Isolation and physico-chemical characterisation of extracellular polymeric substances produced by the marine bacterium Vibrio parahaemolyticus

A marine bacterial strain identified as Vibrio parahaemolyticus by 16S rRNA gene (HM355955) sequencing and gas chromatography (GC) coupled with MIDI was selected from a natural biofilm by its capability to produce extracellular polymeric substances (EPS). The EPS had an average molecule size of 15.278 μm and exhibited characteristic diffraction peaks at 5.985°, 9.150° and 22.823°, with d-spacings of 14.76661, 9.29989 and 3.89650 Å, respectively. The Fourier-transform infrared spectroscopy (FTIR) spectrum revealed aliphatic methyl, primary amine, halide groups, uronic acid and saccharides. Gas chromatography mass spectrometry (GCMS) confirmed the presence of arabinose, galactose, glucose and mannose. ¹HNMR (nuclear magnetic resonance) revealed functional groups characteristic of polysaccharides. The EPS were amorphous in nature (CI_xrd 0.092), with a 67.37% emulsifying activity, thermostable up to 250°C and displayed pseudoplastic rheology. MALDI-TOF–TOF analysis revealed a series of masses, exhibiting low-mass peaks (m/z) corresponding to oligosaccharides and higher-mass peaks for polysaccharides consisting of different ratios of pentose and hexose moieties. This is the first report of a detailed characterisation of the EPS produced by V. parahaemolyticus, which could be further explored for biotechnological and industrial use.     

Production of Extracellular Polysaccharide by a Rhizobium Species from Root Nodules of the Leguminous Tree Dalbergia lanceolaria

The ability of the Rhizobium D1 10 species, which was isolated from the root nodules of the leguminous forest tree Dalbergia lanceolaria, for the production of extracellular polysaccharides (EPS) was investigated. High amounts of EPS (765 μg/mL) were produced by the bacteria (Rhizobium D1 10) in yeast extract mannitol medium. Both growth and EPS production started simultaneously, but the EPS production was at its maximum in the stationary phase of growth at 32 h. The EPS production was maximal when the medium was supplemented with mannitol (2 %), thiamine hydrochloride (1 μg/mL) and KNO₃ (0.1 %), which was accompanied by a great increase in the production compared to the control. The EPS contained xylose, rhamnose, glucose, galactose and arabinose. The possible role of rhizobial EPS production in root nodule symbiosis is discussed.     

Production of extracellular polysaccharides by a Rhizobium species from the root nodules of Melilotus alba

The Rhizobium sp., isolated from the root nodules of the leguminous fodder herb Melilotus alba, produced large amounts of extracellular polysaccharides (EPS) (963.5 μg/ml) in a yeast extract mannitol medium. Growth and EPS production started simultaneously, but EPS production reached its maximum during the stationary phase of growth of the bacteria, at 20 hours. EPS production was increased with all of the thirteen sugars tested. Different nitrogen sources, such as nitrates, glutamic acid, casamino acid and L-asparagine, increased the EPS production although it was inhibited by glycine, nitrite and ammonium salts. Among the vitamins and metal ions, only pyridoxal phosphate and ZnSO₄ promoted EPS production. Attempts were made to optimize the cultural requirements for growth and maximum EPS production. Maximum EPS production (1457.0 μg/ml) was obtained when the medium was supplemented with glucose (1%), pyridoxal phosphate (2 μ g/ml), ZnSO₄ × 7 H₂O (10 μg/ml) and glutamic acid (0.1%). Under these conditions, the production was increased by 254.3% compared to the control. The EPS contained arabinose, xylose and rhamnose monomers. The presence of arabinose and xylose in the EPS produced by a Rhizobium sp. was uncommon.     

Production of extracellular polysaccharides by a Rhizobium species from root nodules of Cajanus cajan

The ability of the Rhizobium sp., isolated from the root nodules of the leguminous pulse yielding shrub Cajanus cajan, to produce extracellular polysaccharides (EPS) was checked. A large amount of EPS (1, 128 μg/ml) was produced by the bacteria in yeast extract mannitol medium. Growth and EPS production started simultaneously, but the production reached its maximum level in the stationary phase of growth at 28 h. The EPS production by this Rhizobium sp. was much higher than by many other strains from nodules of Cajanus cajan which took a much longer time to reach maximum EPS production than this strain. The maximum EPS production (2,561 μg/ml) was obtained when the medium was supplemented with mannitol (1%), cetyl pyridinium chloride (2 μg/ml) and KNO₃ (0.2%), in which the production was increased by 276% compared to the control. The EPS production rose in the period up to 65 h with increased mannitol concentration. The EPS contained arabinose, xylose and rhamnose monomers. The possible role of rhizobial EPS production in root nodule symbiosis is discussed.     

