  • selectable markers include genes encoding dihydrofolate reductase or genes conferring neomycin resistance for eukaryotic cell culture, genes conferring tetracycline or ampicillin resistance in E. coli, and the S. cerevisiae TRPl gene.
  • Promoter regions can be selected from any desired gene using chloramphenicol fransferase (CAT) vectors or other vectors with selectable markers.
  • CAT chloramphenicol fransferase
  • Vectors for expressing the polypeptide or fragment thereof in eukaryotic cells can also contain enhancers to increase expression levels.
  • Enhancers are cis-acting elements of DNA, usually from about 10 to about 300 bp in length that act on a promoter to increase its transcription. Examples include the SV40 enhancer on the late side of the replication origin bp 100 to 270, the cytomegalo virus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancers.
  • a nucleic acid sequence can be inserted into a vector by a variety of procedures. In general, the sequence is ligated to the desired position in the vector following digestion of the insert and the vector with appropriate restriction endonucleases.
  • blunt ends in both the insert and the vector may be ligated.
  • a variety of cloning techniques are known in the art, e.g., as described in Ausubel and Sambrook. Such procedures and others are deemed to be within the scope of those skilled in the art.
  • the vector can be in the form of a plasmid, a viral particle, or a phage.
  • Other vectors include chromosomal, non-chromosomal and synthetic DNA sequences, derivatives of SV40; bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
  • a variety of cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by, e.g., Sambrook.
  • Particular eukaryotic vectors include pSV2CAT, ⁇ OG44, pXTl, pSG (Sfratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia).
  • any other vector may be used as long as it is replicable and viable in the host cell.
  • the nucleic acids of the invention can be expressed in expression cassettes, vectors or viruses and transiently or stably expressed in plant cells and seeds.
  • One exemplary transient expression system uses episomal expression systems, e.g., cauliflower mosaic virus (CaMV) viral RNA generated in the nucleus by transcription of an episomal mini-chromosome containing supercoiled DNA, see, e.g., Covey (1990) Proc. Natl. Acad. Sci. USA 87:1633-1637.
  • coding sequences, i.e., all or sub-fragments of sequences of the invention can be inserted into a plant host cell genome becoming an integral part of the host chromosomal DNA.
  • Sense or antisense transcripts can be expressed in this manner.
  • a vector comprising the sequences (e.g., promoters or coding regions) from nucleic acids of the invention can comprise a marker gene that confers a selectable phenotype on a plant cell or a seed.
  • the marker may encode biocide resistance, particularly antibiotic resistance, such as resistance to kanamycin, G418, bleomycin, hygromycin, or herbicide resistance, such as resistance to chlorosulfuron or Basta.
  • Expression vectors capable of expressing nucleic acids and proteins in plants are well known in the art, and can include, e.g., vectors from Agrobacterium spp., potato virus X (see, e.g., Angell (1997) EMBO J. 16:3675-3684), tobacco mosaic virus (see, e.g., Casper (1996) Gene 173:69-73), tomato bushy stunt virus (see, e.g., Hillman (1989) Virology 169:42-50), tobacco etch virus (see, e.g., Dolja (1997) Virology 234:243-252), bean golden mosaic virus (see, e.g., Morinaga (1993) Microbiol Immunol.
  • potato virus X see, e.g., Angell (1997) EMBO J. 16:3675-3684
  • tobacco mosaic virus see, e.g., Casper (1996) Gene 173:69-73
  • tomato bushy stunt virus see, e.g., Hillman (1989)
  • cauliflower mosaic virus see, e.g., Cecchini (1997) Mol. Plant Microbe Interact. 10:1094-1101
  • maize Ac/Ds fransposable element see, e.g., Rubm (1997) Mol. Cell. Biol. 17:6294-6302; Kunze (1996) Cun. Top. Microbiol. Immunol. 204:161-194)
  • Spm maize suppressor-mutator fransposable element
  • the expression vector can have two replication systems to allow it to be maintained in two organisms, for example in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification.
  • the expression vector can contain at least one sequence homologous to the host cell genome. It can contain two homologous sequences which flank the expression construct.
  • the integrating vector can be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector. Constructs for integrating vectors are well known in the art.
  • Expression vectors of the invention may also include a selectable marker gene to allow for the selection of bacterial strains that have been transformed, e.g., genes which render the bacteria resistant to drugs such as ampicillin, chloramphemcol, erythromycin, kanamycin, neomycin and tetracycline.
  • selectable markers can also include biosynthetic genes, such as those in the histidine, tryptophan and leucine biosynthetic pathways.
  • Exemplary insect cells include Drosophila S2 and Spodoptera Sf9.
  • Exemplary animal cells include CHO, COS or Bowes melanoma or any mouse or human cell line. The selection of an appropriate host is within the abilities of those skilled in the art. Techniques for transforming a wide variety of higher plant species are well known and described in the technical and scientific literature. See, e.g., Weising (1988) Ann. Rev. Genet. 22:421-477, U.S. Patent No. 5,750,870.
  • the vector can be introduced into the host cells using any of a variety of techniques, including transformation, fransfection, transduction, viral infection, gene guns, or Ti-mediated gene transfer. Particular methods include calcium phosphate transfection, DEAE-Dexfran mediated transfection, hpofection, or elecfroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)).
  • the nucleic acids or vectors of the invention are introduced into the cells for screening, thus, the nucleic acids enter the cells in a manner suitable for subsequent expression of the nucleic acid.
  • the method of introduction is largely dictated by the targeted cell type. Exemplary methods include CaPO precipitation, liposome fusion, Hpofection (e.g., LIPOFECTINTM), elecfroporation, viral infection, etc.
  • the candidate nucleic acids may stably integrate into the genome of the host cell (for example, with retroviral introduction) or may exist either transiently or stably in the cytoplasm (i.e. through the use of traditional plasmids, utilizing standard regulatory sequences, selection markers, etc.).
  • the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting fransformants or amplifying the genes of the invention.
  • the selected promoter may be induced by appropriate means (e.g., temperature shift or chemical induction) and the cells may be cultured for an additional period to allow them to produce the desired polypeptide or fragment thereof.
  • Cells can be harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract is retained for further purification.
  • Microbial cells employed for expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. Such methods are well known to those skilled in the art.
  • the expressed polypeptide or fragment thereof can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography.
  • Protein refolding steps can be used, as necessary, in completing configuration of the polypeptide. If desired, high performance hquid chromatography (HPLC) can be employed for final purification steps.
  • HPLC high performance hquid chromatography
  • Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts and other cell lines capable of expressing proteins from a compatible vector, such as the C127, 3T3, CHO, HeLa and BHK cell lines. The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
  • the polypeptides produced by host cells containing the vector may be glycosylated or may be non-glycosylated.
  • Polypeptides of the mvention may or may not also include an initial methionine amino acid residue.
  • Cell-free translation systems can also be employed to produce a polypeptide of the invention.
  • Cell-free translation systems can use mRNAs transcribed from a DNA construct comprising a promoter operably linked to a nucleic acid encoding the polypeptide or fragment thereof.
  • the DNA construct may be linearized prior to conducting an in vitro transcription reaction.
  • the franscribed mRNA is then incubated with an appropriate cell-free translation extract, such as a rabbit reticulocyte extract, to produce the desired polypeptide or fragment thereof.
  • the expression vectors can contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
  • nucleic acids of the invention and nucleic acids encoding the polypeptides of the mvention, or modified nucleic acids of the invention can be reproduced by amplification.
  • -Amplification can also be used to clone or modify the nucleic acids of the invention.
  • the invention provides amplification primer sequence pairs for amplifying nucleic acids of the mvention.
  • Amplification reactions can also be used to quantify the amount of nucleic acid in a sample (such as the amount of message in a cell sample), label the nucleic acid (e.g., to apply it to an anay or a blot), detect the nucleic acid, or quantify the amount of a specific nucleic acid in a sample.
  • message isolated from a cell or a cDNA library are amplified.
  • Amplification methods are also well known in the art, and include, e.g., polymerase chain reaction, PCR (see, e.g., PCR PROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, Academic Press, N.Y. (1990) and PCR STRATEGIES (1995), ed.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • transcription amplification see, e.g., Kwoh (1989) Proc. Natl. Acad. Sci. USA 86:1173
  • self-sustained sequence replication see, e.g., Guatelli (1990) Proc. Natl. Acad. Sci. USA 87:1874)
  • Q Beta replicase amplification see, e.g., Smith (1997) J. Clin. Microbiol.
  • the invention provides nucleic acids and polypeptides having various sequence identities to the exemplary sequences of the invention.
  • the extent of sequence identity may be determined using any computer program and associated parameters, including those described herein, such as BLAST 2.2.2. or FASTA version 3.0t78, with the default parameters.
  • Homologous sequences also include RNA sequences in wliich uridines replace the tliymines in the nucleic acid sequences.
  • the homologous sequences may be obtained using any of the procedures described herein or may result from the conection of a sequencing enor. It will be appreciated that the nucleic acid sequences as set forth herein can be represented in the traditional single character format (see, e.g., Stryer,
  • sequence comparison programs identified herein are used in this aspect of the invention. Protein and/or nucleic acid sequence identities (homologies) may be evaluated using any of the variety of sequence comparison algorithms and programs known in the art. Such algorithms and programs include, but are not limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85(8):2444-2448, 1988; Altschul et al., J. Mol. Biol. 215(3):403- 410, 1990; Thompson et al, Nucleic Acids Res.
  • subsequences ranging from about 20 to 600, about 50 to 200, and about 100 to 150 are compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequence for comparison are well known in the art.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482, 1981, by the homology alignment algorithm of
  • Such alignment programs can also be used to screen genome databases to identify polynucleotide sequences having substantially identical sequences.
  • a number of genome databases are available, for example, a substantial portion of the human genome is available as part of the Human Genome Sequencing Project (Gibbs, 1995).
  • Several genomes have been sequenced, e.g., M. genitalium (Fraser et al., 1995), M. jannaschii
  • BLAST, BLAST 2.0 and BLAST 2.2.2 algorithms are also used to practice the invention. They are described, e.g., in Altschul (1977) Nuc. Acids Res. 25:3389- 3402; Altschul (1990) J. Mol. Biol. 215:403-410. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is refened to as the neighborhood word score threshold (Altschul (1990) supra).
  • HSPs high scoring sequence pairs
  • W wordlength
  • E expectation
  • B BLOSUM62 scoring matrix
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873).
  • One measure of similarity provided by BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a references sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
  • protein and nucleic acid sequence homologies are evaluated using the Basic Local Alignment Search Tool ("BLAST").
  • BLAST Basic Local Alignment Search Tool
  • five specific BLAST programs can be used to perfoim the following task: (1) BLASTP and BLAST3 compare an amino acid query sequence against a protein sequence database; (2) BLASTN compares a nucleotide query sequence against a nucleotide sequence database; (3) BLASTX compares the six- frame conceptual translation products of a query nucleotide sequence (both strands) against a protein sequence database; (4) TBLASTN compares a query protein sequence against a nucleotide sequence database translated in all six reading frames (both sfrands); and, (5) TBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.
  • the NCBI BLAST to determine if a nucleic acid has the requisite sequence identity to be within the scope of the invention, the NCBI BLAST
  • BLAST 2.2.2 program In this exemplary aspect of the invention, all default values are used except for the default filtering setting (i.e., all parameters set to default except filtering which is set to OFF); in its place a "-F F" setting is used, which disables filtering. Use of default filtering often results in Karlin- Altschul violations due to short length of sequence.
  • the default values used in this exemplary aspect of the invention include: 5 "Filter for low complexity: ON
  • Word Size 3 Matrix: Blosum62 Gap Costs: Existence: 11 Extension: 1" o
  • Other default settings can be: filter for low complexity OFF, word size of 3 for protein, BLOSUM62 matrix, gap existence penalty of -11 and a gap extension penalty of -1.
  • An exemplary NCBI BLAST 2.2.2 program setting has the "-W" option default to 0. This means that, if not set, the word size defaults to 3 for proteins and 11 for nucleotides. 5 Computer systems and computer program products
  • the sequence of the invention can be stored, recorded, and manipulated on any medium which can be read and accessed by a computer. Accordingly, the invention provides computers, computer systems, computer readable 0 mediums, computer programs products and the like recorded or stored thereon the nucleic acid and polypeptide sequences of the invention.
  • the words "recorded” and “stored” refer to a process for storing information on a computer medium. A skilled artisan can readily adopt any known methods for recording information on a computer readable medium to generate manufactures comprising one or more of the nucleic acid 5 and/or polypeptide sequences of the invention.
  • Computer readable media include magnetically readable media, optically readable media, electronically readable media and magnetic/optical media.
  • the computer 0 readable media may be a hard disk, a floppy disk, a magnetic tape, CD-ROM, Digital
  • FIG. 1 One example of a computer system 100 is illustrated in block diagram form in Figure 1.
  • a computer system refers to the hardware components, software components, and data storage components used to analyze a nucleotide or polypeptide sequence of the invention.
  • the computer system 100 can include a processor for processing, accessing and manipulating the sequence data.
  • the processor 105 can be any well-known type of central processing unit, such as, for example, the Pentium III from Intel Corporation, or similar processor from Sun, Motorola, Compaq, AMD or International Business Machines.
  • the computer system 100 is a general purpose system that comprises the processor 105 and one or more internal data storage components 110 for storing data, and one or more data retrieving devices for retrieving the data stored on the data storage components.
  • the computer system 100 includes a processor 105 connected to a bus which is connected to a main memory 115 (preferably implemented as RAM) and one or more internal data storage devices 110, such as a hard drive and/or other computer readable media having data recorded thereon.
  • the computer system 100 can further include one or more data retrieving device 118 for readmg the data stored on the internal data storage devices 110.
  • the data retrieving device 118 may represent, for example, a floppy disk drive, a compact disk drive, a magnetic tape drive, or a modem capable of connection to a remote data storage system (e.g., via the internet) etc.
  • the internal data storage device 110 is a removable computer readable medium such as a floppy disk, a compact disk, a magnetic tape, etc. containing confrol logic and/or data recorded thereon.
  • the computer system 100 may advantageously include or be programmed by appropriate software for reading the control logic and/or the data from the data storage component once inserted in the data retrieving device.
  • the computer system 100 includes a display 120 which is used to display output to a computer user. It should also be noted that the computer system 100 can be linked to other computer systems 125a-c in a network or wide area network to provide centralized access to the computer system 100. Software for accessing and processing the nucleotide or amino acid sequences of the invention can reside in main memory 115 during execution. In some aspects, the computer system 100 may further comprise a sequence comparison algorithm for comparing a nucleic acid sequence of the invention. The algorithm and sequence(s) can be stored on a computer readable medium.
  • sequence comparison algorithm refers to one or more programs which are implemented (locally or remotely) on the computer system 100 to compare a nucleotide sequence with other nucleotide sequences and/or compounds stored within a data storage means.
  • the sequence comparison algorithm may compare the nucleotide sequences of the mvention stored on a computer readable medium to reference sequences stored on a computer readable medium to identify homologies or structural motifs.
  • FIG. 2 is a flow diagram illustrating one aspect of a process 200 for comparing a new nucleotide or protein sequence with a database of sequences in order to determine the homology levels between the new sequence and the sequences in the database.
  • the database of sequences can be a private database stored within the 5' computer system 100, or a public database such as GENBANK that is available through the Internet.
  • the process 200 begins at a start state 201 and then moves to a state 202 wherein the new sequence to be compared is stored to a memory in a computer system 100.
  • the memory could be any type of memory, including RAM or an internal storage device.
  • the process 200 then moves to a state 204 wherein a database 0 of sequences is opened for analysis and comparison.
  • the process 200 then moves to a state 206 wherein the first sequence stored in the database is read into a memory on the computer.
  • a comparison is then performed at a state 210 to determine if the first sequence is the same as the second sequence. It is important to note that this step is not limited to performing an exact comparison between the new sequence and the first 5 sequence in the database.
  • Well-known methods are known to those of skill in the art for comparing two nucleotide or protein sequences, even if they are not identical.
  • gaps can be infroduced into one sequence in order to raise the homology level between the two tested sequences.
  • the parameters that control whether gaps or other features are infroduced into a sequence during comparison are normally entered by the 0 user of the computer system.
  • a determination is made at a decision state 210 whether the two sequences are the same.
  • the term "same” is not limited to sequences that are absolutely identical. Sequences that are within the homology parameters entered by the user will be marked as "same" in the process 200. If a determination is made that the two sequences are the same, the process 200 moves to a state 214 wherein the name of the sequence from the database is displayed to the user.
  • This state notifies the user that the sequence with the displayed name fulfills the homology constraints that were entered.
  • the process 200 moves to a decision state 218 wherein a determination is made whether more sequences exist in the database. If no more sequences exist in the database, then the process 200 terminates at an end state 220. However, if more sequences do exist in the database, then the process 200 moves to a state 224 wherein a pointer is moved to the next sequence in the database so that it can be compared to the new sequence. In this manner, the new sequence is aligned and compared with every sequence in the database.
  • one aspect of the invention is a computer system comprising a processor, a data storage device having stored thereon a nucleic acid sequence of the invention and a sequence comparer for conducting the comparison.
  • the sequence comparer may indicate a homology level between the sequences compared or identify structural motifs, or it may identify structural motifs in sequences which are compared to these nucleic acid codes and polypeptide codes.
  • Figure 3 is a flow diagram illustrating one embodiment of a process 250 in a computer for determining whether two sequences are homologous.
  • the process 250 begins at a start state 252 and then moves to a state 254 wherein a first sequence to be compared is stored to a memory.
  • the second sequence to be compared is then stored to a memory at a state 256.
  • the process 250 then moves to a state 260 wherein the first character in the first sequence is read and then to a state 262 wherem the first character of the second sequence is read.
  • the sequence is a nucleotide sequence, then the character would normally be either A, T, C, G or U.
  • the sequence is a protein sequence, then it can be a single letter amino acid code so that the first and sequence sequences can be easily compared.
  • a determination is then made at a decision state 264 whether the two characters are the same.
  • the process 250 moves to a state 268 wherein the next characters in the first and second sequences are read. A deten-aination is then made whether the next characters are the same. If they are, then the process 250 continues this loop until two characters are not the same. If a dete nination is made that the next two characters are not the same, the process 250 moves to a decision state 274 to determine whether there are any more characters either sequence to read. If there are not any more characters to read, then the process 250 moves to a state 276 wherein the level of homology between the first and second sequences is displayed to the user.
  • the level of homology is dete ⁇ -nined by calculating the proportion of characters between the sequences that were the same out of the total number of sequences in the first sequence. Thus, if every character in a first 100 nucleotide sequence aligned with an every character in a second sequence, the homology level would be 100%.
  • the computer program can compare a reference sequence to a sequence of the invention to determine whether the sequences differ at one or more positions.
  • the program can record the length and identity of inserted, deleted or substituted nucleotides or amino acid residues with respect to the sequence of either the reference or the invention.
  • the computer program may be a program which determines whether a reference sequence contains a single nucleotide polymorphism (SNP) with respect to a sequence of the invention, or, whether a sequence of the invention comprises a SNP of a known sequence.
  • the computer program is a program which identifies SNPs.
  • the method may be implemented by the computer systems described above and the method illusfrated in Figure 3.
  • the method can be performed by reading a sequence of the invention and the reference sequences tlirough the use of the computer program and identifying differences with the computer program.
  • the computer based system comprises an identifier for identifying features within a nucleic acid or polypeptide of the invention.
  • An "identifier" refers to one or more programs which identifies certain features within a nucleic acid sequence.
  • an identifier may comprise a program which identifies an open reading frame (ORF) in a nucleic acid sequence.
  • Figure 4 is a flow diagram illustrating one aspect of an identifier process 300 for detecting the presence of a feature in a sequence.
  • the process 300 begins at a start state 302 and then moves to a state 304 wherein a first sequence that is to be checked for features is stored to a memory 115 in the computer system 100.
  • the process 300 then moves to a state 306 wherein a database of sequence features is opened.
  • a database would include a list of each feature's attributes along with the name of the feature. For example, a feature name could be
  • the features may be structural polypeptide motifs such as alpha helices, beta sheets, or functional polypeptide motifs such as enzymatic active sites, helix-turn-helix motifs or other motifs known to those skilled in the art.
  • the process 300 moves to a state 308 wherein the first feature is read from the database.
  • a comparison of the attribute of the first feature with the first sequence is then made at a state 310.
  • a determination is then made at a decision state 316 whether the attribute of the feature was found in the first sequence. If the attribute was found, then the process 300 moves to a state 318 wherein the name of the found feature is displayed to the user.
  • the process 300 then moves to a decision state 320 wherein a determination is made whether move features exist in the database. If no more features do exist, then the process 300 terminates at an end state 324. However, if more features do exist in the database, then the process 300 reads the next sequence feature at a state 326 'and loops back to the state 310 wherein the attribute of the next feature is compared against the first sequence. If the feature attribute is not found in the first sequence at the decision state 316, the process 300 moves directly to the decision state 320 in order to determine if any more features exist in the database.
  • the invention provides a computer program that identifies open reading frames (ORFs).
  • a polypeptide or nucleic acid sequence of the invention can be stored and manipulated in a variety of data processor programs in a variety of formats.
  • a sequence can be stored as text in a word processing file, such as MicrosoftWORD or WORDPERFECT or as an ASCII file in a variety of database programs familiar to those of skill in the art, such as DB2, SYBASE, or ORACLE.
  • many computer programs and databases may be used as sequence comparison algorithms, identifiers, or sources of reference nucleotide sequences or polypeptide sequences to be compared to a nucleic acid sequence of the invention.
