WO1989009270A1 - Method for altering surface charge of microorganisms - Google Patents

Abstract

The present invention is a method to produce polysaccharides with altered functional groups on their surface having modified surface charge as a result of the altered functional groups. These functional groups include, but are not limited to, negatively charged pyruvyl and succinyl moieties and positively charged amino groups. The polysaccharides can be produced by either: (1) mutation and selection of organisms synthesizing polysaccharides with modified surface charge or (2) engineering of specific genes within organisms to alter the charged groups attached to the polysaccharides during synthesis. Mutation of Zoogloea ramigera polysaccharide, as measured by the change in pyruvate content, is demonstrated. The isolation, characterization and manipulation of the gene for the pyruvyl transferase enzyme, which adds pyruvyl moities to the exopolysaccharide produced by Zoogloea ramigera, leading to an exopolysaccharide having a modified surface charge, is detailed. The modified exopolysaccharides have new applications due to the differences in surface charge. The modified enzymes and sequences regulating their expression can be used to produce novel polysaccharides. Further, studies of the structural-functional relationships of the modified exopolysaccharide can lead to a better understanding of secondary and tertiary structure in polysaccharides.

Description

METHOD FOR ALTERING SURFACE CHARGE OF MICROORGANISMS Background of the Invention

This is a continuation-in-part application of U.S. Serial No. 891,136 filed July 28, 1986 by Anthony J. Sinskey, Donald Davidson Easson, Jr., Oliver P. Peoples and Chokyun Rha and U.S. Serial No. 035,604 filed April 7, 1987 by Donald D. Easson Jr., Oliver P. Peoples, and Anthony J. Sinskey, both entitled "Method to Control and Produce Novel Biopolymers". Biopolymers have applications in many industries, including the food, cosmetic, chemical, biomedical, waste treatment and oil industries. Biotechnology is particularly useful in increasing the applicability and utilization of biologically synthesized polymers. For example, the availability of recombinant and classical techniques to manipulate the genetics of an organism allows the development of strategies to alter the polymer structure, in vivo, and hence its function. The application of these technologies to the production of microbial polysaccharides can lead to the structural manipulation at the genetic level and the development of unique, well-defined polymers for specific functional applications.

The synthesis of bacterial exopolysaccharides, with the exception of dextrans, levans and mutan, involves activated sugars in the form of nucleotide diphosphate intermediates. Monosaccharides are transferred from their sugar nucleotide derivatives to the growing polysaccharide chain, usually via isoprenoid lipid intermediates. A possible biosynthetic pathway for a hypothetical glucose and galactose polysaccharide from glucose is shown in Figure 1. Glucose is transported into the cell as glucose-6-phosphate, which is then converted to glucose-1-phosphate by phosphoglucomutase. A sugar nucleotide diphosphate is formed by a nucleotidyl transfer to the sugar phosphate, forming, in this case, UDP-glucose.

At this stage, the activated sugar can be interconverted into a variety of sugar nucleotides. Reactions that accomplish these interconversions include epimerization, dehydration and decarboxylation. Almost without exception, the presence of a particular monosaccharide in a polysaccharide requires the existence of that particular monosaccharide nucleotide diphosphate as a precursor. In this example, UDP-glucose is epimerized to UDP-galactose. The reactions leading to polymerization of the sugar nucleotide are varied and not completely understood; however, there is strong evidence supporting the presence of lipid intermediates in some systems. It has been proposed that sugar-1-phosphate is transferred to isoprenoid lipid phosphate, followed by the addition of sugars from sugar nucleotides to form the lipid bound oligosaccharide repeat units. Multiples of the repeat unit are then formed by transfer to the reducing end of the growing polysaccharide. A system such as this has been confirmed to carry out xanthan biosynthesis. The exopolysaccharide must subsequently be released so that the isoprenoid lipid can be returned to the pool. The polysaccharide then passes through the hydrophobic outer membrane. The probable mechanism involves the adhesion sites of Gram-negative bacteria, known as Bayer sites, in which the outer and inner membranes are associated with each other. It is proposed that exopolysaccharides are exported through these channels in a way that is analogous to the import of some substrates through porins. This scheme is a compilation of proposed pathways for several expolysaccharide producing organisms. One variation on the scheme is the production of alginate by Azotobacter vinelandii in which there is no evidence of the involvement of lipid intermediates. In this pathway, GDP-mannuronic acid is polymerized to polymannuronic acid by alginic acid polymerase followed by epimerization to alginic acid. The environment surrounding the organism plays an important role in determining the character of the polysaccharide. Traditionally, manipulation of the nutrient and oxygen supply has been the primary means for modification of the exopolysaccharide structure and rheological properties. For example, variable culture conditions causes variability in pyruvate content in xanthan gum, affecting viscosity, thermostability and salt compatibility.

