The production-influencing factors of extracellular polysaccharide (EPS) from a strain of lactic acid bacteria and EPS extraction

The influencing factors of extracellular polysaccharide (EPS) produced from a strain of lactic acid bacteria (LAB L15) were studied by using the phenol-H₂SO₄ method. It was demonstrated that the strain produced EPS at the most amount when it was incubated for 40–48 h and when the pH value was 4 under 30°C. Glucose was the most suitable carbon source for LAB-producing EPS. The rough EPS was obtained from L15 culture after centrifugation, dialysis, deprotein, decoloration, and ethanol-precipitation. The sample was at least composed of two polysaccharides that were completely different in molecular weight and the amount. The purified EPS was passed through the SephadexG-200 column and it showed that it was a sample purified by thin layer chromatography. __________ Translated from Microbiology, 2005, 32(4): 85–90 [译自: 微生物学通报, 2005, 32(4): 85–90]     

Isolation and characterization of mucous exopolysaccharide (EPS) produced by Vibrio furnissii strain VB0S3

Marine bacterial strains were isolated from coastal regions of Goa and screened for the strains that produce the highest amount of mucous exopolysaccharide (EPS). Our screening resulted in the identification of the strain Vibrio furnissii VB0S3 (hereafter called VB0S3), as it produced the highest EPS in batch cultures during the late logarithmic growth phase. The isolate was identified as VB0S3 based on morphological and biochemical properties. Growth and EPS production were studied in mineral salts medium supplemented with NaCl (1.5%) and glucose (0.2%). The exopolymer was recovered from the culture supernatant by using three volumes of cold ethanol precipitation and dialysis procedure. Chemical analyses of EPS revealed that it is primarily composed of neutral sugars, uronic acids, and proteins. Fourier-transform infrared (FT-IR) spectroscopy revealed the presence of carboxyl, hydroxyl, and amide groups, which correspond to a typical heteropolymeric polysaccharide, and the EPS also possessed good emulsification activity. The gas chromatographic analysis of an alditol-acetate derivatized sample of EPS revealed that it was mainly composed of galactose and glucose. Minor components found were mannose, rhamnose, fucose, ribose, arabinose, and xylose. EPS was readily isolated from culture supernatants, which suggests that the EPS was a slime-like exopolysaccharide. This is the first report of exopolysaccharide characterization that describes the isolation and characterization of an EPS expressed by Vibrio furnissii strain VB0S3. The results of the study contribute significantly and go a long way towards an understanding of the correlation between growth and EPS production, chemical composition, and industrial applications of the exopolysaccharide in environmental biotechnology and bioremediation.     

Purification and properties of extracellular polysaccharide (EPS) antigens produced by different mould species

Extracellular polysaccharide (EPS) antigens produced by different mould species were purified and partially characterized. Purification included (NH₄)₂SO₄ treatment, Sepharose CL-4B column chromatography and Con A-sepharose chromatography. The EPS of Penicillium digitatum, Mucor racemosus and Cladosporium cladosporioides showed high antigenic capacities. Immunologically the EPS were partially genus-specific, but cross-reactivity was observed. The EPS antigens produced by species of Penicillium, Aspergillus repens and Geotrichum candidum lost their immunological activity upon heating (100C) at pH 18, while the EPS antigen of M. racemosus, Rhizopus oligosporus and C. cladosporioides were stable under the same conditions. The dominant monosaccharides present in the EPS antigen were mannose, galactose and glucose. The EPS obtained from cultures of M. racemosus and R. oligosporus also contained rhamnose. In the EPS produced by Penicillium spp. and A. repens the galactose residues were determined to be immunodominant.     

