WO2002008431A1 - Compositions et procédés d'utilisation de ces compositions pour modifier les génomes de micro-organismes - Google Patents

Compositions et procédés d'utilisation de ces compositions pour modifier les génomes de micro-organismes Download PDF

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WO2002008431A1
WO2002008431A1 PCT/US2001/007324 US0107324W WO0208431A1 WO 2002008431 A1 WO2002008431 A1 WO 2002008431A1 US 0107324 W US0107324 W US 0107324W WO 0208431 A1 WO0208431 A1 WO 0208431A1
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sequence
sequences
transposon
vector
interest
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PCT/US2001/007324
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English (en)
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Kevin P. Francis
Anthony F. Purchio
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Xenogen Corporation
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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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries

Definitions

  • the present invention generally relates to transposon cassettes, methods of making same, and methods of use thereof.
  • Bioluminescent bacteria are widely found in both marine and terrestrial environments. Interestingly, all identified species of naturally occurring marine and terrestrial bioluminescent bacteria are Gram-negative. To date, at least eleven species in four Gram-negative genera have been described: Vibrio, Photobacterium, Shewanella (Altermonas) and Photorhabdus (Xenorhabdus). In all these species, the five genes responsible for bioluminescence are clustered in the lux operon (luxCDABE).
  • luxCDABE lux operon
  • the bioluminescence (emitted blue-green light having a wavelength of about 490 nm) is thought to result from a luciferase-catalyzed oxidation of reduced flavin mononucleotide (FMNH 2 ) and a long-chain fatty aldehyde.
  • the luciferase enzyme is encoded by two subunits (luxAB), whereas the fatty acid reductase polypeptides responsible for the biosynthesis of the aldehyde substrate for the luminescent reaction are encoded by the three genes luxCDE.
  • luxCDE genes flank the luxAB genes, with transcription in the order luxCDABE.
  • luxA-E are essential for the biosynthesis of light (reviewed by Meighen, E., (1993,The FASEB Journal 7:1016-1022 andUlitzur, S., (1997), J. Biolumin Chemilumin 12:179-192).
  • Patent 5,650,135 make possible the detection of bioluminescent bacteria in a living animal without dissecting or otherwise opening the animal up (“in vivo momtoring") - the light is detected through muscle, skin, fur & other traditionally "opaque” tissues using a highly sensitive camera
  • a non-bioluminescent Gram-negative bacterium can typically be engineered to have bioluminescence properties by cloning into it a luxCDABE operon (under control of a suitable promoter) from a bioluminescent species (see, e.g., Contag, et al, U.S. Patent Serial Number 5,650,135)
  • previous attempts to make bioluminescent Gram positive bacteria have met with limited success.
  • one approach employed an expression cassette encoding a functional LuxAB fusion protein (Jacobs, M., et al, (1991) Mol. Gen. Genet. 230:251-256).
  • a Gram-positive ribosome binding site (RBS) was inserted upstream of luxA, with the luxB gene cloned in frame downstream of luxA.
  • the present invention provides, inter alia, such methods, transposon cassettes, and other tools useful for generating bioluminescent bacteria, for example, Gram- positive bacteria and related organisms, suitable for studies relating to infection and/or pathogenesis.
  • the present invention includes a transposon cassette for use in modifying a genome of a target organism.
  • the target organism can be, for example, a gram-positive bacterium.
  • the transposon cassette comprises a polynucleotide sequence comprising first and second transposon inverted repeat sequences flanking an internal polynucleotide sequence, wherein the internal polynucleotide sequence comprises a first sequence of interest encoding polypeptide sequences present in a first orientation.
  • the first sequence of interest typically lacks control sequences capable of promoting transcription in the target organism.
  • polynucleotide sequences derived from the transposon are obtained from a transposon derived from a gram-positive bacterium, for example, IMOOl or 7W917.
  • the transposon cassette may further comprise transposase encoding sequences, wherein the transposase is capable of inducing transposition mediated by the transposon inverted repeat sequences.
  • the transposon cassette of the present invention can be used to modify the genomes of a variety of microorganisms including, but not limited to Gram-positive bacteria (e.g., Staphylococus spp, Streptococcus spp., Enterococcus spp., Bacillus spp., Clostridium spp., Mycobacterium spp., Corynebacterium spp., Listeria spp., Propriobacterium spp., Micrococcus spp, Lactobacillus pp., and Lactococcus spp.); Gram-negative bacteria (e.g., Vibrio spp., Brucella spp., Bordetella spp., Campylobacter spp., Pseudomonas spp., Escherichia spp., Enterobacter spp., Klebsiella spp., Serratia spp., Citro
  • the first sequence of interest comprises at least one polynucleotide sequence encoding light generating polypeptide sequences.
  • light generating polypeptide encoding sequences include, but are not limited to lux and/or luc genes, for example, (a) a polynucleotide encodmg luxA, and luxB gene products; (b) a polynucleotide encoding luxA, luxB, luxC, luxD and luxE gene products; (c) a polynucleotide encoding luxY gene product; and (d) a polynucleotide encoding luc gene product.
  • the first sequence of interest may further comprise at least one Gram-positive ribosome binding site sequence.
  • the polynucleotide sequence encoding light generating polypeptide sequences may encode luxA and luxB gene products.
  • the polynucleotide sequence encoding light generating polypeptide sequences may also comprise at least one Gram-positive ribosome binding site sequence upstream of at least one of the polynucleotide sequences encoding each of the luxA and luxB gene products.
  • the polynucleotide sequence encoding light generating polypeptide sequences further comprises a polynucleotide encoding luxC, luxD, and luxE gene products, which may also include at least one Gram-positive ribosome binding site sequence upstream of at least one, several, or all of the polynucleotide sequences encoding each of the luxA, luxB, luxC, luxD and luxE gene products.
  • the polynucleotide sequence encoding light generating polypeptide sequences further comprises a polynucleotide sequence encoding the luxY gene product, which may include at least one Gram-positive ribosome binding site sequence upstream of the luxY gene product.
  • the transposon cassette of the present invention may further comprise at least one coding sequence for a selectable marker.
  • the selectable marker encodes a polypeptide conferring antibiotic resistance.
  • Exemplary antibiotics that may be used for selection include, but are not limited to actinomycin, ampicillin, chloramphenicol, erythromycin, gentamycin sulfate, hygromycin, kanamycin, neomycin, penicillin, polymixin B sulfate and streptomycin sulfate.
  • Sequences encoding polypeptides which confer antibiotic resistance may further comprises at least one Gram-positive ribosome binding site sequence upstream the coding sequence for the selectable marker.
  • the first sequence of interest comprises the following: (a) a polynucleotide sequence encoding light generating polypeptide sequences comprising a polynucleotide encoding luxA, luxB, luxC, luxD and luxE gene products, and further comprising at least one Gram- positive ribosome binding site sequence upstream of each of the polynucleotide sequences encoding each of the luxA, luxB, luxC, luxD and luxE gene products; and, (b) a coding sequence for a selectable marker encoding a polypeptide conferring kanamycin resistance.
  • the internal polynucleotide sequence further comprises a transposase coding sequence operably linked to a promoter functional in the target organism.
  • the transposase is capable of inducing transposition mediated by the transposon inverted repeats.
  • the transposase coding sequence is in a second orientation relative to polypeptide coding sequences of the first sequence of interest encoding polypeptide sequences.
  • Transcription termination sequences may be included at one of both ends of the transposase coding sequences.
  • at least one transcription termination control sequence may be interposed between the first sequence of interest encoding polypeptide sequences and the transposase coding sequence which is operably linked to a promoter functional in the target organism.
  • Exemplary first and second transposon inverted repeat sequences can be derived from T ⁇ 4001.
  • a corresponding transposase coding sequence may also be derived from Tn4001.
  • the present invention further includes a vector comprising, (a) a vector backbone and (b) a transposon cassette of the present invention as described, for example, above.
  • the transposase which facilitates transposition may typically be included within the transposon cassette, or alternately, may be provided on the vector backbone.
  • the transposase coding sequence is operably linked to a promoter functional in the target organism. In preferred embodiments this promoter does not affect transcription of any coding sequences in the transposon cassette.
  • the vector backbone may further comprise at least one origin of replication that is functional in at least one target host cell, including, but not limited to, a Gram- positive origin of replication, a Gram-negative origin of replication, and/or an origin of replication that is functional in more than one type of target host cell.
  • origins of replication may be wild-type or conditional (e.g., temperature-sensitive).
  • Vector backbones comprising, for example, a Gram-positive and a Gram-negative origin of replication are useful for shuttling vector constructs between different types of microorganisms.
  • the vector backbone may further comprise a selectable marker sequence of interest operably linked to a promoter functional in a target organism, wherein the promoter does not affect transcription of any coding sequences in the transposon cassette.
  • a selectable marker coding sequence may, for example, encode a polypeptide conferring antibiotic resistance.
  • Useful selection antibiotics include, but are not limited to actinomycin, ampicillin, chloramphenicol, erythromycin, gentamycin sulfate, hygromycin, kanamycin, neomycin, penicillin, polymixin B sulfate and streptomycin sulfate.
  • the vector backbone may comprises at least one polynucleotide sequence encoding light generating polypeptide sequences operably linked to a promoter functional in a target organism of interest, wherein the promoter does not affect transcription of any coding sequences in the transposon cassette.
  • Such light generating polypeptide encodmg sequences may be used, for example, to directly screen for microorganisms transformed by vectors of the present invention.
  • the present invention includes the use of such polynucleotide sequence encoding light generating polypeptide sequences in any vector backbone to allow screening of transformed microorganisms for the presence of the vector carrying such sequences.
  • the transposon cassette contains a polynucleotide sequence encoding a first light generating polypeptide sequences wherein the light generating first polypeptide produced from coding sequences within the transposon cassette produce bioluminescence of a characteristic first wavelength.
  • the backbone vector may then comprise a polynucleotide sequence encoding a light generating second polypeptide sequences, wherein the second light generating polypeptide produces a characteristic second wavelength of bioluminescence that is detectably different from the first light generating polypeptide encoded by the transposon cassette.
  • Various exemplary light generating polypeptide sequences have been discussed above and are further discussed hereinbelow.
  • Exemplary vector backbones include, but are not limited to, pAUL-A, pE194, and pSK.
  • the backbone vectors of the present invention may comprise at least one transcription termination sequence in the vector backbone adjacent the transposon cassette, such that the transcription termination sequence essentially prevents transcription originating from any promoter present in the vector from reading through into the transposon cassette sequences.
  • the backbone vector may comprise two transcription termination sequences in the vector backbone wherein the transcription termination sequences flank the transposon cassette, such that the transcription termination sequences essentially prevent read-through transcription originating from any promoter present in the vector into the transposon cassette sequences.
  • Yet another aspect of the present invention includes a method for modifying a microorganism having a genome.
  • the microorganism is transformed with any of the vectors of the present invention.
  • the method may also include the step of culturing the transformed microorganism under conditions that facilitate transposition of the transposon cassette from the vector into the genome of the microorganism.
  • the present invention also includes a cell carrying any of the vectors of the present invention, a cell produced by any of the methods of the present invention, as well as a modified host-cell carrying at least one transposon cassette of transposon cassettes of the present invention wherein expression of the first sequence within the transposon cassette is mediated by a transcriptional promoter endogenous to the target organism.
  • Such cells may include, but are not limited to Gram-negative bacteria (e.g., Vibrio spp., Brucella spp., Bordetella spp., Campylobacter spp., Pseudomonas spp., Escherichia spp., Enterobacter spp., Klebsiella spp., Serratia spp., Citrobacter spp., Proteus spp., Salmonella spp., Shigella spp., and Yersinia spp.; Gram-positive bacteria (e.g., Staphylococus spp, Streptococcus spp., Enterococcus spp., Bacillus spp., Clostridium spp., Mycobacterium spp., Corynebacterium spp., Listeria spp., Propriobacterium spp., Micrococcus spp, Lac
  • Cells modified by the methods and compositions of the present invention may, for example, exhibit constitutive, inducible, and/or repressible bioluminescence.
  • the modified cell may exhibit bioluminescence upon infecting an animal susceptible to infection by the cell.
  • One method of the present invention is directed to a method of isolating cells capable of exhibiting bioluminescence.
  • a target cell of choice e.g., a selected microorganism such as a gram-positive bacteria
  • a vector of the present invention may, for example, comprise, (i) a vector backbone compatible with the cell, and (ii) a transposon cassette as described herein, wherein the transposon cassette comprises a first sequence of interest encoding light generating polypeptide sequences lacking control sequences capable of promoting transcription in the target cell.
  • the transformed cells are cultured under conditions permitting transposition of the transposon cassette. Transformed cells are then optically detected as cells (or colonies of cells) exhibiting bioluminescence.
  • the cells exhibiting bioluminescence are then physically isolated and clonal isolates may be obtained (i.e., individual cells that have grown to form colonies).
  • the present invention further includes another method of isolating cells capable of exhibiting bioluminescence.
  • the cells of interest e.g., a microorganism
  • the vector backbone comprises a polynucleotide encoding light generating polypeptide sequences operably linked to a promoter functional in the cells, wherein the light generating polypeptide produces bioluminescence of a characteristic first wavelength
  • a transposon cassette of the present invention e.g., a transposon cassette of the present invention.
  • the transposon cassette may include a polynucleotide sequence comprising first and second transposon inverted repeat sequences flanking an internal polynucleotide sequence, wherein the internal polynucleotide sequence comprises a first sequence of interest encoding light generating polypeptide sequences present in a first orientation the light generating polypeptide producing bioluminescence of a characteristic second wavelength.
  • the first sequence of interest lacks control sequences capable of promoting transcription in the target organism.
  • the internal polynucleotide sequence may further comprise a transposase coding sequence operably linked to a promoter functional in the target organism. The transposase being capable of inducing transposition mediated by the transposon inverted repeats.
  • the transposase coding sequence is in a second orientation relative to polypeptide coding sequences of the first sequence of interest encoding polypeptide sequences.
  • one or more transcription termination sequences may be placed adjacent the transposase, and/or the transposase may be on the vector backbone instead of within the transposon cassette.
  • Transformed cells are identified by their ability to produce bioluminescence of the characteristic first wavelength. The transformed cells are cultured under conditions permitting transposition of the transposon cassette.
  • Transposants are optically detected, i.e., they are capable of producing bioluminescence of the characteristic second wavelength. These bioluminescent cells and/or colonies, which bioluminesce at the characteristic second wavelength, are isolated (e.g., by physical manipulation).
  • the present invention also includes methods of identifying active host-cell gene promoters.
  • the host-cell of interest is transformed with a vector of the present invention carrying a transposon cassette of the present invention.
  • the transformed cells are cultured under conditions permitting transposition of the transposon cassette.
  • These transformed host-cells are then screened for expression of the first sequence of interest encoding polypeptide sequences.
  • the active host-cell gene promoter mediating expression of the first sequence of interest encoding polypeptide sequences is then identified.
  • Another method of identifying active host-cell gene promoters infecting a first animal with a microorganism carrying a vector of the present invention.
  • the vector may, for example, comprise (a) a vector backbone compatible with the microorganism, and (b) a transposon cassette comprising a polynucleotide sequence comprising first and second transposon inverted repeat sequences flanking an internal polynucleotide sequence, wherein the internal polynucleotide sequence comprises a first sequence of interest encoding (i) light generating polypeptide sequences present in a first orientation, and (ii) a polypeptide sequence conferring antibiotic resistance, the first sequence of interest lacking control sequences capable of promoting transcription in the target organism.
  • the internal polynucleotide sequence further comprises a transposase coding sequence operably linked to a promoter functional in the target organism, where the transposase is capable of inducing transposition mediated by the transposon inverted repeats.
  • the transposase coding sequence may be in a second orientation relative to polypeptide coding sequences of the first sequence of interest.
  • at least one transcription termination sequence may be placed adjacent the tranposase coding sequence.
  • the transposase may be placed on the vector backbone instead of within the transposon cassette.