Zoogloea ramigera, which produces an exopolysaccharide having interesting rheological behavior and metal adsorption properties, is an especially useful organism for demonstrating a method for the production of polysaccharide with modified surface charge and the effect on the structural and functional properties of the polysaccharide. Z . ramigera is a gram-negative, rod-shaped, floc-forming, motile, non-spore forming obligate aerobe isolated from sewage and aerobic waste treatment facilities that is generally thought to be the major contributor to flocculation in activated sludge. They are distinguished from other gram-negative pseudomonads by the presence of the exocellular polysaccharide which causes flocculation,^" called "zooglan", and occurs, in some strains, as a zoogloeal or capsule-like matrix. In nature, the polysaccharide is thought to function to concentrate nutrients around the cell floes enabling them to grow in nutrient deficient environments. Cell floes of Z.. ramigera accumulate heavy metal ions. Z. ramigera is also known to accumulate poly-3-hydroxy butyric acid (PHB) granules intracellularly.

Several isolates of Z . ramigera have been reported. Three strains contained in the American Type Culture Collection, Rockville, MD (ATCC) , Z.. ramigera strains I-16-M, 106 and 115, flocculate and produce different extracellular polysaccharides. Z_.. ramigera strains have been studied for their role in aerobic waste treatment, the adsorption of heavy metals, the production of PHB, and the production of extracellular polysaccharides. In addition, purified polysaccharide has been characterized to some extent, both chemically and physically. Z.. ramigera isolate 115 is a zoogloeal matrix-forming strain which, when grown in a nitrogen limiting medium, converts 60% (w/w) of the available glucose substrate into exopolymer with yields of more than 15 g/1 in batch culture having been reported. Isolate I-16-M produces a non-capsular exopolysaccharide that contains glucose, galactose and mannose. Isolate 106 produces an extracellular polysaccharide that contains amino sugars.

As described in U.S. Serial No. 891,136 filed July 28, 1986, genes involved in exopolysaccharide synthesis can be isolated by constructing a library of the organism's DNA, introducing the DNA into polysaccharide deficient mutants, and screening for restored polysaccharide production. Isolation of the genes provides a means for the modification of the rate and product of expression of the genes and, ultimately, the construction of novel polymers using the isolated genes.

In U.S. Serial No. 035,604 filed April 7, 1987, two new strains of Z. ramigera. 115SL and 115SLR, derived from 115, which do not produce an exopolysaccharide capsule layer, were described. Although the organisms produce exopolysaccharide having very similar monosaccharide composition to the 115 exopolysaccharide, the pyruvate content of the 115SL and 115SLR polysaccharide is much higher. From an applications perspective, the new strains have advantages over 115 since the organisms can receive foreign DNA using conventional techniques such as conjugation. Conjugation is not possible in 115 due to the capsule layer of polysaccharide enclosing the cells.

In addition to enhancing control of the production of known polysaccharides, modification at the genetic level of polysaccharide producing organisms would enable production of novel polysaccharides and development of new applications for the polysaccharides thereby produced.

It is therefore an object of the present invention to provide a method for producing modified and novel polysaccharides having a selected surface charge.

It is a further object of the present invention to provide means for mutating and screening organisms for the production of polysaccharides having .modified or novel structure and function, particularly through alteration of the charged groups on the polysaccharid .

It is a still further object of the present invention to provide such a method in which polysaccharide structure and function is controlled through genetic manipulation of the producing organism.

Summary of the Invention

The present invention is a method to produce polysaccharides with altered functional groups on their surface resulting in variable or zero charge density. These functional groups include, but are not limited to, negatively charged pyruvyl and succinyl moieties and positively charged amino groups. There are two variations of the method to produce polysaccharides having modified surface charge: (1) mutation and screening for organisms synthesizing polysaccharides with modified surface charge and (2) engineering of specific genes within organisms to alter the charged groups attached to the polysaccharides during synthesis.

Organisms can be mutated using radiation or chemical mutagens. Organisms are screened on the basis of the measured charge or relative percent of charged groups (for example, the percent pyruvate) .

Organisms, can be engineered to alter the surface charge of the expressed polysaccharide by isolation and modification of the genes for specific enzymes. For example, the identification of the transcription initiation site of the pyruvyl transferase (pyv) gene, as well as other genes, allows the replacement of the wild-type promoter with a regulatable, high level expression promoter. Levels of pyruvyl transferase and the other enzymes can then be varied to produce exopolysaccharides with varying degrees of pyruvate substitution and varying physical properties. The isolation, characterization and manipulation of the gene for an enzyme which adds pyruvyl moieties to an exopolysaccharide produced by Zoogloea ramigera, leading to an exopolysaccharide having a modified surface charge, is detailed. The modified exopolysaccharide has new applications due to the differences in charge and rheological properties. Further, studies of the structural-functional relationships of the modified exopolysaccharide which can lead to a better understanding of secondary and tertiary structure in polysaccharides are now possible.