Structural and Functional Properties of Exopolysaccharide Excreted by a Novel <Emphasis Type="Italic">Bacillus anthracis</Emphasis> (Strain PFAB2) of Hot Spring Origin

Exopolysaccharide produced by a unique avirulent Bacillus anthracis strain PFAB2 of hot spring origin has been characterized and its functional properties are investigated which is a first report. Maximum yield of EPS is 7.66 g/l with 2% glucose and 1% peptone as optimum carbon and nitrogen source respectively. The EPS is found to be a homopolymer consisting of only glucose as principle monosaccharide component. Through ¹H NMR study, different dextran-like proton peaks are observed. Molecular weight of the EPS resembles low molecular weight bacterial origin polysaccharides. Melting transition of the EPS has started after 276 °C which indicates good thermal stability. The EPS also shows potent antioxidant activity in terms of DPPH and ABTS mediated free radical scavenging property compared to standard ascorbic acid. Emulsifying property of the EPS is also observed and has shown good emulsification of vegetable oils. The polysaccharide forms a thermo resistant gel during the heating phase, with G′ higher than G″ indicating excellent shear-thinning behaviour and viscoelastic nature of the EPS.     

Yield and physicochemical properties of EPS from Halomonas sp. strain TG39 identifies a role for protein and anionic residues (sulfate and phosphate) in emulsification of n‐hexadecane

In this study, we investigated the yield and physicochemical properties of the high molecular weight extracellular polymeric substance (HMW–EPS) produced by Halomonas sp. strain TG39 when grown on different types and ratios of substrates. Glucose (1% w/v) and a peptone/yeast extract ratio of 5.1 (0.6% w/v final concentration) yielded an EPS fraction (HMW‐glucose) exhibiting the highest anionic activity (20.5) and specific emulsifying activity (EI₂₄ = 100%) compared to EPS produced by cells grown on mannitol, sucrose, malt extract or no carbon source. The HMW–EPS fractions were capable of binding ≈255–464 mg of methylene blue (MB) per gram of EPS, which represents the highest reported binding of MB by a bacterial EPS. A comparative evaluation of these properties to those of commercial hydrocolloids indicated that the combined effect of protein and anionic residues of the HMW–EPS contributed to its ability to emulsify n‐hexadecane. Liquid chromatography revealed the HMW‐glucose EPS to be a heterogeneous polymer with a polydispersity index of 1.8. This work presents evidence of a correlation between the anionic nature and protein content of bacterial EPS with its emulsifying qualities, and identifies EPS produced by strain TG39 as a high MB‐binding bacterial sorbant with potential biotechnological application. Biotechnol. Bioeng. 2009;103: 207–216. © 2008 Wiley Periodicals, Inc.     

Studies on the Extracellular Polysaccharides (EPS) Produced in vitro by Pseudomonas phaseolicola

Native EPS produced by Pseudomonas syringae pv. phaseolicola in vitro was separated by ion exchange chromatography on DEAE fractogel into three different polysaccharide fractions. A neutral polysaccharide eluting with the void volume yielded only fructose upon hydrolysis and exhibited an IR spectrum similar to authentic levan. At about 300 mM KCl a mannuronan eluted. Comparison with authentic alginate by IR spectroscopy, elution behaviour during DEAE-fractogel column chromatography, and monomer composition (mannuronic acid and traces of guluronic acid) confirmed the identity of this fraction as a bacterial alginate. It contained about 56 mol% acetyl groups. A third polysaccharide eluted at about 160 mM KCl. Its monomeric composition (rhamnose, fucose, glucose, and amino sugars), elution behaviour upon DEAE-fractogel column chromatography, and TLC patterns, closely resembled the sugar moiety of lipopolysaccharides (LPS) from, Pseudomonas syringae pv. phaseolicola. The protein component of crude EPS represented a fourth macromolecular fraction. It was not covalently linked to any of the polysaccharides since it could be removed from the EPS by phenol extraction.     