  • transposants are then screened in vitro to identify transposants that do not exhibit constitutive bioluminescence. Such transposants are then used to infect a second animal and are screened for their ability to exhibit bioluminescence in vivo upon infection of the second animal. Transposants exhibiting bioluminescence in vivo upon infection of the second animal are then isolated. The active gene promoter associated with the first sequence of interest in the transposants exhibiting bioluminescence in vivo upon infection of the second animal is then identified, for example, by sequencing genomic DNA adjacent the transposon cassette sequences. Alternatively, the transposants exhibiting bioluminescence in vivo upon infection of the second animal are isolated and used to infect third and fourth animals. The infected third animal may be treated with a compound of interest.
  • the treated, infected third animal and the untreated, infected fourth animal are then monitored for bioluminescence in vivo. Bioluminescence from the third and fourth animals are then compared to determine whether the compound of interest detectably affects in vivo bioluminescence in the third animal relative to the fourth, wherein reducing or eliminating in vivo bioluminescence in the third animal relative to the fourth indicates pharmacological effectiveness against the microorganism of interest in the animal.
  • a further aspect of the present invention includes a method of monitoring the proliferation of a microorganism of interest in a medium of interest.
  • a microorganism of interest is transformed with a vector of the present invention, comprising a transposon cassette of the present invention.
  • the transposon cassette typically includes a first sequence of interest encodmg light generating polypeptide sequences lacking control sequences capable of promoting transcription in the target organism.
  • the transposase may be contained within the transposon cassette or on the vector backbone.
  • the transformed microorganisms are cultured under conditions permitting transposition and screened for transposants capable of exhibiting bioluminescence.
  • the medium of interest is inoculated with bioluminescent transposants.
  • the medium is then sampled and the samples monitored for the degree of bioluminescence over time, wherein an increase in the degree of bioluminescence over time is correlated to proliferation of the microorganism in the sample.
  • the method may further comprise adding a compound of interest to the medium, and evaluating the effect of the compound on proliferation of the microorganisms .
  • Figure 1 presents a schematic diagram of the pAUL-A backbone vector.
  • Figure 2 presents a schematic diagram of the pMGC57 backbone vector.
  • Figure 3 presents a schematic diagram of the pAUL-A vector containing a transposon cassette of the present invention.
  • the vector backbone region labeled "lacZ represents the lacZ operon; the regions labeled “T” represent transcription termination regions; "MCS” indicates the multiple cloning site present in the lacZ operon, (individual restriction endonuclease sites present therein are indicated);
  • "ori” represents the Gram-negative origin of replication;
  • ori ts represents a temperature-sensitive pE194 Gram-positive origin of replication (restriction endonuclease recognition sites present therein are indicated); and
  • EM R represents the erythromycin resistance gene.
  • the regions labeled 'TR Tn4001 " represent inverted repeats; luxA-E is the re-engineered lux cassette; (see Example 3); "kanamycin” is the kanamycin resistance gene; “Tranposase Tn4001” is the gene encoding the transposase enzyme of the same denomination.
  • Figure 4 presents data showing relative light unit expression from a variety of heat shock isolates constructed by the methods of the present invention.
  • Figure 5 depicts construction of the plasmid pAUL-A Tn4001 luxABCDE Km R .
  • 'TR refers to inverted repeat
  • Km R refers ti kanamycin resistant gene
  • Em R refers to erythromycin resistant gene
  • tap refers to transposase gene
  • TT refers to transcription terminator
  • MCS multiple cloning site.
  • Figure 6 is a graph depicting the effects of kanamycin concentration on the selection of promoter strength. S. pneumoniae strain Xen 7 was grown to mid exponential phase and 10 CFUs were plated on chocolate agar plates supplemented with varying concentration of kanamycin.
  • Open squares (D) represent the total CFUs on the plates. Solid circles ( • ) represent the ratio of light vs total CFUs. The numbers of CFUs were the average of 5 plates at each kanamycin concentration.
  • Figure 7, panels A-E are graphs depicting relative light units (RLUs) emitted by bioluminescent S. pneumoniae strains during in vitro growth. Three hundred microliters of overnight culture was inoculated in 30 ml BHI and grown at 37°C and 5% CO 2 . At 1 hr intervals, both the optical density (OD 600 nm, open circles O) and the number of relative light units (RLUs in million units, solid squares ⁇ ) from a 1 ml culture volume were determined for each of the specified strains.
  • RLUs relative light units
  • RLU mean thoracic bioluminescence
  • Figure 9 is a schematic depicting the construct designated pLTVI.
  • nucleic acid molecule and “polynucleotide” are used interchangeably to and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown.
  • Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • mRNA messenger RNA
  • transfer RNA transfer RNA
  • ribosomal RNA ribozymes
  • cDNA recombinant polynucleotides
  • branched polynucleotides branched polynucleotides
  • plasmids vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • a polynucleotide is typically composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T) uracil (U) for thymine (T) when the polynucleotide is RNA).
  • A adenine
  • C cytosine
  • G guanine
  • T thymine
  • U thymine
  • T uracil
  • T thymine
  • the term polynucleotide sequence is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.
  • a “coding sequence” or a sequence which "encodes” a selected polypeptide is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences (or “control elements”).
  • the boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus.
  • a coding sequence can include, but is not limited to, cDNA from viral, procaryotic or eucaryotic mRNA, genomic DNA sequences from viral, eucaryotic, or procaryotic DNA, and even synthetic DNA sequences.
  • a transcription termination sequence may be located 3' to the coding sequence.
  • control elements include, but are not limited to, transcription regulators, such as promoters, transcription enhancer elements, transcription termination signals, and polyadenylation sequences; and translation regulators, such as sequences for optimization of initiation of translation, e.g., Shine-Dalgarno (ribosome binding site) sequences, and translation termination sequences.
  • transcription regulators such as promoters, transcription enhancer elements, transcription termination signals, and polyadenylation sequences
  • translation regulators such as sequences for optimization of initiation of translation, e.g., Shine-Dalgarno (ribosome binding site) sequences, and translation termination sequences.
  • Promoters can include inducible promoters (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), repressible promoters (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and constitutive promoters.
  • a double-stranded DNA molecule comprises two strands of DNA having "opposite orientations," one strand being designated 5' to 3'; the second strand being its complement.
  • a first coding sequence in a first strand and a second coding sequence in the complementary strand have "opposite" orientations relative to each other, that is, the first and second coding sequences are in opposite orientations relative to each other.
  • isolated polynucleotide is a nucleic acid molecule separate and discrete from the whole organism with which the molecule is found in nature; or a nucleic acid molecule devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences (as defined below) in association therewith.
  • a "polypeptide” is used in it broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or other peptidomimetics. The subunits may be linked by peptide bonds or by other bonds, for example ester, ether, etc.
  • amino acid refers to either natural and or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.
  • a peptide of three or more amino acids is commonly called an oligopeptide if the peptide chain is short. If the peptide chain is long, the peptide is typically called a polypeptide or a protein.
  • operably linked refers to an a ⁇ angement of elements wherein the components so described are configured so as to perform their usual function.
  • a given promoter that is operably linked to a coding sequence e.g., a reporter gene
  • the promoter or other control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof.
  • intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked" to the coding sequence.
  • Recombinant as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation: (1) is not associated with all or a portion of the polynucleotide with which it is associated in nature; and/or (2) is linked to a polynucleotide other than that to which it is linked in nature.
  • the term "recombinant” as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide.
  • Recombinant host cells “host cells,” “cells,” “cell lines,” “cell cultures,” and other such terms denoting procaryotic microorganisms or eucaryotic cell lines cultured as unicellular entities, are used interchangeably, and refer to cells which can be, or have been, used as recipients for recombinant vectors or other transfer DNA, and include the progeny of the original cell which has been transformed. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent, due to accidental or deliberate mutation.
  • Progeny of the parental cell which are sufficiently similar to the parent to be characterized by the relevant property, such as the presence of a nucleotide sequence encoding a desired peptide, are included in the progeny intended by this definition, and are covered by the above terms.
  • Techniques for determining nucleic acid and amino acid "sequence identity" also are known in the art. Typically, such techniques include determining the nucleotide sequence of the mRNA for a gene and/or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence.
  • identity refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) can be compared by detem ining their "percent identity.” The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100. An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in
  • a preferred method of establishing percent identity in the context of the present invention is to use the MPSRCH package of proGrams copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, CA). From this suite of packages the Smith- Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the "Match" value reflects "sequence identity.”
  • Other suitable proGrams for calculating the percent identity or similarity between sequences is generally known in the art, for example, another alignment proGram is BLAST, used with default parameters.
  • homology can be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments.
  • Two DNA, or two polypeptide sequences are "substantially homologous" to each other when the sequences exhibit at least about 80%-85%, preferably at least about 90%, and most preferably at least about 95%-98% sequence identity over a defined length of the molecules, as determined using the methods above.
  • substantially homologous also refers to sequences showing complete identity to the specified DNA or polypeptide sequence. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system.
  • a “gene” refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular product (e.g., a polypeptide, protein or RNA). Typically, the term “gene” includes control sequences associated with the expression of the product. Any of the polynucleotide sequences described herein may be used to identify larger fragments or full-length coding sequences of the genes with which they are associated. Methods of isolating larger fragment sequences are known to those of skill in the art. Two nucleic acid fragments are considered to "selectively hybridize” as described herein. The degree of sequence identity between two nucleic acid molecules affects the efficiency and strength of hybridization events between such molecules.
  • a partially identical nucleic acid sequence will at least partially inhibit a completely identical sequence from hybridizing to a target molecule. Inhibition of hybridization of the completely identical sequence can be assessed using hybridization assays that are well known in the art (e.g., Southern blot, Northern blot, solution hybridization, or the like, see Sambrook, et al., Molecular Cloning: A Laboratory Manual. Second Edition, (1989) Cold Spring Harbor, N.Y.). Such assays can be conducted using varying degrees of selectivity, for example, using conditions varying from low to high stringency.
  • the absence of non-specific binding can be assessed using a secondary probe that lacks even a partial degree of sequence identity (for example, a probe having less than about 30% sequence identity with the target molecule), such that, in the absence of non-specific binding events, the secondary probe will not hybridize to the target.
  • a partial degree of sequence identity for example, a probe having less than about 30% sequence identity with the target molecule
  • a nucleic acid probe When utilizing a hybridization-based detection system, a nucleic acid probe is chosen that is complementary to a target nucleic acid sequence, and then by selection of appropriate conditions the probe and the target sequence "selectively hybridize,” or bind, to each other to form a hybrid molecule.
  • a nucleic acid molecule that is capable of hybridizing selectively to a target sequence under "moderately stringent” typically hybridizes under conditions that allow detection of a target nucleic acid sequence of at least about 10-14 nucleotides in length having at least approximately 70% sequence identity with the sequence of the selected nucleic acid probe.
  • Stringent hybridization conditions typically allow detection of target nucleic acid sequences of at least about 10-14 nucleotides in length having a sequence identity of greater than about 90-95% with the sequence of the selected nucleic acid probe.
  • Hybridization conditions useful for probe/target hybridization where the probe and target have a specific degree of sequence identity can be dete ⁇ nined as is known in the art (see, for example, Nucleic Acid Hybridization: A Practical Approach, editors B.D. Hames and S.J. Higgins, (1985) Oxford; Washington, DC; IRL Press).
  • stringency conditions for hybridization it is well known in the art that numerous equivalent conditions can be employed to establish a particular stringency by varying, for example, the following factors: the length and nature of probe and target sequences, base composition of the various sequences, concentrations of salts and other hybridization solution components, the presence or absence of blocking agents in the hybridization solutions (e.g., formamide, dextran sulfate, and polyethylene glycol), hybridization reaction temperature and time parameters, as well as, varying wash conditions.
  • the selection of a particular set of hybridization conditions is selected following standard methods in the art (see, for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual. Second Edition, (1989) Cold Spring Harbor, N.Y.).
  • Encoded by refers to a nucleic acid sequence which codes for a polypeptide sequence, wherein the polypeptide sequence or a portion thereof contains an amino acid sequence of at least 3 to 5 amino acids, more preferably at least 8 to 10 amino acids, and even more preferably at least 15 to 20 amino acids from a polypeptide encoded by the nucleic acid sequence. Also encompassed are polypeptide sequences which are immunologically identifiable with a polypeptide encoded by the sequence.
  • Polynucleotide refers to a polynucleotide of interest or fragment thereof which is essentially free, e.g., contains less than about 50%, preferably less than about 70%, and more preferably less than about 90%, of the protein with which the polynucleotide is naturally associated.
  • Techniques for purifying polynucleotides of interest include, for example, disruption of the cell containing the polynucleotide with a chaotropic agent and separation of the polynucleotide(s) and proteins by ion-exchange chromatography, affinity chromatography and sedimentation according to density.
  • a “vector” is capable of transferring gene sequences to target cells (e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes).
  • a “vector construct” refers to a nucleic acid vector capable of transferring sequences of interest into target cells.
  • Nucleic acid vectors can be transiently present in or capable of replication in target cells. Transient vectors typically do not have an origin of replication that can function in the target cell, or one which does not function under certain conditions in the target cell (a "conditional” origin of replication).
  • Nucleic acid expression vector refers to an assembly which is capable of directing the expression of a sequence or gene of interest.
  • the nucleic acid expression vector includes a promoter which is operably linked to the sequences or gene(s) of interest.
  • Other control elements may be present as well.
  • the plasmid construct may also include one or more bacterial origin(s) of replication, one or more selectable markers, a signal which allows the plasmid construct to exist as single-stranded DNA (e.g., a Ml 3 origin of replication), a multiple cloning site, and a "mammalian" origin of replication (e.g., a SV40 or adeno virus origin of replication).
  • an “expression cassette” comprises any nucleic acid construct which contains polynucleotide gene(s) or sequence(s) capable of being expressed in a cell.
  • Expression cassettes may contain, in addition to polynucleotide gene(s) or sequence(s) of interest, additional transcriptional, translational or other regulatory or control elements.
  • Such cassettes are typically constructed into a “vector,” “vector construct,” or “expression vector,” (i.e., a "nucleic acid expression vector) in order to transfer the expression cassette into target cells.
  • an expression cassette typically has no transcriptional promoter, but does have translation enhancing sequences, such as Shine-Dalgarno sequences.
  • a "transposon” as used herein defines a polynucleotide which comprises a repeated element capable of relocating from one genetic locus to another (e.g., from a chromosomal point to another chromosomal point, or from an episomal point to a chromosomal point), i.e., a transposon is a mobile genetic element.
  • a "transposon cassette” comprises a minimum unit required for transposition (e.g., first and second transposon inverted repeat sequences flanking an internal polynucleotide sequence comprising at least a transposase capable of inducing transposition mediated by said transposon inverted repeats).
  • a transposon cassette may be first and second transposon inverted repeat sequences flanking an internal polynucleotide sequence, where the transposase function is provided in trans or is encoded external to the transposon cassette (i.e., outside of the two transposon inverted repeats flanking the internal polynucleotide sequence).
  • a transposon cassette comprises at least two inverted repeat sequences flanking an internal region. The internal region may contain transposase coding sequence and/or other sequences of interest.
  • a schematic representation of a transposon cassette is as follows: IR-internal region-IR, where IR represents the inverted repeats.
  • a representation is as follows: JR-tnp-TK, where tnp represents a transposase gene.
  • IR- sequence of interest - tnp-IR represents yet another embodiment, capable of inducing transposition mediated by the IR sequences. Further sequences, 5' and 3' of the inverted repeats may be included in the transposon cassette where indicated.
  • a transposon cassette that is "functional" in a host organism is one that is capable of undergoing transposition in that organism.
  • the term "transposant” typically refers to a cell in which a transposon has integrated into the cell's genome.
  • gram-positive transposon refers to a transposable element derived from a gram-positive bacteria, for example transposons Tn4001 and Tn917.
  • Gram-positive is a taxonomic feature referring to bacteria which resist decolorization with any standard Gram-staining dyes.
  • Gram-negative bacteria are easily decolorized with certain organic solvents such as ethanol or acetone.
