Identification of a slime exopolysaccharide depolymerase in mucoid strains ofPseudomonas aeruginosa

Strains ofPseudomonas aeruginosa recovered from pulmonary infections in cystic fibrosis (CF) patients are often mucoid in appearance owing to the secretion of a viscous slime exopolysaccharide (EPS). Unlike most mucoid isolates, strains WcM#2, P10, and P11 produce mucoid colonies after 24 h of incubation at 37°C, which become nonmucoid upon further incubation; this suggests the presence of a slime-degrading enzyme or depolymerase. Using both qualitative and quantitative assays, the presence of a slime EPS depolymerase was confirmed in each of these three strains as well as in four of four additional mucoid strains. Depolymerase activity was lower but still detectable in four of four nonmucoid strains. Enzyme preparations from strains WcM#2, P10, and P11 were active on most, but not all, slime EPS preparations fromP. aeruginosa strains, as well as sodium alginate; greater activity was observed on substrates after deacetylation. Comparisons are made between the enzyme described in this study and previous reports of slime EPS depolymerase in mucoid strains ofP. aeruginosa.     

Extracellular Polysaccharide of Erwinia amylovora: a Correlation with Virulence

The extracellular polysaccharides produced as slime or capsule layers by bacterial pathogens of animals and plants have been often implicated as factors essential to pathogenesis. In the present study, virulence of the plant pathogen Erwinia amylovora was correlated with the ability to produce extracellular polysaccharide (EPS). EPS production by a series of field isolates and bacterio-phage-resistant mutants differing in the extent to which they cause symptoms in host tissues was examined by quantitation with a modified Laurell rocket immunoelectrophoresis assay. The amount of EPS produced as an easily removed capsular layer or slime on solid nutrient agar approximated the capacity to exhibit symptoms in host inoculation tests. Features common to the virulent isolates are mucoid colony morphology, sensitivity to EPS-specific bacteriophages [S3 and PEal(h)], and ability to produce a characteristic EPS. Mutants selected for resistance to S3 or nonmucoid colony morphology are deficient in EPS production and have lost the ability to multiply in host tissue and cause symptoms. We conclude that EPS may be directly involved in symptom expression and provide a function essential to the growth of the pathogen in host tissues.     

Studies on the Extracellular Polysaccharides (EPS) Produced in vitro by Pseudomonas phaseolicola I. Indications for a Polysaccharide Resembling Alginic Acid in Seven P. syringae Pathovars

Extracellular polysaccharides (EPS) produced by Pseudomonas syringae pv. phaseolicola are obviously composed of two main components: the long known levan consisting of fructose, and a mannuronan consisting mainly of mannuronic acid (manA), thus resembling alginic acid (alginate). The identification of manA was established by TLC utilizing different developing systems, and by cellulose acetate electrophoresis in different buffers. References were authentic uronic acids and hydrolyzed authentic alginate. A rough quantification of the “alginate” present in crude EPS was achieved with a selective colour reaction which largely excluded compounds other than uronic acids. Levan was only synthesized with sucrose as primary carbon source. When grown on several other sugars and related compounds “alginate” was the predominant component of the EPS. Additionally, rhamnose, fucose, glucose and amino sugars were found in some instances in hydrolysates of crude EPS, suggesting the release of lipopolysaccharides (LPS) from the bacterial cell walls during culture. Growth on carbon sources not related to sugars resulted in these “LPS” as the main constituent of EPS. After cultivation with sucrose, the “alginate” was restricted to the “slime” fraction of the EPS. In the “capsular” fraction, levan was predominating. A screening program revealed the capacity to synthesize the “alginate” in six additional P. syringae pathovars: pisi, lachrymans, aptata, tomato, syringae, and glycinea. All of the strains tested so far produced levan from sucrose, however, the “alginate” was formed not by all of them. There was a tendency that fresh isolates produced more “alginate” than strains subcultured for an extended time in vitro. This was also true for the total amount of EPS.     