Brief Description of the Drawings

Figure 1 is a biosynthetic pathway for a hypothetical glucose and galactose polysaccharide from glucose.

Figure 2 is the strategy for the cloning of Z. ramigera polysaccharide genes.

Detailed Description of the Invention The method of the present invention is to provide polysaccharides having modified surface charge due to variations in the negatively charged pyruvyl and succinyl moieties and positively charged amino groups on the polysaccharide which are the result of alteration of the genetic structure of the organisms rather than the environment in which the organism is grown. Pyruvyl groups contribute to the polysaccharide a -negative charge which plays a large role in determining its functional properties. Control of pyruvylation at the genetic level allows the production of polysaccharides with varying charge densities having a range of functional properties. Similarly, control of the amount of succinyl group substitution leads to polysaccharides with variable succinate content and a range of different functional properties.

Regulation of positive charge density by controlling the amino group content of a particular polysaccharide can be done by regulating a specific amino transferase or transaminase that adds an amino group to the polysaccharide. It can also be accomplished by controlling the amount of aminosugars in the polysaccharide or by manipulating the rate of aminosugar synthesis.

The development of this system allows the manipulation of the number of positively and negatively charged functional groups to produce novel polysaccharides with virtually any charge density and distribution, having unique physical properties. There are two variations of the method to produce polysaccharides having modified surface charge: (1) mutation of organisms and screening for production of polysaccharides having modified surface charge and (2) engineering of specific genes within organisms synthesizing exopolysaccharides to alter the charged groups attached to the polysaccharides during synthesis.

Organisms can be mutated using radiation or chemical mutagens or by techniques such as transposon mutagenesis. Organisms are screened on the basis of the measured charge or relative percent of charged groups (for example, the percent pyruvate) .

Organisms can be engineered to alter the surface charge of the expressed polysaccharide by isolation and modification of the genes for specific enzymes. For example, the identification of the transcription initiation site of the pyv gene, as well as other genes, allows the replacement of the wild-type promoters with a regulatable, high level expression promoter. Levels of pyruvyl transferase and the other enzymes can then be varied to produce exopolysaccharides with varying degrees of pyruvate substitution and varying physical properties. Example 1: Modification of Zoogloea Polysaccharide Surface Charge by Mutation.

When grown in a nitrogen limiting medium, Z. ramigera 115 converts approximately 60% of the available glucose substrate into a heteropolysaccharide composed of glucose and galactose in a molar ratio of 2:1 and approximately 2.5% pyruvate by weight. This heteropolysaccharide is termed "zooglan". Previous efforts to alter the percent pyruvate in zooglan have centered around alteration of the conditions under which the organism is grown. As demonstrated below, the percent pyruvate in the polymer can be altered at the genetic level by mutation using ultraviolet radiation, chemical mutagenesis (nitrosoguanidine) , or transposon mutagenesis. The following methods were used to produce and characterize mutants synthesizing polysaccharide with altered pyruvate content. Media and Culture Conditions.

Z.. ramigera cultures are stored frozen at -70°C in trypticase soy broth (TSB) containing 7% (v/v) dimethyl sulfoxide (DMSO) and 15% (v/v) glycerol. TSB and a defined medium as described by Norber and

Enfors, Appl.Microbiol. 44,1231-1237 (1982), are used for routine cultivation of the various Z.ramigera strains. The defined medium has the following composition: glucose, 25 g. ; 2HPO4, 2 g; KH2PO , -■- •?' NH4CI, 1 g; MgS0₄: 7H₂0, 0.2g; yeast extract (Difco Laboratories), 0.01 g in 1 liter distilled water. Glucose, g≤4: 7H2O, yeast extract and salts are autoclaved separately.

100 ml cultures of Z.. ramigera for exopolysaccharide (EPS) isolation are grown on a rotary shaker (200 rpm) at 30°C in 500 ml baffled shaker flasks for 1 to 2 weeks. Cell growth is measured using the Bio-Rad Protein Assay (Bio-Rad Laboratories) after cells are lysed by heating at 90°C for 10 min in 1 N NaOH (Gerhardt, Manual of Methods for General Microbiology (American Society of Microbiology 1981) ) .

__.• coli strains are grown in LB (Luria-Bertani) medium, 1% (w/v) NaCl, 1% (w/v) tryptone (Difco) and 0.5% (w/v) yeast extract (Difco). Exopolysaccharide Isolation.