  • the ability of bacteria to retain or resist staining generally reflects the structure of the cell wall and it has been suggested that Gram-negative bacteria have more extensive peptidoglycan crosslinking and less permeable cells walls than their Gram-negative counterparts.
  • Non-limiting examples of Gram-positive bacteria include: Stapholococcus, Streptococcus, certain Bacillus, Anthrax, Mycobacterium, etc.
  • Light-generating is defined as capable of generating light through a chemical reaction or through the absorption of radiation.
  • Light is defined herein, unless stated otherwise, as electromagnetic radiation having a wavelength of between about 300 nm and about 1100 nm.
  • Vehicle light is defined herein, unless stated otherwise, as electromagnetic radiation having a wavelength of between about 400 nm and about 750 nm.
  • Light-generating protein is defined as a protein or polypeptide capable of generating light through a chemical reaction (e.g., bioluminescence, as generated by luciferase) or through the absorption of radiation (e.g., fluorescence, as generated by Green Fluorescent Protein).
  • “Lux” refers to prokaryotic genes associated with luciferase and photon emission.
  • Luc refers to eukaryotic genes associated with luciferase and photon emission.
  • Animal typically refers to a non-human animal, including, without limitation, farm animals such as cattle, sheep, pigs, goats and horses; domestic animals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; rabbits; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like. The term does not denote a particular age. Thus, adult, newborn individuals, and fetuses are intended to be covered.
  • analyte refers to any compound or substance whose effects (e.g., induction or repression of a specific promoter) can be evaluated using the test animals and methods of the present invention.
  • Such analytes include, but are not limited to, chemical compounds, pharmaceutical compounds, polypeptides, peptides, polynucleotides, and polynucleotide analogs.
  • Many organizations e.g., the National Institutes of Health, pharmaceutical and chemical corporations have large libraries of chemical or biological compounds from natural or synthetic processes, or fermentation broths or extracts. Such compounds/analytes can be employed in the practice of the present invention.
  • bioluminescent bacteria As discussed above, the synthesis of light in naturally occurring bioluminescent bacteria is encoded by five genes. In one embodiment, these genes are clustered in an operon (luxCDABE) that can be moved into non-bioluminescent bacteria to produce a bioluminescent phenotype.
  • luxCDABE operon
  • all identified species of naturally occurring marine and terrestrial bioluminescent bacteria are Gram-negative; the transformation of Gram- positive bacteria to a bioluminescent phenotype has been limited, due in part to the differing genetics of these two bacterial groups.
  • the constructs and methods of the present invention facilitate the ability to confer bioluminescence properties on a bacterium of one's choice, so that the bacterium could, for example, be followed with in vivo monitoring in various models of infection or used in the tracking of bacteria, e.g., in the food industries.
  • it would be desirable to confer such bioluminescence properties on Gram positive bacteria because many bacteria pathogenic to mammals are in fact Gram- positive.
  • infections caused by Stapholococcus, a Gram-positive cocci are ubiquitous and include, e.g., abscesses, mastitis, pneumonia, bacteremia, osteomyletis, enterocolitis and toxic shock syndrome (TSS).
  • TSS toxic shock syndrome
  • Streptococcus is the primary cause of pharyngeal infections (strep throat).
  • Gram-positive bacilli such as Anthrax and Listeria (which causes meningitis) can cause severe, and even fatal infections in humans and other mammals.
  • transposon cassettes e.g., T «4001, Tn917, and others
  • T transposon cassettes
  • microorganisms of interest including, but not limited to, Gram-negative bacteria (including Heliobacter pylori and others) and Gram-positive bacteria (including related organisms such as Chlamydia and Treponema pallidum).
  • the present invention relates to the re-engineering of the entire Photorhabdus luminescens lux operon to first remove the endogenous promoter sequences associated with the lux operon genes, and finally introduce Gram-positive control elements immediately upstream of each of the lux operon genes, in particular, Shine-Dalgarno sequences (i.e., ribosome binding sites).
  • the luciferase enzyme is encoded by luxA and luxB, whereas the enzymes responsible for the aldehyde biosynthesis are encoded by the three genes luxC, luxD and luxE.
  • aldehyde can rapidly diffuse across cellular membranes and is commercially available (e.g., Sigma), the genes encoding the synthesis of this substrate (luxCDE) a e not an absolute necessity for bioluminescence and can be substituted by the addition of this compound, or a suitable luciferin, exogenously.
  • luxCDE a substrate for generating a bioluminescent Gram-positive bacterium therefore, it is only necessary to ensure that the cell can synthesize a functional luciferase.
  • a novel promoterless luxABCDE cassette was further modified by addition of a promoterless kanamycin resistance gene immediately adjacent to the 3' end of the coding strand of the operon. While the use of kanamycin as a selectable marker is explicitly described herein, the use of other selectable marker sequences appropriate to the target host cell are also contemplated and may be used in the practice of the present invention.
  • genes useful for conferring antibiotic resistance to the following antibiotics are non-limiting examples of those that may be used: actinomycin, ampicillin, chloramphenicol, erythromycin; gentamicin sulfate; hygromycin, neomycin, penicillin, polymixin B sulfate and stretomycin sulfate.
  • Transformation of organisms of interest to a bioluminescent phenotype was then accomplished by integrating the cassette into the genome of those organisms by means of a transposon.
  • the novel luxABCDE km R cassette was inserted into the
  • Tn4001 transposon and cells of interest were then transformed with a shuttle vector carrying the Tn4001 luxABCDE km R construct.
  • Transposition events subsequent to transformation resulted in integration of the luxABCDE km R cassette into the genome of the host organism and resulted in the ability of the organism to bioluminesce; integration of the otherwise promoterless cassette behind an active promoter sequence yielded host organisms having a bioluminescent phenotype.
  • the kanamycin resistance gene facilitated selection of those integrants.
  • several different genera of Gram-positive bacteria were made brightly bioluminescent, either constitutively or inducibly.
  • the present invention generally relates to transposon constructs comprising, a promoterless coding sequence of interest, e.g., a light-generating protein coding cassette.
  • transposon constructs comprising, a promoterless coding sequence of interest, e.g., a light-generating protein coding cassette.
  • These transposon constructs can be inserted into a suitable backbone (e.g., a shuttle vector) and thereby confer the ability to produce the product of the coding sequence of interest (which, in the case of a light-generating protein, also confers the ability to produce light in a cell or animal) upon integration of the coding sequence of interest, (e.g., light-generating protein coding cassette) behind an active promoter region in the host cell genome.
  • the transposon cassettes described herein allow, for the first time, more than minimal amount of light to be produced from Gram-positive bacteria at physiological temperatures.
  • the transposon cassette to include a promoterless selectable marker coding sequence downstream of the coding sequence of interest and operably linked thereto.
  • the selectable marker is typically located in close proximity to the coding sequence of interest and the open reading frame of the selectable marker is in the same 5' to 3' orientation as that of the coding sequence of interest.
  • the light generating protein coding cassette contains promoterless bacterial lux genes recombinantly engineered to promote functional expression of lux for example, by arranging the genes in the order luxABCDE. Thus, this cassette rearranges the unmodified order of these genes, namely luxCABDE.
  • this coding cassette does not require the addition of exogenous substrate. Moreover, the rearrangement of genes together with the introduction of Gram-positive Shine- Dalgarno sequences confers a greater light-generating ability upon integration than the unmodified order.
  • a Gram-positive Shine-Dalgarno sequence (“SD”) is preferably inserted before (typically 5' to) one, more than one, or all of the lux genes coding sequences (e.g., SD-luxA-SD-luxB-SD-luxC-SD-luxD-SD-luxE).
  • Another cassette provided by the present invention includes polynucleotides encoding luxAB, but not including the substrate encoding genes.
  • exogenous substrate for example, aldehyde
  • the luxAB coding cassettes typically include a DNA sequence which enhances translation between the genes encoding for luxA and luxB (for example, Gram-positive Shine-Dalgarno sequences).
  • Yet another light generating protein coding cassette of the present invention includes polynucleotides encoding functional luc, an eukaryotic luciferase gene.
  • the coding cassette includes polynucleotides encoding luc and control elements, such as Shine-Dalgarno sequences, for example, from Gram-positive bacteria.
  • the transposon cassettes described herein may be introduced into a plasmid or vector, e.g., various shuttle vector backbones. Also provided are methods of modifying Gram-positive organisms to a bioluminescent phenotype using those shuttle vector constructs.
  • Target host cells are transformed with shuttle vector DNA to provide the opportunity for a transposition of the transposon cassette; subsequent integration of the transposon cassette downstream of an active promoter region in the host cell genome gives rise to a bioluminescent phenotype, which may be constitutive, inducible or repressible depending on the properties of the promoter region behind which the transposon cassette has integrated.
  • the bioluminescent transposants are themselves useful in a number of applications.
  • pathogenic transposants can be used to study the promoters active during pathogenesis.
  • experimental animals are infected with pathogenic bacteria transformed with a transposon cassette of the present invention (e.g, using pAUL-ATn ⁇ OOi luxABCDE km R shuttle vector construct), and a selection is applied to select for transposants.
  • a selection is applied to select for transposants.
  • cells from those colonies are re-injected into other experimental animals, where induction of light production in vivo, for example during the initial stages of infection, indicates a bacterial promoter turned on in response to infection of the host animal.
  • the promoter sequences which mediate bioluminescence in a transposant may be identified.
  • bacteria are isolated from experimental animals that exhibit bioluminescence attributable to the activity of promoters induced by infection.
  • the promoters are then isolated by, for example, inverse PCR amplification using primers based on known gene sequences adjacent or in close proximity to the active bacterial promoters, for example, gene sequences present in the coding sequence of interest, such as, one of the lux genes or kanamycin resistance sequence of the integrated transposon cassette.
  • the amplification products are then sequenced by methods known in the art.
  • chromosomal DNA may be directly isolated from a single bacterial colony of interest, and then sequenced using primers based on gene sequences adjacent or in close proximity to the active bacterial promoters.
  • colonies that exhibit bioluminescence attributable to the activity of promoters induced by infection can be used to identify effective pharmaceutical agents.
  • transposant cells which exhibit bioluminescence attributable to the activity of promoters induced by infection are used to infect experimental and control animals. The experimental animals are then treated with a pharmaceutical agent of interest. Both the experimental animals and the controls are monitored for bioluminescence, and effective agents identified as those which extinguish bioluminescence.
  • transposants may be used as a means of monitoring bacterial growth in foodstuffs, and further as a means of identifying agents or conditions which suppress or encourage that growth.
  • Advantages of the present invention include, but are not limited to, (i) transforming a variety of organisms, including Gram-positive bacteria and organisms such as Chlamydia, Treponema pallidum, as well as Gram-negative and others to a bioluminescent phenotype; (ii) obtaining high levels of light generating protein expression in those transformed organisms, which, for example, permits more sensitive detection of bioluminescence both in vitro and in vivo; (iii) integration of the transposon cassette into the host chromosome such that the cassette becomes operably linked to host cell promoters, which, for example, permits identification of promoters involved in pathogenesis; and stable light production from such organsims at physiological temperatures (e.g., 37°C-42°C).
  • transposons derived from gram- positive bacteria including but not limited to, transposons _Tn4001, Tn917, 7n5401 and Tn5706, as well as sequences comprising such transposons, including, but not limited to IS sequences and transposase encoding sequences.
  • transposons _Tn4001, Tn917, 7n5401 and Tn5706, as well as sequences comprising such transposons, including, but not limited to IS sequences and transposase encoding sequences.
  • Tn4001 is employed.
  • 7 ⁇ 4001 a class I composite-type transposon originally isolated from Staphyloccus aureus (GeneBank Accession No. M18086, base pair 1-1,324 of the sequence; see Byrne, M.E., Rouch, D.A., and Skurray, R.A. (1989) Gene 81:361-367).
  • This element is capable of inserting with a high degree of randomness into the bacterial chromosome of Gram-positive organisms.
  • Experiments performed in support of the present invention indicate that the transposon functions in Gram-negative host cells as well.
  • the components of the Tn4001 transposon include (1) two identical copies of the IS256 insertion sequence, present as inverted repeats (IR's) which define an insertion sequence therebetween, and (2) a transposase gene located within the inverted repeats, which defines an insertion .
  • the transposon may include sequences outside of the IR regions downstream of the 3' IR region.
  • the inverted repeats are considered to be the boundary of a functional transposon unit. This basic structure may be further modified and placed into a variety of vector backbones, as discussed below.
  • additional sequences of interest may be inserted between the inverted repeats (e.g., 5 ' - IR - sequence of interest - IR 3 '); alternately, they may be inserted following the 3'-IR sequence.
  • the additional sequences of interest lack an associated promoter region.
  • the additional sequences of interest are preferably introduced into the 2n4001 transposon such that the direction of transcription for the inserted sequences is opposite that of the direction of the transposase coding sequence, and is therefore not under influence of transposase promoter. Hence, the sequences will not be transcribed unless and until integration of the insertion sequence behind an active or activatable promoter region occurs.
  • the coding sequences for the open reading frames for the sequences of interest and the transposase are in opposite orientations in the DNA.
  • the tranposase sequence may be present in the same orientation as the sequence of interest, provided that it is located downstream of that sequence, in order to avoid read-through from the endogenous transposase promoter and expression of the light generating protein gene product prior to integration.
  • polynucleotide sequences encoding a light generating protein which proteins are discussed below, may be inserted between the inverted repeats.
  • the light-generating protein sequences are typically employed in the absence of a promoter contained within the tranposable element.
  • the light-generating protein sequence is inserted adjacent or in close proximity to the 3' end of the 5' inverted repeat sequence, e.g., 5'-IR-light generating sequences...-IR-3'. Further, the sequence is inserted in an orientation opposite that of the tranposase sequence, such that even with transcription of the transpose sequence there is no transcription of the sequence of interest (e.g., light-generating polypeptide coding sequences) prior to integration into the genome of an organism of interest adjacent an active host promoter region.
  • the sequence of interest e.g., light-generating polypeptide coding sequences
  • the transposon may be further modified by inserting polynucleotide sequences which code for one or more selective markers between the IR sequences.
  • the marker employed is chosen to be functional in the target organism of interest, and serves to identify candidate transposition events where expression of the selectable marker protein is related to transposition events of interest.
  • transcription of the selectable marker is also in the opposite direction relative to transcription of the transposase sequences. Accordingly, there is no transcription of the selectable marker prior to integration into the genome of an organism of interest adjacent an active host promoter region.
  • an antibiotic resistance genes may be employed as a selective marker.
  • a promoter-less kanamycin resistance gene is employed as a selective marker in the practice of the present invention.
  • a promoterless nucleotide sequence encoding kanamycin resistance is inserted downstream of, and is operably linked to, the light-generating protein genes.
  • the km R sequence is inserted, either immediately adjacent to the 3' end of the sequence encoding a light generating protein or in close proximity thereto with the light-generating protein coding sequence, for example, 5 '-IR-light generating polypeptide-km...--IR-3 '.
  • This configuration provides a means for the selection of cells wherein the transposable element has integrated into the bacterial chromosome, as well as a means for distinguishing among integrants located adjacent to either constitutively- or nonconstitutively-active promoters, as will be discussed below.
  • sequences of interest may be inserted outside of the inverted repeats.
  • a sequence of interest may be inserted downstream of the 3' inverted repeat sequence.
  • the transposase sequence can be moved outside of the transposable element defined between the IR regions, and placed downstream of the 3 '-IR sequence on a vector backbone.
  • the practice of the present invention will typically employ nucleotide sequences encoding light generating proteins, and the property of bioluminescence which transcription of those sequences will confer.
  • Bioluminescence provides a powerful reporter system for studying bacterial infection (e.g., U.S. Patent No. 5,650,135).
  • Luciferase is a term applied to members of a family of diverse enzymes which share the property of producing light when provided with a substrate (e.g. , luciferin, long-chain aldehyde or colentrazine), an energy source (e.g., ATP or FMNH ) and oxygen.
  • Luciferases can be broadly classified into eukaryotic luciferases and prokaryotic luciferases.
  • Eukaryotic luciferase ("luc") is typically encoded by a single gene (see, e.g., de Wet, J.R, et al, (1985), Proc.