Purification and properties of extracellular polysaccharide (EPS) antigens produced by different mould species

Extracellular polysaccharide (EPS) antigens produced by different mould species were purified and partially characterized. Purification included (NH4)2SO4 treatment, Sepharose CL-4B column chromatography and Con A-sepharose chromatography. The EPS of Penicillium digitatum, Mucor racemosus and Cladosporium cladosporioides showed high antigenic capacities. Immunologically the EPS were partially genus-specific, but cross-reactivity was observed. The EPS antigens produced by species of Penicillium, Aspergillus repens and Geotrichum candidum lost their immunological activity upon heating (100 degrees C) at pH 1.8, while the EPS antigen of M. racemosus, Rhizopus oligosporus and C. cladosporioides were stable under the same conditions. The dominant monosaccharides present in the EPS antigen were mannose, galactose and glucose. The EPS obtained from cultures of M. racemosus and R. oligosporus also contained rhamnose. In the EPS produced by Penicillium spp. and A. repens the galactose residues were determined to be immunodominant.     

Isolation and characterization of exopolysaccharide produced by Vibrio harveyi strain VB23

AIMS: The aim of the study was to isolate and characterize exopolysaccharide (EPS) produced by Vibrio harveyi strain VB23. METHODS AND RESULTS: Growth and EPS production by V. harveyi strain VB23, was studied in mineral salts medium supplemented with NaCl (1.5%) and glucose (0.2%). The rate of EPS production in batch cultures was highest during the late log phase of growth when compared with stationary growth phase. The exopolymer was recovered from the culture supernatant by using a cold ethanol precipitation-dialysis procedure. Chemical analyses of EPS revealed that it is primarily composed of neutral sugars, uronic acids, proteins and sulfates. The purified EPS revealed prominent functional reactive groups, such as hydroxyl, carboxylic and amides, which correspond to a typical heteropolymeric polysaccharide and the EPS, also possessed good emulsification activity. The gas chromatographic analysis of an alditol acetate-derivatized sample of EPS revealed that it is composed primarily of galactose and glucose. Minor components found were rhamnose, fucose, ribose, arabinose, xylose and mannose. CONCLUSIONS: The EPS produced by V. harveyi strain VB23 is a heteropolysaccharide possessing good emulsification activity. EPS was readily isolated from culture supernatants, which suggests that the EPS was a slime-like EPS. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of EPS characterization in luminous V. harveyi bacteria, which describes the isolation and characterization of an EPS expressed by V. harveyi. The results of the study contributes significantly towards an understanding of the chemical composition and applications of the EPS in environmental biotechnology and bioremediation.     

Isolation of an extracellular polysaccharide (EPS) depolymerase produced byBradyrhizobium japonicum

Bradyrhizobium japonicum is capable of producing an acidic, high molecular weight, extracellular polysaccharide (EPS). An enzyme exhibiting EPS depolymerase activity was detected in cell lysates ofB. japonicum strain 2143. The depolymerase was active against the EPS produced by strain 2143 and the closely related EPS produced by strain 311b 110. Depolymerase activity was characterized by its ability to decrease the viscosity of EPS solutions, to decrease the molecular weight of EPS, and to catalyze the release of reducing groups from EPS. The depolymerase exhibited a sharp activity optimum at pH 6 and had a molecular weight of approximately 45 kD as determined by gel permeation chromatography. Analysis of depolymerase-treated EPS indicates that the enzyme acts as an endo-depolymerase, producing a relatively narrow size range of high molecular weight products.Contribution from the Missouri Agricultural Experiment Station, Journal Series Number 10:959.     

Structural elucidation of the EPS of slime producing Brevundimonas vesicularis sp. isolated from a paper machine

The slime forming bacteria Brevundimonas vesicularis sp. was isolated from a paper mill and its EPS was produced on laboratory scale. After production, the exopolysaccharide (EPS) was purified and analysed for its purity and homogeneity, HPSEC revealed one distinct population with a molecular mass of more than 2,000 kDa. The protein content was around 9 w/w%. The sample was analysed to determine its chemical structure. The EPS was found to consist of rhamnose, glucose, galacturonic acid and glucuronic acid. Due to the presence of uronic acids the molar ratio between the four sugars found varies from 3:5:2:4 by sugar composition analyses after methanolysis to 1:1:1:1 found by NMR. A repeating unit with a molecular mass of 678 Da was confirmed by MALDI-TOF mass spectrometry after mild acid treatment. 13C and 1H hetero- and homonuclear 2D NMR spectroscopy of the native and partial hydrolysed EPS revealed a repeating unit, no non-sugar substituents were present.     