Exopolysaccharide from Z.. ramigera 115 was purified by the addition of concentrated NaOH to the cell culture to a final concentration of 0.2 M, followed by the addition of 3 volumes of ethanol to precipitate the polymer and other materials which were collected and redissolved in half the original volume of water. Protein was removed by extracting twice with phenol, followed by extraction with- ether to remove excess phenol. The aqueous phase was dialyzed, lyophilized and ground to yield a fine white powder. Exopolysaccharide from Z . ramigera 115SL and its derivatives was purified by removing cells by centrifugation followed by precipitation with 2 volumes of ethanol. Exopolysaccharide precipitate was collected, redissolved, dialyzed, lyophilized and ground to yield a fluffy, white material.

Carbohydrate Determination - Hexose Assay. Total carbohydrate concentration in culture broths and polymer solutions was determined by the Phenol reaction (Gerhardt, 1981) . Glucose, galactose and xanthan gum (Sigma Chem. Co.) were used as standards.

Protein Determination.

Total protein concentration in culture broths and polymer solutions was determined using the Bio-Rad Protein Assay (Bio-Rad Laboratories, 1979) . Lysozyme was used as the standard. Cellular protein was released by boiling in 0.2 N NaOH.

Compositional Analysis of Polysaccharides. Purified polysaccharide was hydrolyzed in 1 M trifluoroacetic acid at 120"C for times varying between 1/2 hour and 2 hours. Monosaccharides in the polysaccharide hydrolysate were separated using a Waters HPLC equipped with a Brownlee Polypore PB, lead loaded cation exchange column, operated at 85°C, with water as the eluent. Detection was by refractive index using a Waters Model 401 Differential Refractometer.

Proton NMR Spectroscopy.

The polysaccharide hydrolysate (10 g) was dissolved in D2O and analyzed using a 500 MHz proton NMR spectrometer at the NMR Facility for Biomolecular Research located at the Francis Bitter National Magnet Laborator , MIT.

Infrared Spectroscopy. Infrared spectra of purified polysaccharide were obtained on a Perkin Elmer Model 283B Infrared Spectrophotomer. Samples were prepared by grinding 1- 5 mg of polysaccharide with 100 mg dry KBr and pressing the mixture into a disk. Size Exclusion Chromatography.

Polysaccharide solutions were analyzed by size exclusion chromatography using a Waters Model 401 HPLC equipped with BioRad Bio-Gel TSK-40 and TSK-60 columns connected in series. Distilled, deionized water containing 0.05% sodium azide was used as the- solvent. Detection of polysaccharides was by different refractive index.

Nitrosoguanidine Mutagenesis.

Cells at a density of 2 x 10⁸ cells/ml were exposed to 50 μg/ml NTG (Gerhardt, 1981) for varying times up to 60 minutes. Cells were plated out at a survival rate of 30-40% ( 35 minute exposure) and screened.

Transposon Mutagenesis. Transposon insertions into the Z. ramigera

115SLR chromosome are made by introducing Tn5_ on the delivery vector pRK602. Tn5 insertions are selected for on plates containing neomycin and rifa picin and obtained at a frequency of approximately 10^~5 insertions/recipient. Exopolysaccharide mutations are screened by visual observation for flocculation in liquid culture, by growth on medium containing Cellufluor (colonies producing exopolysaccharide fluoresce under UV light) , by composition analysis. Tn5 mutagenesis was carried out with a culture of Z. ramigera 115SLR grown up in Trypticase soy broth to a density of approximately 5 x 10⁹ cells per ml. A 20 ml portion of this flocculated cell suspension was sonicated for 2 min to break up the cell floes. Transfer of pRK602 (Cm^r,Nm^r,PRK2013 nm:Tn9 containing Tn5) into Z. ramigera was carried out using E. coli MM294A(pRK602) as the only donor. Tn5 insertions into the Z. ramigera chromosome were selected for by growth on neomycin (50 μg/ml) and rifa picin (100 μg/ml) . Analysis of Pyruvate Content.

Pyruvate content was analyzed using lactate dehydrogenase. Samples of zooglan (approximately 5 mg) were dissolved in 1 N HCl (2.0 ml) and heated to 100°C for 3 h. 10% Na₂C0₃ (2.0 ml) was added and the samples diluted to 20 ml with deionized water. Three parallel samples of each of these solutions were assayed for pyruvate using a pyruvate diagnostic kit (Pyruvate/lactate, Kit no. 726-uv, Sigma Diagnostics, St. Louis, MO 63178) . Absorbance was measured at 340 nm using a Gilford 2600 spectrophotometer, and pyruvate concentration was determined from a standard curve, using the average of the three parallels. Results. The pyruvic acid content of zooglans produced by strain 115 and mutants of 115 is shown in Table 1. 115SL was produced from 115 by chemical mutagenesis. 115SLR is a spontaneous rifampicin resistant mutant. The remaining mutants resulted from transposon mutagenesis. The pyruvate content varies from 3.6 to 1.7% by weight. TABLE 1: Pyruvate Content of Zooglan

Bacterial strain Pyruvate (%)

115 (ATCC 25935) 2.5

115SL (ATCC 53589) 3.1

115SLR* P4-2 2.5

115SLR P B176 2.5

115SLR PN 232 1.7

M 22 "Bright smooth" 3.3

M 24 "Bright rough" 2.7

M6 "Dark" 3.6

* Z. ramigera 115SLR is ATCC 53590

Example 2: Modification of Polysaccharide Surface Charge by Genetic Engineering.

