Two new bacterial strains designated Zoogloea ramigera 115SL and Zoogloea ramigera 115SLR, a rifampicin resistant derivative of 115SL, have been developed. These strains are derived from the wild type Zoogloea ramigera 115, ATCC 25935. The two new strains produce a novel exopolysaccharide (EPS) and have several desirable characteristics that are absent from the parent strain, including improved culture properties, since they do not produce an EPS capsule layer like that of the parent 115 strain. The 115SL EPS is instead excreted as a slime layer which is not confined to the immediate area surrounding the cells. Since cells are not trapped within a floc where they grow at a reduced rate or die because of nutrient starvation, the new strains have more consistent and reproducible growth cycles and increased growth rates. As a consequence, exopolysaccharide production is more consistent and titers are higher. The separation of the EPS from the cells is also much easier and more economical. The other very important characteristic of strains 115SL and 115SLR is that they are able to receive foreign DNA using conventional techniques due to the absence of the capsule layer. This facilitates the application of recombinant DNA technology to control and produce novel expolysaccharides.

The present invention is in the field of genetically engineered organisms, 
particularly genetically modified exopolysaccharide producing strains of 
Zoogloea ramigera. 
Polysaccharide biopolymers have found applications in many industries, 
including the food, cosmetic, chemical, biomedical, waste treatment and 
oil industries. 
Flocculation is one important commercial use of biopolymers. Several types 
of floc-forming bacteria have been identified. The most efficient are the 
cellulose, or cellulose-like, producing bacteria such as certain species 
of Pseudomonas, Aerobacter, Agrobacterium, Azotobacter and Zoogloea. 
Flocculation of these bacteria appears to occur when cells become embedded 
in a network of exopolysaccharide fibrils. Other floc-forming bacteria 
produce capsular polysaccharides enclosing large packets of cells which 
lead to floc formation. An example of this phenomenon occurs with Zoogloea 
ramigera 115. In some cases these exocellular polysaccharides have metal 
ion binding properties. 
Z. ramigera 115, designated ATCC 25935 by the American Type Culture 
Collection, Rockville, Md., is an exopolysaccharide matrix forming strain 
which, when grown in a nitrogen limiting medium, converts 60% (w/w) of the 
available glucose substrate into a water soluble capsular branched 
heteropolysaccharide composed of glucose and galactose in a molar ratio of 
2:1 with approximately 3% to 5% pyruvate. The negatively charged carboxyl 
groups of the pyruvate are thought to be primarily responsible for the 
biopolymer's high affinity for heavy metal ions. 
Due to the unique rheological and strong metal binding properties of the 
Zoogloea ramigera exocellular polysaccharide, it is desirable to isolate, 
characterize, express and modify the exopolysaccharide genes produced by 
Zoogloea ramigera strains. It is also desirable to provide the genes or 
complementary nucleotide sequences for production of exocellular 
polysaccharides produced by Zoogloea ramigera for use in synthesis of 
novel polysaccharides. 
It is therefore an object of the present invention to provide nucleotide 
sequences which can be used to produce novel biopolymers, especially the 
Zoogloea ramigera exopolysaccharides. 
It is still further object of the present invention to produce a system for 
production of biopolymers with enhanced rate and level of synthesis and 
greater ease in isolation of the biopolymers. 
SUMMARY OF THE INVENTION 
Two new bacterial strains designated Zoogloea ramigera 115SL and Zoogloea 
ramigera 115SLR have been developed. They have been designated ATCC 53589 
and ATCC 53590, respectively, as deposited on Mar. 5, 1987 with the 
American Type Culture Collection in Rockville Md. Strain 115SLR is a 
rifampicin resistant derivative of 115SL. These strains are derived from 
the wild type Zoogloea ramigera 115, ATCC 25935. The two new strains 
produce a novel exopolysaccharide (EPS) and have several desirable 
characteristics that are absent from the parent strain. 
The new strains, 115SL and 115SLR, have improved culture properties, as 
compared to the parent 115 strain, since they do not produce an EPS 
capsule layer like that of strain 115. The 115SL EPS is excreted as a 
slime layer which is not confined to the immediate area surrounding the 
cells. The result is that strains 115SL and 115SLR do not flocculate 
during growth, unlike 115 which forms large cell flocs during the growth 
phase. Since cells are not trapped within a floc where they grow at a 
reduced rate or die because of nutrient starvation, the new strains have 
the advantage of more consistent and reproducible growth cycles and 
increased growth rates. 
As a consequence of the higher cell density and healthier cultures, 
exopolysaccharide production is more consistent and titers are higher. The 
separation of the EPS from the cells is also much easier and more 
economical. 
In contrast to strain 115, a very important characteristic of strains 115SL 
and 115SLR is that they are able to receive foreign DNA using conventional 
techniques such as by conjugation, due to the absence of the capsule 
layer. This facilitates the application of recombinant DNA technology to 
control and produce novel expolysaccharides. 