  • the programs and databases used to practice the invention include, but are not limited to: MacPattem (EMBL), DiscoveryBase (Molecular Applications Group), GeneMine (Molecular Applications Group), Look (Molecular Applications Group), MacLook (Molecular Applications Group), BLAST and BLAST2 (NCBI), BLASTN and BLASTX (Altschul et al, J. Mol. Biol. 215: 403, 1990), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85: 2444, 1988), FASTDB (Brutiag et al. Comp. App. Biosci. 6:237-245, 1990), Catalyst (Molecular Simulations Inc.), Catalyst/SHAPE (Molecular Simulations Inc.), Cerius2.DB Access (Molecular Simulations Inc.), HypoGen (Molecular Simulations Inc.), Insight II, (Molecular
  • Motifs which may be detected using the above programs include sequences encoding leucine zippers, helix-turn-helix motifs, glycosylation sites, ubiquitination sites, alpha helices, and beta sheets, signal sequences encoding signal peptides which direct the secretion of the encoded proteins, sequences implicated in transcription regulation such as homeoboxes, acidic stretches, enzymatic active sites, substrate binding sites, and enzymatic cleavage sites.
  • the invention provides isolated or recombinant nucleic acids that hybridize under stringent conditions to an exemplary sequence of the mvention, e.g., a sequence as set forth in SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:ll, SEQ ID NO:13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID
  • the stringent conditions can be highly stringent conditions, medium stringent conditions, low stringent conditions, including the high and reduced stringency conditions described herein. In one aspect, it is the stringency of the wash conditions that set forth the conditions which determine whether a nucleic acid is with i the scope of the invention, as discussed below.
  • nucleic acids of the invention as defined by their ability to hybridize under stringent conditions can be between about five residues and the full length of nucleic acid of the mvention; e.g., they can be at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or more, residues in length. Nucleic acids shorter than full length are also included.
  • nucleic acids of the invention can be useful as, e.g., hybridization probes, labeling probes, PCR oligonucleotide probes, iRNA, antisense or sequences encoding antibody binding peptides (epitopes), motifs, active sites and the like.
  • nucleic acids of the invention are defined by their ability to hybridize under high stringency comprises conditions of about 50% formamide at about 37°C to 42°C.
  • nucleic acids of the invention are defined by their ability to hybridize under reduced stringency comprising conditions in about 35% to 25% formamide at about 30°C to 35°C.
  • nucleic acids of the invention are defined by their ability to hybridize under high stringency comprising conditions at 42°C in 50% formamide, 5X SSPE, 0.3% SDS, and a repetitive sequence blocking nucleic acid, such as cot-1 or salmon sperm DNA (e.g., 200 n/ml sheared and denatured salmon sperm DNA).
  • nucleic acids of the invention are defined by their ability to hybridize under reduced stringency conditions comprising 35% formamide at a reduced temperature of 35°C.
  • the filter may be washed with 6X SSC, 0.5%> SDS at 50°C. These conditions are considered to be “moderate” conditions above 25% formamide and “low” conditions below 25% formamide.
  • 6X SSC 0.5%> SDS at 50°C.
  • “moderate” hybridization conditions is when the above hybridization is conducted at 30% formamide.
  • a specific example of “low stringency” hybridization conditions is when the above hybridization is conducted at 10% formamide.
  • the temperature range conesponding to a particular level of stringency can be further nanowed by calculating the purine to pyrimidine ratio of the nucleic acid of interest and adjusting the temperature accordingly.
  • Nucleic acids of the invention are also defined by their ability to hybridize under high, medium, and low stringency conditions as set forth in Ausubel and Sambrook. Variations on the above ranges and conditions are well known in the art. Hybridization conditions are discussed further, below.
  • the above procedure may be modified to identify nucleic acids having decreasing levels of homology to the probe sequence.
  • less stringent conditions may be used.
  • the hybridization temperature may be decreased in increments of 5°C from 68°C to 42°C in a hybridization buffer having a Na + concenfration of approximately 1M.
  • the filter may be washed with 2X SSC, 0.5% SDS at the temperature of hybridization. These conditions are considered to be “moderate” conditions above 50°C and "low” conditions below 50°C.
  • a specific example of “moderate” hybridization conditions is when the above hybridization is conducted at 55°C.
  • a specific example of "low stringency" hybridization conditions is when the above hybridization is conducted at 45°C.
  • the hybridization may be carried out in buffers, such as 6X SSC, containing formamide at a temperature of 42°C.
  • concentration of formamide in the hybridization buffer may be reduced in 5% increments from 50% to 0% to identify clones having decreasing levels of homology to the probe.
  • the filter may be washed with 6X SSC, 0.5% SDS at 50°C. These conditions are considered to be “moderate” conditions above 25% formamide and “low” conditions below 25% formamide.
  • 6X SSC 0.5% SDS at 50°C.
  • wash conditions used to identify nucleic acids within the scope of the invention include, e.g.: a salt concenfration of about 0.02 molar at pH 7 and a temperature of at least about 50°C or about 55°C to about 60°C; or, a salt concentration of about 0.15 M NaCI at 72°C for about 15 minutes; or, a salt concentration of about 0.2X SSC at a temperature of at least about 50°C or about 55°C to 5 about 60°C for about 15 to about 20 minutes; or, the hybridization complex is washed twice with a solution with a salt concentration of about 2X SSC containing 0.1% SDS at room temperature for 15 minutes and then washed twice by 0.
  • Oligonucleotides probes and methods for using them
  • the mvention also provides nucleic acid probes that can be used, e.g., for identifying nucleic acids encoding a polypeptide with a biofilm-control enzyme activity or fragments thereof or for identifying biofilm-control enzyme genes.
  • the 5 probe comprises at least 10 consecutive bases of a nucleic acid of the mvention.
  • a probe of the invention can be at least about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150 or about 10 to 50, about 20 to 60 about 30 to 70, consecutive bases of a sequence as set forth in a nucleic acid of the invention.
  • the probes identify a nucleic acid by binding and/or hybridization.
  • the 0 probes can be used in anays of the invention, see discussion below, including, e.g., capillary anays.
  • the probes of the invention can also be used to isolate other nucleic acids or polypeptides.
  • the probes of the invention can be used to determine whether a biological sample, such as a soil sample, contains an organism having a nucleic acid sequence of the invention or an organism from which the nucleic acid was obtained.
  • a biological sample potentially harboring the organism from which the nucleic acid was isolated is obtained and nucleic acids are obtained from the sample.
  • the nucleic acids are contacted with the probe under conditions which permit the probe to specifically hybridize to any complementary sequences present in the sample. Where necessary, conditions wliich permit the probe to specifically hybridize to complementary sequences may be detera-iined by placing the probe in contact with complementary sequences from samples known to contain the complementary sequence, as well as control sequences which do not contain the complementary sequence.
  • Hybridization conditions such as the salt concenfration of the hybridization buffer, the formamide concenfration of the hybridization buffer, or the hybridization temperature, may be varied to identify conditions which allow the probe to hybridize specifically to complementary nucleic acids (see discussion on specific hybridization conditions).
  • Hybridization may be detected by labeling the probe with a detectable agent such as a radioactive isotope, a fluorescent dye or an enzyme capable of catalyzing the formation of a detectable product.
  • detectable agent such as a radioactive isotope, a fluorescent dye or an enzyme capable of catalyzing the formation of a detectable product.
  • Many methods for using the labeled probes to detect the presence of complementary nucleic acids in a sample are familiar to those skilled in the art. These include Southern Blots, Northern Blots, colony hybridization procedures, and dot blots. Protocols for each of these procedures are provided in Ausubel and Sambrook.
  • more than one probe may be used in an amplification reaction to determine whether the sample contains an organism containing a nucleic acid sequence of the invention (e.g., an organism from wliich the nucleic acid was isolated).
  • the probes comprise oligonucleotides.
  • the amplification reaction may comprise a PCR reaction. PCR protocols are described in Ausubel and Sambrook (see discussion on amplification reactions). In such procedures, the nucleic acids in the sample are contacted with the probes, the amplification reaction is performed, and any resulting amplification product is detected.
  • the ampHf ⁇ cation product may be detected by performing gel electrophoresis on the reaction products and staining the gel with an intercalator such as ethidium 004/066945
  • one or more of the probes may be labeled with a radioactive isotope and the presence of a radioactive amplification product may be detected by autoradiography after gel electrophoresis.
  • Probes derived from sequences near the 3' or 5' ends of a nucleic acid sequence of the invention can also be used in chromosome walking procedures to identify clones containing additional, e.g., genomic sequences. Such methods allow the isolation of genes which encode additional proteins of interest from the host organism.
  • nucleic acid sequences of the invention are used as probes to identify and isolate related nucleic acids.
  • the so-identified related nucleic acids may be cDNAs or genomic DNAs from organisms other than the one from which the nucleic acid of the invention was first isolated.
  • a nucleic acid sample is contacted with the probe under conditions which permit the probe to specifically hybridize to related sequences.
  • Hybridization of the probe to nucleic acids from the related organism is then detected using any of the methods described above.
  • the conditions used to achieve a particular level of stringency can vary, depending on the nature of the nucleic acids being hybridized.
  • the length, degree of complementarity, nucleotide sequence composition (e.g., GC v. AT content), and nucleic acid type (e.g., RNA v. DNA) of the hybridizing regions of the nucleic acids can be considered in selecting hybridization conditions.
  • An additional consideration is whether one of the nucleic acids is immobilized, for example, on a filter. Hybridization can be carried out under conditions of low stringency, moderate stringency or high stringency.
  • a polymer membrane containing immobilized denatured nucleic acids is first prehybridized for 30 minutes at 45°C in a solution consisting of 0.9 M NaCI, 50 mM NaH 2 PO4, pH 7.0, 5.0 mM Na 2 EDTA, 0.5% SDS, 10X Denhardt's, and 0.5 mg/ml polyriboadenylic acid. Approximately 2 X 10 7 cpm (specific activity 4-9 X 10 8 cpm/ug) of 32 P end-labeled oligonucleotide probe can then added to the solution.
  • the membrane is washed for 30 minutes at room temperature (RT) in IX SET (150 mM NaCI, 20 mM Tris hydrochloride, pH 7.8, 1 mM Na 2 EDTA) containing 0.5% SDS, followed by a 30 minute wash in fresh IX SET at Tm-10°C for the oligonucleotide probe.
  • IX SET 150 mM NaCI, 20 mM Tris hydrochloride, pH 7.8, 1 mM Na 2 EDTA
  • the membrane is then exposed to auto-radiographic film for detection of hybridization signals.
  • nucleic acids having different levels of homology to the probe can be identified and isolated.
  • Stringency may be varied by conducting the hybridization at varying temperatures below the melting temperatures of the probes.
  • the melting temperature, Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly complementary probe.
  • Very stringent conditions are selected to be equal to or about 5°C lower than the Tm for a particular probe.
  • the melting temperature of the probe may be calculated using the following exemplary formulas.
  • Prehybridization may be carried out in 6X SSC, 5X Denhardt's reagent, 0.5%> SDS, lOO ⁇ g denatured fragmented salmon sperm DNA or 6X SSC, 5X Denhardt's reagent, 0.5% SDS, lOO ⁇ g denatured fragmented salmon sperm
  • Hybridization is conducted by adding the detectable probe to the prehybridization solutions listed above. Where the probe comprises double stranded DNA, it is denatured before addition to the hybridization solution. The filter is contacted with the hybridization solution for a sufficient period of time to allow the probe to hybridize to cDNAs or genomic DNAs containing sequences complementary thereto or homologous thereto. For probes over 200 nucleotides in length, the hybridization may be carried out at 15-25°C below the Tm. For shorter probes, such as oligonucleotide probes, the hybridization may be conducted at 5- 10°C below the Tm. In one aspect, hybridizations in 6X SSC are conducted at approximately 68°C.
  • hybridizations in 50% formamide containing solutions are conducted at approximately 42°C. All of the foregoing hybridizations would be considered to be under conditions of high stringency.
  • the filter is washed to remove any non- specifically bound detectable probe.
  • the stringency used to wash the filters can also be varied depending on the nature of the nucleic acids being hybridized, the length of the nucleic acids being hybridized, the degree of complementarity, the nucleotide sequence composition (e.g., GC v. AT content), and the nucleic acid type (e.g., RNA v. DNA).
  • Examples of progressively higher stringency condition washes are as follows: 2X SSC, 0.1% SDS at room temperature for 15 minutes (low stringency); 0.1X SSC, 0.5% SDS at room temperature for 30 minutes to 1 hour (moderate stringency); 0.1X SSC, 0.5% SDS for 15 to 30 minutes at between the hybridization temperature and 68°C (high stringency); and 0.15M NaCI for 15 minutes at 72°C (very high stringency).
  • a final low stringency wash can be conducted in 0. IX SSC at room temperature.
  • the examples above are merely illusfrative of one set of conditions that can be used to wash filters.
  • One of skill in the art would know that there are numerous recipes for different stringency washes.
  • Nucleic acids which have hybridized to the probe can be identified by autoradiography or other conventional techniques. The above procedure may be modified to identify nucleic acids having decreasing levels of homology to the probe sequence. For example, to obtain nucleic acids of decreasing homology to the detectable probe, less stringent conditions may be used. For example, the hybridization temperature may be decreased in increments of 5°C from 68°C to 42°C in a hybridization buffer having a Na+ concenfration of approximately 1M. Following hybridization, the filter may be washed with 2X SSC, 0.5% SDS at the temperature of hybridization. These conditions are considered to be “moderate” conditions above 50°C and “low” conditions below 50°C. An example of “moderate” hybridization conditions is when the above hybridization is conducted at 55°C. An example of "low stringency" hybridization conditions is when the above hybridization is conducted at 45°C.
  • the hybridization may be carried out in buffers, such as 6X SSC, containing formamide at a temperature of 42°C.
  • concentration of formamide in the hybridization buffer may be reduced in 5%> increments from 50%o to 0% to identify clones having decreasing levels of homology to the probe.
  • the filter may be washed with 6X SSC, 0.5% SDS at 50°C.
  • 6X SSC 0.5% SDS at 50°C.
  • probes and methods of the invention can be used to isolate nucleic acids having a sequence with at least about 99%, 98%, 97%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50% homology to a nucleic acid sequence of the invention comprising at least about 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or more consecutive bases thereof, and the sequences complementary thereto. Homology may be measured using an alignment algorithm, as discussed herein.
  • the homologous polynucleotides may have a coding sequence which is a naturally occurring allelic variant of one of the coding sequences described herein.
  • allelic variants may have a substitution, deletion or addition of one or more nucleotides when compared to a nucleic acid of the invention.
  • probes and methods of the invention can be used to isolate nucleic acids which encode polypeptides having at least about 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50% sequence identity (homology) to a polypeptide of the invention comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acids, as determined using a sequence alignment algorithm (e.g., such as the FASTA version 3.0t78 algorithm with the default parameters, or a BLAST 2.2.2 program with exemplary settings as set forth herein).
  • a sequence alignment algorithm e.g., such as the FASTA version 3.0t78 algorithm with the default parameters, or a BLAST 2.2.2 program with exemplary settings as set forth herein.
  • the invention provides nucleic acids complementary to (e.g., antisense sequences to) the nucleic acid sequences of the invention.
  • Antisense sequences are capable of inhibiting the transport, splicing or transcription of biofilm-control enzyme- encoding genes.
  • the inhibition can be effected through the targeting of genomic DNA or messenger RNA.
  • the transcription or function of targeted nucleic acid can be inhibited, for example, by hybridization and/or cleavage.
  • One particularly useful set of inhibitors provided by the present invention includes oligonucleotides which are able to either bind biofilm-confrol enzyme gene or message, in either case preventing or inhibiting the production or function of biofilm-confrol enzyme.
  • the association can be through sequence specific hybridization.
  • Another useful class of inhibitors includes oligonucleotides which cause inactivation or cleavage of biofilm-control enzyme message.
  • the oligonucleotide can have enzyme activity which causes such cleavage, such as ribozymes.
  • the oligonucleotide can be chemically modified or conjugated to an enzyme or composition capable of cleaving the complementary nucleic acid. A pool of many different such oligonucleotides can be screened for those with the desired activity.
  • the invention provides various compositions for the inhibition of enzyme expression on a nucleic acid and/or protein level, e.g., antisense, iRNA and ribozymes comprising sequences of the invention and antibodies of the invention.
  • the invention provides antisense oligonucleotides capable of binding biofilm-control enzyme message which can inhibit proteolytic activity by targeting mRNA.
  • Strategies for designing antisense oligonucleotides are well described in the scientific and patent literature, and the skilled artisan can design such biofilm-confrol enzyme oligonucleotides using the novel reagents of the invention.
  • gene walking/ RNA mapping protocols to screen for effective antisense oligonucleotides are well known in the art, see, e.g., Ho (2000) Methods Enzymol. 314:168-183, describing an RNA mapping assay, which is based on standard molecular techniques to provide an easy and reliable method for potent antisense sequence selection. See also Smith (2000) Eur. J. Pharm. Sci. 11:191-198.
  • Naturally occurring nucleic acids are used as antisense oligonucleotides.
  • the antisense oligonucleotides can be of any length; for example, in alternative aspects, the antisense oligonucleotides are between about 5 to 100, about 10 to 80, about 15 to 60, about 18 to 40. The optimal length can be determined by routine screening.
  • the antisense oligonucleotides can be present at any concentration. The optimal concenfration can be determined by routine screening.
  • a wide variety of synthetic, non- naturally occurring nucleotide and nucleic acid analogues are known which can address this potential problem.
  • peptide nucleic acids containing non-ionic backbones, such as N-(2-aminoethyl) glycine units can be used.
  • Antisense oligonucleotides having phosphorothioate linkages can also be used, as described in WO 97/03211; WO 96/39154; Mata (1997) Toxicol Appl Pharmacol 144:189-197; Antisense Therapeutics, ed. Agrawal (Humana Press, Totowa, N J., 1996).
  • Antisense oligonucleotides having synthetic DNA backbone analogues provided by the invention can also include phosphoro-dithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3'-thioacetal, methylene(methylimino), 3'-N-carbamate, and morpholino carbamate nucleic acids, as described above.
  • Combinatorial chemistry methodology can be used to create vast numbers of oligonucleotides that can be rapidly screened for specific oligonucleotides that have appropriate binding affinities and specificities toward any target, such as the sense and antisense biofilm-control enzyme sequences of the invention (see, e.g., Gold (1995) J. of Biol. Chem. 270:13581-13584).
  • the invention provides ribozymes capable of binding biofilm-control enzyme message. These ribozymes can inhibit biofilm-control enzyme activity by, e.g., targeting mRNA. Strategies for designing ribozymes and selecting the biofilm-confrol enzyme-specific antisense sequence for targeting are well described in the scientific and patent literature, and the skilled artisan can design such ribozymes using the novel reagents of the invention. Ribozymes act by binding to a target RNA through the target RNA binding portion of a ribozyme which is held in close proximity to an enzymatic portion of the RNA that cleaves the target RNA.
  • the ribozyme recognizes and binds a target RNA tlirough complementary base-pairing, and once bound to the conect site, acts enzymatically to cleave and inactivate the target RNA. Cleavage of a target RNA in such a manner will destroy its ability to direct synthesis of an encoded protein if the cleavage occurs in the coding sequence. After a ribozyme has bound and cleaved its RNA target, it can be released from that RNA to bind and cleave new targets repeatedly.
  • a ribozyme can be advantageous over other technologies, such as antisense technology (where a nucleic acid molecule simply binds to a nucleic acid target to block its transcription, translation or association with another molecule) as the effective concentration of ribozyme necessary to effect a therapeutic freatment can be lower than that of an antisense oligonucleotide.
  • antisense technology where a nucleic acid molecule simply binds to a nucleic acid target to block its transcription, translation or association with another molecule
  • This potential advantage reflects the ability of the ribozyme to act enzymatically.
  • a single ribozyme molecule is able to cleave many molecules of target RNA.
  • a ribozyme is typically a highly specific inhibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding, but also on the mechanism by which the molecule inhibits the expression of the RNA to which it binds. That is, the inhibition is caused by cleavage of the RNA target and so specificity is defined as the ratio of the rate of cleavage of the targeted RNA over the rate of cleavage of non-targeted RNA. This cleavage mechamsm is dependent upon factors additional to those involved in base pairing. Thus, the specificity of action of a ribozyme can be greater than that of antisense oligonucleotide binding the same RNA site.
  • the ribozyme of the invention e.g., an enzymatic ribozyme RNA molecule
  • hammerhead motifs are described by, e.g., Rossi (1992) Aids Research and Human Retroviruses 8:183; hairpin motifs by Hampel (1989) Biochemistry 28:4929, and Hampel (1990) Nuc. Acids Res.
  • a ribozyme of the invention e.g., an enzymatic RNA molecule of this invention, can have a specific substrate binding site complementary to one or more of the target gene RNA regions.
  • a ribozyme of the invention can have a nucleotide sequence within or surrounding that subsfrate binding site which imparts an RNA cleaving activity to the molecule.
  • RNA interference RNA interference
  • RNA interference RNA interference
  • dsRNA double-stranded RNA
  • RNAi's of the invention are used in gene-silencing therapeutics, see, e.g., Shuey (2002) Drug Discov. Today 7:1040-1046.
  • the invention provides methods to selectively degrade RNA using the RNAi's of the invention.
  • RNAi molecules of the invention can be used to generate a loss-of-function mutation in a ceH, an organ or an animal.
  • Methods for making and using RNAi molecules for selectively degrade RNA are well known in the art, see, e.g., U.S. Patent No. 6,506,559; 6,511,824; 6,515,109; 6,489,127. Modification of Nucleic Acids
  • the invention provides methods of generating variants of the nucleic acids of the invention, e.g., those encoding a biofilm-control enzyme of the invention or an antibody of the invention. These methods can be repeated or used in various combinations to generate biofilm-confrol enzymes having an altered or different activity or an altered or different stability from that of a biofilm-control enzyme encoded by the template nucleic acid. These methods also can be repeated or used in various combinations, e.g., to generate variations in gene/ message expression, message translation or message stability.