  • luciferase system An exemplary eukaryotic organism containing a luciferase system is the North American firefly Photinus pyralis. Firefly luciferase has been extensively studied, and is widely used in ATP assays. cDNAs encoding luciferases from Pyrophorus plagiophthalamus, another species of click beetle, have been cloned and expressed (Wood, K.V., et al. (1989) Science 244:700-702).
  • This beetle is unusual in that different members of the species emit bioluminescence of different colors.
  • the last class (593 nm) may be particularly advantageous for use as a light-generating moiety with the present invention, because the emitted light has a wavelength that penetrates tissues more easily than shorter wavelength light.
  • Bacterial luciferase (“lux”) is typically made up of two subunits ( ⁇ and ⁇ ) encoded by two different genes (luxA and luxB) on the lux operon.
  • Bacterial lux is present in certain bioluminescent Gram- negative bacteria (e.g., Photorhabdus luminescens) and the wild-type operon is ordered CD ABE.
  • luxY isolated from Vibrio fischeri strain Y- 1
  • YFP yellow fluorescent protein
  • a variety of luciferase encoding genes have been identified including, but not limited to, the following: B.A. Sherf and K.V. Wood, U.S. Patent No. 5,670,356, Kazami, J., et al., U.S. Patent No. 5,604,123, S. Zenno, et al. U.S. Patent No.
  • gene cassettes comprising polynucleotides encoding both the structural and substrate-encoding lux gene-products are provided.
  • rearranging the lux genes for example, from the wild-type CABDE to ABCDE, and inserting Gram-positive Shine-Dalgarno sequences before one or more of the lux genes, confers on the resulting luciferase an enhanced ability to produce light .
  • Suitable Gram-positive Shine-Dalgarno sequences e.g., Example 1, Table 1 will be known to those of skill in the art in view of the teachings of the specification, and are also described in the Examples below.
  • the luxABCDE cassettes express not only luciferase, but also the biosynthetic enzymes necessary for the synthesis of the lux luciferase's substrate ⁇ aldehyde. Accordingly, oxygen is the only extrinsic requirement for bioluminescence when this expression cassette is used.
  • luxABCDEY cassettes are provided. Adding the luxY gene to the luxABCDE gene cassette results in broadening the range of wavelength of light emitted during bioluminescence towards the red end of the visible light spectrum. Given that longer-wavelength light more easily penetrates living tissue as compared to light of shorter wavelengths, selected embodiments of the luxABCDE gene cassette of the present invention will therefore additionally include the luxY coding sequence, as a means of increasing the sensitivity of applications which employ bioluminescence as a reporter means.
  • transposon constructs of the present invention confer the ability of bioluminescence in the organisms of interest only upon integration of the insertion sequence into the host cell genome behind an active promoter sequence.
  • Those transposon constructs employ light generating polypeptide sequences, e.g., luxABCDE and luxABCDEY, that do not include an integral promoter.
  • luxABCDE and luxABCDEY light generating polypeptide sequences
  • the luxAB cassettes typically contain a Gram positive ribosome binding site (also referred to as a "Shine- Dalgarno" sequence) operably linked upstream of each of the polynucleotides encoding luxA and B.
  • Host organisms carrying the luxAB cassette exhibit bioluminescence when provided with exogenous aldehyde substrate.
  • these luxAB cassettes confer higher levels of luciferase activity than found in previously known constructs, particularly when expressed in Gram-positive bacteria such as Stapholococcus or Streptococcus.
  • the present invention also includes gene cassettes that allow for expression of eukaryotic luciferase.
  • the luc expression cassette is employed in the absence of a promoter contained within the transposable region.
  • the lux and luc genes can also be combined for use in the transposon cassette constructs of the present invention.
  • one of the light- generating protein coding cassettes may be present in the vector backbone in place of an antibiotic resistance gene, and the other as a means of identifying active host cell promoters.
  • a vector construct may include both a promoterless luxAB coding cassette in the first sequence of interest of the transposon cassette, and a luc coding cassette operably linked to a promoter functional in the target organism on the vector.
  • Organisms transformed with such a construct could then be identified upon supplying the luc substrate, upon which the transformed organisms would exhibit bioluminescence of a wavelength characteristic of the luc gene product; transposants in which the luxAB cassette has successfully integrated behind an active promoter may then be identified by the bioluminescence exhibited upon supplying aldehyde.
  • the luxABCDE coding cassette may be used in the transposon construct and the luxABCDEY coding cassette on the vector backbone, obviating the need for addition of an exogenous substrate while maintaining the ability to discern the source of the light being produced based on the emitted wavelength.
  • sequences producing two or more light- generating polypeptides producing emitted light at two or more wavelengths may be provided as the first sequence of interest in separate transposon cassettes; the transposon cassettes may in turn be provided in either a single vector backbone or in multiple vector backbones.
  • Such constructs may be used to produce, for example, single microorganisms bearing multiple transposon cassettes of the present invention, where the transposon cassettes may each encode light generating polypeptides that emit light at different wavelengths.
  • the practice of the present invention may employ selectable markers, both as a component of the Tn4001 transposon cassette, as discussed above, and as an element of the vector backbone, as discussed below.
  • selectable markers may be used; with the choice of which marker to employ being made on the basis of whether it is functional in the target organism of interest.
  • a selectable marker is used in the transposon cassette it does not have a transcriptional promoter operably linked to it. This allows for selection of transposons that have integrated adjacent operative promoters in the host cell genome.
  • a selectable marker when used in a backbone vector carrying a transposon cassette, such a selectable • marker is typically operably linked to a functional transcriptional promoter in order to allow selection for the presence and/or absence of the vector.
  • the selectable marker in the transposon cassette is different from the selectable marker present in the backbone vector (e.g., kanamycin resistance coding sequences in the transposon (no endogenous promoter), a promoter operably linked to erythromycin resistance coding sequences in the backbone vector).
  • the present invention may employ an antibiotic effective against the host organism, and the genes or set of genes that confer resistance against that antibiotic, as a selectable marker.
  • an antibiotic resistance coding sequence is typically inserted immediately adjacent to the luxABCDE cassette within the JR. regions of the transposon, in order to facilitate selection of host cells that have undergone transposition events.
  • kanamycin and the kanamycin resistance gene are particularly preferred.
  • a variety of other antibiotic resistance genes may also be advantageously employed.
  • genes include, but are not limited to genes encoding products which confer resistance to the following antibiotics: actinomycin; ampicillan, chloramphenicol; erythromycin; gentamicin sulfate; hygromycin; neomycin; penicillin;; polymixin B sulfate; and streptomycin sulfate.
  • other selection systems may be used, including but not limited to, metabolic selection, repressor proteins, and others.
  • the selectable marker employed as part of the transposon will typically be inserted in the same orientation as the luxABCDE cassette, i.e., in the orientation opposite that of the transposase gene (such that transcription of the transposase gene does not result in transcription of the selectable marker/lux genes).
  • the selectable marker sequence will typically be inserted downstream of the luxABCDE cassette, either immediately adjacent or in close proximity to that cassette, such that the sequences are operably linked in that transcription of the two is coupled, preferably tightly coupled.
  • the encoded marker protein will not be expressed unless and until integration of the transposable sequence behind an active promoter region in the host organism's genome (i.e., operably linked to an endogenous promoter).
  • transposon cassettes are typically cloned into shuttle vectors for ease of manipulation and isolation of large quantities of vector DNA.
  • shuttle vectors are commonly available, e.g., pAUL-A (Chakraborty, et al. (1992) J. Bacteriol. 174:568-574) (a schematic diagram of the vector is presented in Figure 1); pE194 (Sozhamannan, s., et al. (1990) J.
  • the shuttle vectors preferably include the following features: (1) a Gram-positive origin of replication, and/or a Gram-negative origin of replication, and/or an origin or replication functional in both types of organisms; (2) polylinkers; and (3) a polynucleotide encoding a selectable marker (e.g., ampicillin, chloramphenicol, erythromycin, and others as discussed supra) which allows selection in the host cells.
  • a selectable marker e.g., ampicillin, chloramphenicol, erythromycin, and others as discussed supra
  • the shuttle vectors will further include (4) transcription termination sequences flanking one or both sides of the transposon cassette. Such transcription termination sequences are used to prevent transcriptional read-through into the coding sequences of the transposon cassette.
  • the presence of a Gram-negative origin of replication permits replication of the vector in Gram-negative organisms, thereby facilitating manipulation of the inserted sequences while avoiding the restriction endonuclease systems of Gram-positive host organisms, as well as permitting isolation of large quantities of vector construct DNA. Manipulation is further facilitated by the presence of the selectable marker coding sequence operably linked to a transcriptional promoter, which permits selection of cells carrying the vector construct.
  • selectable marker coding sequences in the transposon cassette are not typically operably linked to a promoter in vector/cassette constructs.
  • an origin of replication may be employed that is functional in both Gram-positive and Gram-negative organisms, e.g., the origins of replication present in certain Streptomyces plasmids (such as, pCKl).
  • the Gram-positive origin of replication may be either continuously active or may instead be conditional, e.g., the temperature-sensitive origin from pE194, as is found in the pAUL-A shuttle vector.
  • An advantage of including a conditional origin of replication in the vector constructs of the present invention is that such elements permit stabilization of the vector construct in the host organism of interest grown under permissive conditions, while permitting the host organism to be "cured" of the vector when grown under restrictive conditions (e.g., temperature elevated to a non- permissive level).
  • a light-based screening method may also be employed.
  • a light generating polypeptide coding sequence is placed under the control of a promoter active in an organism of interest.
  • control elements may be constitutive or conditional.
  • the luxABCDE cassette, operably linked to a promoter sequence functional in the target organism of interest may be introduced into a suitable vector.
  • the organism of interest is then transformed and the resulting organisms are screened for their ability to produce light.
  • the production of light is used to identify transformants of interest.
  • Light producing colonies (or patches of bacteria) are typically cloned (i.e., physically isolated) by standard methods (e.g., dilution plating, for example, using microtiter wells, or streaking for single colonies).
  • One aspect of the present invention provides a vector comprising light-generating polypeptide sequences operably linked to promoter sequences functional in a target organism of interest.
  • Using such a vector provides means for the transformation of organisms for which no selectable marker (such as a drug resistance marker) is available.
  • vector backbones possess transcription termination sequences flanking one or both sides of the transposon cassette, for the purpose of preventing expression of the light generating polypeptide prior to integration of the cassette into the host organism's DNA.
  • Such regions are known in the art. See e.g., Henkin, T.M., (1996) Ann Rev Genet 30:35-37; MacDonald L.E., et al. (1993) J Mol Biol. 232:1030-1037; Jeng, S.T., et al. (1997) Can J Microbiol 43:1147-1156.
  • the luciferase expression cassettes are inserted into the pAUL-A shuttle vector.
  • This vector backbone contains, inter alia, (1) a Gram negative origin of replication, (2) a conditional (temperature- sensitive) Gram-positive origin of replication, and (3) an erythromycin-resistance coding sequence, in addition to (4) a lacZ coding sequence containing (5) a polylinker region and (6) transcription termination regions.
  • a Gram negative origin of replication (2) a conditional (temperature- sensitive) Gram-positive origin of replication
  • an erythromycin-resistance coding sequence in addition to (4) a lacZ coding sequence containing (5) a polylinker region and (6) transcription termination regions.
  • the gene cassettes, transposon cassettes and shuttle vector constructs described herein can be assembled utilizing methodologies known in the art of molecular biology (see, for example, Ausubel, F.M., et al, Current Protocols in Molecular Biology. John Wiley and Sons, Inc., Media, PA (1995), or Sambrook, et al.) in view of the teachings of the present specification.
  • gene cassettes comprising sequences encoding light-generating polypeptides (e.g., lux genes) are assembled.
  • the sequences are assembled from polynuclotides encoding lux or luc genes by operably linking these polynucleotides to translational regulatory elements (e.g., Gram-positive Shine- Dalgarno sequences).
  • translational regulatory elements e.g., Gram-positive Shine- Dalgarno sequences.
  • Short, random nucleotide sequences, selectable markers, and the like can also be introduced into the expression cassettes at suitable positions.
  • a preferred method of obtaining polynucleotides, suitable regulatory sequences and short, random nucleotide sequences is PCR. General procedures for PCR as taught in MacPherson et al., PCR: A PRACTICAL APPROACH.
  • PCR conditions for each application reaction may be empirically determined. A number of parameters influence the success of a reaction. Among these parameters are annealing temperature and time, extension time, Mg2+ and ATP concentration, pH, and the relative concentration of primers, templates and deoxyribonucleotides. Exemplary primers are described below in Example 1. After amplification, the resulting fragments can be detected by agarose gel electrophoresis followed by visualization with ethidium bromide staining and ultraviolet illumination. Another method for obtaining polynucleotides, for example, short, random nucleotide sequences, is by enzymatic digestion.
  • a blunt-cutting four-nucleotide recognition restriction enzyme such as Alul, HaeY ⁇ and Sau3Al
  • Lux gene cassettes are inserted into transposon sequences typically between the first and second inverted repeats, and transposon constructs are inserted into vector backbones, using methods known in the art.
  • insert and vector DNA can be contacted, under suitable conditions, with a restriction enzyme to create complementary or blunt ends on each molecule that can pair with each other and be joined with a ligase.
  • synthetic nucleic acid linkers can be ligated to the termini of a polynucleotide. These synthetic linkers can contain nucleic acid sequences that correspond to a particular restriction site in the vector DNA. Other means are known and available in the art as well.
  • the transposase coding sequences are located between the first and second inverted repeats of the transposon. In other embodiments, however, the transposase coding sequences and associated promoter may be moved from within the confines of the transposable sequences (typically defined as sequences framed by the first and second inverted repeat sequences) and placed instead on the vector backbone.
  • the luciferase vector constructs such as the ones described above and in the Examples, can be used to transform a variety of procaryotic host cells, including Gram- negative bacteria, Gram-positive bacteria, and other genera not included in either of the preceding classifications (e.g., Rickettsia spp.; Rochalimaea spp,; Coxiella spp.;
  • Treponema spp. including Treponema pallidum, the organism which causes syphilis; Mycoplasma spp., and Chlamydia spp.).
  • constructs of the present invention may be used to transform organisms including but not limited to the following: Clostridium spp., Vibrio spp., Brucella spp., Bordetella spp.,
  • Campylobacter spp. Pseudomonas spp., Escherichia spp., Enter obacter spp., Klebsiella spp., Serratia spp., Citrobacter spp., Proteus spp., Salmonella spp., Shigella spp., and Yersinia spp.
  • constructs of the present invention may be used to transform organisms including but not limited to the following:
  • Micrococcaceae Members of the Gram-positive cocci families Micrococcaceae (Micrococcus spp., Stomatococcus spp., Planococcus spp., and Staphylococcus spp.), Deinococcaceae (Deinococcus spp.), and species of other cocci genera including: Streptococcus spp (e.g., pyogenic hemolytic streptococci spp., oral streptococci spp., Enterococci spp., lactic acid streptococci spp., anaerobic Streptococci spp., and other species of Streptococci); Leuconostoc spp., Pediococcus spp., Aerococcus spp., Gemella spp., Peptococcus spp., Peptostreptococcus spp., Ruminococcus spp., Coprococc
  • Endospore-forming Gram-positive rods and cocci including: Bacillus spp., Sporolactobacillus spp., Clostridium spp., Desulfotomaculum spp., Sporosarcina spp., and species of the genus Oscillospira.
  • Regular, nonsporing, Gram-positive rods including Lactobacillus spp., Listeria spp.
  • Organisms of the family Mycobacteriaceae i.e., Mycobacterium spp.
  • the nocardioforms including Nocardia spp., Rhodococcus spp., Nocardioides spp., Pseudonocardia spp., Oerskovia spp., Saccharopolyspora spp., Micropolyspora spp., Promicromonospora spp., and species of the genus Intrasporangium.
  • Organisms of especial interest include: Clostridium spp., Vibrio spp., Brucella spp., Bordetella spp., Campylobacter spp., Pseudomonas spp., Escherichia spp., Enter obacter spp., Klebsiella spp., Serratia spp., Citrobacter spp., Proteus spp., Salmonella spp., Shigella spp., and Yersinia spp.