The following non-limiting example details the isolation, characterization and manipulation of the gene for the pyruvyl transferase enzyme which adds pyruvyl moieties to an exopolysaccharide produced by Zoogloea ramigera.

Isolation of Exopolysaccharide Biosynthetic Genes.

Control of a polysaccharide's structure by manipulation of the genes that code for its biosynthesis first requires isolation and characterization of the gene. This involves cloning the polysaccharide genes directly in Z_.. ramigera, as shown schematically in Figure 2. The requirements to accomplish this are 1) a method for introducing foreign DNA into Z_.. ramigera. 2) the construction of a Z . ramigera gene library, 3) the isolation of polysaccharide mutant strains, and 4) the subsequent complementation of those mutants using the Z . ramigera gene library.

This procedure was carried out in strain 115 using the following materials and methods.

TABLE 2. Bacterial Strains and Plasmids

Strain/ Source or Plasmid Relevant Characteristics Reference

Z . ramigera

115 Wild type ATCC 25935

115SL 115, slime forming mutant (NTG) ATCC 53589

115SLR 115SL, Rf^r (spontaneous) ATCC 53590

E. coli HB101 hsdS20 (r Bm~_rB) , recA13_, proA2 Maniatis et al. , rpsL20, (Sm^r) , supE44. 1982

MM294A pro-82 , thi-1, endAl, hsdR17 , Leigh e supE44 al. , 1985

DH5 endAl. hsdR17 (r~_] ,m⁺ _k) , supE44, BRL thi-1. recAl, gyrA96. relAl

DH5α end Al, hsdR17 (r~_] ,m⁺ _] ) , supE44 , BRL thi-1, recAl, gyrA96. relAl,

___(argF-laczva)U169, Φ80dlacZ_aM15

Plasmids pUC8 Ap^r Vieira and Messing, 1982 pRK2013 Nm^r Figurski et al.

1979 pLAFR3 Tc^r, cosmid vector Staskawic pRK602 Cm^r, Nm^r Leigh et pRK2013 nm::Tn9. containing Tn5 al.. 1985

Maniatis et al.. Molecular Cloning.

(Cold Spring Harbor Laboratory, Cold

Spring Harbor, NY 1982) ; Leigh et al., Proc.Natl.Acad.Sci.USA 82,6231-

6235 (1985) ; Vieira and

Messing, Gene 19,259-268 (1982) ;

Figurski et al.

Proc.Natl.Acad.Sci.USA 76,1648-1652

(1979) ; Leigh et al.,

Proc.Natl.Acad.Sci.USA 82,6231-6235

(1985) DNA Manipulations: Method for introducing foreign

DNA into Z . ramigera.

Chromosomal DNA was isolated from Z . ramigera 115 as follows: 200 ml of mid log phase cells were harvested by centrifugation (8,000 rpm, GSA rotor, 10 min), washed in 80 ml of 20 mM Tris HCl, pH 8.2 and re-harvested as described above. Cell pellets were resuspended in 10 ml of 10 mM Tris. HCl, pH 3.2, and 10 ml of PEG 8000 (24% w/v) and 2 ml of lysozyme (25 mg/ml added. The mixture was then incubated for 30 min at 37°C. Spheroplasts were then collected by centrifugation (10,000 rpm, 8x50 rotor, 10 min) , resuspended in 5 ml of TE buffer (10 mM Tris HCl, pH 8.2, 1 mM EDTA), 300 μl of 10% (w/v) SDS added followed by a 10 min incubation at 55°C to lyse the cells. The solution was diluted by adding 10 ml of TE and treated with RNase A (150 μl of a 5 mg/ml stock solution) for 15 min at 37°C. Proteinase K, 50 μl of a 10 mg/ml solution, was added followed by a 1 h incubation at 45°C. The DNA was then purified by cesium chloride gradient centrifugation as follows: The solution was added to 40.8 g of CsCl and the total volume made up to 36 ml with TE buffer and the density adjusted to 1.70; gradients were centrifuged at 46,000 rpm for 18 h at room temperature in a VTi 50 rotor; the gradients^' were then fractionated, DNA containing fractions pooled, the CsCl diluted out with TE and DNA recovered by precipitation with 2 volumes of ethanol. The precipitated DNA was washed 3 times with 70% (v/v) ethanol, dried, resuspended in TE buffer at a final concentration of 1 μg/μl and stored at 4°C.