  • the genetic composition of a cell is altered by, e.g., modification of a homologous gene ex vivo, followed by its reinsertion into the cell.
  • a nucleic acid of the invention can be altered by any means. For example, random or stochastic methods, or, non-stochastic, or "directed evolution," methods, see, e.g., U.S. Patent No. 6,361,974. Methods for random mutation of genes are well known in the art, see, e.g., U.S. Patent No. 5,830,696.
  • mutagens can be used to randomly mutate a gene. Mutagens include, e.g., ultraviolet light or gamma irradiation, or a chemical mutagen, e.g., mitomycin, nitrous acid, photoactivated psoralens, alone or in combination, to induce DNA breaks amenable to repair by recombination.
  • Other chemical mutagens include, for example, sodium bisulfite, nitrous acid, hydroxylamine, hydrazine or formic acid.
  • Other mutagens are analogues of nucleotide precursors, e.g., nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine. These agents can be added to a PCR reaction in place of the nucleotide precursor thereby mutating the sequence.
  • Intercalating agents such as proflavine, acriflavine, quinacrine and the like can also be used. Any technique in molecular biology can be used, e.g., random PCR mutagenesis, see, e.g., Rice (1992) Proc. Natl.
  • nucleic acids e.g., genes
  • can be reassembled after random, or "stochastic," fragmentation see, e.g., U.S. Patent Nos. 6,291,242; 6,287,862; 6,287,861; 5,955,358; 5,830,721; 5,824,514; 5,811,238; 5,605,793.
  • modifications, additions or deletions are introduced by enor-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, gene site saturated mutagenesis (GSSM), synthetic ligation reassembly (SLR), recombination, recursive sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-contaiiiing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chi
  • Oligonucleotide- directed mutagenesis a simple method using two oligonucleotide primers and a single- stranded DNA template" Methods in Enzymol. 154:329-350); phosphorothioate-modified DNA mutagenesis (Taylor et al. (1985) "The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA” Nucl. Acids Res. 13: 8749-8764; Taylor et al. (1985) "The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA” Nucl.
  • Non-stochastic, or "directed evolution,” methods include, e.g., saturation mutagenesis (GSSM), synthetic ligation reassembly (SLR), or a combination thereof are used to modify the nucleic acids of the invention to generate biofilm-control enzymes with new or altered properties (e.g., activity under highly acidic or alkaline conditions, high temperatures, and the like).
  • Polypeptides encoded by the modified nucleic acids can be screened for an activity before testing for proteolytic or other activity. Any testing modality or protocol can be used, e.g., using a capillary anay platform. See, e.g., U.S. Patent Nos. 6,361,974; 6,280,926; 5,939,250.
  • codon primers containing a degenerate N,N,G/T sequence are used to introduce point mutations into a polynucleotide, e.g., a biofilm-control enzyme or an antibody of the invention, so as to generate a set of progeny polypeptides in which a full range of single amino acid substitutions is represented at each amino acid position, e.g., an amino acid residue in an enzyme active site or ligand binding site targeted to be modified.
  • oligonucleotides can comprise a contiguous first homologous sequence, a degenerate N,N,G/T sequence, and, optionally, a second homologous sequence.
  • the downstream progeny translational products from the use of such oligonucleotides include all possible amino acid changes at each amino acid site along the polypeptide, because the degeneracy of the N,N,G/T sequence includes codons for all 20 amino acids.
  • one such degenerate oligonucleotide (comprised of, e.g., one degenerate N,N,G/T cassette) is used for subjecting each original codon in a parental polynucleotide template to a -full range of codon substitutions.
  • At least two degenerate cassettes are used - either in the same oligonucleotide or not, for subjecting at least two original codons in a parental polynucleotide template to a full range of codon substitutions.
  • more than one N,N,G/T sequence can be contained in one oligonucleotide to introduce amino acid mutations at more than one site.
  • This plurality of N,N,G/T sequences can be directly contiguous, or separated by one or more additional nucleotide sequence(s).
  • oligonucleotides serviceable for introducing additions and deletions can be used either alone or in combination with the codons containing an N,N,G/T sequence, to introduce any combination or pennutation of amino acid additions, deletions, and/or substitutions.
  • simultaneous mutagenesis of two or more contiguous amino acid positions is done using an oligonucleotide that contains contiguous N,N,G/T triplets, i.e. a degenerate (N,N,G/T)n sequence.
  • degenerate cassettes having less degeneracy than the N,N,G/T sequence are used. For example, it may be desirable in some instances to use (e.g.
  • a degenerate triplet sequence comprised of only one N, where said N can be in the first second or third position of the triplet.
  • Any other bases including any combinations and permutations thereof can be used in the remaining two positions of the triplet.
  • degenerate triplets allows for systematic and easy generation of a full range of possible natural amino acids (for a total of 20 amino acids) into each and every amino acid position in a polypeptide (in alternative aspects, the methods also include generation of less than all possible substitutions per amino acid residue, or codon, position). For example, for a 100 amino acid polypeptide, 2000 distinct species (i.e. 20 possible amino acids per position X 100 amino acid positions) can be generated.
  • an oligonucleotide or set of oligonucleotides containing a degenerate N,N,G/T triplet 32 individual sequences can code for all 20 possible natural amino acids.
  • Nondegenerate oligonucleotides can optionally be used in combination wifli degenerate primers disclosed; for example, nondegenerate oligonucleotides can be used to generate specific point mutations in a working polynucleotide. This provides one means to generate specific silent point mutations, point mutations leading to conesponding amino acid changes, and point mutations that cause the generation of stop codons and the conesponding expression of polypeptide fragments.
  • each saturation mutagenesis reaction vessel contains polynucleotides encodmg at least 20 progeny polypeptide (e.g., biofilm-control enzymes) molecules such that all 20 natural amino acids are represented at the one specific amino acid position conesponding to the codon position mutagenized in the parental polynucleotide (other aspects use less than all 20 natural combinations).
  • the 32-fold degenerate progeny polypeptides generated from each saturation mutagenesis reaction vessel can be subjected to clonal amplification (e.g. cloned into a suitable host, e.g., E. coli host, using, e.g., an expression vector) and subjected to expression screening.
  • an individual progeny polypeptide is identified by screening to display a favorable change in property (when compared to the parental polypeptide, such as increased proteolytic activity under alkaline or acidic conditions), it can be sequenced to identify the conespondingly favorable amino acid substitution contained therein.
  • favorable amino acid changes may be identified at more than one amino acid position.
  • One or more new progeny molecules can be generated that contain a combination of all or part of these favorable amino acid substitutions. For example, if 2 specific favorable amino acid changes are identified in each of 3 amino acid positions in a polypeptide, the permutations include 3 possibilities at each position (no change from the original amino acid, and each of two favorable changes) and 3 positions. Thus, there are 3 x 3 x 3 or 27 total possibilities, including 7 that were previously examined - 6 single point mutations (i.e. 2 at each of three positions) and no change at any position.
  • site-saturation mutagenesis can be used together with another stochastic or non-stochastic means to vary sequence, e.g., synthetic ligation reassembly (see below), shuffling, chimerization, recombination and other mutagenizing processes and mutagenizing agents.
  • This invention provides for the use of any mutagenizing process(es), including saturation mutagenesis, in an iterative manner.
  • the invention provides a non-stochastic gene modification system termed "synthetic ligation reassembly,” or simply “SLR,” a “directed evolution process,” to generate polypeptides, e.g., biofilm-control enzymes or antibodies of the invention, with new or altered properties.
  • SLR is a method of hgating oligonucleotide fragments together non-stochastically. This method differs from stochastic oligonucleotide shuffling in that the nucleic acid building blocks are not shuffled, concatenated or chimerized randomly, but rather are assembled non-stochastically. See, e.g., U.S. Patent Application Serial No.
  • SLR comprises the following steps: (a) providing a template polynucleotide, wherein the template polynucleotide comprises sequence encoding a homologous gene; (b) providing a plurality of building block polynucleotides, wherein the building block polynucleotides are designed to cross-over reassemble with the template polynucleotide at a predetermined sequence, and a building block polynucleotide comprises a sequence that is a variant of the homologous gene and a sequence homologous to the template polynucleotide flanking the variant sequence; (c) combining a building block polynucleotide with a template polynucleotide such that the building block polynucleotide cross-over reassembles with the template polynucle
  • SLR does not depend on the presence of high levels of homology between polynucleotides to be rearranged.
  • this method can be used to non-stochastically generate libraries (or sets) of progeny molecules comprised of over 10100 different chimeras.
  • SLR can be used to generate libraries comprised of over 101000 different progeny chimeras.
  • aspects of the present invention include non-stochastic methods of producing a set of finalized chimeric nucleic acid molecule shaving an overall assembly order that is chosen by design. This method includes the steps of generating by design a plurality of specific nucleic acid building blocks having serviceable mutually compatible ligatable ends, and assembling these nucleic acid building blocks, such that a designed overall assembly order is achieved.
  • the mutually compatible ligatable ends of the nucleic acid building blocks to be assembled are considered to be "serviceable" for this type of ordered assembly if they enable the building blocks to be coupled in predetermined orders.
  • the overall assembly order in which the nucleic acid building blocks can be coupled is specified by the design of the Hgatable ends. If more than one assembly step is to be used, then the overall assembly order in which the nucleic acid building blocks can be coupled is also specified by the sequential order of the assembly step(s).
  • the annealed building pieces are freated with an enzyme, such as a ligase (e.g. T4 DNA ligase), to achieve covalent bonding of the building pieces.
  • a ligase e.g. T4 DNA ligase
  • the design of the oligonucleotide building blocks is obtained by analyzing a set of progenitor nucleic acid sequence templates that serve as a basis for producing a progeny set of finalized chimeric polynucleotides.
  • These parental oligonucleotide templates thus serve as a source of sequence information that aids in the design of the nucleic acid building blocks that are to be mutagenized, e.g., cbimerized or shuffled.
  • the sequences of a plurality of parental nucleic acid templates are aligned in order to select one or more demarcation points.
  • the demarcation points can be located at an area of homology, and are comprised of one or more nucleotides.
  • demarcation points are preferably shared by at least two of the progenitor templates.
  • the demarcation points can thereby be used to dehneate the boundaries of oligonucleotide building blocks to be generated in order to rearrange the parental polynucleotides.
  • the demarcation points identified and selected in the progenitor molecules serve as potential chimerization points in the assembly of the final chimeric progeny molecules.
  • a demarcation point can be an area of homology (comprised of at least one homologous nucleotide base) shared by at least two parental polynucleotide sequences.
  • a demarcation point can be an area of homology that is shared by at least half of the parental polynucleotide sequences, or, it can be an area of homology that is shared by at least two thirds of the parental polynucleotide sequences. Even more preferably a serviceable demarcation points is an area of homology that is shared by at least three fourths of the parental polynucleotide sequences, or, it can be shared by at almost all of the parental polynucleotide sequences. In one aspect, a demarcation point is an area of homology that is shared by all of the parental polynucleotide sequences.
  • a ligation reassembly process is performed exhaustively in order to generate an exhaustive library of progeny chimeric polynucleotides.
  • all possible ordered combinations of the nucleic acid building blocks are represented in the set of finalized chimeric nucleic acid molecules.
  • the assembly order i.e. the order of assembly of each building block in the 5' to 3 sequence of each finalized chimeric nucleic acid
  • the assembly order is by design (or non-stochastic) as described above. Because of the non-stochastic nature of this invention, the possibility of unwanted side products is greatly reduced.
  • the ligation reassembly method is performed systematically.
  • the method is performed in order to generate a systematically compartmentalized library of progeny molecules, with compartments that can be screened systematically, e.g. one by one.
  • this invention provides that, through the selective and judicious use of specific nucleic acid building blocks, coupled with the selective and judicious use of sequentially stepped assembly reactions, a design can be achieved where specific sets of progeny products are made in each of several reaction vessels. This allows a systematic examination and screening procedure to be performed. Thus, these methods allow a potentially very large number of progeny molecules to be examined systematically in smaller groups.
  • the progeny molecules generated preferably comprise a library of finalized chimeric nucleic acid molecules having an overall assembly order that is chosen by design.
  • the saturation mutagenesis and optimized directed evolution methods also can be used to generate different progeny molecular species.
  • the invention provides freedom of choice and control regarding the selection of demarcation points, the size and number of the nucleic acid building blocks, and the size and design of the couplings. It is appreciated, furthermore, that the requirement for intermolecular homology is highly relaxed for the operability of this invention. In fact, demarcation points can even be chosen in areas of little or no intermolecular homology. For example, because of codon wobble, i.e. the degeneracy of codons, nucleotide substitutions can be infroduced into nucleic acid building blocks without altering the amino acid originally encoded in the conesponding progenitor template. Alternatively, a codon can be altered such that the coding for an originally amino acid is altered.
  • This invention provides that such substitutions can be introduced into the nucleic acid building block in order to increase the incidence of intermolecular homologous demarcation points and thus to allow an increased number of couplings to be achieved among the building blocks, which in turn allows a greater number of progeny chimeric molecules to be generated.
  • the synthetic nature of the step in which the building blocks are generated allows the design and introduction of nucleotides (e.g., one or more nucleotides, wliich may be, for example, codons or introns or regulatory sequences) that can later be optionally removed in an in vitro process (e.g. by mutagenesis) or in an in vivo process (e.g. by utilizing the gene splicing ability of a host organism). It is appreciated that in many instances the introduction of these nucleotides may also be desirable for many other reasons in addition to the potential benefit of creating a serviceable demarcation point.
  • nucleotides e.g., one or more nucleotides, wliich may be, for example, codons or introns or regulatory sequences
  • a nucleic acid building block is used to introduce an infron.
  • functional infrons are infroduced into a man-made gene manufactured according to the methods described herein.
  • the artificially introduced infron(s) can be functional in a host cells for gene splicing much in the way that naturaUy-occurring infrons serve functionaUy in gene splicing.
  • the invention provides a non-stochastic gene modification system termed "optimized directed evolution system" to generate polypeptides, e.g., biofilm-confrol enzymes or antibodies of the invention, with new or altered properties.
  • Optimized directed evolution is directed to the use of repeated cycles of reductive reassortment, recombination and selection that allow for the directed molecular evolution of nucleic acids through recombination.
  • Optimized directed evolution allows generation of a large population of evolved chimeric sequences, wherein the generated population is significantly enriched for sequences that have a predetermined number of crossover events.
  • One method for creating a chimeric progeny polynucleotide sequence is to create oligonucleotides conesponding to fragments or portions of each parental sequence. Each oligonucleotide preferably includes a unique region of overlap so that mixing the oligonucleotides together results in a new variant that has each oligonucleotide fragment assembled in the conect order.
  • oligonucleotides generated for each parental variant bears a relationship to the total number of resulting crossovers in the chimeric molecule that is ultimately created.
  • three parental nucleotide sequence variants might be provided to undergo a ligation reaction in order to find a chimeric variant having, for example, greater activity at high temperature.
  • a set of 50 oligonucleotide sequences can be generated conesponding to each portions of each parental variant. Accordingly, during the ligation reassembly process there could be up to 50 crossover events within each of the chimeric sequences.
  • each of the generated chimeric polynucleotides will contain oligonucleotides from each parental variant in alternating order is very low. If each oligonucleotide fragment is present in the ligation reaction in the same molar quantity it is likely that in some positions oligonucleotides from the same parental polynucleotide will ligate next to one another and thus not result in a crossover event. If the concenfration of each oligonucleotide from each parent is kept constant during any ligation step in this example, there is a 1/3 chance (assuming 3 parents) that an oligonucleotide from the same parental variant will ligate within the chimeric sequence and produce no crossover.
  • a probability density function can be determined to predict the population of crossover events that are Hkely to occur during each step in a ligation reaction given a set number of parental variants, a number of oligonucleotides conesponding to each variant, and the concentrations of each variant during each step in the ligation reaction.
  • the statistics and mathematics behind dete ⁇ n ⁇ iing the PDF is described below. By utilizing these methods, one can calculate such a probability density function, and thus enrich the chimeric progeny population for a predetermined number of crossover events resulting from a particular ligation reaction.
  • a target number of crossover events can be predetermined, and the system then programmed to calculate the starting quantities of each parental oligonucleotide during each step in the ligation reaction to result in a probability density function that centers on the predetermined number of crossover events.
  • These methods are directed to the use of repeated cycles of reductive reassortment, recombination and selection that allow for the directed molecular evolution of a nucleic acid encoding a polypeptide through recombination.
  • This system allows generation of a large population of evolved chimeric sequences, wherem the generated population is significantly enriched for sequences that have a predetermined number of crossover events.
  • a crossover event is a point in a chimeric sequence where a shift in sequence occurs from one parental variant to another parental variant. Such a point is normally at the juncture of where oligonucleotides from two parents are ligated together to form a single sequence.
  • the method allows calculation of the conect concentrations of oligonucleotide sequences so that the final chimeric population of sequences is enriched for the chosen number of crossover events. This provides more control over choosing chimeric variants having a predetermined number of crossover events.
  • the population of chimerics molecules can be enriched for those variants that have a particular number of crossover events.
  • each of the molecules chosen for further analysis most likely has, for example, only three crossover events.
  • the boundaries on the functional variety between the chimeric molecules is reduced. This provides a more manageable number of variables when calculating which oligonucleotide from the original parental polynucleotides might be responsible for affecting a particular trait.
  • the method creates a chimeric progeny polynucleotide sequence by creating oligonucleotides conesponding to fragments or portions of each parental sequence.
  • Each pligonucleotide preferably includes a unique region of overlap so that mixing the oligonucleotides together results in a new variant that has each oligonucleotide fragment assembled in the conect order. See also USSN 09/332,835.
  • the number of oligonucleotides generated for each parental variant bears a relationship to the total number of resulting crossovers in the chimeric molecule that is ultimately created.
  • three parental nucleotide sequence variants might be provided to undergo a ligation reaction in order to find a chimeric variant having, for example, greater activity at high temperature.
  • a set of 50 oligonucleotide sequences can be generated conesponding to each portions of each parental variant. Accordingly, during the ligation reassembly process there could be up to 50 crossover events within each of the chimeric sequences.
  • each of the generated chimeric polynucleotides will contain oligonucleotides from each parental variant in alternating order is very low. If each oligonucleotide fragment is present in the ligation reaction in the same molar quantity it is likely that in some positions oligonucleotides from the same parental polynucleotide will ligate next to one another and thus not result in a crossover event. If the concenfration of each oligonucleotide from each parent is kept constant during any ligation step in this example, there is a 1/3 chance (assuming 3 parents) that an oligonucleotide from the same parental variant will ligate within the chimeric sequence and produce no crossover.
  • a probability density function can be determined to predict the population of crossover events that are likely to occur during each step in a ligation reaction given a set number of parental variants, a number of oligonucleotides conesponding to each variant, and the concentrations of each variant during each step in the ligation reaction.
  • PDF probability density function
  • a target number of crossover events can be predetermined, and the system then programmed to calculate the starting quantities of each parental oligonucleotide during each step in the ligation reaction to result in a probability density function that centers on the predetennined number of crossover events.
  • aspects of the invention include a system and software that receive a desired crossover probability density function (PDF), the number of parent genes to be reassembled, and the number of fragments in the reassembly as inputs.
  • PDF crossover probability density function
  • the output of this program is a "fragment PDF" that can be used to determine a recipe for producing reassembled genes, and the estimated crossover PDF of those genes.
  • the processing described herein is preferably performed in MATLABa (The Mathworks, Natick, Massachusetts) a programming language and development environment for technical computing.
  • these processes can be iteratively repeated.
  • a nucleic acid or, the nucleic acid
  • This process can be iteratively repeated until a desired phenotype is engineered.
  • an entire biochemical anabolic or catabolic pathway can be engineered into a cell, including, e.g., biofilm-control activity.
  • a particular ohgonucleotide has no affect at all on the desired trait (e.g., a new biofilm-control enzyme phenotype)
  • it can be removed as a variable by synthesizing larger parental oligonucleotides that include the sequence to be removed. Since incorporating the sequence within a larger sequence prevents any crossover events, there will no longer be any variation of this sequence in the progeny polynucleotides.
  • This iterative practice of detennining which oligonucleotides are most related to the desired trait, and which are unrelated, allows more efficient exploration all of the possible protein variants that might be provide a particular trait or activity.
  • the invention provides a method for producing a hybrid polynucleotide from at least a first polynucleotide (e.g., a biofilm-control enzyme of the invention) and a second polynucleotide (e.g., an enzyme, such as a biofilm-confrol enzyme of the invention or any other biofilm-control enzyme, or, a tag or an epitope).
  • a first polynucleotide e.g., a biofilm-control enzyme of the invention
  • a second polynucleotide e.g., an enzyme, such as a biofilm-confrol enzyme of the invention or any other biofilm-control enzyme, or, a tag or an epitope.
  • the invention can be used to produce a hybrid polynucleotide by introducing at least a first polynucleotide and a second polynucleotide which share at least one region of partial sequence homology into a suitable host cell.
  • hybrid polynucleotide is any nucleotide sequence which results from the method of the present invention and contains sequence from at least two original polynucleotide sequences.
  • hybrid polynucleotides can result from intermolecular recombination events which promote sequence integration between DNA molecules.
  • hybrid polynucleotides can result from intramolecular reductive reassortment processes which utilize repeated sequences to alter a nucleotide sequence within a DNA molecule.
  • the invention also provides additional methods for making sequence variants of the nucleic acid (e.g., biofilm-control enzyme) sequences of the invention.
  • the mvention also provides additional methods for isolating biofilm-confrol enzymes using the nucleic acids and polypeptides of the invention.