  • Transformation methods for both prokaryotic cells and eukaryotic cells are known in the art (e.g., Sambrook, et al.) and include, but are not limited to, calcium phosphate precipitation, microinjection or electroporation.
  • Vectors containing the appropriate regulatory elements and multiple cloning sites are widely commercially available (e.g., Stiatagene, La Jolla, Calif; Clontech, Palo Alto, Calif.) and can be used as backbone vectors to carry the transposon cassette sequences.
  • the luciferin substrate is aldehyde. Based on the luciferase being used the appropriate luciferin substrate is selected. When administered to cells, aldehyde may be applied in the atmosphere surrounding the culture media as a vapor or directly to the culture media as a liquid or solid.
  • each transposon cassette may encode a light generating polypeptide which emits light at a different characteristic wavelength relative to each other.
  • transposon cassettes carrying light generating polypeptides which emit light at the characteristic wavelength may be used.
  • Combinations of transposon cassettes having a variety of such mixtures of light generating polypeptides which emit light at a different characteristic wavelengths may be constructed in view of the teachings of the present specification.
  • Microorganisms carrying the transposon constructs described herein are particularly useful for non-invasive imaging in whole animals.
  • Non-invasive imaging in whole animals is described in co-owned U.S. Patent No. 5,650,135, by Contag, et al. (See, also, Contag, et al, (1998) Nature Medicine 4(2):245-247; Contag, et al, (1996) OSA Tops on Biomedical Optical Spectroscopy and Diagnostics 3:220-224; Contag, et al, (1997) Photochemistry and Photobiology, 66(4):523-531; and Contag, et al, (1995) Mol. Microbiol. 18:593-603.
  • the conjugates contain a biocompatible entity (e.g., a transformed bacterium carrying a transposon of the present invention integrated into its genome) and a light-generating moiety (e.g., a luciferase enzyme).
  • a biocompatible entity e.g., a transformed bacterium carrying a transposon of the present invention integrated into its genome
  • a light-generating moiety e.g., a luciferase enzyme
  • Light-emitting conjugates are typically administered to a subject by any of a variety of methods, allowed to localize within the subject, and imaged. Since the imaging, or measuring photon emission from the subject, may last up to tens of minutes, the subject is typically, but not necessarily, immobilized during the imaging process. Imaging of the light-emitting entities involves the use of a photo detector capable of detecting extremely low levels of light—typically single photon events—and integrating photon emission until an image can be constructed. Examples of such sensitive photo detectors include devices that intensify the
  • a photon emission image is generated, it is typically expressed as a pseudocolor image superimposed on a "photographic" reflected light image of the subject to provide a frame of reference for the source of the emitted photons (i.e. localize the light-emitting conjugates with respect to the subject).
  • a "composite" image is then analyzed to determine the location and/or level of expression of a reporter gene in the subject.
  • the Tn4001 lux bn R cassettes described herein are useful in evaluating various procaryotic cells in an animal.
  • the cassettes described can be integrated into the genome of pathogenic bacteria (e.g., Gram-positive bacteria) previously described and subsequently introduced into a whole animal. The animal can then be used to follow the infection process in vivo and to evaluate potential anti-infective drugs, such as new antibiotics, for their efficacy in inhibiting the infection.
  • the expression cassettes described herein are useful in non-invasive imaging and/or detecting of light-emitting conjugates in mammalian subjects infected with bacteria carrying a luciferase expression cassette.
  • the lux transposon cassettes can be used to screen agents useful in inhibiting the growth and/or proliferation of pathogenic bacteria.
  • E. coli libraries containing bacteria expressing surface-bound antibodies which can be screened to identify a colony expressing an antibody against a selected antigen (Stiatagene, La Jolla, Calif.).
  • Bacteria from this colony can then be transformed with a lux transposon cassette of the present invention, and transformants having undergone at least one transposition event can be utilized in the methods of the present invention, as described above, to localize the antigen in a mammalian host.
  • the transformed cells may be administered to a test subject such that they become uniformly distributed in the subject.
  • the substrate is aldehyde.
  • the substrate may also be administered to the whole animals. Appropriate concentrations for the substrate can be empirically determined for each line of test animal constructed.
  • the substrate typically, luciferin or aldehyde
  • the routes of administration of the substrate can be as described for the analyte.
  • Preferred routes of administration for the substrate include, but are not limited to, intravenous or topical administration or by providing substrate in the atmosphere, for example, as a vapor.
  • sequences of interest present in the transposon cassettes of the present invention lack the associated promoter sequences necessary to allow their transcription in the target organism. Manifestation of the phenotypic characteristics encoded by the sequences of interest in organisms transformed with the Tn4001 lux km R shuttle vector constructs are therefore likely due to incorporation of the transposable unit within the genome of the target organism. In particular, the sequences of interest have likely integrated in such as way as to have become operably linked to an active promoter present in the host organism's genome (i.e., a promoter endogenous to the host organism).
  • the transposon cassette of the present invention is useful in a wide variety of applications. For example, they may be employed in methods of modifying a target organism.
  • p AUL- A Tn4001 luxABCDE km R shuttle vector constructs are used to modify an organism of interest.
  • Bacterial cells of interest are transformed with the shuttle vector construct via, for example, electroporation, or phage-mediated transduction or conjugation.
  • Transformed cells carrying the shuttle vector may be selected and maintained by means of growing the electroporated cells on media containing erythromycin.
  • Cells wherein the luxABCDE km R coding cassette has integrated behind active promoter regions in the host organism's genome may be selected and maintained by means of culturing erythromycin-resistant transformants on media containing kanamycin: integrant cells will give rise to bioluminescent, kanamycin-resistant colonies. See Examples 6 and 7.
  • Organisms modified with the pAUL-A Tn4001 luxABCDEY km R shuttle vector construct may be modified as above with the exception that the bioluminescence exhibited by those cells is of a different wavelength.
  • a pAUL-A shuttle vector comprising the luxAB transposon cassette is employed as above, with the exception that organisms capable of exhibiting the bioluminescent phenotype will be detected by first exposing them to exogenous aldehyde substrate, as described above.
  • Constitutivefy bioluminescent organisms may be prepared and identified by this method. See Examples 5 and 6, infra. Alternatively, conditionally bioluminescent organisms may also be identified as above, with the exception that selection of the conditionally bioluminescent transposants is accomplished by first replica-plating erythromycin-resistant transformants on culture media containing kanamycin, then growing the plates of a replica pair under restrictive and permissive conditions, respectively, and finally identifying colonies which exhibit a bioluminescent, kanamycin-resistant phenotype only when grown under permissive conditions. See Example 7.
  • conditional promoters identified by this method are heat- shock promoters, where the restrictive temperature is typically 42°C and the permissive temperature can be, for example, room temperature to 37°C.
  • Conditional promoters may also be associated with growth phase, stage of infection, and quorum sensing, pathogenicity, stage of life cycle (cyst formation or reproduction), growth conditions, etc.
  • Genes specifically involved in the pathogenesis of various organisms of interest may be identified using the shuttle vector constructs of the present invention in conjunction with in vivo expression technology.
  • a strain of pathogenic bacteria transformed with the Tn4001 lux km R shuttle vector construct and grown under conditions for maintaining the construct, is injected into an experimental animal, such as a mouse.
  • Transposants which have integrated the lux km R cassette next to promoters active in, for example, the early stages of pathogenesis are then selected for by injecting the animal with kanamycin shortly after infection, plating on non-selective media, and selecting dark colonies.
  • This step insures that the transposition of the transposon cassette into the genome has not resulted in the association of the transposon with a constitutive promoter.
  • bacterial promoters active in pathogenesis are identified by monitoring the animals for light production. See Example 9. Owing to the close-coupling of the lux km R cassette with active promoter regions in the bioluminescent transposants of interest, it is possible to identify and sequence both the promoter region and the gene coding sequence normally associated with those promoters in the host cell genome by methods known to persons of ordinary skill in the art in view of the teachings of the present specification.
  • PCR amplification and subsequent sequencing of the amplification products may be employed, using primers based on gene sequences present in the integrated luxABCDE km R cassette.
  • the promoter may also be sequenced by directly isolating chromosomal DNA from a single bioluminescent bacterial colony of interest, and then sequencing using primers specific for regions of the kanamycin resistance gene adjacent or in close proximity to the promoter sequence of interest.
  • the associated gene sequences may also be isolated and sequenced by these methods. See Example 10.
  • Integrants wherein the lux km R cassette is operably linked to promoters active during pathogenesis may be used to identify effective pharmaceutical agents and determine their point of action. After isolating a panel of transposants corresponding to the family of promoters activated during pathogenesis (for example, different stages of infection or different stages of the infectious organisms' life cycle) by the methods described in Example 9, that panel is used to infect multiple groups of experimental animals, each group corresponding to a particular promoter/gene of interest. The group is then treated with the pharmaceutical agent of interest, and both the experimental animals and infected, untreated control animals are then monitored for bioluminescence.
  • the relative strength of promoter activity may be determined, either by means of replica plating onto culture media containing increasing concentrations of antibiotic, or by employing "crippled" antibiotic genes as the selective marker in the transposon cassette.
  • a modified neomycin resistance gene can be employed where in order to get resistance to the antibiotic a high-level of expression of the neomycin resistance gene is required.
  • the crippled selectable marker is a neomycin resistance (Neo r ) sequence in which amino acid residue 182 (Glu) is mutated to Asp. (Yanofsky, et al., (1990) PNAS USA 87:3435-39). Use of such crippled selectable markers improves the strength of the selection, because more of the enzyme is required to produce antibiotic resistance. See Example 12.
  • the constructs of the present invention are useful in methods designed to monitor bacterial growth in foodstuffs.
  • the contamination of foodstuffs, cosmetics, pharmaceuticals and the like by undesirable micro-organisms represents a significant threat to public health.
  • a number of methods to monitor the presence of such microorganisms in, for example, foodstuffs, water supplies and on food preparation surfaces have been developed. These methods generally rely on conventional microbiological techniques, including the growth of micro-organisms on selective nutrient solid support media or alternatively in selective nutrient media. Morphological analyses are usually carried out. Testing methods such as these are hindered by the fact that results may not be obtained for 24 to 48 hours or more. Further, the process is complex and laborious.
  • Organisms modified to produce light as taught by the present invention can be used to monitor the presence of microorganisms, for example, in foodstuffs, in drinking water, and on food preparation surfaces.
  • Potential applications in this context include, but are not limited to, the following:
  • a light-generating organism of the present invention can be used to, for example, seed a food product. Then the food product can be treating using a particular sterilization method (e.g., X-rays, heat processing, pasturizing, microwaves, etc.). The efficacy of the sterilization method can then be evaluated by examining light production (or the absence thereof) from microorganisms in the food product.
  • the light-generating organisms of the present invention may be used to evaluate the proliferation of such organisms in any selected medium, as well as, whether treatments (e.g., the addition of compounds such as antimicrobials) are efficacious at stopping the proliferation of such organisms in the selected medium.
  • modified microorganisms produced using the constructs and methods of the present invention may be used in conjunction with the in vivo expression technology methods described below. See Example 13. Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.
  • Exogenous aldehyde substrate was added prior to imaging plates or cultures of bacteria not containing the luxCDE genes.
  • n-decyl aldehyde decanal; Sigma Chemical Company
  • aldehyde vapor imaging an intensified CCD camera
  • Competent Cells Preparation of Competent Cells. Unless indicated otherwise, bacterial cells were transformed as follows. Bacterial cultures were grown overnight in LB. Five ml of each culture were used to inoculate fresh 500 ml volumes of LB. These cultures were shaken at 37°C until an O.D (600 nm) of approximately 0.6 was reached. The cells were then chilled on ice for 30 min before being harvested by centrifugation at 3,000 x g for 10 min at 4°C. The cells were resuspended in 50 ml of either cold 0.5 M sucrose (S. aureus) or ddH O (E. coli), before being re-centrifuged and resuspended in 5ml of either cold 0.5 M sucrose (S. aureus) or ddH O (E. coli), before being re-centrifuged and resuspended in 5ml of either cold 0.5 M sucrose (S.
  • Plasmid DNA was purified using a Qiagen column, dialyzed, and electroporated into competent cells using a "GenePulser" (BioRad). The settings were 25 ⁇ F, 2.5 kV, and either 100 ohms resistance for S. aureus, or 400 ohm resistance for E. coli and S. pneumoniae. The cells were left to recover in 1 ml of culture medium 2 hr at 37°C before being plated on a suitable agar containing the requisite selection antibiotic.
  • the amount of light generated by a sample was quantified using either an intensified photon-counting camera (Hamamatsu Photonics Model 2400-32) or a cooled integrating camera.
  • an intensified photon-counting camera Hamamatsu Photonics Model 2400-32
  • a cooled integrating camera the particular instrument used was selected from among three makes/models: (1) Princeton Instruments Model LN/CCD 1340-1300-EB/l; (2) Roper model LN-1300EB cooled CCD camera (available from Roper Scientific, Inc., Arlington, Arizona); and (3) Spectral Instruments model 600 cooled CCD camera (available from Spectral Instruments, Inc., Arlington, Arizona).
  • the preferred apparatus was the Hamamatsu Photonics camera number XEN-3 and the Princeton Instruments camera number XEN-5, respectively, both located at Xenogen Corporation, Alameda, California. Both types of cameras use a charge-coupled device array (CCD array), to generate a signal proportional to the number of photons per selected unit area.
  • CCD array charge-coupled device array
  • the selected unit area may be as small as that detected by a single CCD pixel, or, if binning is used, that detected by any selected group of pixels.
  • This signal may optionally be routed through an image processor, such as the Argus available from Hamamatsu Photonics, and is then transmitted to a computer (either a PC running Windows NT (Dell Computer Corporation; Microsoft Corporation, Redmond, WA) or a Macintosh (Apple Computer, Cupertino, CA) running an image-processing software application, such as "Livinglmage” (Xenogen Corporation, Alameda, CA).
  • an image processor such as the Argus available from Hamamatsu Photonics
  • the data generally take the form of (x, y, z) values, where x and y represent the spatial coordinates of the point or area from which the signal was collected, and z represents the amount of signal at that point or area, expressed as "Relative Light Units (RLUs).
  • RLUs Relative Light Units
  • the data are typically displayed as a "pseudocolor” image, where a color spectrum is used to denote the z value (amount of signal) at a particular point.
  • the pseudocolor signal image is typically superimposed over a reflected light or "photographic" image to provide a frame of reference.
  • the RLU signal values from any camera can be compared to the RLUs from any other camera that has been calibrated using the same photo-emission standard. Further, after calibrating the photo-emission standard for an absolute photon flux (photons emitted from a unit area in a unit of time), one of skill in the art can convert the RLU values from any such camera to photon flux values, which then allows for the estimation of the number of photons emitted by a transformed cell in the sample per unit time. E.
  • the five genes of the Photorhabdus luminescens lux operon, luxA-E were PCR amplified using the polymerase chain reaction (PCR; Mullis, et al., U.S. Patent No. 4,683,195; Mullis, U.S. Patent No. 4,683,202) to incorporate the sequence of the Gram-positive ribosome binding site (RBS) AGGAGG (SEQ ID NO: 1) such that this site was at least seven nucleotides upstream of each start codon.
  • RBS Gram-positive ribosome binding site
  • Each of the lux genes was amplified individually using the primer sets shown in Table 1, below.
  • nucleotides highlighted in bold show the position and sequence of the different restriction endonuclease sites (identified in far-right column) incorporated to facilitate cloning.
  • Gram-positive RBSs, and start/stop codons (ATG/TAA) are underscored by solid and broken lines, respectively.