Exopolysaccharide production by a new Halomonas strain CRSS isolated from saline lake Cape Russell in Antarctica growing on complex and defined media

A haloalkalophilic Halomonas strain CRSS, isolated from salt sediments in Antarctica, produced exocellular polysaccharides (EPS) up to 2.9gg^-1 dry cells. Acetate was the most efficient carbon source for EPS production. The composition of media strongly affected the nature of the polymers; a mannan and a xylo-mannan, were obtained when cells were grown on complex media. Acetate was the most efficient carbon source for EPS production and in presence of this substrate, a new polysaccharide, a fructo-glucan, was produced. The EPS fraction was composed by glucose, fructose, glucosamine and galactosamine in relative proportions of 1:0.7:0.3:trace.Revisions requested; Revisions received 6 September 2004     

Exopolysaccharide production by a unicellular freshwater cyanobacterium Cyanothece sp. isolated from a rice field in Vietnam

A unicellular cyanobacterium that produces a large amount of exopolysaccharide (EPS) was isolated from a rice field in Phu Tho Province, Vietnam. Morphological characteristics and phylogenetic analysis using a partial sequence of the 16S rRNA gene revealed that the isolate was closely related to the genus Cyanothece. The isolate, named Cyanothece sp. Viet Nam 01, grew at a wide range of temperatures (25–40 °C), but was not viable below 20 °C. The isolate had an ability of aerobic nitrogen fixation. The EPS was purified using NaOH extraction and ethanol precipitation, and the absolute molecular weight was estimated to be 4.5?×?10⁴ kDa. The pattern of the Fourier transform infrared spectrum indicated that the EPS had carbonyl and sulfate groups, as well as the typical functional groups of sugars. The uronic acid and sulfur contents were 23 and 8.4 mol% per total monosaccharide, respectively. The EPS constituent monosaccharides were rhamnose, glucose, galactose, xylose, mannose, fucose, arabinose, ribose, and unknown sugar, with molar compositions of 38.6:13.8:4.8:4.8:2.4:3.5:2.0:0.6:6.5, respectively.     

Isolation and physico-chemical characterisation of extracellular polymeric substances produced by the marine bacterium Vibrio parahaemolyticus

A marine bacterial strain identified as Vibrio parahaemolyticus by 16S rRNA gene (HM355955) sequencing and gas chromatography (GC) coupled with MIDI was selected from a natural biofilm by its capability to produce extracellular polymeric substances (EPS). The EPS had an average molecule size of 15.278 μm and exhibited characteristic diffraction peaks at 5.985°, 9.150° and 22.823°, with d-spacings of 14.76661, 9.29989 and 3.89650 ?, respectively. The Fourier-transform infrared spectroscopy (FTIR) spectrum revealed aliphatic methyl, primary amine, halide groups, uronic acid and saccharides. Gas chromatography mass spectrometry (GCMS) confirmed the presence of arabinose, galactose, glucose and mannose. (1)HNMR (nuclear magnetic resonance) revealed functional groups characteristic of polysaccharides. The EPS were amorphous in nature (CI(xrd) 0.092), with a 67.37% emulsifying activity, thermostable up to 250°C and displayed pseudoplastic rheology. MALDI-TOF-TOF analysis revealed a series of masses, exhibiting low-mass peaks (m/z) corresponding to oligosaccharides and higher-mass peaks for polysaccharides consisting of different ratios of pentose and hexose moieties. This is the first report of a detailed characterisation of the EPS produced by V. parahaemolyticus, which could be further explored for biotechnological and industrial use.     

The extracellular polysaccharide produced by <Emphasis Type="Italic">Rhizobium</Emphasis> sp. isolated from the root nodules of <Emphasis Type="Italic">Phaseolus mungo</Emphasis>

The symbiont isolated from root nodules of Phaseolus mungo L., a widely grown legume in India was identified as a Rhizobium sp. a Rhizobium sp. close to R. multihospitium based on a biochemical and 16S rRNA gene-based phylogenetic approach. This Rhizobium sp. was able to produce large amounts of extracellular polysaccharides (EPS) in a yeast extract mannitol (YEM) broth medium. Both growth and EPS production started simultaneously though each had different stationary phases. EPS production increased enormously with supplementation by the preferred carbon, nitrogen and vitamin sources. Attempts were made to optimize the cultural requirements for maximum growth and maximum EPS production. The EPS produced by the symbiont contained large amount of mannose together with small amounts of arabinose and xylose. The possible role of EPS production on the Rhizobium—root nodule symbiosis is briefly discussed.     