  • the invention provides for variants of a biofilm-control enzyme coding sequence (e.g., a gene, cDNA or message) of the invention, which can be altered by any means, including, e.g., random or stochastic methods, or, non-stochastic, or "directed evolution," methods, as described above.
  • the isolated variants may be naturally occurring. Variant can also be created in vitro.
  • Variants may be created using genetic engineering techniques such as site directed mutagenesis, random chemical mutagenesis, Exonuclease III deletion procedures, and standard cloning techniques. Alternatively, such variants, fragments, analogs, or derivatives may be created using chemical synthesis or modification procedures. Other methods of making variants are also familiar to those skilled in the art. These include procedures in which nucleic acid sequences obtained from natural isolates are modified to generate nucleic acids which encode polypeptides having characteristics which enhance their value in industrial or laboratory applications. In such procedures, a large number of variant sequences having one or more nucleotide differences with respect to the sequence obtained from the natural isolate are generated and characterized. These nucleotide differences can result in amino acid changes with respect to the polypeptides encoded by the nucleic acids from the natural isolates.
  • the reaction may be performed using 20 fmoles of nucleic acid to be mutagenized, 30 pmole of each PCR primer, a reaction buffer comprising 50mM KCI, lO M Tris HCl (pH 8.3) and 0.01% gelatin, 7mM MgCl 2 , 0.5mM MnCl 2 , 5 units of Taq polymerase, 0.2mM dGTP, 0.2mM dATP, ImM dCTP, and ImM dTTP.
  • PCR may be performed for 30 cycles of 94°C for 1 min, 45°C for 1 min, and 72°C for 1 min. However, it will be appreciated that these parameters may be varied as appropriate.
  • the mutagenized nucleic acids are cloned into an appropriate vector and the activities of the polypeptides encoded by the mutagenized nucleic acids is evaluated. Variants may also be created using oligonucleotide directed mutagenesis to generate site-specific mutations in any cloned DNA of interest. Oligonucleotide mutagenesis is described, e.g., in Reidhaar-Olson (1988) Science 241:53-57. Briefly, in such procedures a plurahty of double stranded oligonucleotides bearing one or more mutations to be introduced into the cloned DNA are synthesized and inserted into the cloned DNA to be mutagenized. Clones containing the mutagenized DNA are recovered and the activities of the polypeptides they encode are assessed.
  • Assembly PCR involves the assembly of a PCR product from a mixture of smaU DNA fragments. A large number of different PCR reactions occur in parallel in the same vial, with the products of one reaction priming the products of another reaction. Assembly PCR is described in, e.g., U.S. Patent No. 5,965,408.
  • Still another method of generating variants is sexual PCR mutagenesis.
  • sexual PCR mutagenesis forced homologous recombination occurs between DNA molecules of different but highly related DNA sequence in vitro, as a result of random fragmentation of the DNA molecule based on sequence homology, followed by fixation of the crossover by primer extension in a PCR reaction.
  • Sexual PCR mutagenesis is described, e.g., in Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751. Briefly, in such procedures a plurality of nucleic acids to be recombined are digested with DNase to generate fragments having an average size of 50-200 nucleotides.
  • Fragments of the desired average size are purified and resuspended in a PCR mixture.
  • PCR is conducted under conditions which facilitate recombination between the nucleic acid fragments.
  • PCR may be performed by resuspending the purified fragments at a concenfration of 10-30ng/:l in a solution of 0.2mM of each dNTP, 2.2mM MgCl 2 , 50mM KCL, lOmM Tris HCl, pH 9.0, and 0.1% Triton X-100.
  • PCR 2.5 units of Taq polymerase per 100:1 of reaction mixture is added and PCR is performed using the following regime: 94°C for 60 seconds, 94°C for 30 seconds, 50-55°C for 30 seconds, 72°C for 30 seconds (30-45 times) and 72°C for 5 minutes.
  • oligonucleotides may be included in the PCR reactions.
  • the Klenow fragment of DNA polymerase I may be used in a first set of PCR reactions and Taq polymerase may be used in a subsequent set of PCR reactions. Recombinant sequences are isolated and the activities of the polypeptides they encode are assessed.
  • Variants may also be created by in vivo mutagenesis.
  • random mutations in a sequence of interest are generated by propagating the sequence of interest in a bacterial strain, such as anE. coli strain, which carries mutations in one or more of the DNA repair pathways.
  • a bacterial strain such as anE. coli strain
  • Such "mutator" strains have a higher random mutation rate than that of a wild-type parent. Propagating the DNA in one of these strains will eventually generate random mutations within the DNA.
  • Mutator strains suitable for use for in vivo mutagenesis are described, e.g., in PCT Publication No. WO 91/16427. Variants may also be generated using cassette mutagenesis.
  • variants are created using exponential ensemble mutagenesis.
  • Exponential ensemble mutagenesis is a process for generating combinatorial libraries with a high percentage of unique and functional mutants, wherein small groups of residues are randomized in parallel to identify, at each altered position, amino acids which lead to functional proteins.
  • Exponential ensemble mutagenesis is described, e.g., in Delegrave (1993) Biotechnology Res. 11:1548-1552. Random and site-directed mutagenesis are described, e.g., in Arnold (1993) Cunent Opinion in Biotechnology 4:450-455.
  • the variants are created using shuffling procedures wherein portions of a plurality of nucleic acids which encode distinct polypeptides are fused together to create chimeric nucleic acid sequences which encode chimeric polypeptides as described in, e.g., U.S. Patent Nos. 5,965,408; 5,939,250 (see also discussion, above).
  • the invention also provides variants of polypeptides of the invention (e.g., biofilm-confrol enzymes) comprising sequences in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (e.g., a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code.
  • a conserved or non-conserved amino acid residue e.g., a conserved amino acid residue
  • Conservative substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics.
  • polypeptides of the invention include those with conservative substitutions of sequences of the invention, including but not limited to the following replacements: replacements of an aliphatic amino acid such as Alanine, Valine, Leucine and Isoleucine with another aliphatic amino acid; replacement of a Serine with a Threonine or vice versa; replacement of an acidic residue such as Aspartic acid and Glutamic acid with another acidic residue; replacement of a residue bearing an amide group, such as Asparagine and Glutamine, with another residue bearing an amide group; exchange of a basic residue such as Lysine and Arginine with another basic residue; and replacement of an aromatic residue such as Phenylalanine, Tyrosine with another aromatic residue.
  • Other variants are those in which one or more of the amino acid residues of the polypeptides of the invention includes a substituent group.
  • polypeptide is associated with another compound, such as a compound to increase the half-life of the polypeptide, for example, polyethylene glycol.
  • Additional variants within the scope of the invention are those in which additional amino acids are used to the polypeptide, such as a leader sequence, a secretory sequence, a proprotein sequence or a sequence which facilitates purification, enrichment, or stabilization of the polypeptide.
  • the variants, fragments, derivatives and analogs of the polypeptides of the invention retain the same biological function or activity as the exemplary polypeptides, e.g., biofilm-confrol enzyme activity, as described herein.
  • the variant, fragment, derivative, or analog includes a proprotein, such that the variant, fragment, derivative, or analog can be activated by cleavage of the proprotein portion to produce an active polypeptide.
  • the invention provides methods for modifying biofilm-control enzyme- encoding nucleic acids to modify codon usage.
  • the invention provides methods for modifying codons in a nucleic acid encoding a biofilm-control enzyme to increase or decrease its expression in a host cell.
  • the invention also provides nucleic acids encoding a biofilm-confrol enzyme modified to increase its expression in a host cell, biofilm-control enzyme so modified, and methods of making the modified biofilm-confrol enzymes.
  • the method comprises identifying a "non-prefened” or a “less prefened” codon in biofilm-confrol enzyme-encoding nucleic acid and replacing one or more of these non-prefened or less prefened codons with a "prefened codon” encoding the same amino acid as the replaced codon and at least one non-prefened or less prefened codon in the nucleic acid has been replaced by a prefened codon encoding the same amino acid.
  • a prefened codon is a codon over-represented in coding sequences in genes in the host cell and a non-prefened or less prefened codon is a codon under-represented in coding sequences in genes in the host cell.
  • Host cells for expressing the nucleic acids, expression cassettes and vectors of the invention include bacteria, yeast, fungi, plant cells, insect cells and mammalian cells. Thus, the invention provides methods for optimizing codon usage in all of these cells, codon-altered nucleic acids and polypeptides made by the codon-altered nucleic acids.
  • Exemplary host cells include gram negative bacteria, such as Escherichia coli and Pseudomonas fluorescens; gram positive bacteria, such as Streptomyces diversa, Lactobacillus gasseri, Lactococcus lactis, Lactococcus cremoris, Bacillus subtilis.
  • Exemplary host cells also include eukaryotic organisms, e.g., various yeast, such as Saccharomyces sp., including Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichiapastoris, and Kluyveromyces lactis, Hansenula polymorpha, Aspergillus niger, and mammalian cells and cell lines and insect cells and cell lines.
  • yeast such as Saccharomyces sp., including Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichiapastoris, and Kluyveromyces lactis, Hansenula polymorpha, Aspergillus niger, and mammalian cells and cell lines and insect cells and cell lines.
  • yeast such as Saccharomyces sp., including Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichiapastoris, and Kluyveromyces lac
  • the codons of a nucleic acid encodmg a biofilm-confrol enzyme isolated from a bacterial cell are modified such that the nucleic acid is optimally expressed in a bacterial cell different from the bacteria from wliich the biofilm-confrol enzyme was derived, a yeast, a fungi, a plant cell, an insect cell or a mammalian cell.
  • Methods for optimizing codons are well known in the art, see, e.g., U.S. Patent No.
  • the invention provides transgenic non-human animals comprising a nucleic acid, a polypeptide (a biofilm-confrol enzyme or an antibody of the mvention), an expression cassette or vector or a fransfected or transformed cell of the mvention.
  • the invention also provides methods of making and using these transgenic non-human animals.
  • the transgenic non-human animals can be, e.g., goats, rabbits, sheep, pigs, cows, rats and mice, comprising the nucleic acids of the mvention. These animals can be used, e.g., as in vivo models to study biofihn-confrol enzyme activity, or, as models to screen for agents that change the biofilm-control enzyme activity in vivo.
  • the coding sequences for the polypeptides to be expressed in the transgenic non-human animals can be designed to be constitutive, or, under the control of tissue-specific, developmental- specific or inducible transcriptional regulatory factors.
  • Transgenic non-human animals can be designed and generated using any method known in the art; see, e.g., U.S. Patent Nos.
  • U.S. Patent No. 6,211 ,428, describes making and using transgenic non-human mammals which express in their brains a nucleic acid construct comprising a DNA sequence.
  • U.S. Patent No. 5,387,742 describes injecting cloned recombinant or synthetic DNA sequences into fertilized mouse eggs, implanting the injected eggs in pseudo-pregnant females, and growing to term transgenic mice whose cells express proteins related to the pathology of Alzheimer's disease.
  • U.S. Patent No. 6,187,992 describes making and using a transgenic mouse whose genome comprises a disruption of the gene encoding amyloid precursor protein (APP).
  • APP amyloid precursor protein
  • the fransgenic or modified animals of the invention comprise a "knockout animal,” e.g., a “knockout mouse,” engineered not to express an endogenous gene, which is replaced with a gene expressing a biofihn-confrol enzyme of the invention, or, a fusion protein comprising a biofilm-confrol enzyme of the invention.
  • the mvention provides fransgenic plants and seeds comprising a nucleic acid, a polypeptide (a biofilm-confrol enzyme or an antibody of the invention), an expression cassette or vector or a fransfected or transformed cell of the invention.
  • the transgenic plant can be dicotyledonous (a dicot) or monocotyledonous (a monocot).
  • the invention also provides methods of making and using these fransgenic plants and seeds.
  • the fransgenic plant or plant cell expressing a polypeptide of the present invention may be constructed in accordance with any method known in the art. See, for example, U.S. Patent No. 6,309,872.
  • Nucleic acids and expression constructs of the invention can be infroduced into a plant cell by any means.
  • nucleic acids or expression constructs can be introduced into the genome of a desired plant host, or, the nucleic acids or expression constructs can be episomes.
  • Infroduction into the genome of a desired plant can be such that the host's a -biofilm-control enzyme production is regulated by endogenous transcriptional or translational control elements.
  • the invention also provides "knockout plants" where insertion of gene sequence by, e.g., homologous recombination, has disrupted the expression of the endogenous gene.
  • biofilm-confrol enzymes of the invention can be used in production of a transgenic plant to produce a compound not naturally produced by that plant. This can lower production costs or create a novel product.
  • the first step in production of a transgenic plant involves making an expression construct for expression in a plant cell.
  • These techniques are well known in the art. They can include selecting and cloning a promoter, a coding sequence for facilitating efficient binding of ribosomes to mRNA and selecting the appropriate gene terminator sequences.
  • a constitutive promoter is CaMV35S, from the cauliflower mosaic virus, which generally results in a high degree of expression in plants. Other promoters are more specific and respond to cues in the plant's internal or external environment.
  • An exemplary light-inducible promoter is the promoter from the cab gene, encoding the major chlorophyll a/b binding protein.
  • the nucleic acid is modified to achieve greater expression in a plant cell.
  • a sequence of the invention is likely to have a higher percentage of A-T nucleotide pairs compared to that seen in a plant, some of which prefer G-C nucleotide pairs. Therefore, A-T nucleotides in the coding sequence can be substituted with G-C nucleotides without significantly changing the amino acid sequence to enhance production of the gene product in plant cells.
  • Selectable marker gene can be added to the gene construct in order to identify plant cells or tissues that have successfully integrated the fransgene. This may be necessary because achieving incorporation and expression of genes in plant cells is a rare event, occr-rring in just a few percent of the targeted tissues or cells.
  • Selectable marker genes encode proteins that provide resistance to agents that are normally toxic to plants, such as antibiotics or herbicides. Only plant cells that have integrated the selectable marker gene will survive when grown on a medium containing the appropriate antibiotic or herbicide. As for other inserted genes, marker genes also require promoter and termination sequences for proper function.
  • making fransgenic plants or seeds comprises incorporating sequences of the invention and, optionally, marker genes into a target expression construct (e.g., a plasmid), along with positioning of the promoter and the terminator sequences.
  • a target expression construct e.g., a plasmid
  • This can involve transferring the modified gene into the plant through a suitable method.
  • a construct may be introduced directly into the genomic DNA of the plant cell using techniques such as elecfroporation and microinjection of plant cell protoplasts, or the constructs can be introduced directly to plant tissue using ballistic methods, such as DNA particle bombardment.
  • Christou 1997 Plant Mol. Biol. 35:197-203; Pawlowski (1996) Mol. Biotechnol. 6:17-30; Klein
  • protoplasts can be immobilized and injected with a nucleic acids, e.g., an expression construct.
  • a nucleic acids e.g., an expression construct.
  • Transformed tissue is then induced to regenerate, usually by somatic embryogenesis. This technique has been successful in several cereal species including maize and rice.
  • Nucleic acids can also be infroduced in to plant ceUs using recombinant viruses.
  • Plant cells can be transformed using viral vectors, such as, e.g., tobacco mosaic virus derived vectors (Rouwendal (1997) Plant Mol. Biol. 33:989-999), see Porta (1996) "Use of viral replicons for the expression of genes in plants," Mol. Biotechnol. 5:209-221.
  • nucleic acids e.g., an expression construct
  • suitable T-DNA flanking regions can be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector.
  • the virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria.
  • Agrobacterium tumefaciens-mediated transformation techniques including disarming and use of binary vectors, are well described in the scientific literature. See, e.g., Horsch (1984) Science 233:496-498; Fraley (1983) Proc. N ⁇ tl. Ac ⁇ d. Sci.
  • the DNA in an A. tumefaciens cell is contained in the bacterial chromosome as well as in another structure known as a Ti (tumor-inducing) plasmid.
  • the Ti plasmid contains a sfretch of DNA termed T-DNA (-20 kb long) that is fransfened to the plant ceU in the infection process and a series of vir (virulence) genes that direct the infection process.
  • tumefaciens can only infect a plant through wounds: when a plant root or stem is wounded it gives off certain chemical signals, in response to which, the vir genes of A. tumefaciens become activated and direct a series of events necessary for the transfer of the T-DNA from the Ti plasmid to the plant's chromosome. The T-DNA then enters the plant cell through the wound.
  • One speculation is that the T-DNA waits until the plant DNA is being replicated or transcribed, then inserts itself into the exposed plant DNA. In order to use A.
  • the tumor-inducing section of T-DNA have to be removed, while retaining the T-DNA border regions and the vir genes.
  • the fransgene is then inserted between the T-DNA border regions, where it is fransfened to the plant cell and becomes integrated into the plant's chromosomes.
  • the mvention provides for the transformation of monocotyledonous plants using the nucleic acids of the invention, including important cereals, see Hiei (1997) Plant Mol. Biol. 35:205-218. See also, e.g., Horsch, Science (1984) 233:496; Fraley (1983) Proc. Natl Acad. Sci USA 80:4803; Thykjaer (1997) supra; Park (1996) Plant Mol. Biol. 32: 1135-1148, discussing T-DNA integration into genomic DNA. See also D'Halluin, U.S. Patent No. 5,712,135, describing a process for the stable integration of a DNA comprising a gene that is functional in a cell of a cereal, or other monocotyledonous plant.
  • the third step can involve selection and regeneration of whole plants capable of transmitting the incorporated target gene to the next generation.
  • Such regeneration techniques rely on manipulation of certain phytohortnones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker that has been introduced together with the desired nucleotide sequences. Plant regeneration from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture,
  • Regeneration can also be obtained from plant callus, explants, organs, or parts thereof. Such regeneration techniques are described generally in Klee (1987) Ann. Rev. of Plant Phys. 38:467-486.
  • tissue culture a process known as tissue culture. Once whole plants are generated and produce seed, evaluation of the progeny begins.
  • the expression cassette After the expression cassette is stably inco ⁇ orated in transgenic plants, it can be infroduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed. Since fransgenic expression of the nucleic acids of the invention leads to phenotypic changes, plants comprising the recombinant nucleic acids of the invention can be sexually crossed with a second plant to obtain a final product. Thus, the seed of the invention can be derived from a cross between two fransgenic plants of the invention, or a cross between a plant of the invention and another plant.
  • the desired effects can be enhanced when both parental plants express the polypeptides of the invention.
  • the desired effects can be passed to future plant generations by standard propagation means.
  • Transgenic plants of the invention can be dicotyledonous or monocotyledonous.
  • monocot fransgenic plants of the invention are grasses, such as meadow grass (blue grass, Pod), forage grass such as festuca, lolium, temperate grass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum, and maize (corn).
  • dicot fransgenic plants of the invention are tobacco, legumes, such as lupins, potato, sugar beet, pea, bean and soybean, and cruciferous plants (family Brassicaceae), such as cauliflower, rape seed, and the closely related model organism Arabidopsis thaliana.
  • the transgenic plants and seeds of the invention include a broad range of plants, including, but not limited to, species from the genera Anacardium, Arachis, Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea, Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lupinus,
  • Lycopersicon Malus, Man ⁇ hot, Majorana, Medicago, Nicotiana, Olea, Oryza, Panieum, Pannisetum, Persea, Phaseolus, Pistachia, Pisum, Pyrus, Prunus, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solanum, Sorghum, Theobromus, Trigonella, Triticum, Vicia, Vitis, Vigna, and Zea.
  • the nucleic acids of the invention are expressed in plants which contain fiber cells, including, e.g., cotton, silk cotton free (Kapok, Ceiba pentandra), desert willow, creosote bush, winterfat, balsa, ramie, kenaf, hemp, roselle, jute, sisal abaca and flax.
  • the transgenic plants of the invention can be members of the genus Gossypium, including members of any Gossypium species, such as G. arbor eum;. G. herbaceum, G. barbadense, and G. hirsutum.
  • polypeptides e.g., enzymes, having biofilm control or modifying activities.
  • the polypeptides of the invention have phosphatase, amidase, deacetylase, esterase and/or glycosidase activities, or related activities, which may include biofilm control or bacterial confrol activities, such as Pseudomonas removal (e.g., removal from biofilms), Pseudomonas prevention, Staphylococcus ("Staph”) removal (e.g., removal from biofilms) or Staphylococcus prevention activities.
  • exemplary polypeptides e.g., enzymes of the mvention have activities as set forth in the following table; for example, the polypeptide having an activity as set forth in SEQ ID NO:46 (and, in one aspect, encoded by a nucleic acid having a sequence as set forth in SEQ ID NO:45) has phosphatase activity, and, Staphylococcus removal and/or Staphylococcus prevention activities; the polypeptide having an activity as set forth in SEQ ID NO:42 (and, in one aspect, encoded by a nucleic acid having a sequence as set forth in SEQ ID NO:41) has amidase activity, and, Staphylococcus removal and/or Staphylococcus prevention activities; the polypeptide having an activity as set forth in SEQ ID NO: 54 (and, in one aspect, encoded by a nucleic acid having a sequence as set forth in SEQ ID NO:53) has deacetylase activity, and, Staphylococcus removal
  • the invention provides isolated or recombinant polypeptides having a sequence identity to an exemplary sequence of the invention, e.g., SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:
  • the identity can be over the full length of the polypeptide, or, the identity can be over a region of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700 or more residues.
  • Polypeptides of the mvention can also be shorter than the full length of exemplary polypeptides.
  • the invention provides polypeptides (peptides, fragments) ranging in size between about 5 residues (amino acids) and the full length of a polypeptide, e.g., an enzyme, such as a biofilm-confrol enzyme; exemplary sizes being of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or more residues, e.g., contiguous residues of an exemplary biofilm-confrol enzyme of the invention.
  • Peptides of the invention can be useful as, e.g., labeling probes, antigens, toleragens, motifs, biofilm-control enzyme active sites.