Chemical Characterization of Exopolysaccharides from Antarctic Marine Bacteria

Exopolysaccharides (EPS) may have an important role in the Antarctic marine environment, possibly acting as ligands for trace metal nutrients such as iron or providing cryoprotection for growth at low temperature and high salinity. Ten bacterial strains, isolated from Southern Ocean particulate material or from sea ice, were characterized. Whole cell fatty acid profiles and 16S rRNA gene sequences showed that the isolates included representatives of the genera Pseudoalteromonas, Shewanella, Polaribacter, and Flavobacterium as well as one strain, which constituted a new bacterial genus in the family Flavobacteriaceae. The isolates are, therefore, members of the “Gammaproteobacteria” and Cytophaga-Flexibacter-Bacteroides, the taxonomic groups that have been shown to dominate polar sea ice and seawater microbial communities. Exopolysaccharides produced by Antarctic isolates were characterized. Chemical composition and molecular weight data revealed that these EPS were very diverse, even among six closely related Pseudoalteromonas isolates. Most of the EPS contained charged uronic acid residues; several also contained sulfate groups. Some strain produced unusually large polymers (molecular weight up to 5.7 MDa) including one strain in which EPS synthesis is stimulated by low temperature. This study represents a first step in the understanding of the role of bacterial EPS in the Antarctic marine environment.     

Isolation and characterization of <Emphasis Type="Italic">Pseudoalteromonas ruthenica</Emphasis> (SBT033), an EPS-producing biofilm bacterium from the seawater intake point of a tropical power station

Biofilm formation in bacteria is closely linked with production of exopolysaccharides (EPS). This study examined the quantitative variations in EPS production and biofilm-forming ability among bacteria isolated from the seawater intake point of a power station located on the east coast of India. Of the 233 isolates obtained from the intake site, 71 bacterial isolates displayed different colony morphological characteristics. Thirteen isolates that produced wide and thick mucoid colonies were further tested for their ability to attach and form biofilms by microtitre plate assay and confocal microscopy. EPS production among the selected bacterial isolates ranged from 826 to 1838 μg ml⁻¹. Strain SBT033, which produced the maximum amount of EPS also displayed the maximum biofilm-forming ability among the 13 isolates. This strain was selected for further characterization using biochemical and molecular methods. The pale orange-pigmented isolate was a Gram negative, aerobic, short rod-shaped and grew well only in the presence of 2% NaCl. On the basis of phenotypic characteristics the isolate SBT033 is shown to belong to the genus Pseudoalteromonas. Analysis of 16S rRNA of the isolate revealed 99% homology with Pseudoalteromonas ruthenica.     

Slime Production by Pseudomonas aeruginosa

Chemical properties and compositions of slimes produced by two Pseudomonas aeruginosa strains of different colonial types were investigated. The main component of the slime from strain IFO 3445 was found to be DNA, contaminated with small amounts of protein. On the other hand, the slime from a mucoid-type strain No. 24 was an alginate-like substance consisting of mannuronic and glucuronic acids, and contained traces of protein and nucleic acid. Slimes from twenty clinical isolates of P. aeruginosa were investigated for their chemical compositions. Slimes from eighteen strains consisted of DNA, while, two strains of a mucoid-type produced slimes composed of polyuronic acid.     

Evaluation of bacteria isolated from rice rhizosphere for biological control of charcoal rot of sorghum caused by <Emphasis Type="Italic">Macrophomina phaseolina</Emphasis> (Tassi) Goid.