Compositional analysis of the exopolysaccharide produced by 115SL and 
115SLR demonstrates that there are no differences between their EPS and 
the EPS of strain 115 with respect to monosaccharide composition. However, 
there is an increase of about 30 to 50% in the pyruvate content in the EPS 
from 115SL and 115SLR.

DETAILED DESCRIPTION OF THE INVENTION 
Isolation of new Z. ramigera strains 115SL and 115SLR and identification of 
their genes for polysaccharide synthesis enable development of strategies 
for the manipulation and control of the biosynthetic pathway for 
exopolysaccharide production at the genetic level. Strategies for 
controlled polymer production and structure include: placing the 
polysaccharide biosynthetic genes under the control of regulatable 
promoters; the introduction of these genes into new host stains to enable 
the development of more economic processes for polysaccharide production; 
mutagenesis of the genes to alter the enzyme activities and the resulting 
polymer structure; and the construction of novel pathways for 
polysaccharide synthesis by "mixing" genes from different strains of Z. 
ramigera or other organisms. 
The novel Z. ramigera strains were derived from Z. ramigera 115 (ATCC 
25935) obtained from the American Type Culture Collection Rockville, 
Maryland, as shown in FIG. 1. The Z. ramigera 115 strain was mutated by 
addition of 40 micrograms nitrosoguanidine (NTG)/ml of cells followed by 
incubation without shaking at 30.degree. C. Samples were treated for 
between zero and 60 minutes to establish a kill curve (concentration of 
cells decreased from 1.times.10.sup.7 at zero minutes to 
6.4.times.10.sup.5 at 60 minutes). The cells that were treated for twenty 
minutes (surviving concentration equal to 6.times.10.sup.6 cells) to 
thirty minutes (surviving concentration equal to 4.times.10.sup.6 cells) 
were then plated out. Cells were screened on plates containing Celluflor 
for loss of fluorescence, indicating loss of polysaccharide production, 
and a change in morpholopy. 
The Zoogloea ramigera strains were characterized by microscopy, 
morphological characterization and determination of the ability to grow 
and produce polysaccharide on different mediums. A complex medium and a 
defined medium were selected that contain all the requirements for growth 
and polysaccharide production and detection. 
The media and culture conditions are as follows: Z. ramigera cultures are 
stored frozen at -70.degree. C. in trypticase soy broth (TSB) medium 
containing 7% DMSO and 15% glycerol. The various Z. ramigera strains are 
routinely cultured in either the TSB medium or a defined medium, described 
by Norberg and Enfors in Appl. Env. Microbiol. 44, 1231-1237 (1982) having 
the following composition: 25 g glucose, 2 g K.sub.2 HPO.sub.4, 1 g 
KH.sub.2 PO.sub.4, 1 g NH.sub.4 Cl; 0.2 g MgSO.sub.4 7H.sub.2 O; 0.01 g 
yeast extract (Difco Laboratories) in one liter distilled water, where the 
glucose, MgSO.sub.4 7H.sub.2 O, yeast extract and salts are autoclaved 
separately. 100 ml cultures of Z. ramigera are grown on a rotary shaker 
(200 rpm) at 30.degree. C. in 500 ml baffled shake flasks for periods up 
to two weeks. 
Cellufluor (Polysciences Chemicals, Warrington, Pa.) is a fluorescent dye, 
disodium salt of 
4,4'-bis-[4-anilino-bis-diethyl-amino-S-triazin-2-ylamin]- 2,2'-stilbene-d 
isulfonic acid, that binds specifically to beta (1-3) and beta (1-4) 
glycosyl linkages and fluoresces when exposed to UV light. Cellufluor is 
added to agar plates, pH 7.4, at a concentration of 200 micrograms/ml and 
used to determine polysaccharide production in Z. ramigera. Strain 115 
flocculates and has a discernable polysaccharide capsule layer surrounding 
the cell flocs, as shown in FIG. 2, as well as a very unique colonial 
morphology. As shown in FIG. 1, the change in appearance is striking. The 
115SL and 115SLR strains of Z. ramigera do not flocculate. 
115SLR is a spontaneous rifampicin resistant mutant of 115SL and is 
particularly useful in selection processes during genetic manipulations. 
For example, it can be used to select against donor strains in experiments 
involving the conjugal transfer of plasmids and plasmid/gene libraries. 
Southern hybridization of .sup.32 P-pHP27, a 115/pLAFR3 recombinant plasmid 
that causes a change in the morphology of Z. ramigera I-16-M, was used to 
confirm that 115SL is derived from 115. 
The 115 polymer is purified by the addition of concentrated NaOH to the 
cell culture at a final concentration of 0.2M, followed by the addition of 
3 volumes of ethanol to precipitate the polymer and other materials. The 
precipitate is collected and redissolved in half the original volume of 
water. Protein is removed by either extracting twice with phenol or by 
ultrafiltration. The aqueous phase is dialyzed, lyophilized and ground to 
yield a fine white powder. 