  • Polypeptides and peptides of the invention can be isolated from natural sources, be synthetic, or be recombinantly generated polypeptides. Peptides and proteins can be recombinantly expressed in vitro or in vivo.
  • the peptides and polypeptides of the invention can be made and isolated using any method known in the art. Polypeptide and peptides of the invention can also be synthesized, whole or in part, using chemical methods well known in the art. See e.g., Caruthers (1980) Nucleic Acids Res. Symp. Ser. 215-223; Horn (1980) Nucleic Acids Res. Symp. Ser.
  • peptide synthesis can be performed using various solid-phase techniques (see e.g., Roberge (1995) Science 269:202; Merrifield (1997) Methods Enzymol. 289:3-13) and automated synthesis maybe achieved, e.g., using the ABI 431 A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer.
  • the peptides and polypeptides of the invention can also be glycosylated.
  • the glycosylation can be added post-translationally either chemicaUy or by cellular biosynthetic mechanisms, wherein the later incorporates the use of known glycosylation motifs, which can be native to the sequence or can be added as a peptide or added in the nucleic acid coding sequence.
  • the glycosylation can be O-linked or N-linked.
  • the peptides and polypeptides of the invention include all “mimetic” and “peptidomimetic” forms.
  • the terms “mimetic” and “peptidomimetic” refer to a synthetic chemical compound which has substantiaUy the same structural and/or functional characteristics of the polypeptides of the invention.
  • the mimetic can be either entirely composed of synthetic, non-natural analogues of amino acids, or, is a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids.
  • the mimetic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantiaUy alter the mimetic 's structure and/or activity.
  • a mimetic composition is within the scope of the invention if it has a biofilm- control enzyme activity.
  • Polypeptide mimetic compositions of the invention can contain any combination of non-natural structural components.
  • mimetic compositions of the invention include one or all of the following three structural groups: a) residue linkage groups other than the natural amide bond ("peptide bond") linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues which induce secondary structural mimicry, i.e., to induce or stabihze a secondary structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix conformation, and the like.
  • a polypeptide of the invention can be characterized as a mimetic when all or some of its residues are joined by chemical means other than natural peptide bonds.
  • peptide bonds can be joined by peptide bonds, other chemical bonds or coupling means, such as, e.g., glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, N,N'-dicyclohexylcarbodiimide (DCC) or N,N'- diisopropylcarbodiimide (DIC).
  • DCC N,N'-dicyclohexylcarbodiimide
  • DIC N,N'- diisopropylcarbodiimide
  • aminomethylene CH 2 -NH
  • ethylene olefin
  • ether
  • a polypeptide of the invention can also be characterized as a mimetic by containing all or some non-natural residues in place of naturally occurring amino acid residues.
  • Non-natural residues are well described in the scientific and patent literature; a few exemplary non-natural compositions useful as mimetics of natural amino acid residues and guidelines are described below.
  • Mimetics of aromatic amino acids can be generated by replacing by, e.g., D- or L- naphylalanine; -Q- or L- phenylglycine; D- or L- 2 thieneylalanine; D- or L-l, -2, 3-, or 4- pyreneylalanine; D- or L-3 thieneylalanine; D- or L-(2-pyridmyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine; D-(trifluoromefhyl)-phenylglycine; D- (trifluoromethyl)-phenylalanine; D-p-fluoro-phenylalanine; D- or L-p- biphenylphenylalanine; D- or L-p-methoxy-bi
  • Aromatic rings of a non-natural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pynolyl, and pyridyl aromatic rings.
  • Mimetics of acidic amino acids can be generated by substitution by, e.g., non-carboxylate amino acids while maintaining a negative charge; (phosphono)alanine; sulfated threonine.
  • Carboxyl side groups (e.g., aspartyl or glutamyl) can also be selectively modified by reaction with carbodiimides (R'-N-C-N-R') such as, e.g., 1- cyclohexyl-3(2-mo holinyl-(4-ethyl) carbodiimide or l-ethyl-3(4-azonia- 4,4- dimetholpentyl) carbodiimide.
  • Aspartyl or glutamyl can also be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
  • Mimetics of basic amino acids can be generated by substitution with, e.g., (in addition to lysine and arginine) the amino acids ontithine, citrulline, or (guamdino)-acetic acid, or (gua-nidino)alkyl-acetic acid, where alkyl is defined above.
  • Nitrile derivative e.g., containing the CN-moiety in place of COOH
  • Asparaginyl and glutaminyl residues can be deaminated to the conesponding aspartyl or glutamyl residues.
  • Arginine residue mimetics can be generated by reacting argrnyl with, e.g., one or more conventional reagents, including, e.g., phenylglyoxal, 2,3-butanedione, 1,2-cyclo- hexanedione, or ninhydrin, preferably under alkaline conditions.
  • Tyrosine residue mimetics can be generated by reacting tyrosyl with, e.g., aromatic diazonium compounds or tefranitromethane.
  • N-acetylimidizol and tetranifromethane can be used to form O- acetyl tyrosyl species and 3 -nitro derivatives, respectively.
  • Cysteine residue mimetics can be generated by reacting cysteinyl residues with, e.g., alpha-haloacetates such as 2- chloroacetic acid or chloroacetamide and conesponding amines; to give carboxymethyl or carboxyamidomethyl derivatives.
  • alpha-haloacetates such as 2- chloroacetic acid or chloroacetamide and conesponding amines
  • Cysteine residue mimetics can also be generated by reacting cysteinyl residues with, e.g., bromo-trifluoroacetone, alpha-bromo-beta-(5- imidozoyl) propionic acid; chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide; methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4 nifrophenol; or, chloro-7-nitrobenzo-oxa-l,3-diazole.
  • cysteinyl residues e.g., bromo-trifluoroacetone, alpha-bromo-beta-(5- imidozoyl) propionic acid
  • chloroacetyl phosphate N-alkylmaleimides
  • 3-nitro-2-pyridyl disulfide methyl 2-pyridyl disulfide
  • Lysine mimetics canbe generated (and amino te-rminal residues can be altered) by reacting lysinyl with, e.g., succinic or other carboxylic acid anhydrides. Lysine and other alpha-ammo-containing residue mimetics can also be generated by reaction with imidoesters, such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitro- benzenesulfonic acid, O-methylisourea, 2,4, pentanedione, and transbiofilm-confrol enzyme-catalyzed reactions with glyoxylate.
  • imidoesters such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitro- benzenesulfonic acid, O-methylisourea, 2,4, pentanedione, and transbiofilm-confrol enzyme-catalyzed reactions with g
  • Mimetics of methionine can be generated by reaction with, e.g., metlfronine sulfoxide.
  • Mimetics of proline include, e.g., pipecolic acid, thiazolidine carboxylic acid, 3- or 4- hydroxy proline, dehydroproline, 3- or 4- methylproline, or 3,3,-dimethylproline.
  • Histidine residue mimetics can be generated by reacting histidyl with, e.g., diethylprocarbonate or para-bromophenacyl bromide.
  • mimetics include, e.g., those generated by hydroxylation of proline and lysine; phosphorylation of the hydroxyl groups of seryl or threonyl residues; methylation of the alpha-amino groups of lysine, arginine and histidine; acetylation of the N-terminal amine; methylation of main chain amide residues or substitution withN-methyl amino acids; or amidation of C-terminal carboxyl groups.
  • a residue, e.g., an amino acid, of a polypeptide of the invention can also be replaced by an amino acid (or peptidomimetic residue) of the opposite chirality.
  • any amino acid naturally occurring in the L-configuration (which can also be refened to as the R or S, depending upon the structure of the chemical entity) can be replaced with the amino acid of the same chemical structural type or a peptidomimetic, but of the opposite chirality, refened to as the D- amino acid, but also can be refened to as the R- or S- form.
  • the mvention also provides methods for modifying the polypeptides of the invention by either natural processes, such as post-translational processing (e.g., phosphorylation, acylation, etc), or by chemical modification techniques, and the resulting modified polypeptides. Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also a given polypeptide may have many types of modifications.
  • Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphatidylinositol, cross-linking cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma- carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristolyation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and fransfer-RNA mediated addition of amino acids to protein such as arginylation.
  • Solid-phase chemical peptide synthesis methods can also be used to synthesize the polypeptide or fragments of the invention. Such method have been known in the art since the early 1960's (Merrifield, R. B., J. Am. Chem. Soc, 85:2149-2154, 1963) (See also Stewart, J. M. and Young, J.
  • a plate of rods or pins is inverted and inserted into a second plate of conesponding wells or reservoirs, which contain solutions for attaching or anchoring an appropriate amino acid to the pin's or rod's tips.
  • amino acids are buUt into desired peptides.
  • FMOC peptide synthesis systems are available. For example, assembly of a polypeptide or fragment can be carried out on a solid support using an Applied Biosystems, Inc. Model 431 ATM automated peptide synthesizer.
  • the invention provides novel biofilm-control or biofilm modifying enzymes, including the exemplary enzymes SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:
  • the biofilm-control compositions of the invention have an amidase activity, e.g., the abihty to hydrolyze amides, including enzymes having secondary amidase activity, including a peptidase, a protease and/or a hydantoinase activity.
  • an amidase activity e.g., the abihty to hydrolyze amides, including enzymes having secondary amidase activity, including a peptidase, a protease and/or a hydantoinase activity.
  • the polypeptides of the invention have cellulase, esterase, glycosidase and/or phosphatase activity.
  • the biofilm-control enzymes of the invention have activities that have been modified from those of the exemplary biofilm-control enzymes described herein.
  • the invention includes biofilm-confrol enzymes with and without signal sequences and the signal sequences themselves.
  • the invention includes immobilized biofilm-confrol enzymes, anti-biofilm-control enzyme antibodies and fragments thereof.
  • the invention provides methods for inhibiting biofilm-confrol enzyme activity, e.g, using dominant negative mutants or anti-biofilm-control enzyme antibodies of the invention.
  • the invention includes heterocomplexes, e.g., fusion proteins, heterodimers, etc., comprising the biofilm-control enzymes of the invention.
  • Biofilm-confrol enzymes of the invention can be used in laboratory and industrial settings to hydrolyze amide compounds for a variety of purposes. These biofilm-control enzymes can be used alone to provide specific hydrolysis or can be combined with other biofilm-confrol enzymes to provide a "cocktail" with a broad spectrum of activity.
  • biofilm-control enzymes of the invention include their use to increase flavor in food (e.g., enzyme ripened cheese), promote bacterial and fungal kiUing, modify and de-protect fine chemical intermediates, synthesize peptide bonds, carry out chiral resolutions, hydrolyze amide-contaming antibiotics or other drugs, e.g., cephalosporin C.
  • food e.g., enzyme ripened cheese
  • promote bacterial and fungal kiUing modify and de-protect fine chemical intermediates
  • synthesize peptide bonds carry out chiral resolutions
  • hydrolyze amide-contaming antibiotics or other drugs e.g., cephalosporin C.
  • Biofilm-confrol enzymes of the invention can have a biofilm-confrol enzyme activity under various conditions, e.g., extremes in pH and/or temperature, oxidizing agents, and the like.
  • the invention provides methods leading to alternative biofilm-control enzyme preparations with different catalytic efficiencies and stabilities, e.g., towards temperature, oxidizing agents and changing wash conditions.
  • biofilm-control enzyme variants can be produced using techniques of site-directed mutagenesis and/or random mutagenesis.
  • directed evolution can be used to produce a great variety of biofilm-control enzyme variants with alternative specificities and stability.
  • the proteins of the invention are also useful as research reagents to identify biofilm-confrol enzyme modulators, e.g., activators or inhibitors of biofilm- confrol enzyme activity. Briefly, test samples (compounds, broths, extracts, and the like) are added to biofilm-control enzyme assays to determine their ability to inhibit hydrolysis. Inhibitors identified in this way can be used in industry and research to reduce or prevent undesired hydrolysis, e.g., proteolysis. As with biofilm-confrol enzymes, inhibitors can be combined to increase the spectrum of activity.
  • the invention also provides methods of discovering new biofilm-confrol enzymes using the nucleic acids, polypeptides and antibodies of the invention.
  • lambda phage libraries are screened for expression-based discovery of biofilm- confrol enzymes.
  • the invention uses lambda phage libraries in screening to allow detection of toxic clones; improved access to substrate; reduced need for engineering a host, by-passing the potential for any bias resulting from mass excision of the library; and, faster growth at low clone densities.
  • Screening of lambda phage libraries can be in liquid phase or in solid phase.
  • the invention provides screening in liquid phase. This gives a greater flexibility in assay conditions; additional subsfrate flexibility; higher sensitivity for weak clones; and ease of automation over solid phase screening.
  • the invention provides screening methods using the proteins and nucleic acids of the invention and robotic automation to enable the execution of many thousands of biocatalytic reactions and screening assays in a short period of time, e.g., per day, as well as ensuring a high level of accuracy and reproducibility (see discussion of arrays, below). As a result, a library of derivative compounds can be produced in a matter of weeks. For further teachings on modification of molecules, including small molecules, see PCT/US94/09174.
  • biofilm-confrol enzyme enzymes which are non-naturally occurring biofilm-control enzyme variants having a different proteolytic activity, stability, substrate specificity, pH profile and/or performance characteristic as compared to the precursor biofilm-confrol enzyme from which the amino acid sequence of the variant is derived.
  • biofilm-control enzyme variants have an amino acid sequence not found in nature, which is derived by substitution of a plurality of amino acid residues of a precursor biofilm-confrol enzyme with different amino acids.
  • the precursor biofilm-control enzyme may be a nat-xrally-occurring biofilm-confrol enzyme or a recombinant biofilm-confrol enzyme.
  • the useful biofilm-confrol enzyme variants encompass the substitution of any of the naturally occurring L-amino acids at the designated amino acid residue positions.
  • the invention also provides polypeptides, e.g., enzymes, such as biofihn- confrol enzymes, comprising signal sequences, and the nucleic acids that encode them.
  • the signal sequences of the invention are identified following identification of novel biofilm-control enzyme polypeptides.
  • the invention provides isolated (including recombinant) signal sequences, which can comprise a heterologous (chimeric) polypeptide comprising a signal sequence of the invention and another polypeptide (which can be or not be a polypeptide of the invention).
  • a signal sequence of the invention comprises or consists of (or consists essentially of) residues 1 to 24 of a polypeptide having a sequence as set forth in SEQ ID NO: 110, encoded, in one aspect, by a nucleic acid having a sequence as set forth in SEQ ID NO: 109; etc.:
  • novel biofilm-control enzyme signal peptides are identified by a method refened to as SignalP.
  • SignalP uses a combined neural network which recognizes both signal peptides and their cleavage sites.
  • biofilm-control enzymes of the invention may not have signal sequences. It may be desirable to include a nucleic acid sequence encoding a signal sequence from one biofilm-control enzyme operably linked to a nucleic acid sequence of a different biofilm-confrol enzyme or, optionally, a signal sequence from a non-biofilm-control enzyme protein may be desired.
  • the invention provides hybrid biofilm-control enzymes, antibodies and fusion proteins, including peptide libraries, comprising sequences of the invention.
  • the peptide libraries of the invention can be used to isolate peptide modulators (e.g., activators or inhibitors) of targets, such as biofilm-control enzyme substrates, receptors, enzymes.
  • the peptide libraries of the invention can be used to identify formal binding partners of targets, such as ligands, e.g., cytokines, hormones and the like.
  • the fusion proteins of the invention e.g., the peptide moiety
  • the invention provides fusions of biofilm-confrol enzymes and antibodies of the invention and other peptides, including known and random peptides. They can be fused in such a manner that the structure of the biofilm-control enzymes is not significantly perturbed and the peptide is metabolically or structurally confonnationally stabUized. This allows the creation of a peptide library that is easily monitored both for its presence within ceUs and its quantity.
  • Amino acid sequence variants of the invention can be characterized by a predetermined nature of the variation, a feature that sets them apart from a naturally occurring form, e.g, an allelic or interspecies variation of a biofilm-control enzyme sequence.
  • the variants of the invention exhibit the same qualitative biological activity as the naturaUy occurring analogue.
  • the variants can be selected for having modified characteristics.
  • the site or region for introducing an amino acid sequence variation is predetermined, the mutation per se need not be predetermined. For example, in order to optimize the performance of a mutation at a given site, random mutagenesis may be conducted at the target codon or region and the expressed biofilm-confrol enzyme variants screened for the optimal combination of desired activity.
  • the invention provides biofilm-confrol enzymes and antibodies where the structure of the polypeptide backbone, the secondary or the tertiary structure, e.g., an alpha-helical or beta-sheet structure, has been modified.
  • the charge or hydrophobicity has been modified.
  • tiie bulk of a side chain has been modified. Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative.
  • substitutions can be made which more significantly affect: the structure of the polypeptide backbone in the area of the alteration, for example a alpha-helical or a beta-sheet structure; a charge or a hydrophobic site of the molecule, which can be at an active site; or a side chain.
  • the invention provides substitutions in polypeptide of the invention where (a) a hydrophilic residues, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g.
  • variants can exhibit the same qualitative biological activity (i.e. biofilm-control enzyme activity) although variants can be selected to modify the characteristics of the biofilm-control enzymes as needed.
  • biofilm-control enzymes and antibodies of the invention comprise epitopes or purification tags, signal sequences or other fusion sequences, etc.
  • the biofilm-control enzymes and antibodies of the invention can be fused to a random peptide to form a fusion polypeptide.
  • fused or “operably linked” herein is meant that the random peptide and the biofihn-confrol enzyme are Hnked together, in such a manner as to minimize the disruption to the stabihty of the biofilm-confrol enzyme structure, e.g., it retains biofilm-control enzyme activity.
  • the fusion polypeptide (or fusion polynucleotide encoding the fusion polypeptide) can comprise further components as well, including multiple peptides at multiple loops.
  • the peptides and nucleic acids encoding them are randomized, either fully randomized or they are biased in their randomization, e.g. in nucleotide/residue frequency generally or per position.
  • Randomized means that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively.
  • the nucleic acids which give rise to the peptides can be chemically synthesized, and thus may incorporate any nucleotide at any position. Thus, when the nucleic acids are expressed to form peptides, any amino acid residue may be incorporated at any position.
  • the synthetic process can be designed to generate randomized nucleic acids, to allow the formation of all or most of the possible combinations over the length of the nucleic acid, thus forming a library of randomized nucleic acids.
  • the library can provide a sufficiently structurally diverse population of randomized expression products to affect a probabilistically sufficient range of cellular responses to provide one or more cells exhibiting a desired response.
  • the mvention provides an interaction library large enough so that at least one of its members will have a structure that gives it affinity for some molecule, protein, or other factor.
  • a variety of apparatus and methodologies can be used to in conjunction with the polypeptides and nucleic acids of the invention, e.g., to screen polypeptides for biofilm-confrol enzyme or antibody activity, to screen compounds as potential modulators, e.g., activators or inhibitors, of a biofilm- confrol enzyme activity, for antibodies that bind to a polypeptide of the invention, for nucleic acids that hybridize to a nucleic acid of the invention, to screen for cells expressing a polypeptide of the invention and the like.
  • a sample screening apparatus can include a plurality of capillaries fonned into an anay of adjacent capUlaries, wherein each capUlary comprises at least one wall defining a lumen for retaining a sample.
  • the apparatus can further include interstitial material disposed between adjacent capillaries in the anay, and one or more reference indicia formed within of the interstitial material.
  • a capUlary for screening a sample wherein the capillary is adapted for being bound in an anay of capillaries, can include a first wall defining a lumen for retaining the sample, and a second wall formed of a filtering material, for filtering excitation energy provided to the lumen to excite the sample.
  • a polypeptide or nucleic acid e.g., a ligand
  • a first component into at least a portion of a capillary of a capillary anay.
  • Each capillary of the capillary anay can comprise at least one wall defining a lumen for retaining the first component.
  • An air bubble can be introduced into the capillary behind the first component.
  • a second component can be infroduced into the capillary, wherein the second component is separated from the first component by the air bubble.
  • a sample of interest can be infroduced as a first liquid labeled with a detectable particle into a capillary of a capillary array, wherein each capillary of the capillary array comprises at least one wall defining a lumen for retaining the first liquid and the detectable particle, and wherein the at least one wall is coated with a binding material for binding the detectable particle to the at least one wall.
  • the method can frirther include removing the first liquid from the capUlary tube, wherein the bound detectable particle is maintained within the capillary, and introducing a second liquid into the capillary tube.
  • the capillary anay can include a plurality of individual capillaries comprising at least one outer wall defining a lumen.
  • the outer wall of the capillary can be one or more walls fused together.
  • the wall can define a lumen that is cylindrical, square, hexagonal or any other geometric shape. so long as the walls form a lumen for retention of a liquid or sample.
  • the capillaries of the capillary anay can be held together in close proximity to form a planar structure.
  • the capillaries can be bound together, by being fused (e.g., where the capiUaries are made of glass), glued, bonded, or clamped side-by-side.
  • the capillary anay can be formed of any number of individual capUlari.es, for example, a range from 100 to 4,000,000 capUlaries.
  • a capUlary anay can form a micro titer plate having about 100,000 or more individual capillaries bound together.
  • Nucleic acids or polypeptides of the invention can be immobilized to or applied to an anay.
  • Arrays can be used to screen for or monitor libraries of compositions (e.g., small molecules, antibodies, nucleic acids, etc.) for their ability to bind to or modulate the activity of a nucleic acid or a polypeptide of the invention.
  • a monitored parameter is transcript expression of a biofilm- confrol enzyme gene.
  • One or more, or, all the transcripts of a cell can be measured by hybridization of a sample comprising transcripts of the ceU, or, nucleic acids representative of or complementary to transcripts of a cell, by hybridization to immobilized nucleic acids on an anay, or "biochip.”