STUDIES ON POLYSACCHARIDES FROM THREE EDIBLE SPECIES OF NOSTOC (CYANOBACTERIA) WITH DIFFERENT COLONY MORPHOLOGIES: COMPARISON OF MONOSACCHARIDE COMPOSITIONS AND VISCOSITIES OF POLYSACCHARIDES FROM FIELD COLONIES AND SUSPENSION CULTURES

Hot water-soluble polysaccharides were extracted from field colonies and suspension cultures of Nostoc commune Vaucher, Nostoc flagelliforme Berkeley et Curtis, and Nostoc sphaeroides Kützing. Excreted extracellular polymeric substances (EPS) were isolated from the media in which the suspension cultures were grown. The main monosaccharides of the field colony polysaccharides from the three species were glucose, xylose, and galactose, with an approximate ratio of 2:1:1. Mannose was also present, but the levels varied among the species, and arabinose appeared only in N. flagelliforme. The compositions of the cellular polysaccharides and EPS from suspension cultures were more complicated than those of the field samples and varied among the different species. The polysaccharides from the cultures of N. flagelliforme had a relatively simple composition consisting of mannose, galactose, glucose, and glucuronic acid, but no xylose, as was found in the field colony polysaccharides. The polysaccharides from cultures of N. sphaeroides contained glucose (the major component), rhamnose, fucose, xylose, mannose, and galactose. These same sugars were present in the polysaccharides from cultures of N. commune, with xylose as the major component. Combined nitrogen in the media had no qualitative influence on the compositions of the cellular polysaccharides but affected those of the EPS of N. commune and N. flagelliforme. The EPS of N. sphaeroides had a very low total carbohydrate content and thus was not considered to be polysaccharide in nature. The field colony polysaccharides could be separated by anion exchange chromatography into neutral and acidic fractions having similar sugar compositions. Preliminary linkage analysis showed that 1) xylose, glucose, and galactose were 1→4 linked, 2) mannose, galactose, and xylose occurred as terminal residues, and 3) branch points occurred in glucose as 1→3,4 and 1→3,6 linkages and in xylose as a 1→3,4 linkage. The polymer preparations from field colonies had higher kinematic viscosities than those from correspondingsuspension cultures. The high viscosities of the polymers suggested that they might be suitable for industrial uses.     

Isolation and Characterization of Exopolysaccharide Secreted by a Toxic Dinoflagellate, <Emphasis Type="Italic">Amphidinium carterae</Emphasis> Hulburt 1957 and Its Probable Role in Harmful Algal Blooms (HABs)

Extracellular polymeric substances (EPS) produced by a toxic dinoflagellate Amphidinium carterae Hulburt 1957 was isolated and characterized. Molecular masses of the EPS were about 233 and 1,354 kDa. Spectral analyses by ¹H nuclear magnetic resonance and Fourier Transformed–Infrared Spectroscopy revealed the characteristic of the functional groups viz. primary amine, carboxyl, halide, and sulfate groups present in the EPS. However, five elements (C, O, Na, S, and Ca) were detected by scanning electron microscopy - energy dispersive X-ray spectroscopy (SEM-EDX) analysis. X-ray diffraction and differential scanning calorimetric analysis confirmed the amorphous nature of EPS, which was comprised of an average particle size of 13.969 μm (d 0.5) with 181 nm average roughness. Two monosaccharide constituents, galactose (73.13%) and glucose (26.87%) were detected by gas chromatography–mass spectroscopy analysis. Thermal gravimetric analysis revealed that degradation of EPS obtained from A. carterae takes place in three steps. The EPS produced by A. carterae was found to be beneficial for the growth of both A. carterae and Bacillus pumilus. The potential heterogeneous properties of EPS may play an important role in harmful algal bloom.     