Since it is not cell-bound, the exopolysaccharide produced by 115SL and 
115SLR is purified without using the alkali treatment or the phenol 
extraction. Not only is the purification process thereby simplified, it 
does not remove alkali-labile acetyl moieties from the purified 115SL and 
115SLR exopolysaccharide. Since all previously used purification 
procedures have included an alkali step, this is believed to be the first 
time the exopolysaccharide has been available in purifed form with acetate 
groups present on the polymer. 
Total carbohydrate concentration in culture broths and polymer solutions is 
determined by the phenol reaction, described by Gerhardt in Manual of 
Methods for General Bacteriol. (Washington Amer. Soc. Microbiol. 1981). 
Glucose, galactose and Xanthan gum (Sigma Chemical Co., St. Louis, Mo.) 
are used as standards. Total protein concentration in culture broths and 
polymer solutions is determined using the Bio-Rad protein assay (Bio-Rad 
Laboratories, Richmond, Calif., 1979). Lysozyme is used as the standard. 
Cellular protein is released by boiling in 0.2N NaOH. 
Purified polysaccharide is hydrolyzed in 1M trifluoroacetic acid at 
120.degree. C. for times varying between 30 minutes and 2 hours. 
Monosaccharides in the polysaccharide hydrolysate are separated using a 
Waters HPLC equipped with a Brownlee Polypore PB, lead loaded cation 
exchange column, operated at 85.degree. C., with water as the eluent. 
Detection is by refractive index using a Waters Model 401 Differential 
Refractometer. The polysaccharide can further characterized by proton NMR 
spectroscopy and infared spectroscopy. Infrared analysis, along with the 
monosaccharide composition data, can be compared to the composition and IR 
scans of polysaccharides from mutant or genetically manipulated strains to 
detect changes in structure. 
The polymer produced by Zoogloea ramigera 115SL consists of glucose and 
galactose in a ratio of approximately 2:1, the same as the 
exopolysaccharide produced by strain 115. The pyruvate content is slightly 
higher in the 115SL and 115SLR strains, however, by approximately 30 to 
50%. 
The isolated exopolysaccharide produced by Z. ramigera 115 SL and 115 SLR 
has a number of uses similar to that of the exopolysaccharide isolated for 
the parent strain. For example, the polymer can be used as a flocculant, 
in the isolation and removal of heavy metal ions, and as a viscosity 
modifying agent. However, although similar to the 115 exopolysaccharide, 
some differences in application are expected due to the slightly higher 
pyruvate level and the presence of the acetate moiety. 
The new strains, 115SL and 115SLR, are useful in ways that the parent 
strain 115 is not. For example, it produces higher, more consistent levels 
of polysaccharide which is more easier purified since the cell floc 
present in 115 is absent. The number of cells which can be grown in 
culture is also greater due to the easier diffusion of nutrients into the 
cell. The differences in cell growth and polymer production are 
demonstrated in FIGS. 3A and 3B. The total amount of EPS (mg/l) produced, 
as well as the total amount released into the medium over time as a 
percentage of the total EPS produced (mg/l), for 115 (FIG. 3A) is 
significantly less than that for 115SL (FIG. 3B). The initial growth curve 
for 115SL (FIG. 3B), shown by the total protein, is also much sharper than 
it is for 115 (FIG. 3B). 
The primary advantage of the new 115SL and 115SLR strains, however, is that 
they can be more easily manipulated genetically. As demonstrated below, 
the 115SL and 115SLR strains can accept foreign or plasmid DNA in a 
relatively high yield by conjugal transfer without extensive manipulation. 
This allows not only the isolation, characterization and modification of 
the genes for exopolysaccharide production but also the insertion of genes 
encoding additional enzymes for synthesis of biopolymers. Methods for 
manipulation and construction of pathways for polymer synthesis are 
described in detail in co-pending application. U.S. Ser. No. 07/3429,594 
filed Mar. 28, 1989, now abandoned. 
As an example of the application of these methods to Z. ramigera 115SL and 
115SLR, transposon mutants of Z. ramigera 115SL were constructed by the 
method described in pending, U.S. Ser. No. 07/329,594 filed Mar. 28, 1989, 
now abandoned and screened for changes in exopolysaccharide composition 
and production, which describes the transfer of a Tn5 containing 
derivative of pRK 2013 in Z. ramigera strains using E. coli MM294A by 
conventional techniques for conjugation. Several Tn5 mutants that were 
non-fluorescent on Cellufluor were recovered. Although not adsorbing 
Cellufluor, the mutants are producing exopolysaccharide. These polymers 
have apparently undergone a structural or compositional change from that 
of the original exopolysaccharide. 
A pLAFR3/115 gene library was then introduced into these mutants. The 
resulting transconjugants were screened for fluorescence on Cellufluor and 
for changes in colony morphology. One class of complemented strains 
regained the ability to fluoresce on Cellufluor. A second class of 
complemented strains had colony morphology identical to that of the 
wild-type 115. 
Modifications and variations of the present invention, a method and means 
for producing genetically manipulated biopolymers including the 
exopolysaccharides produced by Zoogloea ramigera strains, will be obvious 
to those skilled in the art from the foregoing detailed description. Such 
modifications and variations are intended to come within the scope of the 
following claims.