  • an “anay” of nucleic acids on a microchip some or all of the transcripts of a cell can be simultaneously quantified.
  • anays comprising genomic nucleic acid can also be used to determine the genotype of a newly engineered strain made by the methods of the invention.
  • Polypeptide anays can also be used to simultaneously quantify a plurality of proteins.
  • the present invention can be practiced with any known “anay,” also refened to as a “microanay” or “nucleic acid anay” or “polypeptide anay” or “antibody anay” or “biochip,” or variation thereof.
  • Arrays are generically a plurahty of “spots” or “target elements,” each target element comprising a defined amount of one or more biological molecules, e.g., oligonucleotides, immobilized onto a defined area of a substrate surface for specific binding to a sample molecule, e.g., mRNA transcripts.
  • any known array and/or method of m- ⁇ king and using anays can be incorporated in whole or in part, or variations thereof, as described, for example, inU.S. Patent Nos. 6,277,628; 6,277,489; 6,261,776; 6,258,606; 6,054,270; 6,048,695; 6,045,996; 6,022,963; 6,013,440; 5,965,452; 5,959,098 5,856,174; 5,830,645; 5,770,456; 5,632,957; 5,556,752; 5,143,854; 5,807,522; 5,800,992 5,744,305; 5,700,637; 5,556,752; 5,434,049; see also, e.g., WO 99/51773; WO 99/09217 WO 97/46313; WO 96/17958; see also, e.g., Johnston (1998) Cun.
  • the invention provides isolated or recombinant antibodies that specifically bind to a biofilm-confrol enzyme of the invention. These antibodies can be used to isolate, identify or quantify the biofilm-control enzymes of the invention or related polypeptides. These antibodies can be used to isolate other polypeptides within the scope the invention or other related biofilm-confrol enzymes.
  • the antibodies can be designed to bind to an active site of a biofilm-confrol enzyme.
  • the invention provides methods of inhibiting biofilm-control enzymes using the antibodies of the invention.
  • the antibodies can be used in immunoprecipitation, staining, immunoaffinity columns, and the like.
  • nucleic acid sequences encoding for specific antigens can be generated by immunization followed by isolation of polypeptide or nucleic acid, amplification or cloning and immobilization of polypeptide onto an anay of the invention.
  • the methods of the invention can be used to modify the structure of an antibody produced by a cell to be modified, e.g., an antibody's affinity can be increased or decreased.
  • the ability to make or modify antibodies can be a phenotype engineered into a cell by the methods of the invention.
  • Antibodies also can be generated in vitro, e.g., using recombinant antibody binding site expressing phage display libraries, in addition to the traditional in vivo methods using animals. See, e.g., Hoogenboom (1997) Trends Biotechnol. 15:62-70; Katz (1997) Annu. Rev. Biophys. Biomol. Struct. 26:27-45.
  • Polypeptides or peptides can be used to generate antibodies which bind specifically to the polypeptides, e.g., the biofilm-confrol enzymes, of the invention.
  • the resulting antibodies may be used in immunoaffinity chromatography procedures to isolate or purify the polypeptide or to determine whether the polypeptide is present in a biological sample.
  • a protein preparation such as an extract, or a biological sample is contacted with an antibody capable of specifically binding to one of the polypeptides of the mvention.
  • the antibody is attached to a solid support, such as a bead or other column matrix.
  • the protein preparation is placed in contact with the antibody under conditions in which the antibody specifically binds to one of the polypeptides of the invention.
  • the abUity of proteins in a biological sample to bind to the antibody may be determined using any of a variety of procedures familiar to those skilled in the art. For example, binding may be determined by labeling the antibody with a detectable label such as a fluorescent agent, an enzymatic label, or a radioisotope. Alternatively, binding of the antibody to the sample may be detected using a secondary antibody having such a detectable label thereon. Particular assays include ELISA assays, sandwich assays, radioimmunoassays, and Western Blots.
  • Polyclonal antibodies generated against the polypeptides of the invention can be obtained by direct injection of the polypeptides into an animal or by admmistering the polypeptides to a non-human animal. The antibody so obtained will then bind the polypeptide itself. In this manner, even a sequence encoding only a fragment of the polypeptide can be used to generate antibodies which may bind to the whole native polypeptide. Such antibodies can then be used to isolate the polypeptide from cells expressing that polypeptide.
  • any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique, the trioma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (see, e.g., Cole (1985) in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
  • Antibodies generated against the polypeptides of the invention may be used in screening for similar polypeptides (e.g., biofilm-confrol enzymes) from other organisms and samples. In such techniques, polypeptides from the organism are contacted with the antibody and those polypeptides which specifically bind the antibody are detected. Any of the procedures described above may be used to detect antibody binding. Kits
  • kits comprising the compositions, e.g., nucleic acids, expression cassettes, vectors, cells, fransgenic seeds or plants or plant parts, polypeptides (e.g., biofilm-control enzymes) and/or antibodies of the invention.
  • the kits also can contain instructional material teaching the methodologies and industrial uses of the invention, as described herein.
  • kits can be for biofilm confrol, biofilm modification, control of microorganisms (e.g., bacterial prevention or removal) increasing flavors in food (e.g., enzyme ripened cheeses), promoting bacterial and fungal killing, modifying and de-protecting fine chemical intermediates, synthesizing peptide bonds, carrying out chiral resolutions, hydrolyzing cephalosporin C using the enzymes of the mvention.
  • microorganisms e.g., bacterial prevention or removal
  • increasing flavors in food e.g., enzyme ripened cheeses
  • modifying and de-protecting fine chemical intermediates modifying and de-protecting fine chemical intermediates
  • synthesizing peptide bonds carrying out chiral resolutions
  • the methods of the invention provide whole cell evolution, or whole ceU engineering, of a cell to develop a new cell strain having a new phenotype, e.g., a new or modified biofilm-control enzyme activity, by modifying the genetic composition of the cell.
  • the genetic composition can be modified by addition to the cell of a nucleic acid of the invention.
  • To detect the new phenotype at least one metabolic parameter of a modified cell is monitored in the cell in a "real time" or "on-line” time frame.
  • a plurality of cells such as a cell culture, is monitored in "real time” or “on-line.”
  • a plurality of metabolic parameters is monitored in “real time” or “on-line.” Metabolic parameters can be monitored using the biofilm-control enzymes of the invention.
  • Metabolic flux analysis is based on a known biochemistry framework.
  • a linearly independent metabolic matrix is constructed based on the law of mass conservation and on the pseudo-steady state hypothesis (PSSH) on the infracellular metabolites.
  • PSSH pseudo-steady state hypothesis
  • pathway components e.g. aUosteric interactions, enzyme-enzyme interactions etc.
  • Metabolic phenotype relies on the changes of the whole metabolic network within a cell. Metabolic phenotype relies on the change of pathway utilization with respect to environmental conditions, genetic regulation, developmental state and the genotype, etc. In one aspect of the methods of the invention, after the on-line MFA calculation, the dynamic behavior of the cells, their phenotype and other properties are analyzed by investigating the pathway utilization.
  • the methods of the invention can help detennine how to manipulate the fermentation by dete ⁇ rnning how to change the substrate supply, temperature, use of inducers, etc. to confrol the physiological state of cells to move along desirable direction.
  • the MFA results can also be compared with franscriptome and proteome data to design experiments and protocols for metabolic engineering or gene shuffling, etc.
  • any modified or new phenotype can be confened and detected, including new or Unproved characteristics in the cell. Any aspect of metabolism or growth can be monitored. Monitoring expression of an mRNA transcript
  • the engineered phenotype comprises increasing or decreasing the expression of an mRNA transcript (e.g., a biofilm-confrol enzyme message) or generating new (e.g., biofilm-confrol enzyme) transcripts in a cell.
  • an mRNA transcript e.g., a biofilm-confrol enzyme message
  • generating new e.g., biofilm-confrol enzyme
  • This increased or decreased expression can be traced by testing for the presence of a biofilm-confrol enzyme of the invention or by biofilm-control enzyme activity assays.
  • mRNA franscripts, or messages also can be detected and quantified by any method known in the art, including, e.g., Northern blots, quantitative amplification reactions, 5 hybridization to anays, and the luce.
  • Quantitative amplification reactions include, e.g., quantitative PCR, including, e.g., quantitative reverse transcription polymerase chain reaction, or RT-PCR; quantitative real time RT-PCR, or "real-time kinetic RT-PCR" (see, e.g., Kreuzer (2001) Br. J. Haematol. 114:313-318; Xia (2001) Transplantation 72:907- 914).
  • the engineered phenotype is generated by knocking out expression of a homologous gene.
  • the gene's coding sequence or one or more transcriptional confrol elements can be knocked out, e.g., promoters or enhancers.
  • the expression of a transcript can be completely ablated or only decreased.
  • the engineered phenotype comprises 5 increasing the expression of a homologous gene. This can be effected by knocking out of a negative confrol element, including a transcriptional regulatory element acting in cis- or trans- , or, mutagenizing a positive control element.
  • a negative confrol element including a transcriptional regulatory element acting in cis- or trans- , or, mutagenizing a positive control element.
  • One or more, or, all the transcripts of a cell can be measured by hybridization of a sample comprising transcripts of the cell, or, nucleic acids representative of or complementary to franscripts of a cell, by hybridization 0 to immobilized nucleic acids on an anay.
  • the engineered phenotype comprises increasing or decreasing the expression of a biofilm confrol enzyme of the invention (e.g., an amidase enzyme) or generating new polypeptides in a cell.
  • a biofilm confrol enzyme of the invention e.g., an amidase enzyme
  • This increased or 5 decreased expression can be traced by determining Uie amount of biofilm-confrol enzyme present or by biofihn-confrol enzyme activity assays.
  • Polypeptides, peptides and amino acids also can be detected and quantified by any method known in the art, including, e.g., nuclear magnetic resonance (TSIMR), spectrophotometry, radiography (protein radiolabeling), electrophoresis, capillary electrophoresis, high performance liquid 0 chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, various immunological methods, e.g.
  • the invention provides methods and compositions (including products of manufacture) for use in a variety of industrial and medical applications, including methods and compositions comprising polypeptides of the invention (e.g., the exemplary SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:
  • SEQ ID NO:212 SEQ ID NO:214; SEQ ID NO:216; SEQ ID NO:218; SEQ ID NO:220 and/or SEQ ID NO:222, or any combination thereof.
  • the following table summarizes properties of polypeptides having SurE activity, or biofilm confrol or biofilm modifying activity, used in exemplary compositions and methods of the invention (for example, the polypeptide having a sequence as set forth in SEQ ID NO: 118 is, in one aspect, encoded by SEQ ID NO: 117, etc.):
  • 22299329 us elongatus BP-1
  • 161 , 162 [Chlorobium tepidum TLSJ. 21674375 TLS survival protein SurE [Deinococcus Deinococcus
  • 181 , 182 [Brucella suis 1330]. 23501772 Brucella suis 1330
  • CMCP6 hypothetical protein [Xylella fastidiosa Xylella fastidiosa
  • the invention provides methods and compositions for treating or coating cooling systems; food and beverage processing systems; industrial processing systems (e.g., for water); pulp and paper mill systems; brewery pasteurizers; sweetwater systems; air washer systems; oil field drilling fluids and muds; petroleum recovery processes; industrial lubricants; cutting fluids; heat transfer systems; gas scrubber systems; latex systems; clay and pigment systems; decorative fountains; water intake pipes; ballast water tanks; and ship reservoirs, and the like.
  • industrial processing systems e.g., for water
  • pulp and paper mill systems e.g., for water
  • brewery pasteurizers e.g., for water
  • sweetwater systems e.g., air washer systems
  • oil field drilling fluids and muds e.g., oil field drilling fluids and muds
  • petroleum recovery processes e.g., industrial lubricants
  • cutting fluids e.g., heat transfer systems; gas scrubber systems; latex systems; clay and pigment systems; decorative fountains; water intake pipes; ballast
  • the invention also provides methods and compositions for treating or coating medical devices, including surgical instruments, implants, valves, sutures, dressings and the like, and medical devices comprising the enzymes of the invention and/or polypeptides having a SurE activity, e.g., as described herein.
  • the invention also provides methods and compositions for treating drugs and pharmaceuticals, including tablets, pills, implants, suppositories, inhalers, sprays, ointments, and the like, using the enzymes of the invention and/or polypeptides having a SurE activity, e.g., as described herein, and drugs and pharmaceuticals comprising the enzymes of the invention and/or polypeptides having a SurE activity, e.g., as described herein.
  • polypeptides e.g., enzymes and antibodies
  • the polypeptides (e.g., enzymes and antibodies) of the invention can be used to remove or confrol biofilms from any medical device, drug or pharmaceutical.
  • the invention provides medical devices, drugs and pharmaceuticals comprising an enzyme of the mvention and/or polypeptides having a SurE activity, e.g., as described herein.
  • the mvention includes all compositions wherein it may be advantageous to prevent or remove a biofilm comprising an enzyme of the invention.
  • compositions can further comprise a antimicrobial agent or a antimicrobial composition e.g., rifamycins (e.g., rifampin), tefracyclines (e.g., minocycline), macrolides (e.g., erythromycin), penicillins (e.g., nafcillin), cephalosporins (e.g., cefazolin), carbepenems (e.g., imipenem), monobactams (e.g., azfreonam), aminoglycosides (e.g., gentamicin), chloramphenicol, sulfonamides (e.g., sulfamefhoxazole), glycopeptides (e.g., vanomycin), metronidazole, clindamycin, mupirocin, quinolones (e.g., ofloxacm
  • rifamycins e.g
  • the invention provides medical devices comprising one or more enzymes of the invention and/or polypeptides having a SurE activity, e.g., as described herein, these medical devices including disposable or permanent catheters, (e.g., central venous catheters, dialysis catheters, long-term tunneled central venous catheters, short-term central venous catheters, peripherally inserted central catheters, peripheral venous catheters, pulmonary artery Swan-Ganz catheters, urinary catheters, and peritoneal catheters), long-term urinary devices, tissue bonding urinary devices, vascular grafts, vascular catheter ports, wound drain tubes, ventricular catheters, hydrocephalus shunts heart valves, heart assist devices (e.g., left ventricular assist devices), pacemaker capsules, incontinence devices, penile implants, small or temporary joint replacements, urinary dilator, cannulas, elastomers, hydrogels, surgical instruments, dental instruments, tubings, such as intravenous tubes
  • the invention provides medical devices comprising one or more enzymes of the invention and/or polypeptides having a SurE activity, e.g., as described herein, these medical devices including any device which may be inserted or implanted into a human being or other animal, or placed at the insertion or implantation site such as the skin near the insertion or implantation site, and wliich include at least one surface which is susceptible to colonization by biofilm embedded microorganisms.
  • the enzymes of the invention and/or polypeptides having a SurE activity can be used in conjunction with (e.g., be coated onto, use to treat) any surface which may be desired or necessary to prevent biofilm embedded microorganisms from growing or proliferating in or on at least one surface of a medical device or a drug or pharmaceutical, or to remove or clean biofilm embedded microorganisms from the at least one surface of a medical device or a drug or pharmaceutical, such as the surfaces of equipment in operating rooms, emergency rooms, hospital rooms, clinics, and bathrooms.
  • the enzymes of the invention and/or polypeptides having a SurE activity can be beneficial on any surface-fluid environment in household, industrial, personal hygiene and medical contexts.
  • the invention provides composition and methods for controlling (e.g., removing or slowing the growth of) or preventing biofilm formation on cooling tower packing materials, which otherwise would result in the loss of heat transfer efficiency and thereby altering system thermodynamics.
  • the invention provides enzymes of the invention and/or polypeptides having a SurE activity, e.g., as described herein, in a variety of enzyme/biocide formulations.
  • the compositions and methods of the invention and/or polypeptides having a SurE activity can be used in the cleaning and decontamination of hard surfaces such as floors, working surfaces, equipment and process machinery.
  • compositions and methods of the invention and/or polypeptides having a SurE activity can be used in conjunction with (e.g., for cooling systems) quaternary ammonium compounds such as cocobenzyl-dimethyl ammonium chloride and other chemicals such as BNPD (2- bromo-2-nifropropane-l,3-diol), DBNPA, glutaraldehyde, active halogens and phenohcs.
  • quaternary ammonium compounds such as cocobenzyl-dimethyl ammonium chloride and other chemicals such as BNPD (2- bromo-2-nifropropane-l,3-diol), DBNPA, glutaraldehyde, active halogens and phenohcs.
  • compositions and methods of the invention and/or polypeptides having a SurE activity can be used in sanitizers and disinfectants, including janitorial/medical products and dairy and food processing products.
  • Enzyme anti-biofilm formulations of the mvention can serve both as cleaning and microbe sanitizing and disinfectant agents on hard surfaces.
  • the compositions and methods of the invention can be used to improve cleaning efficiency and decrease mechanical contact requirement and to decrease chemical usage.
  • compositions and methods of the invention and/or polypeptides having a SurE activity can be used in dairy and food processing products to dislodge and remove microbes, to decrease pitch deposits, to decrease chemical usage and to enable use of equipment and environment-friendly chemicals.
  • compositions and methods of the invention and/or polypeptides having a SurE activity can be to increase usage of low temperature cleaners and to set higher cleanliness standards.
  • the compositions and methods of the invention and/or polypeptides having a SurE activity can be used in conjunction with quaternary ammonium compounds, miscellaneous biocides such as glycine-based amphoterics, glyoxal, biguanides, foUowed by active halogens, phenolics, organic acids/salts, and organosulfur chemicals, and amine-based chemicals and organic acids/salts.
  • anti- biofilm enzymes can be used in products such as surgical implants, bone fixtures and catheters.
  • the compositions and methods of the invention and/or polypeptides having a SurE activity e.g., as described herein, can be used to dislodge and remove plaque from dental and oral surfaces, to prevent tartar formation and to decrease toxicity and skin irritation.
  • the invention provides methods for treating (including removing, slowing the growth of or preventing the growth of) biofilms comprising contacting a composition (e.g., a water freatment device, a water conduit such as a pipe, a medical device, a drug, etc.) by contacting the composition with at least one polypeptide (e.g., antibody or enzyme) of the invention and/or polypeptides having a SurE activity, e.g., as described herein.
  • the methods can comprise soaking, rinsing, flushing, submerging or washing with a composition (e.g., a solution, fluid, gas, spray) comprising at least one polypeptide of the invention.
  • the composition can be contacted with a biofilm control composition of the mvention for a period of time sufficient to remove some, or, substantially all, of the biofilm, including, e.g., embedded microorganisms.
  • the composition can be submerged in a biofilm confrol composition of the invention for at least 1, 5, 10, 15, 20, 30, 40, 50 or 60 minutes.
  • a composition e.g., medical device, water pipe
  • the biofilm confrol composition of the invention may be poured into the pipe or tubing and both ends of the pipe or tube sealed or clamped such that the biofilm control composition of the mvention is retained within the lumen of the pipe or tube.
  • the pipe or tube is then allowed to remained filled with the biofilm confrol composition of the invention for a period of time sufficient to remove some or, substantially all, of the biofilm embedded microorganisms, e.g., from at least one surface.
  • the freatment can last from at least about 1 minute to about 48 or more hours.
  • the pipe or tubing may be flushed by pouring the biofilm control composition of the invention into the lumen of the pipe or tubing for an amount of time sufficient to prevent, or remove, substantially all biofilm embedded microorganism growth.
  • the methods and compositions of the invention, and/or polypeptides having a SurE activity find use to remove or prevent biofilms and their associated microorganisms in order to decrease conosion of a conosion- sensitive material, e.g., a tank, e.g., ballast tanks.
  • compositions and methods of the invention, and/or polypeptides having a SurE activity can also be used in combination with other biofilm controlling methods and compositions.
  • coating ballast tanks with non-toxic, conosion-resistant epoxy or methylsilicone polymers which have been reported to help control fransferable biodiversity that appears in biofilms in ballast tanks.
  • Biocides and ozone or UV treatments that can also be combined with methods and compositions of the invention.
  • compositions of the invention, and/or polypeptides having a SurE activity can be used in combination with a water filter, purifier, or sterilizer, to remove, decrease or kill, the microorganisms and pathogens associated with the biofilm.
  • a water filter, purifier, or sterilizer to remove, decrease or kill, the microorganisms and pathogens associated with the biofilm.
  • This is particular useful with the present invention that allows the biofilm to release from its host surface, or for the microorganisms associated with the biofilm to enter a mobile growth (planktonic) state and release from the biofilm.
  • Such released organisms are more readily treatable by filters or other liquid purifying or sterilizing means.
  • enzymes of the invention include esterase SEQ ID NOs: 29, 30, 85, 86, glycosidase SEQ ID NOs: 7, 8, 67, 68, 1, 2, 95, 96, 59, 60, 23, 24, 105, 106, 43, 44, 51, 52, 57, 58, 71, 72, 93, 94, 103, 104, 89, 90, deacetylase SEQ ID NOs: 15, 16, 53, 54, amidase SEQ ID NOs: 107, 108.
  • enzymes of the invention include esterase SEQ ID NOs: 49, 50, deacetylase SEQ ID NOs: 53, 54, amidase SEQ ID NOs: 41, 42, 115, 116, 9, 10, 77, 78, phosphatase SEQ ID NOs: 5, 6, 211, 212, 11, 12, 45, 46, glycosidase SEQ ID NOs: 83, 84, 89, 90, 113, 114, 73, 74, 101,
  • enzymes of the invention that shown significant activity in biofilm prevention, particularly for Staphylococcus biofilms, include esterase SEQ ID NOs: 79, 80, 55, 56, 49, 50, glycosidase SEQ ID NOs: 31, 32, 57, 58, 83, 84, 89, 90, 113, 114, 73, 74, 7, 8, amidase SEQ ID NOs: 41, 42, 91, 92, 115, 116, 39, 40, 9, 10, 77, 78, phosphatase SEQ ID NOs: 27, 28, 81, 82, 21, 22, 45, 46.