A total of 360 bacteria, isolated from the rhizospheres of a system of rice intensification (SRI) fields, were characterized for the production of siderophore, fluorescence, indole acetic acid (IAA), hydrocyanic acid (HCN) and solubilization of phosphorus. Of them, seven most promising isolates (SRI-156, -158, -178, -211, -229, -305 and -360) were screened for their antagonistic potential against Macrophomina phaseolina (causes charcoal rot in sorghum) by dual culture assay, blotter paper assay and in greenhouse. All the seven isolates inhibited M. phaseolina in dual culture assay, whereas six isolates solubilized phosphorous (except SRI-360), all seven produced siderophore, four produced fluorescence (except SRI-178, -229 and -305), six produced IAA (except SRI-305) and five produced HCN (except SRI-158 and -305). In the blotter paper assay, no charcoal rot infection was observed in SRI-156-treated sorghum roots, indicating complete inhibition of the pathogen, while the roots treated with the other isolates showed 49–76% lesser charcoal rot infection compared to the control. In the antifungal activity test (in green house on sorghum), all the isolates increased shoot dry mass by 15–23% and root dry mass by 15–20% (except SRI-158 and -360), over the control. In order to confirm the plant growth-promoting (PGP) traits of the isolates, the green house experiment was repeated but, in the absence of M. phaseolina. The results further confirmed the PGP traits of the isolates as evidenced by increases in shoot and root dry mass, 22–100% and 5–20%, respectively, over the control. The sequences of 16S rDNA gene of the isolates SRI-156, -158, -178, -211, -229, -305 and -360 were matched with Pseudomonas plecoglossicida, Brevibacterium antiquum, Bacillus altitudinis, Enterobacter ludwigii, E. ludwigii, Acinetobacter tandoii and P. monteilii, respectively in BLAST analysis. This study indicates that the selected bacterial isolates have the potential for PGP and control of charcoal rot disease in sorghum.     

Characterization of<Emphasis Type="Italic"> Aureobasidium pullulans</Emphasis> isolated from airborne spores in Thailand

Isolates from air in several locations in Thailand were identified as Aureobasidium pullulans PR with dark pigmentation (Loei province), A. pullulans SU with an unusual conidial apparatus (Chiangmai province), and A. pullulans CU with burgundy-red pigmentation (from a shady area in Bangkok). The internal transcribed spacer sequences of the rDNA of A. pullulans SU and A. pullulans CU confirmed that they were A. pullulans. Both A. pullulans CU and A. pullulans PR preferred 30 °C and pH 7.5 for exopolysaccharide (EPS) production, while A. pullulans SU preferred 25 °C and pH 6.5. All three isolates preferred glucose over sucrose and (NH₄)₂SO4 over peptone for EPS production. Under optimal conditions, A. pullulans PR produced EPS yields of up to 0.225 g g⁻¹, followed by A. pullulans CU (0.185 g g⁻¹) and A. pullulans SU (0.158 g g⁻¹). Amylase activities were detected during the course of EPS production but gradually decreased as the EPS yields increased. IR spectra suggest that the EPS from these isolates was pullulan. EPS from the three isolates were partially sensitive to pullulanase. Electronic Publication     

Polysaccharide-producing bacteria isolated from paper machine slime deposits

Development of novel enzymatic methods for slime deposit control in paper mills requires knowledge of polysaccharide-producing organisms and the polysaccharide structures present in deposits. In this work, 27 polysaccharide-producing bacteria were isolated from slime samples collected from different parts of a paper machine. Most of the isolates produced polysaccharides in liquid culture and nine of them were selected for production of polysaccharides for characterisation. The selected isolates belonged to seven different genera: Bacillus, Brevundimonas, Cytophaga, Enterobacter, Klebsiella, Paenibacillus and Starkeya. Using ribotyping, partial 16S rDNA sequencing, physiological tests and fatty acid analysis, four of the nine isolates: Bacillus cereus, Brevundimonas vesicularis, K. pneumoniae and P. stellifer were identified to the species level. Production of polysaccharides by the selected isolates varied between 0.07 and 1.20 g L^–1, the highest amount being produced by B. vesicularis. The polysaccharides were heteropolysaccharides with varying proportions of galactose, glucose mannose, rhamnose fucose and uronic acids.     