  • thermocycler Techne Progene, Princeton
  • Reactions were carried out in 50 ⁇ l volumes containing 5 ⁇ l of 10X PCR buffer (supplied with Taq DNA polymerase obtained from Roche Molecular Biochemicals (Switzerland), 2.0 mM MgCl 2 , 50 pmol of each oligonucleotide primer (Operon; see Table 1 for sequences), 0.2 mM of each deoxynucleotide triphosphate (dATP, dCTP, dGTP, dTTP;
  • Xenorhabdus luminescens luxCDABE cassette is available from GenBank, under accession number M90092.1 (GL155411; XENLABCDEB) (Meighen,E.A. and
  • the luxA PCR product was digested with BamH VSal I and ligated into pBluescript SK " at the BamH VSal I sites (directionally orientated downstream of the IPTG-inducible lacZ promoter), generating plasmid SK ' Q luxA.
  • Plasmid pSK ' G+luxA was then electroporated into DH5 ⁇ E. coli (Stiatagene), and the cells were plated on LB agar plates containing 100 ⁇ g/ml ampicillin.
  • luxCDE cassette Assembly of a separate luxCDE cassette in pBluescript SK " was achieved by the sequential cloning of luxC, luxD and luxE essentially as described in Example 2 for the generation of the luxAB cassette.
  • the luxC-E PCR amplification products were individually digested with the compatible enzymes SaR and Xhol, and each step of the cloning procedure was confirmed by PCR of the E. coli transformants.
  • the fidelity of the final luxCDE cassette was confirmed by inserting this sequence, cut with Sal llXho I, at the Sal I site downstream of the luxAB genes in oSK ' G+luxAG+luxB, generating pSK " luxABCDE.
  • the construct was inserted into the Gram- positive/negative shuttle vector pMK4 (Sullivan, M.A., et al. (1984) Gene 29:21-26) and tested for bioluminescence in different S. aureus strains using random host DNA fragments as promoters.
  • the luxABCDE cassette was cut from pBluescript SK " using the restriction enzymes BamHI and Sail, and ligated with similarly cut pMK4 plasmid DNA (orientated so that no promoter lay upstream of this cassette; Figure 1). This ligation was electroporated into E. coli DH5 ⁇ and luxABCDE clones, grown overnight at 37°C on LB containing 100 ⁇ g/ml ampicillin (the Gram-negative selectable marker found on pMK4), were selected by screening for colonies emitting low levels of bioluminescence (light barely detectable using the Hamamatsu ICCD camera).
  • genomic DNA from a clinical isolate of methicillin resistant S. aureus was cut with Sau3A in a partial digest and ligated into pMK4 luxABCDE plasmid DNA cut with BamHI (the Ban ⁇ H ⁇ enzyme was left active to reduce background from re-ligation of the empty vector DNA).
  • MRSA methicillin resistant S. aureus
  • DH5 ⁇ and the resulting transformants were plated on LB containing 100 ⁇ g/ml ampicillin. The colonies were scraped from the plates, pooled, and their plasmid DNA extracted (Plasmid Spin Miniprep Kit, Qiagen, Valencia, CA). This plasmid DNA was electroporated into competent S. aureus RN4220 cells and the resulting transformants selected on LB containing 5 ⁇ g/ml chloramphenicol (the Gram-positive selectable marker found on pMK4).
  • the luxABCDE bn R cassette was constructed as follows: A Gram-positive kanamycin cassette from DL289 (Buckley, N.D., et al. (1995) J. Bacteriol. 177:5028- 5034) was PCR amplified using the primers KanF2 (5'-CTG TAG ACT CGA GGA GGG AAA TAA TAA ATG GC; SEQ ID NO:12; the bolded letters represent aXhoI site) and KanR2 (5'-CAG AGT GTC GAC AGT TGC GGA TGT AC; SEQ ID NO:13; the bolded letters represent a Sail site).
  • KanF2 5'-CTG TAG ACT CGA GGA GGG AAA TAA TAA ATG GC; SEQ ID NO:12; the bolded letters represent aXhoI site
  • KanR2 5'-CAG AGT GTC GAC AGT TGC GGA TGT AC; SEQ ID NO:13; the bolded letters
  • Amplification was carried out for 30 heating/cooling cycles of 15 seconds at 95°C, 30 seconds at 50°C, and 2 minutes at 72°C.
  • the resulting amplification product provided a promoter-less bn R antibiotic resistance gene.
  • the amplification product was then cut and ligated into the Sail site of the pSK-luxAB CDE plasmid construct (prepared in Example 3, above) directly downstream of the luxABCDE cassette.
  • the pSK luxABCDE km R plasmid construct was electroporated into E. coli DH5 ⁇ cells and the transformed bacteria were plated onto LB plates containing
  • the luxABCDE km R cassette was cut from the pSK luxABCDE km R construct, prepared above, using SpeUSaR. The ends of the fragments were filled in with nucleotides to generate blunt-ended molecules. These molecules were ligated into the EcoRY site of the plasmid pMGC57 ( Figure 2; Lyon et al. (1998) EMBO J. 17:6263-6275) and the constructs electroporated into DH5 ⁇ cells. The transformed bacteria were plated onto LB media containing 15 ⁇ g/ml chloramphenicol.
  • the resulting transformants were screened for light production, chloramphenicol-resistance (CmR) and kanamycin- resistance (KanR).
  • DNA was prepared from light-generating, CmR, KanR colonies.
  • the correct orientation of the luxABCDE km R cassette i.e., the location of the 5' end of the luxA sequence relative to the 5' end of the Tn4001 transposon, was confirmed by restriction digestion (XhoUNdel and XhoI/EcoRY) and restriction pattern analysis, as well as, by PCR analysis of DNA.
  • PCR was carried out using the primers MGC-CAT- Fl (5'-GGT GTC CCT GTT GAT ACC G-3', SEQ ID NO: 14) and LuxA-Rev (5'- CCA CAC TCC TCA GAG ATG CG-3', SEQ ID NO: 15) under conditions detailed supra. The correct orientation was identified by fragment size.
  • Tn4001 as well as its related insertion sequences (IS) are known and available as GenBank Accession Numbers X53951; X53952; Ml 8086; and M29261 and are further described in Hahn et al. (1999) Plasmid 41:120-124.
  • Tn4001 luxABCDE hn R Shuttle Vector The Tn4001 cassette containing the lux and km R genes (designated IR luxABCDE km R tnp IR, where, IR represents inverted repeats and tnp represents the gene encoding the Tn4001 transposase) was inserted into a broad-range shuttle vector having a gram negative origin of replication and a gram positive origin of replication (either constitutive or conditional, e.g., temperature sensitive).
  • a shuttle vector is the pAUL-A vector (Chakraborty, et al. (1992) J. Bacteriol.
  • transposon cassettes of the present invention are schematically represented as follows.
  • the inverted repeats (IR) generally indicate the ends of the transposable element. Accordingly one designation for the transposon is IR- tnp -IR, where tnp designates the gene encoding the transposase.
  • Further elements can be added to the transposon and are indicated similarly, e.g., addition of a luxABCDE km R cassette is rendered schematically as IR luxABCDE km R tnp IR.
  • the IR luxABCDE km R tnp IR cassette was cut from pMGC57 using the enzymes EcoRI/Xhol and ligated into the Eco US ⁇ K sites of the pAUL-A shuttle vector ( Figure 3) to give the shuttle vector construct pAUL-A Tn OOl luxABCDE km R .
  • E. coli cells (DH ⁇ 5) were transformed with the shuttle vector construct by electroporation, plated onto LB plates containing erythromycin at a concentration of 150 ⁇ g/ml, and incubated at 37°C overnight. The resulting transformants were screened for light production, erythromycin-resistance (EmR) and kanamycin- resistance (KanR).
  • EmR RN4220 colonies were then pooled directly from plates and plasmid DNA prepared from the pool. This DNA was then electroporated into S. aureus 8325-
  • the luxABCDE lan R cassette in a broad-range shuttle vector having a Gram-positive origin of replication is capable of transposing into the bacterial chromosome of a Gram-positive bacterial strain.
  • the transposase gene (tnp) is located within the transposable unit.
  • the transposase gene may be deleted from the transposon itself and placed on the vector backbone.
  • the transposase might be introduced into the vector backbone at a unique restriction site in the multiple cloning site (MCS) region.
  • MCS multiple cloning site
  • the plasmids pSK and pE194 were used in combination.
  • the Tn4001 cassette was moved from ⁇ MGC57 into pSK as follows: The
  • ⁇ n4001 cassette was cut from pMGC57 using XhoIlPstl and ligated with similarly cut pSK. This ligation was electroporated into competent E. coli DH5 and transformants selected on LB containing 100 ⁇ g/ml ampicillin, IPTG, X-gal. White colonies were screened by PCR using the primers M13R and TNP-R2 (sequence in Tn4001 transposase gene). Colonies indicated to be positive by PCR were plasmid prepped and these DNA's cut with i ⁇ oI/ st/to confirm that they contained the correct size fragment.
  • the luxABCDE bn R cassette was then moved into pSK Tn4007 so that it lay between the two IR sequences.
  • the ampicillin cassette was removed from pSK luxABCDE km R using the enzymes Ahdl/Kpnl (in order to aid subsequent cloning of the lux cassette into pSK Tn400I).
  • the luxABCDE km R cassette was then excised from the ampicillin deleted pSK backbone with B ⁇ mRVXhol.
  • the pSK ⁇ n4001 DNA was then cut with EcoRV and ligated with the blunt-ended luxABCDE hn R cassette.
  • the ligation was electroporated into competent E. coli DH5 ⁇ and transformants selected on LB containing 100 ⁇ g/ml ampicillin. Light transformants were patched on either chloramphenicol or kanamycin. The correct orientation of the positive clones was confirmed by PCR using the primers M13F and LuxA-R.
  • pSK Tn4001 luxABCDE bn R was linearized with S ⁇ cl and blunt-ended. Finally, this DNA was then ligated with blunt-ended C/ ⁇ /-cut pE194, resulting in the pSK Tn4001 luxABCDE km R pE194 shuttle vector construct. This construct was electroporated into competent E. coli DH5 . Transformants were selected on LB containing 150 ⁇ g/ml erythromycin. Resulting light colonies were then patched onto LB containing 100 ⁇ g/ml ampicillin and LB containing either 50 ⁇ g/ml kanamycin or 15 ⁇ g/ml chloramphenicol.
  • a Tn4001 luxABCDE hn R shuttle vector was constructed wherein the Gram- negative origin was located inside of the two IR sequences. Firstly, the Gram-negative Tn4001 containing plasmid pMGC57 was fused with the Gram-positive erythromycin resistance plasmid pE194. pMGC57 was cut with Pstl/BamBI and blunt-ended. At the same time pE194 was cut with Clal and blunt-ended. These two linearized plasmids were then ligated and this mix was electroporated into competent E. coli DH5 ⁇ and transformants selected on LB containing 150 ⁇ g/ml erythromycin.
  • the chloramphenicol resistance cassette and the Gram-negative origin were removed from the pMGC57/pE194 composite.
  • Plasmid DNA purified from a number of above erythromycin resistant colonies was cut with KpnIIXh ⁇ l (sites flanking the chloramphenicol resistance cassette and the Gram-negative origin) and a proportion of each digest was run on an agarose gel to identify a plasmid of the correct size. The remainder of a plasmid digest appearing to be the correct size was then blunt- ended and ligated. This ligation was electroporated into S. aureus RN4220 and plated onto chocolate agar containing 0.3 ⁇ g/ml erythromycin.
  • the pSK luxABCDE km plasmid was introduced within the IR of Tn4001. Plasmid DNA purified from 10 of the pMGC57/pE194 ori cm R S. aureus clones was cut with EcoRV and a proportion of each digest was run on an agarose gel to identify a plasmid of the correct size. The remainder of a plasmid digest showing the correct size (approximately 6 kb, #10) was then ligated with blunt-ended BamHI cut pSK luxABCDE km DNA. This ligation was electroporated into DH5 ⁇ and transformants selected on LB containing 150 ⁇ g/ l erythromycin.
  • Bioluminescent colonies were then patched in duplicate onto LB plates containing either 50 ⁇ g/ml kanamycin or 100 ⁇ g/ml ampicillin to confirm the activity of the latter antibiotic cassettes. Since the probability of pSK luxABCDE km ligating into the former plasmid in the correct orientation should be approximately 50%, it was decided that such variants could be selected by phenotype in S. aureus (only plasmids with lux in the correct orientation imparting bioluminescent upon transposition).
  • Plasmid DNA was purified from the two positive clones (from the original tiansformant grown on 0.3 ⁇ g/ml erythromycin) and this was used to transform E. coli and S. aureus RN4220 (again). Transformants of E. coli were selected on LB containing either 150 ⁇ g/ml erythromycin, 50 ⁇ g/ml kanamycin or 100 ⁇ g/ml ampicillin. Although numerous transformants could be recovered on both erythromycin and kanamycin plates, none could be recovered on plates containing ampicillin, indicating the plasmid to have rea ⁇ anged.
  • Plasmid DNA was purified from these two RN4220 transformants, and this was electroporated into 8325-4, selecting for transformants on chocolate plates containing 0.3 ⁇ g/ml erythromycin. Again the transformants were patched onto chocolate plates containing 0.3 ⁇ g/ml erythromycin and then streaked onto BHI containing 200 ⁇ g/ml kanamycin.
  • L. monocytogenes was screened for transformants where expression of KanR and light production were heat inducible.
  • One milliliter of an overnight culture grown at 30°C of L. monocytogenes transformed with pAUL-A IR luxABCDE km R tnp IR was inoculated into 30 ml of Brain Heart Infusion (Gibco BRL, Gaithersburg, Maryland) containing 200 ⁇ g/ml kanamycin. This culture was then incubated overnight at 42°C, centrifuged, and the cell pellet resuspended and plated for single colonies on BHI plates containing 200 ⁇ g/ml kanamycin. These plates were incubated at 42°C and then screened for light-generating colonies.
  • Light-generating colomes were patched onto two blank BHI plates (i.e., plates containing no kanamycin). One set of plates were incubated at 30°C and the other set at 42°C. Comparison of the patches for bioluminescence at the two temperatures, revealed a number of isolates that were strongly induced for light production at 42°C. Some representative data for such isolates were presented in Figure 4. In the figure, the X-axis shows a number of bacterial isolates and the Y-axis shows relative light units (RLU) for each bacterial isolate, cultures of which were grown at three temperatures (30°C, 37 °C, and 42 °C).
  • RLU relative light units
  • Exponential cultures of the aforementioned bacteria carrying the Tn4000I cassette of the present invention were grown at 37°C in growth media containing a selection agent, pelleted and then resuspended in phosphate-buffered saline (PBS).
  • Bacterial concentrations were estimated spectrophotometrically by absorbance at 600 nm and adjusted to an appropriate concentration by dilution with PBS. The cells were then held on ice for a short period until the mice were ready to be inoculated. Cell numbers were verified by plating dilutions of inoculum onto agar containing the appropriate growth medium.
  • mice were anesthetized with ketamine (100 mg/ml) and xylazine (20 mg/ml), mixed at a 4:1 ratio v/v just before use. 100 mg of ketamine/xylazine mixture (dose based on ketamine concentration) per kg body weight was injected intramuscularly into the right hamstring muscle. After anesthesia was established, the mice were injected in the left anterior tibialis with the bacterial inoculum. Mice were imaged at 0, 4, 8, and 24 hours post-infection using a CCD camera. At each time point, both dorsal and ventral images were taken. All images were collected for 5 minutes.
  • Isolates obtained by the methods of the present invention were tested (a total of 12 S. aureus 8325-4 isolates; 3 S. pneumoniae D39 isolates; and 2 L. monocytogenes isolates). All gave bioluminescence levels that were detectable in vivo, thereby demonstrating that bioluminescent bacteria can be effectively monitored in living animals in real time.
  • mice were sacrificed and the infected thigh muscles (both the anterior tibialis and the quadricep muscle) were surgically removed.
  • the muscle tissue was homogenized in 500 ⁇ l PBS using a loose Dounce Homogenizer.
  • the tissue and bacterial suspension was then diluted in PBS by 10 5 , 10 6 , and 10 7 to a final volume of 1 ml for each dilution.
  • lOO ⁇ l of each dilution was plated out in duplicate onto media plates and incubated overnight at 37°C. The following morning, colonies were counted and the numbers of CFU for each tissue sample were estimated.