For large numbers of samples where only small amounts of DNA are required, 10 ml lots of cells were harvested by centrifugation (10,000 x g, 10 min), washed in 10 ml of 20 mM Tris HCl, pH 8.2 and re- harvested as described above. Cell pellets were resuspended in 1 ml of 20 mM Tris HCl, pH 8.2, and 1 ml of PEG 8000 (24% w/v) and 0.2 ml of lysozyme (25 mg/ml) added. The mixture was then incubated for 30 min at 37°C. Spheroplasts were collected by centrifugation (10,000 x g, 10 min) resuspended in 250 μl of TE buffer (10 mM Tris HCl, pH 8.2, ImM EDTA) , 15 μl of 10% (w/v) sodium dodecylsulfate added followed by a 10 min incubation at 55°C to lyse the cells. The solution was diluted by adding 500 μl of TE and treated with RNase A (7.5 μl of a 5 mg/ml stock solution) for 15 min at 37°C. Proteinase K, 2.5 μl of a 10 mg/ml solution, was added followed by a 1 h incubation at 45"C. Following phenol extraction to remove proteins, the DNA was recovered by ethanol precipitation and resuspended in TE buffer at a final concentration of 1 μg/μl. Plasmid DNA was prepared from E. coli using the methods of Birnboim and Poly, Nucleic Acids Res.7.1513-1523 (1979) as modified by Ish-Horowicz and Burke, Nucleic Acids Res. 9,2989-2998 (1981). Restriction endonucleases and T4 DNA ligase were purchased from IBI, New Haven, CT. Calf intestinal alkaline phosphatase (CIP) was obtained from Boehringer Mannheim (Indianapolis, IN) . All enzymes were used under manufacturer's recommended conditions. DNA polymerase I was obtained from Amersham (Arlington Heights, IL) .

Construction of Z. ramigera Gene Libraries.

MobI partial digestion conditions for Z . ramigera DNA were determined to yield fragments in the size range 15-28 kb, as described by Maniatis et al. (1982) . 40 μg lots of DNA were digested for 1 h with appropriate amounts of enzyme, followed by the addition of EDTA to 25 mM and incubation at 68°C for 10 min. After recovery by ethanol precipitation the DNA was treated with CIP and electrophoresed on a

0.75% (w/v) agarose gel. Agarose containing fragments in the size range 15-28 kb were excised and the DNA electroeluted, ethanol precipitated and resuspended in TE at a concentration of 1 μg/μl. Ligation reactions contained 1 μg of

Hindlll/BamHI cut vector, 1 μg of EcoRI/BamHI cut vector and.2 μg of target DNA in a total volume of 10 μl and were incubated for 12-16 hours at 14°C. Ligated DNA was packaged using in vitro packaging extracts prepared from E. coli BHB2688 and BHB2690 as described by Ish-Horowicz and Burke (1981) . Recombinant phage particles were transduced into E. coli DH5 as described by Maniatis et al. (1982) and plated on LB agar containing tetracycline (10 μg/ml) . Ligated DNA was transformed into E. coli DH5α cells and plated on LB agar plates containing tetracycline (10 μg/ml) and Xgal (50 μg/ml) . Clones containing recombinant plasmids were isolated by screening for white colonies and confirmed by restriction analysis of their plasmid DNA.

Conjugation in Z . ramigera.

Transfer of recombinant DNA molecules into Z.. ramigera 115SLR_.was accomplished using the conjugative plasmid pRK2013 as follows: E. coli containing the Z . ramigera gene library was grown up in LB broth containing tetracycline (5 μg/ml) . E. coli containing pRK2013 was grown up in LB broth containing neomycin (25 μg/ml) . Z. ramigera 115SLR was grown up in TSB containing rifampicin (50 μg/ml) . All three cultures were grown to a density of approximately 2 x 10⁹ cells/ml. Equal amounts (0.5 ml) of each were mixed after washing. The mixture was deposited dropwise onto the center of a single 100 mm TS agar plate, allowed to dry and incubated overnight at 30°C. Cells were resuspended in 1 ml TSB and dilutions were plated on TS agar containing tetracycline (10 μg/ml) and rifampicin (50 μg/ml) .

Transposon Tn5 mutagenesis was carried out using cultures of Z.. ramigera grown up in TSB to a density of approximately 5xl0⁹ cells/ml. Transfer of pRK602 into Z . ramigera 115SLR was carried out as described above using E. coli MM294A (pRK602) as the only donor. Tn5 insertions into the Z . ramigera chromosome were selected for by growth on neomycin (50 μg/ml) and rifampicin (50 μg/ml) .