Sensitivity of capsular-producing Streptococcus thermophilus strains to bacteriophage adsorption

Aims: To determine whether the presence and type of exopolysaccharides (EPS), slime‐EPS or capsular, and the structural characteristics of the polymers produced by Streptococcus thermophilus strains could interfere with or be involved in phage adsorption. Methods and Results: Phage–host interactions between eight EPS‐producing Strep. thermophilus strains (CRL419, 638, 804, 810, 815, 817, 821, 1190) and five streptococcus specific phages (φYsca, φ3, φ5, φ6, φ8) isolated from Argentinean faulty fermentation failed yoghurts were evaluated. No relationship was found between the EPS chemical composition and the phage sensitivity/resistance phenotype. In general, the capsular‐producing strains were more sensitive to phage attacks than the noncapsular‐producing strains. Streptococcus thermophilus CRL1190 (capsular‐producing) was the only strain sensitive to all bacteriophages and showed the highest efficiency of plating. Phage adsorption to a capsular‐negative, EPS low‐producing mutant of strain CRL1190 was reduced, especially for φYcsa and φ8. Conclusions: The presence of capsular polysaccharide surrounding the cells of Strep. thermophilus strains could play a role in the adsorption of specific phages to the cells. Significance and Impact of the Study: Capsular‐producing Strep. thermophilus strains should be evaluated for their bacteriophage sensitivity if they are included in starter cultures for the fermented food industry.     

Genetic and morphological comparisons ofGlomerella (Colletotrichum) isolates from maize and from sorghum

Various morphological and genetic characteristics were compared among six isolates ofColletotrichum from maize and six from sorghum. For the first time, a teleomorph was induced in sorghum isolates by pairing them on autoclaved sorghum leaves in a humidity chamber. The sorghum teleomorph was morphologically similar toGlomerella graminicola andGlomerella tucumanensis, the teleomorphs ofColletotrichum isolates from maize and from sugarcane, respectively. Mating tests demonstrated thatGlomerella isolates from maize and sorghum were not interfertile. Several small but consistent differences in the morphologies of the isolates from maize and from sorghum were observed which agreed with earlier reports. DNA fingerprints detected as restriction fragment length polymorphisms of mitochondrial DNA and random polymorphic DNA (RAPD) produced from nuclear DNA by the polymerase chain reaction could be used to reliably and unambiguously distinguish members of the two groups of isolates. Results of a statistical analysis of similarity of the RAPD fingerprints suggested that maize and sorghum isolates ofColletotrichum are only about 45% similar (±10%) and represent two distinct and separate genetic lineages. We conclude that isolates ofColletotrichum from maize and sorghum are sibling species since they are morphologically very similar but reproductively completely isolated.     

Optimization of exopolysaccharide production and diesel oil emulsifying properties in root nodulating bacteria

Bioremediation, a strategy mediated by microorganisms, is a promising way used in the degradation or removal of organic contaminants from soil or aquatic system. Exopolysaccharide (EPS) which was produced by a variety of Gram-negative bacteria has been demonstrated to be a potential bioemulsifier used in the degradation of hydrocarbons. In the present study, attempts were made to optimize the production of EPS from our newly isolates by adjusting the culture conditions and medium components. Besides, the performance of diesel oil emulsification using partially purified EPS derived from different conditions was also demonstrated. Out of 40 root nodulating bacteria the better emulsifying abilities were recorded from three strains namely Rhizobium miluonense CC-B-L1, Burkholderia seminalis CC-IDD2w and Ensifer adhaerens CC-GSB4, as can be seen from their emulsification index (E₂₄) 66, 64 and 60%, respectively. These three strains produced 212, 203 and 198 mg l⁻¹ of EPS, respectively, in yeast extract mannitol (YEM) medium. After modifying culture conditions, better performances can be achieved from these three strains, with increases of 21.7, 21.4, 16.7% in the EPS production and 12.1, 10.9, 8.3% in E₂₄, respectively. When considered for strain CC-B-L1 and CC-IDD2w, the addition of 1.5% (v/v) of mannitol and 0.1% (v/v) of asparagine in YEM enhanced 42.9 and 34.7% in EPS production along with 28.8 and 37.5% higher in E₂₄. The supplement of 2.0% (v/v) glucose and 0.2% (v/v) asparagine in YEM increased 65.2% of EPS and 38.3% of E₂₄ in strain CC-GSB4. This is the first report demonstrating the optimization of diesel emulsification by EPS from root nodulating isolates, and these microbial agents might be used in the remediation of hydrocarbon contaminated soils in a near future.     