Effects of yeast extract on the production and the quality of the exopolysaccharide, zooglan, produced by Zoogloea ramigera 115SLR

Although many studies have examined the influence of culture conditions on the production and composition of polysaccharides, little is known about the factors influencing the quality of exopolysaccharides (EPS). In this work we studied the effect of yeast extract on the production, composition and molecular weight of the EPS zooglan produced by Zoogloea ramigera 115SLR. This bacterium was grown on a new completely defined synthetic medium and on a medium containing yeast extract. Growth and polysaccharide production performances were comparable on the two media with a glucose to exopolysaccharide conversion yield of 35% (g/g). The polysaccharides produced on these two media have an identical composition but a different molecular weight and molecular weight distribution. The yeast extract medium leads to a more homogeneous polysaccharide solution. Received: 12 June 1998 / Received revision: 19 September 1998 / Accepted: 11 October 1998     

Cryoprotective properties and preliminary characterization of exopolysaccharide (P-Arcpo 15) produced by the Arctic bacterium Pseudoalteromonas elyakovii Arcpo 15

Twenty-two bacterial strains that secrete exopolysaccharides (EPS) were isolated from marine samples obtained from the Chukchi Sea in the Arctic Ocean; of these, seven strains were found to be capable of producing cryoprotective EPS. The ArcPo 15 strain was isolated based on its ability to secrete large amounts of EPS, and was identified as Pseudoalteromonas elyakovii based on 16S rDNA analysis. The EPS, P-ArcPo 15, was purified by protease treatment and gel filtration chromatography. The purified EPS (P-ArcPo 15) had a molecular mass of 1.7 × 10⁷ Da, and its infrared spectrum showed absorption bands of hydroxyl and carboxyl groups. The principal sugar components of P-ArcPo 15 were determined to be mannose and galacturonic acid, in the ratio of 3.3:1.0. The cryoprotective properties of P-ArcPo 15 were characterized by an Escherichia coli viability test. In the presence of 0.5% (w/v) EPS, the survival percentage of E. coli cells was as high as 94.19 ± 7.81% over five repeated freeze–thaw cycles. These biochemical characteristics suggest that the EPS P-ArcPo 15 may be useful in the development of cryoprotectants for biotechnological purposes, and we therefore assessed the utility of this novel cryoprotective EPS.     

Chemical composition of extracellular polysaccharides of cowpea rhizobia

The extracellular polysaccharides (EPS) of six strains of cowpea rhizobia were examined. The strains (MI50A, M6-7B, IRC253) produced polysaccharides containing glucose, galactose and mannose in a molar ratio of 2:1.1:1, 1:1.3:3.1 and 1:1.3:3.5 respectively. Two strains (513-B and Ez-Aesch) produced polysaccharides containing galactose and mannose in a molar ratio of 2:3. Mannose was the only sugar detected in the EPS of strain IRC291. Pyruvate, acetate, glucuronic acid and galacturonic acid were not detected in any strain.Abbreviations EPS Extracellular polysaccharide - YEMA yeast-extract mannitol agar - YEMB yeast extract mannitol broth     

DAILY FLUCTUATIONS OF EXOPOLYMERS IN CULTURES OF THE BENTHIC DIATOMS CYLINDROTHECA CLOSTERIUM AND NITZSCHIA SP. (BACILLARIOPHYCEAE)1

Dynamics in the production of extracellular polymeric substances (EPS) were investigated for the benthic diatoms Cylindrotheca closterium (Ehrenberg) and Nitzschia sp. The effect of growth phase and light:dark conditions were examined using axenic cultures. Two EPS fractions were distinguished. Soluble EPS was recovered from the culture supernatant and represented polysaccharides that were only loosely associated with the cells. Bound EPS was extracted from the cells using warm (30° C) water and was more closely associated with the diatom aggregates. Concentrations of EPS exceeded internal concentrations of sugar throughout growth, indicating that EPS production is important in these organisms. Soluble and bound EPS revealed distinct differences in daily dynamics during the course of growth. Soluble EPS was produced continuously once cultures entered the stationary phase. During the stationary phase, chl a‐normalized EPS production rates equaled 6.4 and 3.4 d ^{? 1} for C. closterium and Nitzschia sp., respectively. In contrast, production of bound EPS occurred only in the light and was highest during the exponential phase. Up to 90% of the attached EPS that was produced in the light was degraded during the subsequent dark period. The monosaccharide distribution of EPS was constant during the course of the experiment. The soluble EPS consisted of high amounts of galactose and glucuronic acid, relative to rhamnose, glucose, xylose/mannose, and galacturonic acid. In contrast, glucose was the dominant monosaccharide present in the bound EPS. These differences suggest that the production of the two distinct EPS fractions is under different metabolic controls and probably serves different cellular functions.     