Preliminary characterization of exopolysaccharides produced by a marine biofilm-forming bacterium Pseudoalteromonas ruthenica (SBT 033)

AIM: The major objective of the present study was the partial characterization of the exopolysaccharides (EPS) produced by a marine biofilm-forming bacterium Pseudoalteromonas ruthenica under shake culture conditions. METHODS AND RESULTS: EPS-producing bacterial cultures were isolated from the sea water collected from the vicinity of coastal electric power station. Zobell marine broth medium was used for growth of the cultures and the EPS produced was quantified using phenol sulfuric acid method. Chemical characterization of the EPS was carried out using Fourier transform infrared spectroscopy (FTIR), and capillary gas chromatography (GC). Further, viscosity and rheological properties of the purified EPS were studied. The FTIR spectrum revealed prominent peaks of various groups of OH and CH(3) bending. GC analysis showed the presence of eight individual sugars. Rheological studies of the aqueous EPS showed good shearing property. CONCLUSIONS: Pseudoalteromonas ruthenica isolated from marine environment produced copious amount of EPS under shake culture conditions. GC analysis of the EPS revealed the presence of eight individual sugars and the EPS had good shearing property. SIGNIFICANCE AND IMPACT OF THE STUDY: The EPS produced by P. ruthenica is pseudoplastic in nature and is stable at higher pH levels. These properties suggest that the EPS may have potential applications in the oil, textiles and food industries.     

Effect of carbohydrates to protein in EPS on sludge settling characteristics

Extracellular polymeric substances (EPS) are believed to play a role in the binding and formation of microbial flocs. However, the precise role is not well known Sludge settling characteristics and the carbohydrate to protein ratio in EPS were tested with various airflow rates in this study. Sludge, was collected from three modified sequencing batch reactors (SBRs), which were operated at 16°C with an airflow rate of 0.8 L/min, 3L/min and 6 L/min, respectively. During the operation, the reactor operated at an airflow rate of 0.8 L/min showed sludge volume index (SVI) of 80 to 90 mL/g and a constant ratio of carbohydrate to protein in the EPS, while a significant increase in the SVI was seen in the other reactors. Sludge bulking increased the amount of carbohydrate in the EPS, while kept protein almost constant in the airflow rate of 3 L/min and 6 L/min. Surface charge also increased with increases in the carbohydrate to protein ratio in the EPS, which weakens the attraction between the EPS and multivalent cations. The ratio of carbohydrate to protein in the EPS was inferred to be essential for bioflocculation.     

Metal-Induced Production of a Novel Bioadsorbent Exopolysaccharide in a Native Rhodotorula mucilaginosa from the Mexican Northeastern Region

There is a current need to develop low-cost strategies to degrade and eliminate industrially used colorants discharged into the environment. Colorants discharged into natural water streams pose various threats, including: toxicity, degradation of aesthetics and inhibiting sunlight penetration into aquatic ecosystems. Dyes and colorants usually have complex aromatic molecular structures, which make them very stable and difficult to degrade and eliminate by conventional water treatment systems. The results in this work demonstrated that heavy metal-resistant Rhodotorula mucilaginosa strain UANL-001L isolated from the northeast region of Mexico produce an exopolysaccharide (EPS), during growth, which has colorant adsorption potential. The EPS produced was purified by precipitation and dialysis and was then physically and chemically characterized by Scanning Electron Microscopy, Fourier Transform Infrared Spectroscopy, and chemical elemental analysis. Here, the ability of the purified EPS produced to adsorb methylene blue (MB), which served as a model colorant, is studied. MB adsorption by the EPS is found to follow Langmuir Adsorption Isotherm kinetics at 25°C. Further, by calculating the Langmuir constant the adsorption capabilities of the EPS produced by the Rhodotorula mucilaginosa strain UANL-001L is compared to that of other adsorbents, both, microbially produced and from agroindustrial waste. The total adsorption capacity of the EPS, from the Rhodotorula mucilaginosa strain UANL-001L, was found to be two-fold greater than the best bioadsorbents reported in the literature. Finally, apart from determining which heavy metals stimulated EPS production in the strain, the optimal conditions of pH, heavy metal concentration, and rate of agitation of the growing culture for EPS production, was determined. The EPS reported here has the potential of aiding in the efficient removal of colorants both in water treatment plants and in situ in natural water streams.     