Colanic acid is an exopolysaccharide common to many enterobacteria isolated from paper-machine slimes

In this study, polysaccharide-producing bacteria were isolated from slimes collected from two Finnish and one Spanish paper mill and the exopolysaccharides (EPSs) produced by 18 isolates were characterised. Most of the isolates, selected on the bases of slimy colony morphology, were members of the family Enterobacteriaceae most frequently belonging to the genera Enterobacter and Klebsiella including Raoultella. All of the EPSs analysed showed the presence of charged groups in the form of uronic acid or pyruvate revealing the polyanionic nature of these polysaccharides. Further results of the carbohydrate analysis showed that the EPS produced by nine of the enterobacteria was colanic acid.     

Identification of bacteria contaminating pulp and a paper machine in a Canadian paper mill

Over 100 bacteria from pulp and slime samples in a Canadian paper mill were identified by partial sequencing of their 16S rDNAs. Seventy-one percent of the isolates could be assigned to a bacterial genus with a high level of confidence. Another 12% exhibited at least 95% similarity within their 16S rDNA sequence with unidentified organisms that originate from warm or wet environments. Pseudomonas, Bacillus, and Pseudoxanthomonas isolates were represented at a relatively high proportion in both pulp and slime samples. This is the first time that Pseudoxanthomonas strains have been isolated from pulp and slime samples on a paper machine. Electronic Publication     

Molecular identification of isolates of <Emphasis Type="Italic">Peronosclerospora sorghi</Emphasis> from maize using PCR-based SCAR marker

The fungus Peronosclerospora sorghi [Weston and Uppal (Shaw)] infects both sorghum and maize and incites downy mildew disease. Pathogenic and molecular variability among isolates of P. sorghi from sorghum and maize has been reported. In the present study we developed a DNA sequence characterized amplified region (SCAR) marker for identification of isolates of P. sorghi from maize by using polymerase chain reaction (PCR). The random amplified polymorphic DNA (RAPD) primer OPB15 consistently amplified a 1,000 base pairs (bp) product in PCR only from DNA of P. sorghi isolates from maize and not from isolates of sorghum. The PCR-amplified 1,000-bp product was cloned and sequenced. The sequence of the SCAR marker was used for designing specific primers for identification of maize isolates of P. sorghi. The SCAR primers amplified a 800 bp fragment only from genomic DNA of maize isolates of P. sorghi. The SCAR primers developed in this study are highly specific and reproducible, and proved to be powerful tool for identification of P. sorghi isolates from maize.     

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.     

Cultural characteristics, morphology, and variation within Claviceps africana and C. sorghi from India

Sorghum ergot in India is caused by Claviceps africana and C. sorghi. The distributions of these two species in India is not known. Eighty-nine sorghum ergot isolates were cultured from young sphacelia obtained from male sterile sorghum plants artificially inoculated using inoculum collected in the field. Based on cultural characteristics, the isolates were separated into two groups which differed distinctly in the morphology of their sphacelia, conidia, and sclerotia. Marked differences also were observed in rates of secondary conidial production and disease spread between the groups. In combination with molecular evidence, our results confirm that the isolates placed in Group I represent C. africana and Group II isolates represent C. sorghi. C. africana was found to be widely distributed in all sorghum growing areas of India. The species first described as occuring in India, C. sorghi, appears to be restricted to a few locations in the states of Maharashtra, Andhra Pradesh, and Karnataka.     