DNA Blotting and Hybridization Analysis. DNA fragments, separated on 1% (w/v) agarose gels, were transferred to nitrocellulose filters by the sandwich blot method (Smith and Summers,

Anal.Biochem. 109,123-129 (1980) based on the technique developed by Southern, J.Mol.Biol. 98,503

(1975) . Filters were hybridized with DNA probes labelled to a high specific activity (0.1-1 x 10⁸ cpm/μg of DNA) with [α~3²P]-dATP, by nick translation

(Ribgy,et al..J. Mol.Biol.113.237 (1977). For the detection of Tn5 insertions, probes were prepared using either the purified Tn5_ sequence on a 5.5 kb Hpal fragment from pRK602 or the entire pRK602 plasmid. Control experiments indicated no detectable homology between the plasmid and wild type Z . ramigera chromosomal DNA. Prehybridizations and hybridizations were carried out at 65°C in sealed polyethylene bags as previously described (Peoples, et al. ,J.Bacteriol. (1987)). Final wash conditions were 2 x SSC (20 x SSC: 3.0 M NaCl, 0.3 M sodium citrate), 0.1% (w/v) sodium dodecylsulfate at 65°C. Results. Morphology of Z. ramigera Strains.

Cell floes of Z.. ramigera isolate 115 were visualized with a light microscope using 1% aqueous crystal violet stain. The cells are grouped together and are surrounded by capsules formed by the exopolysaccharide matrix. A similar preparation of mutant 115SL does not have a capsule, and does not flocculate but does produce extracellular polysaccharide.

Differences in the colony morphology of 115 and 115SL were also observed. Isolate 115 colonies had an irregular form and a high dome shape (pulvinate) with a rough and bumpy surface. The morphology of the 115 colonies, except for size, did not change throughout the development of the colony. Colonies were tough, not readily broken up, and entire colonies were easily lifted from plates. The colonies of isolate 115SL were circular, glistening, had a mucoid appearance and were viscous.

Cloning of Z. ramigera 115 EPS Biosynthetic Genes.

Genetic Transfer in Z. ramigera 115SLR. Several broad host range cloning vectors have been obtained and transferred into Z.. ramigera 115SLR by conjugation. The procedure used was a triparental mating between two E . coli strains and the Z.. ramigera recipient. The donor was E. coli HB101 harboring the broad host range vector, pLAFR3. This cosmid confers tetracycline resistance and is mobilizable but not self-transmissible. The mating requires a second E. coli strain which contains the "helper" plasmid pRK2013 carrying the RK2 transfer functions ligated to a ColEl replicon which can mobilize pLAFR3 into Z . ramigera. The transconjugants were selected for by growth on appropriate medium. The transfer of pLAFR3 to Z . ramigera 115SLR occurred at a frequency of approximately 10~⁴ transconjugants/recipient.

Construction of Z . ramigera Gene Libraries. The cosmid pLAFR3, derived from RK2 via pRK290, was used to construct Z . ramigera 115 gene libraries. This procedure increases the cloning efficiency by ensuring that only recombinant molecules can be packaged. A "right" and "left" arm are created which can only be packaged if they ligate to an insert molecule that is between 15 and 28 Kb, thus a high frequency of recombinants are obtained. The recombinant molecules were packaged in vitro and transduced into E. coli DH5. This procedure was carried out for both I-16-M and 115 and yielded approximately 10⁵ recombinants/μg of insert DNA in each case.

Isolation of the 115 Pryuvyl Transferase Gene. The pyruvyl transferase (pyv) gene of Xanthamonas ca pestris (described by Harding et al., J.Bacteriol. 169(6), 2854-2861 (1987) was obtained from Kelco Division of Merck, San Diego, CA, on plasmid pNH232. The pyv gene was subcloned into pUC8 for use as a hybridization probe to detect and identify homologous sequences in Z . ramigera 115 DNA. A restriction enzyme could not be found that excised the pyv gene intact so it was subcloned in two PstI fragments. These subclones were designated pPYV1.4 and pPYV2.2. ³²P-labelled pPYV1.4 DNA hybridized to plasmid DNA of pHP27, pEXlF and pEX3B, plasmids containing genes involved in exopolysaccharide biosynthesis, digested with EcoRl. pEX3B contains a 0.9 kb PstI fragment that is hybridized by pPYV1.4. pEX3B was deposited in E. coli DH5_* with the American Type Culture Collection, Rockville, MD on February 10, 1988 and assigned ATCC 67626. This 0.9 kb PstI fragment was isolated and subcloned into pUC8 yielding the plasmid pEX0.9 which was labelled with ³²P and hybridized to the same Southern blot of pHP27, pEXlF and pEX3B DNAs after the filter was boiled to remove the first probe. pEX0.9 hybridized to the same size PstI fragment as hybridized by pPYV1.4.