  • the regulation of both the lux and the kanamycin resistance genes in the IR luxABCDE km R tnp IR vector constructs of the present invention are tightly coupled, owing to their close physical association in the luxABCDE km R cassette. Accordingly, the constructs of the present invention can be used to identify genes specifically involved in the pathogenicity of Gram-positive bacteria using the following in vivo expression technology (IVET) protocols.
  • a pathogenic strain of gram positive bacteria for example, S. aureus, is transformed with an IR luxABCDE km R tnp IR vector construct of the present invention.
  • the transformed strain is grown under conditions to maintain the shuttle vector construct, for example, under conditions which select for erythromycin resistance when using the pAUL-A IR luxABCDE lan R tnp TR. construct.
  • An experimental animal such as a mouse, is inj ected with the transformed bacteria; shortly thereafter, the animal is injected with kanamycin to select for bacteria that have acquired kanamycin resistance.
  • This step selects for transposants which have integrated the IR luxABCDE km R tnp IR cassette next to active bacterial promoters. These KanR bacteria are extracted from the animal and plated onto non- selective media, and dark colonies are selected. This step selects for integrants located adjacent to or near bacterial promoters that are not constitutively active.
  • the dark colonies are then cultured and injected into experimental animals, which are monitored for bacterially-mediated light production. Induction of light production in vivo in these isolates suggests the identification of a bacteria promoter turned on in response to infection of its mammalian host (i.e., a promoter involved in establishment of infection).
  • promoters active at other stages in pathogenicity and/or life cycle of a light-generating microorganism obtained by the methods of the present invention, can be similarly evaluated.
  • bacterial genes active during chronic infection but not active during the establishment of infection may be identified by comparing in vivo light production during early stages of infection versus in vivo light production during chronic infection.
  • a first animal is infected with pathogenic bacteria transformed with a shuttle vector construct of the present invention, for example, the pAUL-A luxABCDE km R shuttle vector described above.
  • a selective agent e.g., kanamycin.
  • This step selects for transposants wherein the expression of bioluminescence and antibiotic resistance is controlled by a promoter active either constitutively or during the chronic stages of infection (vs. during the initial stages). Bacteria are then extracted and replica-plated onto selective and non-selective media.
  • Bacteria are isolated from experimental animals that exhibit bioluminescence attributable to the activity of either constitutive, repressible or inducible promoters. The promoters are then identified and/or isolated by a number of methods which are well known in the art.
  • Promoters associated with known genes in the target organism may be identified by genetic mapping techniques. For example, the genomic location of the integrated luxABCDE km R cassette is mapped and the map position compared to the known genetic map of the organism to identify the gene at the corresponding position. Such mapping is carried out by standard genetic methods known in the art. To exemplify, an apparently constitutively expressed luxABCDE lm R cassette is mapped to the physical genomic location known to co ⁇ espond to the gene encoding glucose-6- phosphate dehydrogenase. The expression of the luxABCDE lan R cassette is, therefore, likely mediated by the glucose-6-phosphate dehydrogenase promoter.
  • PCR amplification of the promoter and subsequent sequencing of the amplification products may be employed.
  • the luxABCDE km R cassette in the bioluminescent transposants of interest integrates adjacent or in close proximity to active bacterial promoters.
  • PCR primers based on DNA sequences present in the integrated lux ABCDE km R cassette, for example, the kanamycin resistance gene can be used in amplification reactions to take advantage of the close proximity of the unknown promoter sequence to the known nucleic acid sequence. Sequencing from known sequences into the associated bacterial sequences reveals the sequence of the promoter.
  • the technique of inverse PCR may also be used.
  • genomic DNA from each of the heat shock inducible strains (HS) obtained in Example 7 above was cut with the four-base recognition restriction enzyme Alul (AG/CT), ligated for 4 hours at RT, and inverse PCR performed using the primers LuxIF3 (GCT TGG TAA CCC TTA TGT CGC) (SEQ ID NO: 16) and LuxR3 (GGG AGG TTG GTA TGT AAG C) (SEQ ID NO: 17).
  • HS1 luxABCDE km R cassettes fused within a single gene of unknown function.
  • a promoter may also be sequenced by directly isolating chromosomal DNA from a single bioluminescent bacterial colony of interest, and then sequencing the DNA using primers specific for regions of the kanamycin resistance gene adjacent or in close proximity to the promoter sequence of interest.
  • the enzyme EcoRV unique site directly downstream of luxB
  • pSK i.e., Sad, Xbal, EcoRI, Sail, Xli ⁇ l or Kpnl
  • Afel blue
  • Hindlll was used in combination with the same pSK MCS enzymes listed above, hi either case (luxAB genes or km R gene), pSK was cut with Smal and the same enzyme as that listed above prior to ligation.
  • Clones containing plasmid DNA with regions of the Tn4001 luxABCDE km R cassettes were selected by either screening for bioluminescence in the presence of exogenous aldehyde (indicating the presence of the luxAB genes from the 5' end of the cassette) or kanamycin resistance (indicating the presence of the km R gene from the 3' end of the cassette).
  • DNA from HS36 and 13 both gave light clones.
  • DNA from HS27, 29, and 31 gave clones which exhibited kanamycin resistance. Plasmid DNA from these clones was sequenced by conventional methods and the sequences obtained were then compared with known sequences as a means of identifying the promoter region with which the luxA or km R coding sequence was associated.
  • HS 36 appears to have the Tn4001 luxABCDE km R cassette fused in a region of the L. monocytogenes chromosome involved with the regulation of hly expression. Interestingly, hly is also temperature regulated and is involved in Listeria pathogenicity.
  • HS 13 has the ⁇ .4001 luxABCDE km R cassette fused at the 3' end of a hisH homolog. Heat induction of this gene has not been reported before. However, in S. typhimurium constitutive expression of histidine operon causes growth inhibition (Fink et al. 1967. J. Mol. Biol. 30:81-95; Flores and Casadesus, 1995. J. Bacteriol. 177:4841- 4850) suggesting that expression of the genes in the operon can be regulated. EXAMPLE 11 Identification of Effective Pharmacological Agents and Determination of their Point of Action
  • a wide variety of pathogenic bacteria of interest may be modified to carry the IR luxABCDE km R IR construct, and the promoters induced during pathogenesis may be identified and isolated as described above in Examples 9 and 10.
  • a panel of transformed cells representing a spectrum of promoters mediating pathogenesis may then be employed as a means for screening potential pharmacological agents, and simultaneously determining a particular agent's point of action.
  • the 7 4001 luxABCDE km R constructs of the present invention may be employed in order to identify novel antibiotics.
  • Multi-drug resistant Gram-positive bacteria such as Staphylococcus, Streptococcus, Enterococcus and Mycobacterium
  • Staphylococcus such as Staphylococcus, Streptococcus, Enterococcus and Mycobacterium
  • Conventional technologies for assessing the bactericidal bacteristatic activity of a drug i.e., plating/growth assays
  • plating/growth assays are extremely laborious and usually require 24 - 48 hrs for completion.
  • assays are of limited use for high-throughput screening and "follow-up" studies in the animal model.
  • TnAOOl luxABCDE constructs of the present invention allow bacterial genes involved with drug resistance, in particular those in Gram-positive bacteria, to be tagged, identified and monitored both in vitro and in vivo.
  • An example of how this technology may be applied to identify novel antibiotics follows.
  • S. aureus containing a promoterless plasmid-based Tn4001::luxA-E kan transposon cassette is cultured on solid non-selective media overnight at 37°C.
  • the culture is then overlain with minimum inhibitory concentrations ("MIC") of different classes of characterized antibiotics (e.g., gentamycin, penicillin etc), in order to induce operons which encode proteins conferring resistance to those antibiotics.
  • MIC minimum inhibitory concentrations
  • the cultures of induced organisms are subsequently overlain with kanamycin to select for organisms wherein the transposon cassettes have integrated behind active promoters, and which are therefore expressing kanamycin resistance.
  • Some of those organisms selected will have the transpsoson cassette integrated behind promoters associated with the antibiotic resistance genes induced by the initial antibiotic treatment.
  • the treated bacteria are plated on non-selective media and colonies that do not produce light (dark colonies) are selected. These colonies should be lux fusions downstream of an antibiotic-induced gene. (Such colonies represent antibiotic-detector strains).
  • the ability of the pure cultures of a particular S. aureus fusion (dark colonies) to be induced ('light-up') when subjected to a MIC of a prior tested class of antibiotic is confirmed by re-treating with that drug.
  • pure cultures of an antibiotic detector strain are exposed to different dilutions of novel compounds suspected of being antibiotics.
  • the tested compound is bactericidal/bacteristatic
  • its efficacy can be tested in animals using both the antibiotic detector strain and different constitutive light strains.
  • the relative strength of promoters which have become operably linked to the lux km R cassette via integration may be determined by replica plating of the transposants onto culture medium of increasing kanamycin concentration. Alternately, the strength of the promoter may be dete ⁇ nined by growing the cells in liquid media where individual liquid cultures contain serial dilutions of the antibiotic. Comparison of the degree of colony growth, and/or the relative intensity of bioluminescence, over increasing antibiotic concentrations, provides an indication of the relative strength of promoter activity.
  • a curve of promoter strengths is established using, for example, integrated luxABCDE km R cassette integrated next to (i) a relatively weak promoter from a gene expressed at low levels, (ii) a house keeping gene promoter, (iii) a highly active promoter, all integrants in the target organism.
  • a bioluminescence curve is established for these integrants where bioluminescence is evaluated for the same number of cells, under the same growth conditions. Comparisons of bioluminescence mediated by promoters in the same target organism is then evaluated by measuring bioluminescence using the same number of cells under the same growth conditions used to establish the curve. This provides a measure of the relative strength of the promoter that is mediating expression of the light-generating polypeptide.
  • bioluminescent colonies were isolated from among colonies plated at high density on solid medium using optical detection and manual selection.
  • S. aureus 8325-4 cells were transformed with pE194 Tn40001 luxABCDE, plated on solid non-selective media plates at a density of 10 4 to 10 cells per plate, and grown overnight at 37°C. Strongly bioluminescent single colonies were detected using an ICCD camera, and those colonies were selected using a disposable micropipette tip; selection of the desired colony was confirmed using the camera. The selected colony was used to inoculate a volume of liquid growth medium, which was then streaked onto fresh media plates. The plates were incubated overnight at 37°C. The process was repeated until isolation of a pure colony was confirmed by observation of essentially uniform light intensity among single colonies on the streaked plates.
  • Streptococcus pneumoniae is the leading cause of invasive bacterial disease in the very young and the elderly, and is the bacterium most responsible for community- acquired pneumonia in the developed world (Schuchat et al. 1997). In the United States pneumococci cause up to 40,000 deaths annually, primarily among the elderly (CDC 1989, 1995). In the developing world they are an important cause of childhood deaths due to secondary respiratory infection following viral disease, killing an estimated one million children annually (Greenwood, 1999; Briles et al. 2000). S. pneumoniae can behave as a transient commensal, colonizing the mucosal epithelium of the nasopharynx of 40% of healthy adults and children, with no adverse effects (Austrian, 1986).
  • pneumococcal otitis media By the age of 5 years, the majority of children in the United States will have experienced at least one case of pneumococcal otitis media. It is estimated that 25 % of all community-acquired pneumonia is due to pneumococcus. While the incidence of memngitis is far less (1 in 100,000 per annum), pneumococcal meningitis has a 25% mortality rate, which is higher than that of other meningeal pathogens (Schuchat et al. 1997). Moreover, 50% of the survivors sustain permanent neurological sequelae (Khoosheh and Tuomanen, 2000).
  • pneumococcal vaccines can be extremely effective, a lack of broad range coverage makes them of limited use in preventative treatments, especially the use of polysaccharide vaccines in infants and children under 2 years of age (Lee and Wang, 1994).
  • Treatment of established pneumococcal infections has also worsened in resent years with the increased occurrence of multidrug resistant strains of S. pneumoniae.
  • About one-third to one-half of pneumococci recovered from humans have become at least partially resistant to penicillin, which may occur in addition to resistance to a number of other common antibiotics (Appelbaum, 1992; Briles et al. 2000).
  • Construction of the Gram-positive lux transposon plasmid, pAUL-A 7w4001 luxABCDE km R Construction of the plasmid pAUL-A 7 001 luxABCDE bn R is shown schematically in Figure 5. Briefly, a Photorhabdus luminescens lux operon, previously altered to be functional in Gram-positive bacteria as described herein, was modified by linking a promoterless Gram-positive kanamycin resistance cassette downstream o ⁇ luxE. The kanamycin resistance gene was PCR amplified from pDL289 (Buckley et al. (1995) J.
  • the amplified kanamycin resistance gene was digested with Xhol and SaR, and ligated into the SaR site of pBluescript II SK " luxABCDE (see, above).
  • the ligation was electroporated into E. coli, spread onto Luria-Bertani (LB) agar containing 50 ⁇ g/ml kanamycin, and incubated overnight at 37°C. Plates with kanamycin resistant colonies were screening for bioluminescence using a photon counting intensified charged couple device (ICCD) camera (model 2400-32, Hamamatsu Photonics, Bridgewater, NJ).
  • ICCD intensified charged couple device
  • Plasmid DNA was isolated from bioluminescent, kanamycin resistant colonies using a standard alkaline lysis procedure (Plasmid Spin Miniprep Kit, Qiagen, Valencia, CA), as were all subsequent plasmid isolations. These plasmids were then screened by PCR using the primers KanF2 and M13F - 5' GTA AAA CGA CGG CCA GT 3' (SEQ ID NO:19) (flanking the multiple cloning site of pBluescript II SK " ), to identify constructs where the luxABCDE cassette was in the same orientation (5' to 3') as the kanamycin resistance gene.
  • a Gram-positive lux transposon was constructed as described in Examples 4 and, to increase the efficiency of transposition in Gram-positive bacteria, was moved onto the temperature sensitive Gram-positive/negative shuttle vector pAUL-A (Chakraborty et al. (1992) J. Bacteriol. 174:568-574) as described in Example 5.
  • E. coli (DH5 ⁇ , Life Technologies, Rockvill, MD) transformed with pAUL-A Tn4001 luxABCDE km R were uniformly bioluminescent, kanamycin resistant and erythromycin resistant (100% of all transformants initially selected on erythromycin). Since the transformation frequency of these cells was similar to that of E. coli transformed with the empty pAUL-A plasmid (approximately 500 CFU/ ⁇ g DNA), and pAUL-A Tn400I luxABCDE km R DNA could be readily extracted from the transformants using standard plasmid isolation procedures, it is unlikely that lux km R gene expression is due to integration of the plasmid into the E. coli chromosome (i.e., episomal). Without being bound by any theory, it is possible that the luxABCDE km R operon is expressed on pAUL-A in E. coli, despite the lack of any apparent promoter upstream of these genes.
  • the pellet was resuspended in 30 ml of supernatant and competence stimulating peptide CSP1 ( ⁇ MRLSKFFRDFILQRKK) (S ⁇ Q ID NO:20) (Havarstein et al. (1995) PNAS USA 92: 11140-11144) was added at a final concentration of 100 ng/ml.
  • the bacteria were heat shocked at 42°C for 10 minutes and the competent cells were harvested by centrifugation.
  • the pellet was washed with 30 ml of ice-cold 0.5 M sucrose, followed by a wash with 30 ml of ice-cold 10% glycerol. The pellet was then resuspended in 10% glycerol (1/100 volume of original culture).
  • Transformants of S. pneumoniae D39 containing the plasmid pAUL-A Tn4001 luxABCDE km R were patched onto chocolate agar plates containing 0.3 ⁇ g/ml erythromycin and incubated overnight. A quantity of each patch (10 ⁇ l loop-full of cell growth consisting of approximately 10 8 - 10 9 cells) was then uniformly streaked over the entire area of a chocolate agar plate containing 400 ⁇ g/ml kanamycin and incubated for 24 to 48 hrs.