THE COMPLEX EXTRACELLULAR POLYSACCHARIDES OF MAINLY CHAIN‐FORMING FRESHWATER DIATOM SPECIES FROM EPILITHIC BIOFILMS1

Diatoms are dominant organisms in phototrophic biofilms in aquatic habitats. They produce copious amounts of extracellular polymeric substances (EPS), which mainly consist of carbohydrates and traces of proteins and glycoproteins. This study focuses on the characterization of EPS from a total of 14 diatoms belonging to the six genera Achnanthes, Cymbella, Fragilaria, Punctastriata, Staurosira, and Pseudostaurosira, all of which were isolated from epilithic biofilms of the littoral zone of Lake Constance. EPS from all isolates were extracted by a sequential extraction procedure resulting in five different fractions. The monosaccharide composition of each fraction was analyzed by HPLC equipped with a pulse amperiometric detector, yielding results similar to those obtained by probing the EPS structures with monomer‐specific fluorophore‐linked lectins. Significant differences in carbohydrate composition occurred in the different fractions of single isolates. Most of the diatom isolates in our study form chain‐like colonies in which the cells are attached to each other by intercellular pads. Here we demonstrate that these pads can be dissolved in hot bicarbonate and that they show a heterogeneous composition of monosaccharides in contrast to other fractions, which mostly were dominated by one or two monosaccharides. Principal component analysis indicates a correlation between carbohydrate composition of EPS fractions and the phylogenetic relationship of the respective species, indicating that EPS analyses under defined culture conditions may support taxonomic analyses.     

Natural occurrence of trichothecene-producing Fusaria isolated from India with particular reference to sorghum

In this study a total of 167 isolates collected from different food materials (68.8% from sorghum and the remaining from various other food materials) were assayed by PCR for amplification of the tri 5 gene present in trichothecene-producing Fusaria. Amplification of the tri 5 fragment was observed in 45 isolates (39 isolates from sorghum and 6 isolates from vegetables). Isolates found positive for presence of the tri 5 gene were classified into different morphological groups based on their cultural and conidial characters; 11 of the tri 5 positive isolates from moldy grains of sorghum, one from each morphology group were selected for further analyses. Five deoxynivalenol producers and three deoxynivalenol and Fusarenon-X producers were detected by analysing culture filtrates of the 11 isolates using GC-MS. One isolate each were identified as producers of NIV alone, or NIV along with DON or DAS toxins. Identification of these isolates to the species level was carried out using spore morphology and sequence comparison of the translation elongation factor 1-alpha (EF-1α) gene against the database as well as using phylogenetic analyses. The isolates were identified as Fusarium proliferatum (6), F. nelsonii (2), F. equiseti (1), F. thapsinum (1) and F. sacchari (1). Amplified Fragment Length Polymorphism (AFLP) based grouping clustered the isolates of same species together. This is the first detailed study of trichothecene production by Fusarium spp. associated with sorghum grain mold in India and the identification of F. nelsonii and F. thapsinum as producers of trichothecenes.     

Production of exopolysaccharides by Antarctic marine bacterial isolates

AIMS: This study was undertaken to examine and characterize Antarctic marine bacterial isolates and the exopolysaccharides (EPS) they produce in laboratory culture. METHODS AND RESULTS: Two EPS-producing bacterial strains CAM025 and CAM036 were isolated from particulate material sampled from seawater and sea ice in the southern ocean. Analyses of 16S rDNA sequences placed these isolates in the genus Pseudoalteromonas. In batch culture, both strains produced EPS. The yield of EPS produced by CAM025 was 30-fold higher at -2 and 10 degrees C than at 20 degrees C. Crude chemical analyses showed that these EPS were composed primarily of neutral sugars and uronic acids with sulphates. Gas chromatographic analysis of monosaccharides confirmed these gross compositional findings and molar ratios of monosaccharides revealed differences between the two EPS. CONCLUSIONS: The EPS produced by Antarctic bacterial isolates examined in this study appeared to be polyanionic and, therefore, 'sticky' with respect to cations such as trace metals. SIGNIFICANCE AND IMPACT OF THE STUDY: As the availability of iron as a trace metal is of critical importance in the southern ocean where it is know to limit primary production, the role of these bacterial EPS in the Antarctic marine environment has important ecological implications.