  • Figure 12 shows enzyme activity against biofilm measured as initial rate of reaction at varying biocide concentrations.
  • biocide Barquat MB-80 commercially obtained from Lonza Group, Brazil
  • Alkyl Dimethyl Benzyl Ammonium Chloride was present at 10, 100 and 1000 ppm in the biofilm removal reactions. Activities are compared to control with no biocide present. Enzymes are designated by their SEQ ID Nos or that of their encoding genes. Barquat MB-80 is often used in paper and pulp water processing.
  • biocide Bardac LF (commercially obtained from Lonza Group, Brazil), N,N-Dioctyl-N,N-dimethylammom ' um chloride, was present at 10, 100 and 1000 ppm in the biofilm removal reactions. Activities are compared to control with no biocide present. Enzymes are designated by their SEQ ID Nos or that of their encoding genes. Bardac LF is often used as a disinfectant in schools, hospitals and institutions.
  • Figure 15 shows enzyme activity against biofilm measured as initial rate of reaction at varying biocide concentrations.
  • biocide Bardac 2280 commercially obtained from Lonza Group, Brazil
  • N,N-Didecyl-N,N-dimethylammom ' um chloride was present at 10, 100 and 1000 ppm in the biofilm removal reactions. Activities are compared to confrol with no biocide present. Enzymes are designated by their SEQ ID Nos or that of their encoding genes.
  • Bardac 2280 is often used as an industrial water freatment and in the pulp and paper industry.
  • Figure 16 shows enzyme activity against biofilm measured as initial rate of reaction at varying biocide concentrations.
  • biocide AqucarTM 515 (Dow Chemical) was present at 10, 100 and 1000 ppm in the biofilm removal reactions. Activities are compared to confrol with no biocide present. Enzymes are designated by their SEQ ID Nos or that of their encoding genes.
  • AqucarTM 515 Water Treatment Microbiocide is an aqueous solution of glutaraldehyde containing 15% active ingredient.
  • FIG 17 shows enzyme activity against biofilm measured as initial rate of reaction at varying biocide concentrations.
  • biocide Dow Antimicrobial 7287 Dow Antimicrobial 7287 (Dow Chemical) was present at 10, 100 and 1000 ppm in the biofilm removal reactions. Activities are compared to control with no biocide present. Enzymes are designated by their SEQ ID Nos or that of their encoding genes.
  • Dow Antimicrobial 7287 contains 20% of the active ingredient 2,2- dibromo-3-nitrilopropionamide, commonly refened to as DBNPA. It is used as a broad- spectrum control of bacteria, fungi, yeast, and algae in water freatment, pulp and paper, reverse osmosis, oil and gas, and metalworking fluid applications.
  • biofilms can be generated using solutions used in actual process conditions, see, e.g., MacDonald, et al. (2000) "The response of a bacterial biofilm community in a simulated industrial cooling water system to treatment with an anionic dispersant.” J. Appl. Microbiol. 89:225-235.
  • Such test biofilms can be created using microbial biofilm inoculum from actual process equipment, ballast tanks, water cooling towers, medical or in-patient devices or implants, etc.
  • an industrial site inoculum can be scraped from above the water line where it may be exposed to light and wetted by cooling tower water.
  • Comparison of 16s RNA and tRFLPs, preferably in combination, analysis of lab generated biofilms with original inoculum can indicate whether species differences exist.
  • tRFLP method PCR amplify of 16S rDNA with one labeled primer and one regular primer, followed by digesting the DNA with enzymes (5 different ones for example), fractionate (e.g., on capillaries) to determine fragment sizes, and identify pattern matches with predicted fragments based on known 16S sequences.
  • the method is faster and less expensive than standard 16S analysis, which involves PCR ampHfy 16S rDNA with regular primers, cloning individual fragments, sequence individual clones (high throughput, 96 well format), and finding top hits using BLAST, followed by a phylogenetic analysis to find how close sequence is to an isolate in database.
  • standard 16S analysis which involves PCR ampHfy 16S rDNA with regular primers, cloning individual fragments, sequence individual clones (high throughput, 96 well format), and finding top hits using BLAST, followed by a phylogenetic analysis to find how close sequence is to an isolate in database.
  • tRFLP provides a qualitative fingerprint of species present, useful for gauging the level of diversity in sample (i.e., the number of phylotypes), provides family or genus level resolution, although can lead to false negatives and positives.
  • 16S sequencing provides a quantitative sampling approach of a finite number of clones, can be manually inspected and match-quality verified, providing genus or species level resolution, with rarely false calls. While neither approach can unambiguously identify a bacterium (e.g. E. coli K12 versus E. coli O157: subspecies-level resolution needed), combined they provide a powerful tool, tRFLP for overall diversity, 16S for bacterial identification. Using the above methods, certain biofilm samples analyzed indicated a connection between disease samples and certain species.
  • Pseudomonas pseudoalcaligenes is found in biofilms and relates to multiple opportunistic infections.
  • Aeromonas hydrophila has been found in biofilm from pneumonia in children with underlying disease.
  • Pseudomonas anguilliseptica a fish pathogen (sea bream)
  • Comamonas testosterone has been isolated from biofilm associated with meningitis.
  • Chryseobacterium meningosepticum (Flavobacterium meningosepticum) has been isolated from biofilm in meningitis in neonatal nurseries. Holophaga sp. has been isolated from a human oral biofilm.
  • biocide dosages used in actual industrial process were tested, including: chlorine at 50-100 ppm, at 1-2 hours contact time, ozone at 10-50 ppm at less than 1 hour contact time, chorine dioxide at 50-100 ppm for 1-2 hours contact time, hydrogen peroxide at 10% (v/v) at 2-3 hours contact time, iodine at 100-200 ppm for 1-2 hours contact time, quaternary ammonia compounds at 300-1000 ppm for 2-3 hours contact time, formaldehyde at 1-2% for 2-3 hours contact time, and anionic and nonionic surfactants at 300-500 ppm for 3-4 hours contact time.
  • compositions and methods of the invention can be used in a variety of medical, food and personal care applications.
  • the invention provides methods and compositions for treating or coating medical devices, including surgical instruments, implants, valves, sutures, dressings and the like, using the enzymes of the invention, and medical devices comprising the enzymes of the invention.
  • the invention also provides methods and compositions for treating drugs and pharmaceuticals, including tablets, pills, implants, suppositories, inhalers, sprays, ointments, and the like, using the enzymes of the invention, and drugs and pharmaceuticals comprising the enzymes of the invention.
  • the polypeptides (e.g., enzymes and antibodies) of the invention can be used to remove or confrol biofilms from any medical device, drug or pharmaceutical.
  • the invention provides medical devices, drugs and pharmaceuticals comprising an enzyme of the invention.
  • the invention includes all compositions wherein it may be advantageous to prevent or remove a biofilm comprising an enzyme of the invention.
  • These compositions can further comprise a antimicrobial agent or a antimicrobial composition e.g., rifamycins (e.g., rifampin), tefracyclines (e.g., minocycline), macrolides (e.g., erythromycin), penicillins (e.g., nafcillin), cephalospoi ⁇ ns (e.g., cefazolin), carbepenems (e.g., ⁇ nipenem), monobactams (e.g., azfreonam), aminoglycosides (e.g., gentamicin), chloramphemcol, sulfonamides (e.g., sulfamefhoxazole
  • the invention provides medical devices comprising one or more enzymes of the invention, these medical devices including any device which may be inserted or implanted into a human being or other animal, or placed at the insertion or implantation site such as the skin near the insertion or implantation site, and wliich include at least one surface which is susceptible to colonization by biofilm embedded microorganisms.
  • the enzymes of the invention can be used in conjunction with (e.g., be coated onto, use to treat) any surface wliich may be desired or necessary to prevent biofilm embedded microorganisms from growing or proliferating in or on at least one surface of a medical device or a drug or pharmaceutical, or to remove or clean biofilm embedded microorganisms from the at least one surface of a medical device or a drug or pharmaceutical, such as the surfaces of equipment in operating rooms, emergency rooms, hospital rooms, clinics, and bathrooms.
  • the invention provides adhesives, such as tapes, comprising at least one enzyme of the invention.
  • Methods for coating compositions, such as medical devices are well known in the art and are described, e.g., in U.S. Patent No. 6,475,434.
  • compositions and solutions including buffer solutions (e.g., phosphate buffered saline), saline, water, polyvinyl, polyethylene, polyurethane, polypropylene, silicone (e.g., silicone elastomers and silicone adhesives), polycarboxylic acids, (e.g., polyacrylic acid, polymethacrylic acid, polymaleic acid, poly- (maleic acid monoester), polyaspartic acid, polyglutamic acid, aginic acid or pectimic acid), polycarboxylic acid anhydrides (e.g., polymaleic anhydride, polymethacrylic anhydride or polyacrylic acid anhydride), polyamines, polyamine ions (e.g., polyethylene imine, polyvinylan-rine, polylysine, poly-(dialkylamineoethyl methacrylate), poly-
  • buffer solutions e.g., phosphate buffered saline
  • saline water
  • polyvinyl poly
  • dialkylaminomethyl styrene or poly-(vinylpyridine)
  • polyammonium ions e.g., poly- (2-methacryloxyethyl trialkyl ammonium ion), poly-(vinylbenzyl trialkyl ammonium ions), poly-(N.N.-alkylypyridinium ion) or poly-(dialkyloctamethylene ammonium ion)
  • polysulfonates e.g. poly-(vinyl sulfonate) or poly-(styrene sulfonate)
  • collodion nylon, rubber, plastic, polyesters, Gortex (polytefrafluoroethylene), DACRONTM
  • polyethylene tefraphthalate polyethylene tefraphthalate
  • TEFLONTM polytefrafluoroethylene polytefrafluoroethylene
  • latex and derivatives thereof, elastomers, gelatin, collagen or albumin, cyanoacrylates, methacrylates, papers with porous barrier films, adhesives, e.g., hot melt adhesives, solvent based adhesives, and adhesive hydrogels, fabrics, and crosslinked and non-crosslinked hydrogels, comprising one or more polypeptide, e.g., enzymes of the invention.
  • compositions and methods of the invention can be used as preservatives in food, medicinal (e.g., drug), hygiene and cosmetic products.
  • the compositions and methods of the invention can be used in personal care products such as toothpastes, chewing gum, mouthwashes, dental appliance cleaners, contact lens cleaners.
  • the compositions and methods of the invention can be used in any skin and tissue related environment, e.g., products used in the medical fields.
  • anti-biofilm enzymes can be used in products such as surgical implants, bone fixtures and catheters.
  • the compositions and methods of the invention can be used to dislodge and remove plaque from dental and oral surfaces, to prevent tartar formation and to decrease toxicity and skin irritation.
  • the invention provides methods for treating (including removing, slowing the growth of or preventing the growth of) biofilms comprising contacting a composition (e.g., a water treatment device, a water conduit such as a pipe, a medical device, a drug, etc.) by contacting the composition with at least one polypeptide (e.g., antibody or enzyme) of the invention.
  • a composition e.g., a water treatment device, a water conduit such as a pipe, a medical device, a drug, etc.
  • the methods can comprise soaking, rinsing, flushing, submerging or washing with a composition (e.g., a solution, fluid, gas, spray) comprising at least one polypeptide of the invention.
  • SurE proteins or the amidases find use to induce, directly or indirectly, the change from sessile to planktonic stage, a growth stage in which the microorganism is typically more susceptible to a biocide or other freatment.
  • these embodiments further biofilm destruction and release, minimizing biofilm growth, increasing the accessibility of biofilm-associated microorganisms and pathogens to biocides, and increase the susceptibility of the microorganism to a biocide or other treatment by changing its growth state, and presumably its morphology and/or physiology.
  • Tissue infections that lead to tissue damage wliich stem form or are exacerbated by microorganisms in biofilms include, (a) respiratory tract infections such as otitis media, cystic fibrosis and related lower respiratory tract infections, (b) gastrointestinal tract infections such as binary tract stentrng and brown stone pigment formation, (c) urinary and genital tract infections such as complicated UTIs such as strivite urolithiasis, and chronic bacterial prostatitis, (d) infections of the locomotive system such as osteomyelitis such as those that are implant related or diabetes and irnmune-deficiency disease related (e.g., AIDS associated), (e) cardiovascular infections such as infective cardioitis, oral microflora sourced, and prosthetic valve endocarditis, and (f) wound infections. Wound infections are particularly amenable since the invention can be topically applied as described herein, for example in the form of creams or ointments or as a component of a bandage or other dressing
  • compositions and methods find use in modifying or treating the devices identified as causally related to fransmitting the infections, such as respiratory-therapy devices, mechamcal ventilation devices, stents, etc. as described herein.
  • compositions, devices and methods are further useful because they can access difficult-to-reach areas of the body, including oral, nasal cavities, and internal cavities, and further are non-toxic, non-staining, catalytic biomolecules.
  • biomolecules of the invention are, or can be modified to be, sufficiently stable to other active components such as biocides, dispersants, surfactants, antibiotics, for medical, marine, agricultural, industrial, commercial, residential and personal care uses as described herein.
  • synergistic effect between the biomolecules of the present invention and these other biofilm-active components can be obtained, which provides a powerful advantage for biomolecules of the invention such embodiments.
  • biomolecules of the invention provide the opportunity to either displace or augment, and thus using lesser amounts, of those other components and agents.
  • Such other methods of biofilm control include agents to prevent or inhibit direct microbe-assisted breakdown of the biocide or antibiotic by hydrolysis or oxidation/reduction, or that result from the ambient environment, e.g., aerobic, anaerobic, reducing/oxidizing, created by the biofilm members and by the macroscopic structuring of the film.
  • Ultrasound treatment has been shown to remove biofilm microorganisms such as Pseudomonas on various surfaces including steel.
  • Jow-sfrength electrical fields (plus or minus 12 V cm “1 ) combined with a low current density (plus or minus 2.1 mA cm “2 ) enhanced the efficacy of gentamicin against the same biofilms.
  • These methods can be combined with the freatment methods of the present invention, both for medical, industrial and other applications.
  • the biomolecules of the invention can be attached to, embedded, adhered, or coated on medical devices and implants as discussed herein, while retaining catalytic activity, they are particularly useful to confrol medical biofilms.
  • the present invention provides a surface of a device or implant that has the property of selective binding, growth, permeability or integration of host or engineered cells while reducing or inhibiting that of a pathogen.
  • a particularly useful embodiment for coating or treating devices and implants is the combination of biocide, e.g., antibiotic, biocidal enzyme (e.g., lysostaphin, nisin), with an enzyme of the invention.
  • biocide e.g., antibiotic
  • biocidal enzyme e.g., lysostaphin, nisin
  • Contact lens storage and/or cleaning solutions can be modified to contain the biomolecules of the invention.
  • mastitis a major reason for culling dairy cows, is biofilm disease susceptible to treatments with compositions of the present invention.
  • the foUowing table provides a partial list of infections amenable to prevention (prophylaxis) or ameUoration (treatment) with the present invention (the foUowing is a table of infections (including human infections) involving biofilms freated (including prophylactic applications) by compositions and methods of the invention):
  • Acidogenic Gram-positive cocci e.g.,
  • Peritoneal dialysis A variety of bacteria and fungi peritonitis
  • Endotracheal tubes A variety of bacteria and fungi Hickman catheters S. epidermidis and C. albicans Central venous catheters S. epidermidis and others Mechanical heart valves S. aureus and S. epidermidis Vascular grafts Gram-positive cocci Biliary stent blockage A variety of enteric bacteria and fungi Orthopedic devices S. aureus and S. epidermidis Penile prostheses S. aureus and S. epidermidis
  • treatment of biofilms is via delivery, administration, or contacting a biofilm, biofilm-involved-pathogen or surface for which biofilm confrol is desired, with an expression host expressing an enzyme of the invention, or a canier of the biofilm-control enzyme gene that can infect the target microbe which will in turn produce the biofilm-control agent of the invention.
  • an expression host expressing an enzyme of the invention, or a canier of the biofilm-control enzyme gene that can infect the target microbe which will in turn produce the biofilm-control agent of the invention.
  • a gene encoding a biofilm-controUing enzyme can be put under the control of an inducible promoter responsive to the presence of a biofihn component or microorganism or other aspect of the environment, such a salinity (as for ballast water treatment).
  • a particularly useful embodiment is a host that carries a gene(s) encoding one or more enzymes of the invention, and is able to infect/transfect/replicate in the biofilm-associated microorganism or pathogen.
  • Bacteriophage are capable of caring such genes of the invention, as disclosed herein, and can selectively infect one or more biofilm-associated microorganisms, whether pathogen and/or a biofilm producing microorganism. In one embodiment, this would lead to expression of the enzymes of the invention by the biofilm itself.
  • such phage can be engineered to deliver or express additional active compounds, or can be co-administered with active drugs or other disinfectant or anti-biofilm agents.
  • a biofilm In early stages a biofilm is comprised of a cell layer attached to a surface. The cells grow and divide, forming a dense mat numerous layers thick. When sufficient numbers of bacteria are present (quorum) they signal each other to reorganize forming an array of pillars and irregular surface structures, all connected by convoluted channels that deliver food and remove waste. The biofilm produces, a glycocalyx matrix shielding them from the environment. This is quorum-sensing. As the biofilm matures, the bacteria become greatly more resistant to antibiotics than when in the planktonic (free cell) state. The host immune system is also significantly less effective against bacteria in the biofilm state.
  • Certain bacterial strains may be able to confer resistance protecting the biofilm from host defense components that would otherwise bind to the surface of viable bacteria and kill them. Yet the bacterial biofilm exudes lipopolysaccharide agitating the host inflammatory response which, in periodontitis, contributes to the tissue destruction.
  • the SurE and amidases may function in the disruption of the biofilm signaling pathway. It would be of substantial benefit to biofilm-control to identify the targets of these enzymes, and further develop biofilm-control agents that affect those targets or signal induction pathways with which control quorum sensing.
  • SurE is beheved to interact with an isoaspartyl methylfransferase (pcm gene product) that functions to convert the isoaspartyl residues of "aging" proteins back to aspartyl residues and thereby functions in protein damage confrol and repair. Genetic analysis of the interaction has shown that surE expression is detrimental to stationary phase cells lacking pcm.
  • the substrate or ligand, or the protein or gene with which it is associated or produces it are targets in assays to develop or screen for interacting molecules that are candidate biofilm-confrol agents.
  • the assay would involve the step of screening for agents that interact with or control the expression or activity of the subsfrate or ligand or their associated protein or gene, and then testing those agents for their abihty to confrol biofilm, preferably for their ability to induce or suppress quorum sensing or inducing biofilm microorganisms to enter or remain in a planktonic state.
  • a method of screening candidate enzymes as biofilm-confrol enzymes comprising the step of identifying one or more candidate enzymes as described herein, assaying the enzyme for a desirable biofilm-control property, selecting the candidate or candidates having a desirable biofilm-confrol property.
  • the candidate enzyme is a homolog, whether annotated as a putative protein, hypothetical or actual protein, of an enzyme of the invention, typically identified through amino acid or nucleic acid sequence comparison.
  • the following example describes an exemplary biofilm micro-assay for evaluating matrix-hydrolyzing enzymes.
  • This exemplary protocol can be used to identify matrix-hydrolyzing enzymes within the scope of the invention.
  • biofilm control assays were carried out by adding the test enzyme and the biofilm-forming bacterial culture to the wells of a 96-well plate at the beginning of the assay. Following overnight incubation in a humidified chamber, the wells of the plate were washed to remove non-adherent cells and then fluorescent staining was used to quantitate cells remaining within the biofilm.
  • biofilm removal assays were performed by adding the test enzyme to wells containing an established early biofilm. Briefly, the biofilm-forming bacterial culture was added to the wells of a 96-well plate and statically incubated in a humidified chamber. Following establishment of the biofilm, the weUs are thoroughly washed to remove non-adherent cells. Next, the test enzyme is added and the plate is returned to the humidified chamber. Following an overnight incubation, the wells of the plate are again thoroughly washed to remove unattached cells. Cells remaining in the biofilm are detected with a fluorescent dye.
  • FIG. 5 illustrates how each enzyme was evaluated in four separate biofilm micro-assays. These assays were designed to measure enzymatic confrol or enzymatic removal of biofilms formed by gram-negative (Pseudomonas fluorescens) and gram-positive (Staphylococcus epidermidis) bacteria.
  • Example 2 Exemplary methods to characterize purified enzymes and test enzyme combinations for additive or synergistic activities
  • This example describes exemplary methods for characterizing purified enzymes and testing enzyme combinations for additive or synergistic activities. These exemplary methods have three parts: part one, micro-scale purification of candidates from secondary screen; part two, basic characterization of purified candidates; and part three, testing of enzyme mixtures for additive or synergistic activities. Following the secondary screen, there were 3 candidate enzymes (from 3 enzyme classes) that demonstrated significant efficacy for removal of Pseudomonas fluorescens biofilms and there were 19 candidate enzymes (from 5 enzyme classes) that demonstrated efficacy against Staphylococcus epidermidis biofilms.
  • Dose response exhibited by each enzyme in either the biofilm confrol or biofilm removal micro-assay can be determined.
  • biochemical characterization assays can be performed with each enzyme. These assays can detennine the pH, temperature (thermal stability and thermal tolerance), and subsfrate profiles for the enzymes.
  • the data obtained during enzyme characterization can be used to select enzyme combinations to be evaluated in biofilm confrol and biofilm removal assays.
  • Buffers and reagents LB, per liter: 10 gm bacto-tryptone, 5 gm yeast exfract, 10 gm NaCI.