  • S. pneumoniae D39 containing the plasmid pAUL-A Tn4001 luxABCDE km R were patched onto chocolate agar plates containing 0.3 ⁇ g/ml erythromycin and incubated overnight. A quantity of each patch (10 ⁇ l loop-full of cell growth consisting of approximately 10 8 - 10 9 cells) was then uniformly streaked over the entire area of a chocolate agar plate containing 400 ⁇ g/ml
  • BHI Brain Heart Infusion
  • erythromycin pelleted, re-suspended in an equal volume of BHI, and 100 ⁇ l volumes of a 10-fold dilution range (10° - 10 "8 in BHI) spread onto chocolate agar plate containing 400 ⁇ g/ml kanamycin and incubated for 24 to 48 hrs.
  • the resulting colonies were screened for bioluminescence using an ICCD camera and the brightest were streaked onto chocolate agar plates containing 400 ⁇ g/ml kanamycin.
  • S. pneumoniae transformants containing pAUL-A Tn400I luxABCDE km R were dark when selected on chocolate plates containing 0.3 ⁇ g/ml erythromycin. Furthermore, the growth rate of an exponentially dividing culture of such a tiansformant was dramatically inhibited by the addition of kanamycin at concentrations of 400 ⁇ g/ml and higher. However, once growth of the culture had reestablished after several hours in the presence of kanamycin, its rate was similar to that found prior to the addition of this antibiotic. Moreover, a low level of bioluminescence could be recorded form the latter culture, indicating that a subpopulation of the original culture, one in which the luxABCDE km R operon had been induced, had been selected for.
  • Promoter strength can be selected for by varying the kanamycin concentration in the medium during the isolation of chromosomal integrants.
  • Tn4001 luxABCDE km R is randomly inserted into the chromosome of S. pneumoniae and is stably maintained.
  • One thousand bioluminescent, kanamycin resistant S. pneumoniae D39 colonies were patched in duplicate onto chocolate plates containing either 0.3 ⁇ g/ml erythromycin or 400 ⁇ g/ml kanamycin. All but 20 colonies grew on both antibiotics, indicating that the vast majority (98%) of the integrants to be episomally based (whole plasmid integrated into the chromosome) as opposed to true transposants.
  • Southern blot analysis of genomic DNA from 10 kanamycin/erythromycin resistant colonies confirmed the entire pAUL-A Tn400I luxABCDE km R construct to have integrated into the S. pneumoniae chromosome.
  • Southern blot analysis (conducted according to the manufacturer's instructions of Alkphos Direct Labeling Kit, Amerhsam Pharmacia Biotech, Piscataway, NJ using a probe amplified with primers XAF (SEQ ID NO:2) and XAR (SEQ ID NO:3))of genomic DNA from 10 kanamycin resistant/erythromycin sensitive colonies showed all ten isolates to be true transposants, and that each strain contained only one copy of the lux transposon construct.
  • inverse PCR was used to facilitate sequencing of the chromosomal lux fusion junctions. Briefly, the genomic DNA sequence lying upstream of each Tn400I luxABCDE km R integration site was obtained using inverse PCR (Ochman et al. (1988) Genetics 120:621-623). Chromosomal DNA from S. pneumoniae Tn4001 luxABCDE km R was digested with a number of different restriction enzymes (both 4 and 6 base pair recognition enzymes) and self-ligated.
  • the upstream region was PCR amplified using the primers R2 - 5' CGT TTC ATT ACC TCT GTT TGA G 3 ' (SEQ ID NO: 18) and XBF - 5 ' GGG AAT TCT CGA GGA GGA GAG AAA GAA ATG AAA TTT GGA 3' (SEQ ID NO:4).
  • Resulting PCR products were purified (PCR purification kit; Qiagen, Valencia, CA) and directly sequenced using the primer R2. Sequencing of the chromosomal lux fusion junctions (gained by this inverse PCR) of each of these transposants confirmed all ten integration sites to be unique, confirming Tn4001 transposition in S. pneumoniae to occur randomly.
  • Xen 7 chromosomal DNA of a highly bioluminescent, kanamycin resistant, erythromycin sensitive D39 transposant, designated Xen 7.
  • the transformation efficiency varied from 50 - 500 CFUs/ ⁇ g of Xen 7 chromosomal DNA.
  • transformants were named Xen 9, Xen 10, Xen 11 and Xen 12, co ⁇ esponding to the strains HUSTMBIG, A66.1, EF3030 and 140301 (obtained from Marc Lipstich, Harvard School of Public Health, Boston, MA and David Briles, University of Alabama at Birmingham, Birmingham, AL), respectively.
  • Xen 9 and Xen 10 were cultured in BHI medium with the presence or absence of kanamycin for two weeks at 37°C, diluting the cells 1 in 1000 into fresh medium every 12 hrs. Chromosomal DNAs were prepared at day 0, 3, 7, 10 and 14, and the ratio of kanamycin resistant colonies vs. total CFUs was examined at the same times. Tn4001 luxABCDE km R in both Xen 9 and Xen 10 was found to be 100% stable, such that after 14 days of continuous culturing without antibiotic selection all CFUs were bioluminescent and the location of the transposons in both strains remained the same and at single copy.
  • Bioluminescent S. pneumoniae A66.1 Xen 10 can be used to accurately monitor pneumococcal drug efficacy studies in vivo in living animals. Initial studies with S. pneumoniae D39 Xen 7 showed this strain to perform poorly in animal infection studies due to its rapid clearance from the mice. Thus, the strains HUSTMBIG, A66.1, EF3030 and 140301 were made bioluminescent and tested in both mouse lung and nasopharyngeal models. Briefly, exponential cultures of S. pneumoniae grown at 37°C in BHI were pelleted and then resuspended in fresh BHI broth.
  • Bacterial concentrations were estimated spectrophotometrically by absorbance at 600 nm and adjusted to approximately 10 7 CFU/ml by dilution BHI. Cell numbers were verified by plating dilutions of inoculum onto BHI agar.
  • Pneumococci were introduced into the lungs of the mice by either direct intratracheal inoculation, or by intranasal administration.
  • the animals were anesthetized with ketamine (100 mg/ml) and xylazine (20 mg/ml), mixed at a 4:1 ratio v/v just before use.
  • the anesthesia mixture was inj ected intramuscularly into the right hamstring muscle at a dose of 100 mg ketamine/kg of body weight.
  • the mice were inoculated with approximately 10 6 CFU in a total volume of 20 ⁇ l, using a 25 ga. Ball-tipped gavage needle. Mice were held in vertical suspension for 10 minutes after inoculation to facilitate deep penetration of the inoculum.
  • mice were infected intianasally with approximately 10 6 CFU by placing 20 ⁇ l of bacterial suspension on the nares and allowing the mice to inhale the inoculum.
  • mice were treated with amoxicillin at 1 mg/kg or 5 mg/kg, given subcutaneously at t - 0, 18, 24, and 42 hours post-infection.
  • mice were imaged for a maximum of 5 min at a number of time points post- infection using an JVISTM CCD camera (Xenogen Corporation, Alameda, CA). Total photon emission from selected and defined areas within the images of each mouse was quantified using the LivinglmageTM software package (Xenogen Corporation, Alameda, CA). The photon signal from the thorax was quantified from the ventral image of each mouse. A66.1 Xen 10 was found to perform best in the mouse pneumococcal lung model, whereas HUSTMBIG Xen 9 and EF3030 Xen 11 performed best in the nasopharyngeal model. Similar to D39 Xen 7, the strain 140301 Xen 12 did not perform well in either model.
  • A66.1 Xen 10 performed best in the mouse pneumococcal lung model, this strain was selected to test in a drug efficacy study. Twelve mice were inoculated with approximately 10 CFU of S. pneumoniae A66.1 Xen 10. The mice were divided into three groups of four animals and two of these groups were treated with an amoxicillin regime at 1 or 5 mg/kg, the third group of animals remaining untreated as controls. In the untreated group of animals, a strong bioluminescent signal could be detected from the thorax of three of the four mice by 20 hr, indicating three of the mice to have an established pneumococcal lung infection. These animals all had ruffled fur and appeared ill, whereas the fourth control animal, showing no bioluminescent signal, appeared healthy.
  • mice were sacrificed and the infected lung tissue was surgically removed and weighed. Both lungs were homogenized together in 500 ⁇ l BHI in a loose Dounce homogenizer. The suspension was serially diluted in BHI and plated in duplicate onto BHI agar. Bacterial burden was estimated from the number of CFU/g of lung tissue. The bacterial load present in the nasal passage of the mice was obtained by washing approximately 0.25 ml of saline through the trachea and collecting the first 0.1 ml of nasal wash as it exited the nares, as described (Lipsitch et al. (2000) Vaccine 18:2895-2901; Wu et al (1997) Microb. Patho 23:127-137).
  • Plating of lung tissue from each of the mice after the final 48 hr imaging time- point showed only the two untreated animals with strong bioluminescent signals to have high numbers of pneumococcus present, one of these animals having 2 x 10 9 CFU/g tissue. No bacteria could be recovered from the untreated mouse that did not have a bioluminescent signal, supporting the assumption that in this host a pneumococcal infection had not been established.
  • the highest number of pneumococci to be isolated from an amoxicillin treated animal was 300 CFU/g tissue, with four of the eight animals showing no bacteria present. All pneumococcus recovered from long-term mouse nasopharyngeal infections are bioluminescent. S.
  • pneumoniae D39 (mostly Xen 7) showed that it was possible to monitor pneumococcal cells in vivo in a mouse thigh model.
  • S. pneumoniae A66.1 Xen 10 performed extremely well in the lungs of mice ( Figure 8) making it a good candidate for in vivo pneumococcal drug efficacy studies.
  • the strains described herein e.g., Streptococcal strains such as HUSTMBIG Xen 9 and EF3030 Xen 11 , which performed best in the mouse nasopharyngeal model
  • HUSTMBIG Xen 9 and EF3030 Xen 11 which performed best in the mouse nasopharyngeal model
  • other gram-positive organisms can provide valuable information for vaccination studies due to their longevity of colonization of the nasal passage.
  • transposon LTV1-Tn9i 7 luxABCDE to engineer bioluminescent MRSA
  • modified transposon TnPi 7 was prepared for use in engineering of bioluminescent methicillin- resistant S. aureus (MRSA).
  • the plasmid pLTVl-Tn 7 (Camilli et al., 1990 J. Bacteriol. 172:3738-44; Watson et al., 1998 Microbiology 144:3159-69; Schwan et al., 1998 Infect Immun. 66:567-72; Sebulsky et al., 2000 J Bacteriol. 182:4394-400; Eichenbaum and Scott, 1997 Gene 186:213-7); Mansilla and Mendoza, 1997 J Bacteriol. 179:976-81; Tran et al., 2000 Mol Microbiol 37:1159-71) is depicted in Figure 9.
  • E. coli strain HBIOI containing plasmid pLTVl-TnPi 7 was purchased from Bacillus Genetic Stock Center (Ohio State University).
  • the plasmid was prepared from E. coli using Qiagen mini-prep column and transformed into competent S. aureus strain RN4220 using electroporation protocol described on page 46.
  • the transformation mixture was plated on BHI plates supplemented with 5 ⁇ g/ml erythromycin and then incubated at 30°C for 24-48 hours.
  • a single colony of RN4220 transformant containing plasmid pLTVl-Tn917 was inoculated in 10-ml BHI medium supplemented with 5 ug/ml erythromycin (erm5) and 15 ⁇ g/ml tetracycline (let 15).
  • the culture was incubated at 37°C at 200 rpm overnight.
  • the culture was diluted 1 :20 in fresh BHI medium with erm5 and grown at non-permissive temperature of 43°C at 200 rpm to OD600 of 0.8.
  • the culture was diluted 1 :20 again in fresh BHI medium with erm5 and grown at 43 °C at 200 rpm overnight.
  • the overnight culture was diluted 10 "1 to 10 "6 and each dilution was plated on BHI plate erm5, cm5 (5 ug/ml chloremphenicol), and tetl 5 respectively. After the plates were incubated at 45°C overnight, total colonies on each plate were counted. An estimated 80% of the colonies showed em R cm R tet s that indicated the transposition of TnPi 7 onto S. aureus chromosome and the loss of plasmid pLTVl backbone containing tetracycline resistant marker.
  • the MRSA strain 16 is resistant to all the antibiotics tested except for chloremphenicol.
  • plasmid pLTVl-Tn917 and its derivatives can be used for engineering of 16 and other MRSA strains.
  • the modified Gram positive luxABCDE operon described above can be cloned into pLTVl-Tn917 in two ways. In one strategy, a 4.3 kb Bsu36 ⁇ /Acc ⁇ l fragment or 3.0 kb Bsu361/Blpl fragment containing lacZ is removed and replaced by Gram positive luxABCDE operon. Upon transposition, the expression of luxABCDE operon depends on the insertion of transposon downstream of promoter on chromosome.
  • luxABCDE operon fused with a l ⁇ iown promoter in front of luxA is cloned into the unique BamHI site in multiple cloning site.
  • the expression of luxABCDE operon is independent of the transposon integration site.

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Abstract

La présente invention concerne des cassettes transposons à luciférase bactérienne convenant pour conférer des propriétés de bioluminescence à une bactérie Gram positive, à des bactéries Gram négatives, et à d'autres organismes d'étude. L'invention concerne également, d'une part des cellules transformées comportant des vecteurs portant les cassettes transposons, d'autre part des cellules dont les génomes ont été modifiés par introduction de telles cassettes, et enfin des procédés permettant de réaliser et d'utiliser de telles cassettes transposons, des vecteurs à cassette transposon, et des cellules contenant le transposon.
PCT/US2001/007324 2000-07-06 2001-03-07 Compositions et procédés d'utilisation de ces compositions pour modifier les génomes de micro-organismes WO2002008431A1 (fr)

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US7138267B1 (en) 2001-04-04 2006-11-21 Epicentre Technologies Corporation Methods and compositions for amplifying DNA clone copy number
US8277797B2 (en) 2006-11-22 2012-10-02 The Regents Of The University Of California Interferon-β production modulating Listeria strains and methods for using same

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US7138267B1 (en) 2001-04-04 2006-11-21 Epicentre Technologies Corporation Methods and compositions for amplifying DNA clone copy number
US8192991B2 (en) 2002-04-30 2012-06-05 The Regents Of The University Of California Methods of eliciting or boosting a cellular immune response
US8865469B2 (en) 2002-04-30 2014-10-21 The Regents Of The University Of California Methods of transforming a Listeria
AU2003237138B2 (en) * 2002-04-30 2008-04-03 The Regents Of The University Of California Site specific Listeria integration vectors and methods for using the same
US7425449B2 (en) 2002-04-30 2008-09-16 The Regents Of The University Of California Site specific Listeria integration vectors and methods for using the same
US7749510B2 (en) 2002-04-30 2010-07-06 The Regents Of The University Of California Methods of eliciting or boosting a cellular immune response
EP1504095A2 (fr) * 2002-04-30 2005-02-09 The Regents of the University of California Vecteurs d'integration de la listeria specifiques d'un site et leurs methodes d'utilisation
US9556441B2 (en) 2002-04-30 2017-01-31 The Regents Of The University Of California Methods of transforming a Listeria
EP1504095A4 (fr) * 2002-04-30 2005-06-22 Univ California Vecteurs d'integration de la listeria specifiques d'un site et leurs methodes d'utilisation
US8679476B2 (en) 2006-11-22 2014-03-25 The Regents Of The University Of California Interferon-β production modulating Listeria strains and methods for using same
US9066900B2 (en) 2006-11-22 2015-06-30 The Regents Of The University Of California Interferon-β production modulating listeria strains and methods for using same
US9381236B2 (en) 2006-11-22 2016-07-05 The Regents Of The University Of California Interferon-β production modulating listeria strains and methods for using same
US8277797B2 (en) 2006-11-22 2012-10-02 The Regents Of The University Of California Interferon-β production modulating Listeria strains and methods for using same
US9895433B2 (en) 2006-11-22 2018-02-20 The Regents Of The University Of California Interferon-β production modulating listeria strains and methods for using same
US10449241B2 (en) 2006-11-22 2019-10-22 The Regents Of The University Of California Interferon-β production modulating Listeria strains and methods for using same

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