Characterization of extracellular polysaccharides from<Emphasis Type="Italic">Nostoc flagelliforme</Emphasis> cells in liquid suspension culture

Nostoc flagelliforme cells were studied with regard to the physico-chemical characterization of the extracellular polysaccharides (EPS) secreted in a liquid suspension culture. The hydrolyzed EPS were determined to be composed of four neutral sugars, which were glucose (43.2%), xylose (20.6%), galactose (29.9%), and mannose (6.3%). The glucuronic acid was the only uronic acid identified in the residue. The apparent molecular weight was estimated at 2.79×10⁵. The Fourier transform infrared spectra showed that the EPS evidenced characteristics typical of non-sulfated polysaccharides. The UV spectrum and Bradford reaction indicated that there were no nucleic acids and proteins in them. The thermal analysis showed a decomposition peak at 245°C on the thermogravimetric (TG) curves. The scanning electron microscope (SEM) analysis indicated that the EPS possessed a porous structure. The observed microstructural irregularities indicated that the polysaccharide was a type of amorphous solid. These results showed that the EPS ofN. flagelliforme cells might be ernployed as a substitute for those normally derived from field colonies. The results of this study may prove to be beneficial to the protection of the natural resource represented byN. flagelliforme.     

Production and Characterization of the Intra‐ and Extracellular Carbohydrates and Polymeric Substances (EPS) of Three Sea‐Ice Diatom Species,and Evidence for a Cryoprotective Role for EPS

Diatoms and their associated extracellular polymeric substances (EPS) are major constituents of the microalgal assemblages present within sea ice. Yields and chemical composition of soluble and cell‐associated polysaccharides produced by three sea‐ice diatoms, Synedropsis sp., Fragilariopsis curta, and F. cylindrus, were compared. Colloidal carbohydrates (CC) contained heteropolysaccharides rich in mannose, xylose, galactose, and glucose. Synedropsis sp. CC consisted mainly of carbohydrates <8 kDa size, with relatively soluble EPS, compared to high proportions of less‐soluble EPS produced by both Fragilariopsis spp. F. curta colloidal EPS contained high concentrations of amino sugars (AS). Both Fragilariopsis species had high yields of hot bicarbonate (HB) soluble EPS, rich in xylose, mannose, galactose, and fucose (and AS in F. cylindrus). All species had frustule‐associated EPS rich in glucose–mannose. Nutrient limitation resulted in declines in EPS yields and in glucose content of all EPS fractions. Significant similarities between EPS fractions from cultures and different components of natural EPS from Antarctic sea ice were found. Increased salinity (52) reduced growth, but increased yields of EPS in Fragilariopsis cylindrus. Ice formation was inhibited byF. cylindrus, EPS, and by enhanced EPS content (additional xanthan gum) down to ?12°C, with growth rate reduced in the presence of xanthan. Differences in the production and composition of EPS between Synedropsis sp. and Fragilariopsis spp., and the association between EPS, freezing and cell survival, supports the hypothesis that EPS production is a strategy to assist polar ice diatoms to survive the cold and saline conditions present in sea ice.     

Vergleich eines "glatten" und eines "rauhen" Stammes von Pseudomonas syringae pv. phaseolicola

Comparison of a “smooth” and a “rough” isolate of Pseudomonas syringae pv. phaseolicola The “smooth” (S) wild strain of Pseudomonas syringae pv. phaseolicola was compared with a “rough” (R) variant of low virulence. Both strains grew nearly equally well on a sucrose containing medium with yeast extract and casamino acids, and the strains did not differ markedly in the quantity of produced EPS (= extracellular polysaccharides). Principally the same results were obtained for high and medium concentrations of sucrose, or when sucrose was replaced by glucose or fructose. However, on glucose and fructose considerably lower quantities of EPS were produced. The biological activity of S-EPS was higher than that of R-EPS. This difference between the EPS preparations was not as marked as leaf inoculation with both bacterial isolates. After prolonged bacterial culture the EPS-production increased further, so that the differences between both strains decreased. A different EPS type was produced on the glycerol containing medium of KING B. Variations in the composition of this medium resulted in different morphology of the agar grown cultures, and the relative differences between S and R bacteria changed. When 62 different physiological tests for both bacterial strains were compared, the “rough” bacteria revealed a lowered range of positive reactions, with a few exceptions. However, it appeared unlikely that the reduced virulence of the “rough” bacteria was due to these differences. Obviously, defects in the extracellular products, but not in levan, were responsible for the reduction of virulence.