Correlation of activities of the enzymes alpha-phosphoglucomutase, UDP-galactose 4-epimerase, and UDP-glucose pyrophosphorylase with exopolysaccharide biosynthesis by Streptococcus thermophilus LY03

The effects of different carbohydrates or mixtures of carbohydrates as substrates on bacterial growth and exopolysaccharide (EPS) production were studied for the yoghurt starter culture Streptococcus thermophilus LY03. This strain produces two heteropolysaccharides with the same monomeric composition (galactose and glucose in the ratio 4:1) but with different molecular masses. Lactose and glucose were fermented by S. thermophilus LY03 only when they were used as sole energy and carbohydrate sources. Fructose was also fermented when it was applied in combination with lactose or glucose. Both the amount of EPS produced and the carbohydrate source consumption rates were clearly influenced by the type of energy and carbohydrate source used, while the EPS monomeric composition remained constant (galactose-glucose, 4:1) under all circumstances. A combination of lactose and glucose resulted in the largest amounts of EPS. Measurements of the activities of enzymes involved in EPS biosynthesis, and of those involved in sugar nucleotide biosynthesis and the Embden-Meyerhof-Parnas pathway, demonstrated that the levels of activity of alpha-phosphoglucomutase, UDP-galactose 4-epimerase, and UDP-glucose pyrophosphorylase are highly correlated with the amount of EPS produced. Furthermore, a weaker relationship or no relationship between the amounts of EPS and the enzymes involved in either the rhamnose nucleotide synthetic branch of the EPS biosynthesis or the pathway leading to glycolysis was observed for S. thermophilus LY03.     

Characteristics of bioemulsifier V2-7 synthesized in culture media added of hydrocarbons: chemical composition, emulsifying activity and rheological properties

The bioemulsifier V2-7 is an exopolysaccharide (EPS) synthesized by strain F2-7 of Halomonas eurihalina and it has the property of emulsifying a wide range of hydrocarbons i.e. n-tetradecane, n-hexadecane, n-octane, xylene mineral light and heavy oils, petrol and crude oil. Characteristics of exopolysaccharide V2-7 produced in media supplemented with various hydrocarbons (n-tetradecane, n-hexadecane, n-octane, xylene, mineral light oil, mineral heavy oil, petrol or crude oil) were studied. Yield production varied from 0.54 to 1.45 g L(-1) according to the hydrocarbon added, in the same way chemical composition, viscosity and emulsifying activity of EPS varied with the culture conditions. Respect to chemical composition, percentage of uronic acids found in exopolymers produced in hydrocarbon media was always higher than that described for V2-7 EPS (1.32%) obtained with glucose. This large amount of uronic acid present could be useful in biodetoxification and waste water treatment. On the other hand, the highest amount of biopolymer was synthesized with mineral light oil, while the most active emulsifiers were those obtained from media added with petrol and n-octane. Furthermore, all EPS were capable of emulsifying crude oil more efficiently than the three chemical surfactants tested as control (Tween 20, Tween 80 and Triton X-100). The capacity of strain F2-7 to grow and produce bioemulsifier in presence of oil hydrocarbons together with the high emulsifying activity and low viscosity power of the biopolymers synthesized in hydrocarbons media could be considered highly beneficial for application of both bioemulsifier and producing strain in bioremediation of oil pollutants.     