Homology to the X. campestris pyv gene was demonstrated by hybridizing both pPYV1.4 DNA and pEX0.9 DNA to Z.. ramigera 115 chromosomal DNA, using a Southern blot of 115 chromosomal DNA digested with BamHI, EcoRl. PstI and Sail probed with ³²P-labelled pPYVl.4 DNA and probed with ³²P-labelled pEX0.9 DNA after boiling to remove the first probe. Comparing both cases, it was found that the two probes hybridize to the same size fragments in each of the four digests. From these results it can be concluded that the cloned segment of Z.. ramigera 115 DNA has sequence homology to the 1.4 kb PstI fragment which contains part of the X. campestris pw gene.

Manipulation of EPS Composition and Structure. The pyv gene, including regulatory sequences, as present in pEX3B, can be inactivated and reintroduced into the 115SL chromosome by recombination. The resulting strain is deficient in pyruvyl transferase and produces a pyruvate-free exopolysaccharide. This polysaccharide would have approximately zero net charge and some unique physical properties. The uncharged chain of the neutral exopolysaccharide can be characterized with respect to its rheological properties. Important information about the secondary and tertiary behavior of this molecule can be determined from this information, helping to define its structure-function relationships.

Modifications and variations of the present invention will be obvious to those skilled in the art form the foregoing detailed description. Such modifications and variations are intended to come within the scope of the appended claims.

We claim:

Claims

1. A method to provide microbial exopolysaccharides -having modified surface charge comprising modifying the nucleic acid sequences encoding or regulating the expression of an enzyme from Zoogloea ramigera selected from the group consisting of pyruvyl transferases, succinyl transferases, amino transferases, amino transaminases, and enzymes responsible for the synthesis and addition of amino sugars to polysaccharide chains.

2. The method of claim 1 wherein the nucleotide sequence encodes or regulates the expression of pyruvyl transferase.

3. The method of claim 1 wherein the nucleic acid sequences are modified by mutating and screening the mutated organisms for altered polysaccharide production.

4. The method of claim 1 wherein the nucleic acid sequences are modified by isolation and addition or deletion of specific nucleotide sequences.

5. The method of claim 1 wherein the nucleic acid sequences regulating expression of the enzymes are modified.

6. The method of claim 5 wherein the promoter sequence is replaced with a sequence causing increased expression of the enzyme.

7. The method of claim 4 further comprising inserting the modified sequences in an organism other than Zoogloea for expression and screening for polysaccharide production.

8. The method of claim 4 further comprising combining the modified sequences with sequences encoding and regulating the expression of other proteins involved in polysaccharide synthesis and screening for polysaccharide production.

9. A nucleotide sequence encoding or regulating the expression of pyruvyl transferase in Zoogloea.

10. The sequence of claim 9 present in plasmid pEX3B deposited in E. coli DH5α with the ATCC on February 10, 1988 and assigned ATCC 67626.

11. The sequence of claim 9 encoding pyruvyl transferase in combination with regulatory sequences altering expression of the enzyme.

12. A microbial exopolysaccharide having modified surface charge produced by modification of nucleic acid sequences encoding or regulating the expression of an enzyme from Zoogloea ramigera selected from the group consisting of pyruvyl transferases, succinyl transferases, amino transferases, amino transaminases, and enzymes responsible for the synthesis and addition of amino sugars to polysaccharide chains.

13. The exopolysaccharide of claim 12 produced by mutation of the Zoogloea and screening for polysaccharide production.

14. The exopolysaccharide of claim 12 produced by isolation and modification of the nucleic acid sequences encoding or regulating expression of the enzymes in Zoogloea.

15. The exopolysaccharide of claim 14 wherein the nucleotide sequence encodes or regulates the expression of pyruvyl transferase in Zoogloea.

16. The exopolysaccharide of claim 15 wherein the sequence is present in plasmid pEX3B deposited in E. coli DH5α with the ATCC on February 10, 1988 and assigned ATCC 67626c

17. The exopolysaccharide of claim 15 wherein the sequence encodes pyruvyl transferase and is in combination with regulatory sequences altering expression of the enzyme.

18. The exopolysaccharide of claim 12 produced by inserting the modified sequences in an organism other than Zoogloea for expression and screening for polysaccharide production.

19. The exopolysaccharide of claim 12 produced by combining the modified sequences with sequences encoding and regulating the expression of other proteins involved in polysaccharide synthesis and screening for polysaccharide production.

20. The exopolysaccharide of claim 12 having approximately zero surface charge due to pyruvyl groups on the polysaccharide surface.