  • TSB per liter: 17 gm bacto-tryptone, 3 gm soybean (casein digest),
  • biofilm control micro-assay The biofilm control micro-assay was used to screen enzymes for their ability to prevent bacterial cells from foi-ming an adherent biofilm in the well of a 96-well microtiter plate. Therefore, in biofilm confrol assays, the enzyme was added to the microtiter plate well at the same time the bacterial culture was added to the well.
  • the biofilm confrol assay reactions were assembled in the wells of a black polystyrene 96-well microtiter plate as follows: 150 ⁇ l 0.05 O.D. ⁇ oo nm ceUs in PfLB (P. fluorescens) or SeTSB (S. epidermidis) was added to each well using a TITERTEK MULTIDROP 384TM liquid dispenser (Lab Systems Inc., Finland). Next, 10 ⁇ l of sample (100 ⁇ g total protein) was added to each well. The 96-well plate, composed of 12 columns of 8 weUs, was loaded with one sample per column (8 replicates per plate) using a multi-channel pipet.
  • the first two columns contained negative control samples, the next column contained a positive enzyme confrol, and the remaining 9 columns contained experimental enzyme samples.
  • the plates were then fransfened to a humidified chamber at 30°C for 22 hr. Following incubation at 30°C, the media was removed from the wells and each well was washed to remove unattached cells. The wells were washed 3 times each with 170 ⁇ l phosphate-buffered saline, pH 7.2 (PBS). Biofilm Staining- Following the final wash, the remaining cells were visualized by adding 170 ⁇ l SYBR green (Molecular Probes, Eugene, Oregon) to each well. SYBER green is a cell permeable fluorescent dye that becomes fluorescent upon binding to DNA.
  • the SYBR green stock solution is diluted 1 : 10,000 in PBS. Fluorescence was measured using an excitation of 485 nm and emission of 535 nm in a SpectraMAX GeminiXS fluorescence plate reader (Molecular Devices, Sunnyvale, California).
  • the biofilm removal assay was used to screen enzymes for their ability to lift and remove adherent bacterial cells that had become established as an early biofilm in the well of a 96-well microtiter plate. Therefore, in biofilm removal assays, the enzyme was added to the microtiter plate well after the bacterial cells had established a biofilm. Biofilm growth.
  • the overnight culture and the 0.05 O.D.600nm culture was prepared as described for the biofilm control micro-assay. Next 150 ⁇ l 0.05 O.D.600nm culture was added to each well of a black polystyrene 96-weU plate and the plate was incubated in a 30°C humidified chamber for 7 hours.
  • the wells of the plate were washed 3 times with 170 ⁇ l SeTSB per wash to remove unattached cells. Following the final wash, 150 ⁇ l fresh SeTSB was added to each well. Enzyme addition. Negative confrol, positive confrol, or enzyme samples were added to the wells of the plate as described for the biofilm confrol micro-assay. The plates were then returned to the humidified chamber for 15 hr. Plate washing and fluorescent visualization of biofihn was performed as described for the biofilm control assay. Development of the biofilm micro-assay
  • biofilm confrol As described above, the primary screen evaluated each enzyme in four separate biofilm micro-assays: P. fluorescens biofilm confrol, P. fluorescens biofilm removal, S. epidennidis biofilm control, and S. epidermidis biofilm removal.
  • Biofilm confrol assays evaluated an enzyme's ability to prevent biofilm formation
  • biofilm removal assays evaluated an enzyme's ability to reduce or eliminate an established biofilm.
  • a total of 426 enzymes were evaluated in these screening assays.
  • Figure 8 shows typical data obtained from one of the screens.
  • Figure 8 iUustrates data obtained from a biofilm removal micro-assay assay using P. fluorescens biofilm.
  • P. fluorescens biofilm control Of the two enzymes with significant activity against P. fluorescens in the biofilm confrol assay, one enzyme is an amidase (encoded by SEQ ID NO: 107, amino acid sequence set forth in SEQ ID NO: 108) and the other is most closely related to a cellulase (encoded by SEQ ID NO: 37, amino acid sequence set forth in SEQ ID NO:38).
  • SEQ ID NOS: 107. 108 This amidase was originally discovered in an activity- based screen of an environmental library constructed from a deep-sea sample.
  • a BLAST search of the pubhc sequence archive shows the polypeptide encoded by SEQ ID NO: 107, the amino acid sequence set forth in SEQ ID NO: 108, is most closely related to a 6-aminohexanoate-cycHc-dimer hydrolase from Deinococcus radiodurans.
  • the new sequence has about 49% sequence identity to the D. radiodurans sequence.
  • the new sequence is an enzyme, a secondary amidase. In one aspect, it is involved in the degradation of the xenobiotic compound nylon-6.
  • biofilm confrol based on the polypeptide encoded by SEQ ID NO: 107 (the amino acid sequence set forth in SEQ ID NO: 108) is due to a signaling event. It may involve cleavage of a homoserine lactone. It may be due to a hydrolytic event that disrupts cell-cell or cell-substrate interactions associated with the exopolymer matrix.
  • the polypeptide encoded by SEQ ID NO: 107 (the amino acid sequence set forth in SEQ ID NO: 108) does not possess a signal sequence. This suggests that it is an infracellular enzyme. Table 1, above, lists some of the properties of the polypeptide encoded by SEQ ID NO: 107 (the amino acid sequence set forth in SEQ ID NO: 108).
  • the polypeptide encoded by SEQ ID NO:37 (the amino acid sequence set forth in SEQ ID NO:38) has not been fully explored and it is possible that this enzyme also displays activity against the exopolymeric matrix of the P. fluorescens biofilm. Therefore, the polypeptide encoded by SEQ ID NO:37 (the amino acid sequence set forth in SEQ ID NO:38) may control biofilm fonnation through disruption of the matrix.
  • An experiment testing if the polypeptide encoded by SEQ ID NO:37 (the amino acid sequence set forth in SEQ ID NO:38) causes increased release of soluble saccharide subunits as measured using a standard reducing sugar assay can be done.
  • polypeptide encoded by SEQ ID NO: 7 (the amino acid sequence set forth in SEQ ID NO:8) is similar to cellulases from fhermophilic organisms. Thus, in one aspect, this enzyme can be used at elevated temperatures for biofilm confrol and removal.
  • P. fluorescens biofilm removal The results of these screening efforts demonstrate that an effective enzyme for P. fluorescens biofilm removal is an esterase, a polypeptide encoded by SEQ ID NO: 13 (the amino acid sequence set forth in SEQ ID NO: 14) (see Table 1).
  • the polypeptide encoded by SEQ ID NO: 13 caused a 74% reduction in biofilm fluorescent staining in the biofilm removal micro-assay. This is a novel enzyme discovered from an environmental library made from a sample of lake sediment.
  • the mechanism of action of the polypeptide encoded by SEQ ID NO: 13 in biofilm removal may be associated with the removal of acyl groups from bacterial polymers hi the exopolymer matrix. These exopolymers are known to be highly acylated and removal of these groups could de-stabihze the matrix.
  • the polypeptide encoded by SEQ ID NO: 13 may have an esterase activity that has effects on cell signaling events that trigger cell detachment.
  • Table 2 summarizes enzymes with activity in the S. epidermidis biofihn micro-assays. Enzymes that show sequence similarity are given a group designation (AMI, AM2, GY1, PHI). Phosphatases having a sequence as set forth in SEQ ID NO:27 (encoded by SEQ ID NO:28) and SEQ ID NO:211 (encoded by SEQ ID NO:212) are not related to the PHI phosphatases and therefore these enzymes do not have group designations.
  • the invention provides several novel amidases with biofilm confrol activity.
  • the seven amidases identified can be divided into 3 groups based on the relatedness of their amino acid sequences.
  • the largest group contains 4 members; they are enzymes most closely related to a family of amidases that contain enantiomer-specific amidases and glutamyl tRNA amidofransferases.
  • the next largest group contains 2 enzymes related to pyrazinamidases and nicatinamidases.
  • the third group contains a single enzyme related to 6-aminohexanoate-cyclic-dimer hydrolase. This enzyme is involved in the degradation of the xenobiotic compound nylon-6.
  • amidases function to prevent or remove biofilms; thus, one possibility is a mechamsm involving the inactivation of the quorum sensing molecules widely believed to be involved in biofilm formation and differentiation.
  • Many gram-negative organisms use N-acyl homoserine lactones as quorum signaling molecules. These molecules contain an acyl chain of varying length attached to the homoserine lactone through an amide linkage. Therefore, an amidase activity could potentially cleave this amide bond and disrupt the quorum signaling pathway.
  • Bacillus subtilis small peptides are involved in quorum sensing.
  • the invention provides several novel phosphatases with biofilm confrol activity. While the invention is not limited to any particular mechanism of action, and the 6 phosphatases function by destabilizing the biofilm matrix, 4 of these enzymes are most closely related to the E. coli stationary survival protein Sur ⁇ .
  • the Sur ⁇ protein is an acid phosphatase that has been suggested to be important for bacterial ceU survival in stationary phase and in media that induce ceU stress (high temperature and high salt). Although the exact role of Sur ⁇ is not known, it is believed that there is an interaction between Sur ⁇ and the product of the pcm gene.
  • the pcm gene encodes an isoaspartyl methylfransferase that functions to convert the isoaspartyl residues of "aging" proteins back to aspartyl residues and thereby functions in protein damage control and repair. Genetic analysis of the interaction has shown that sur ⁇ expression is detrimental to stationary phase ceUs lacking pcm.
  • glycosidases that demonstrate efficacy in the biofilm assays are also likely broad specificity enzymes that can hydrolyze components of the biofilm matrix. These enzymes were discovered in a screen for beta-glucosidase activity, but sequence analysis shows them to be related to glucosidases, mannosidases, and galactosidases. The esterases identified could function either by disrupting cellular signaling or by destabilizing the biofilm matrix through removal of acyl groups from exopolysaccharide polymers.
  • the methods use appropriate sunogate substrates to characterize basic biochemical properties of the hits.
  • AU of the hits can be characterized in standard biochemical assays to determine pH performance, thermal tolerance, thermal stability, and commercial biocide tolerance.
  • the amidases can be assayed using N-acyl homoserine lactones as substrates to look at their potential to degrade these quorum-signaling molecules.
  • the cellulases and glycosidases can be evaluated for their ability to degrade the biofihn matrix. These assays can measure soluble sugar release from the biofilm matrix using standard reducing sugar determination assays.
  • the commercial biocide tolerance assays can be performed to determine enzyme activity half-life in the presence of commercial biocides.
  • the invention provides enzyme-biocide formulations wherein the enzyme activity significantly increases the efficacy of the biocide in biofilm control. Consequently, less biocide is required in the application.
  • the invention provides final characterization assays to evaluate the levels of expression of select hits in a variety of host/vector combinations. These can include Gram-negative, Gram-positive, and yeast expression systems.
  • enzymes can be expressed both as infracellular and secreted enzymes in the Gram-positive and yeast hosts because glycosylation frequently improves the stability of the expressed enzyme. These methods can identify a suitable host-vector system for cost-effective expression scale-up.
  • a flexible laboratory reactor system is used for simulating biofilm development in various medical and industrial environments.
  • One exemplary reactors is the drip-flow reactor shown in Figure 10. It can be used to grow robust, mixed species biofilms like those that develop in paper mills. The effect of enzyme samples on these biofilms can be measured by a combination of microscopic imaging and plate counting. Up to 20 tests can be performed. Each test canbe performed in either of two j formats. The first format is a prevention mode in wliich the enzyme is added periodically beginning early in the experiment. The purpose of this design is to test whether the treatment is able to retard or prevent a biofilm from forming on an initially clean surface. The second format is a removal mode. Mature biofilms are established, then treated once with the enzyme. Loss of biomass is measured.
  • the drip-flow biofilm reactor system is a continuous flow reactor in which growth medium is delivered dropwise over four stainless steel coupons contained in separate parallel chambers. Each chamber measures 10.1 cm long by 1.9 cm wide by 1.9 cm deep.
  • the chambered reactor is fabricated from polycarbonate plastic. Each of the chambers is fitted with an individual removable plastic lid that can be affixed with thumbscrews.
  • the reactor is placed on a stand that inclines the device at an angle of 10° from horizontal. Acid-washed steel slides are placed in the reactor and the reactor assembly is wrapped and autoclaved.
  • rubber tubing is attached to the effluent port of the sterilized reactor. This tubing is clamped off and then each chamber is separately inoculated.
  • An inoculum is obtained from a paper mill source.
  • the inoculum is allowed to stand, without flow, in each chamber for 24 h.
  • Each chamber is then be drained and the flow of medium initiated at a flow rate of 50 ml h "1 .
  • the medium can be either paper mill "white water” amended with yeast extract or simply a dilute nutrient broth.
  • the growth temperature can be 35-45°C to reflect the environment in a paper mill. This procedure can be modified, in accord with the known microbiology and chemistry of water in paper manufacture to obtain a workable method. Treatment protocols and controls
  • the medium delivered to the reactor can be amended with the desired enzyme concentrations.
  • the biofilm can therefore be continuously exposed to these chemicals as it forms. After 5 days of biofilm development, the biofilm is harvested for analysis.
  • the reactors can be operated as described above but with no freatment chemical for 5 days. After this time it is anticipated that the biofilm will have accumulated to a level of approximately 10 8 viable bacteria per square centimeter.
  • the enzyme formulation can then be delivered to the fouled surface for a 1 h contact period. After this treatment, the system can be briefly rinsed with buffer and the biofilm harvested for analysis.
  • the negative confrol is simply no chemical addition in the prevention experiments and treatment with water alone in the removal experiments.
  • a positive confrol experiment can be conducted using 10 mg/1 thiocarbamate for prevention experiments and a 10 minute contact time with 10,000 mg/1 thiocarbamate for removal experiments.
  • Thiocarbamate is a biocide commonly used to control biofouling in paper manufacturing equipment. Analytical methods Sample coupons can be scraped and the biofilm microorganisms dispersed before enumeration by serial dilution and plating. Microorganisms can be plated on R2A agar and incubated at room temperature for 6 days. Visual observations of the extent of fouling on sample disks are recorded.
  • biofilm The presence of biofilm is clearly visible on untreated coupons after several days.
  • a duplicate coupon can be stained with a commercial viability indicator (BacLight, Molecular Probes) and examined by confocal scanning laser microscopy.
  • the thickness of the biofilm can be determined by image analysis. The relative intensities of red and green staining will provide a qualitative indication of viability.
  • Data analysis The log reduction in viable cell numbers in a freated biofilm relative to an untreated control can be calculated. Visible fouling of the untreated and treated samples can be scored numerically as 1 (no visible fouling) to 5 (heavily fouled). Mean biofilm thickness and standard deviation can be reported in microns. The ratio of thickness of a freated biofilm to thickness of an untreated biofilm can be calculated. A representative microscope image can be saved for each specimen.
  • Enzyme/Biocide synergies Effective enzyme-biocide mixtures that aUow the reduction of needed chemical biocide for biofilm control:
  • the invention provides biocidal agents as candidates for coformulation with optimized, biofilm active enzymes, e.g., see Table 2.
  • a matrix can be established that combines each chemical biocide with the best performing enzymes that result from bench scale applications. Enzyme+biocide combinations can be initially evaluated using the high-throughput, biofilm micro-assay, as described herein. Any combinations that appear to work particularly well together relative to only enzyme and only biocide controls can be characterized further. Selected sets of enzyme-biocide combinations can be subjected to another round of bench scale testing on mixed-species, "pulp and paper" biofilms. Directed Evolution for stability and efficacy; Gene Site Saturation Mutagenesis (GSSM) for stability optimization; Gene Reassembly of parental genes for enhanced biofilm hydrolysis:
  • GSSM Gene Site Saturation Mutagenesis
  • Enzymes wliich show hydrolytic efficacy toward complex biofilms and which show positive results in biocide synergy experiments may not have optimal phenotypes for ultimate industrial use in formulation.
  • Industrial application may require temperature and pH stability as well as optimal turnover to ensure efficacy.
  • coformulation with a chosen biocide may necessitate stability enhancement towards biocide inactivation of the protein catalyst.
  • use of the enzyme in a pulp processing context could require product application at alkaline pH, temperatures near 80°C and coformulation with oxidizing biocides.
  • Genes coding for lead enzymes can be optimized for performance under industrial process conditions.
  • the GSSM and GeneReassembly technologies can be used to create clone variant libraries which will be screened for performance using the assay paradigms for simple biofilms of the invention.
  • a clone triage screening regime can be implemented to identify candidate enzymes.
  • the high throughput assay can be modified to accommodate targeted temperature and pH conditions.
  • AdditionaUy clones can be screened for turnover in the presence of biocide. Clones which shown improvement can be characterized for efficacy toward complex films.
  • Further triage can screen lead enzymes for synergy with biocide in complex biofilms.
  • Candidate enzymes captured from the screening triage can be formulated for commercialization.
  • Final evaluation of product candidate optimize host/vector system for overexpression; product ready for overproduction and formulation:
  • Heterologous expression of evolved enzyme(s) can be carried out in a microbial host in order to produce large quantities of enzyme(s) for water freatment.
  • Prokaryotic and eukaryotic expression systems can be used.
  • Enzyme(s) can be expressed either infracellularly (cytoplasmic) or extracellularly (active secretion).
  • An anay of host- vector combinations can be available for evaluation.
  • Initial expression studies can be carried out in E.coli using a strong, regulated promoter in order to allow high-level expression of the enzyme(s). Following the assessment of activity and quantity of enzyme produced in E.coli, other expression hosts will be evaluated, if needed, to develop a commercially viable production process.
  • a number of bench (1- 30L), pilot (30-500L) and commercial scale (>50,000L) fermentors can be used.
  • Advanced cell-monitoring tools can be used to improve the physiology of the expression host to achieve high-level expression.
  • Recovery of the infracellularly expressed enzyme (s) can be carried out using cell concentration, disruption and filtration. Down-stream products are recovered.

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Abstract

L'invention concerne des enzymes ayant une activité désacétylase, amidase, cellulase, estérase, glycosidase, xylanase, amylase, transaminase, laminarinase, bêta-galactosidase, bêta-mannosidase, pullulanase et/ou phosphatase, ainsi que les acides nucléiques codant pour ces enzymes et les anticorps se liant spécifiquement à ces enzymes. L'invention concerne des enzymes pour la commande de biofilms, des polynucléotides et des procédés pour produire et utiliser ces polynucléotides et polypeptides. L'invention concerne également des produits comprenant ces compositions de commande de biofilms. Selon un mode de réalisation de l'invention, les compositions de commande de biofilms selon l'invention ont une activité amidase, par exemple la capacité à hydrolyser des amides, et comprennent des enzymes ayant une activité amidase secondaire, notamment une activité peptidase, protéase et/ou hydantoinase. Selon un autre mode de réalisation de l'invention, les compositions de commande de biofilms selon l'invention ont une activité amidase, cellulase, estérase, glycosidase, xylanase, amylase, transaminase, laminarinase, bêta-galactosidase, bêta-mannosidase, pullulanase et/ou phosphatase.
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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007095398A2 (fr) 2006-02-14 2007-08-23 Verenium Corporation Xylanases, acides nucléiques codant pour elles et leurs procédés de préparation et d'utilisation
WO2009020459A3 (fr) * 2006-08-04 2009-04-23 Verenium Corp Glucanases, acides nucléiques codant ceux-ci et procédés pour les fabriquer et les utiliser
WO2009100456A2 (fr) 2008-02-08 2009-08-13 Prothera, Inc. Inhibition et traitement de biofilms gastro-intestinaux
WO2010051288A1 (fr) 2008-10-27 2010-05-06 Revivicor, Inc. Ongulés immunodéprimés
JP2011510613A (ja) * 2007-10-03 2011-04-07 ヴェレニウム コーポレイション キシラナーゼ、キシラナーゼをコードする核酸並びにそれらを製造及び使用する方法
EP2527456A1 (fr) 2004-10-22 2012-11-28 Revivicor Inc. Porcs transgéniques déficients en chaîne légère d'immunoglobuline endogène
US20130330783A1 (en) * 2005-03-15 2013-12-12 Bp Corporation North America Inc. Cellulases, nucleic acids encoding them and methods for making and using them
US8728769B2 (en) 2002-06-14 2014-05-20 Bp Corporation North America Inc. Xylanases, nucleic acids encoding them and methods for making and using them
CN107820052A (zh) * 2017-11-10 2018-03-20 北京优易惠技术有限公司 基于评标业务数据和视频数据的融合方法及系统
CN113278671A (zh) * 2021-07-13 2021-08-20 山东省科学院菏泽分院 一种利用碎米制备大米活性肽的方法
CN113930462A (zh) * 2021-10-19 2022-01-14 江南大学 高丝氨酸内酯信号分子促进碳链延长微生物合成己酸的方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5942430A (en) * 1996-02-16 1999-08-24 Diversa Corporation Esterases

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE GENPEPT [Online] 21 August 1997 ROBERTSON ET AL: 'Alignment with SEQ ID NO:1', XP002990393 Database accession no. (AAT9334) & WO 97 30160 A1 *

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US11078516B2 (en) 2008-02-08 2021-08-03 Prothera, Inc. Inhibition and treatment of gastrointestinal biofilms
WO2010051288A1 (fr) 2008-10-27 2010-05-06 Revivicor, Inc. Ongulés immunodéprimés
CN107820052A (zh) * 2017-11-10 2018-03-20 北京优易惠技术有限公司 基于评标业务数据和视频数据的融合方法及系统
CN113278671A (zh) * 2021-07-13 2021-08-20 山东省科学院菏泽分院 一种利用碎米制备大米活性肽的方法
CN113930462A (zh) * 2021-10-19 2022-01-14 江南大学 高丝氨酸内酯信号分子促进碳链延长微生物合成己酸的方法

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