Production of a Novel Extracellular Polysaccharide by Lactobacillus sake 0-1 and Characterization of the Polysaccharide

A novel exopolysaccharide (EPS) produced by Lactobacillus sake 0-1 (CBS 532.92) has been isolated and characterized. When the strain was grown on glucose, the produced EPS contained glucose and rhamnose in a molar ratio of 3:2 and the average molecular mass distribution (M(infm)) was determined at 6 x 10(sup6) Da. At a concentration of 1%, the 0-1 EPS had better viscosifying properties than xanthan gum when measured over a range of shear rates from 0 to 300 s(sup-1), while shear-thinning properties were comparable. Rheological data and anion-exchange chromatography suggested the presence of a negatively charged group in the EPS. Physiological parameters for optimal production of EPS were determined in batch fermentation experiments. Maximum EPS production was 1.40 g (middot) liter(sup-1), which was obtained when L. sake 0-1 had been grown anaerobically at 20(deg)C and pH 5.8. When cultured at lower temperatures, the EPS production per gram of biomass increased from 600 mg at 20(deg)C to 700 mg at 10(deg)C but the growth rate in the exponential phase decreased from 0.16 to 0.03 g (middot) liter(sup-1) (middot) h(sup-1). EPS production started in the early growth phase and stopped when the culture reached the stationary phase. Growing the 0-1 strain on different energy sources such as glucose, galactose, mannose, fructose, lactose, and sucrose did not alter the composition of the EPS produced.     

The ++Sinorhizobium meliloti lon protease is involved in regulating exopolysaccharide synthesis and is required for nodulation of alfalfa

While screening for Sinorhizobium meliloti Pho regulatory mutants, a transposon mutant was isolated that constitutively expressed higher levels of acid and alkaline phosphatase enzymes. This mutant was also found to form pseudonodules on alfalfa that were delayed in appearance relative to those formed by the wild-type strain, it contained few bacteroids, and it did not fix nitrogen. Sequence analysis of the transposon insertion site revealed the affected gene to have high homology to Lon proteases from a number of organisms. In minimal succinate medium, the mutant strain was found to grow more slowly, reach lower maximal optical density, and produce more extracellular polysaccharide (EPS) than the wild-type strain. The mutant fluoresced brightly on minimal succinate agar containing calcofluor (which binds to EPSI, a constitutively expressed succinoglycan), and gas chromotographic analysis of purified total EPS showed that the glucose-to-galactose ratio in the lon mutant total EPS was 5.0 +/- 0.2 (mean +/- standard error), whereas the glucose-to-galactose ratio in the wild-type strain was 7.1 +/- 0.5. These data suggested that in addition to EPSI, the lon mutant also constitutively synthesized EPSII, a galactoglucan which is the second major EPS known to be produced by S. meliloti, but typically is expressed only under conditions of phosphate limitation. (13)C nuclear magnetic resonance analysis showed no major differences between EPS purified from the mutant and wild-type strains. Normal growth, EPS production, and the symbiotic phenotype were restored in the mutant strain when the wild-type lon gene was present in trans. The results of this study suggest that the S. meliloti Lon protease is important for controlling turnover of a constitutively expressed protein(s) that, when unregulated, disrupts normal nodule formation and normal growth.     

Correlation of Activities of the Enzymes α-Phosphoglucomutase, UDP-Galactose 4-Epimerase, and UDP-Glucose Pyrophosphorylase with Exopolysaccharide Biosynthesis by Streptococcus thermophilus LY03

The effects of different carbohydrates or mixtures of carbohydrates as substrates on bacterial growth and exopolysaccharide (EPS) production were studied for the yoghurt starter culture Streptococcus thermophilus LY03. This strain produces two heteropolysaccharides with the same monomeric composition (galactose and glucose in the ratio 4:1) but with different molecular masses. Lactose and glucose were fermented by S. thermophilus LY03 only when they were used as sole energy and carbohydrate sources. Fructose was also fermented when it was applied in combination with lactose or glucose. Both the amount of EPS produced and the carbohydrate source consumption rates were clearly influenced by the type of energy and carbohydrate source used, while the EPS monomeric composition remained constant (galactose-glucose, 4:1) under all circumstances. A combination of lactose and glucose resulted in the largest amounts of EPS. Measurements of the activities of enzymes involved in EPS biosynthesis, and of those involved in sugar nucleotide biosynthesis and the Embden-Meyerhof-Parnas pathway, demonstrated that the levels of activity of α-phosphoglucomutase, UDP-galactose 4-epimerase, and UDP-glucose pyrophosphorylase are highly correlated with the amount of EPS produced. Furthermore, a weaker relationship or no relationship between the amounts of EPS and the enzymes involved in either the rhamnose nucleotide synthetic branch of the EPS biosynthesis or the pathway leading to glycolysis was observed for S. thermophilus LY03.     

Production of exopolysaccharide by Lactobacillus rhamnosus R and analysis of its enzymatic degradation during prolonged fermentation

The potential of Lactobacillus rhamnosus R for producing exopolysaccharide (EPS) when grown on basal minimum medium supplemented with glucose or lactose was investigated. EPS production by L. rhamnosus R is partially growth associated and about 500 mg of EPS per liter was synthesized with both sugars. The product yield coefficient (Y(EPS/S)) was 3.15 (0.0315 g of EPS [g of lactose](-1)) and 2.88 (0.0288 g of EPS [g of glucose](-1)). It was clearly shown that the amount of EPS produced declined upon prolonged fermentation. Degradation of EPS in fermentation processes was also assessed by measuring its molecular weights and viscosities. As these reductions might have a negative effect on the yield and viscosifying properties of EPS, it was essential to examine possible causes related to this breakdown. The decrease in viscosities and molecular weights of EPS withdrawn at different cultivation times permitted us to suspect the presence of a depolymerizing enzyme in the fermentation medium. Our study on enzymatic production profiles showed a large spectrum of glycohydrolases (alpha-D-glucosidase, beta-D-glucosidase, alpha-D-galactosidase, beta-D-galactosidase, beta-D-glucuronidase, and some traces of alpha-L-rhamnosidase). These enzymes were localized, two of them (alpha-D-glucosidase and beta-D-glucuronidase) were partially purified and characterized. When incubated with EPS, these enzymes were capable of lowering the viscosity of the polymer as well as liberating some reducing sugars. Upon prolonged incubation (27 h), the loss of viscosity was increased up to 33%.     

Identification of L-altrose in the extracellular polysaccharide from Butyrivibrio fibrisolvens strain CF3

Abstract Butyrivibrio fibrisolvens strain CF3, a stricyly anaerobic bacterial isolate from the ovine cecum, produces extracellular polysaccharides (EPS) when grown on a defined medium containing glucose as the carbon source. EPS were purified from culture supernatants and their monosaccharide composition was determined by two different procedures. Analysis of EPS hydrolysates by thin-layer chromatography (TLC) yielded spots coincident with standard glucose, altrose, and 1,6-anhydroaltrose. These results were corroborated by both gas-liquid chromatography (GLC) and GLC-mass spectroscopy (GLC-MS) of alditol acetates prepared from EPS hydrolysates. Purification of the altrose from EPS hydrolysates was accomplished by preparative paper and column chromatography. Polarimetry demonstrated the isolated altrose to have the L -configuration. The occurrence of this hexose in nature has not yet been reported.     

Altered exopolysaccharides of Bradyrhizobium japonicum mutants correlate with impaired soybean lectin binding, but not with effective nodule formation

 The exact mechanism(s) of infection and symbiotic development between rhizobia and legumes is not yet known, but changes in rhizobial exopolysaccharides (EPSs) affect both infection and nodule development of the legume host. Early events in the symbiotic process between Bradyrhizobium japonicum and soybean (Glycinemax [L.] Merr.) were studied using two mutants, defective in soybean lectin (SBL) binding, which had been generated from B. japonicum 2143 (USDA 3I-1b-143 derivative) by Tn5 mutagenesis. In addition to their SBL-binding deficiency, these mutants produced less EPS than the parental strain. The composition of EPS varied with the genotype and with the carbon source used for growth. When grown on arabinose, gluconate, or mannitol, the wild-type parental strain, B. japonicum 2143, produced EPS typical of DNA homology group I Bradyrhizobium, designated EPS I. When grown on malate, strain 2143 produced a different EPS composed only of galactose and its acetylated derivative and designated EPS II. Mutant 1252 produced EPS II when grown on arabinose or malate, but when grown on gluconate or mannitol, mutant 1252 produced a different EPS comprised of glucose, galactose, xylose and glucuronic acid (1:5:1:1) and designated EPS III. Mutant 1251, grown on any of these carbon sources, produced EPS III. The EPS of strain 2143 and mutant 1252 contained SBL-binding polysaccharide. The amount of the SBL-binding polysaccharide produced by mutant 1252 varied with the carbon source used for growth. The capsular polysaccharide (CPS) produced by strain 2143 during growth on arabinose, gluconate or mannitol, showed a high level of SBL binding, whereas CPS produced during growth of strain 2143 on malate showed a low level of SBL binding. However, the change in EPS composition and SBL binding of strain 2143 grown on malate did not affect the wild-type nodulation and nitrogen fixation phenotype of 2143. Mutant 1251, which produced EPS III, nodulated 2 d later than parental strain 2143, but formed effective, nitrogen-fixing tap root nodules. Mutant 1252, which produced either EPS II or III, however nodulated 5–6 d later and formed few and ineffective tap root nodules. Restoration of EPS I production in mutant 1252 correlated with restored SBL binding, but not with wild-type nodulation and nitrogen fixation. Received: 6 October 1999 / Accepted: 18 November 1999     

Demonstration of exopolysaccharide production by enterohemorrhagicEscherichia coli

EnterohemorrhagicEscherichia coli O157H7 produces visibly slimy colonies when grown on Sorbitol/MacConkey or Maloney's agar plates at room temperature, indicative of exopolysaccharide (EPS) production. Eighteen of 27 (67%) wild-typeE. coli O157H7 isolates produced enough EPS to be visually distinguishable. Of five strains that showed no visible EPS production on these media, four (80%) did produce slimy colonies on media containing higher salt concentrations. Measurements of EPS production by colorimetric determination of uronic acid indicated that EPS production was affected by growth temperature, atmosphere, and medium. Wild-typeE. coli O157H7 strain 932 produced the greatest amounts of EPS when grown anaerobically at 37°C, whereas its plasmid-cured derivative 932P produced large quantities of EPS when grown aerobically at room temperature. Electron micrographs revealed thin, flexible fibers extending from the bacterial cell surface. Cells of strain 932P grown aerobically at room temperature were completely encased in a thick EPS matrix. Chemical analysis of purified EPS revealed that it is very similar or identical to colanic acid.E. coli O157H7 adheres better to INT 407 cells when grown under conditions that favor high EPS production than when grown under conditions that repress EPS production.     

野油菜黄单胞菌野油菜致病变种中一个与EPS合成有关的新基因的鉴定

用转座子Tn5gusA5对野油菜黄单胞菌野油菜致病变种(Xanthomonas campestris pv.campestris,简称Xcc)野生型菌株8004进行诱变,分离到一批胞外多糖(EPS)合成减少的突变体。采用TAIL-PCR(thermal asymmetric interlaced PCR)分析突变体的Tn5gusA5插入位点,发现其中一株编号为151D09的突变体的插入位点位于Xcc 8004菌株的基因组编号为XC3695的ORF内,该ORF功能尚未见报道。序列分析表明,该ORF演绎的编码产物与Serratia marcescens的kdtX基因和Klebsiella pneumoniae的waaE基因演绎的编码产物分别具有52%和50%的相似性,并具有第2家族糖基转移酶的功能域, 因此暂将该ORF命名为waxE基因。用同源双交换方法构建了waxE基因的缺失突变体,并采用PCR和Southern杂交的方法对突变体进行了验证。waxE基因缺失突变体在营养丰富培养基的生长繁殖不受影响,但其EPS产量与野生型菌株8004相比,降低35%左右,并且一段PCR合成的包含waxE基因的DNA片段能反式互补waxE基因缺失突变体,恢复缺失突变体的EPS产量,表明Xcc waxE基因与EPS的生物合成有关。