Regulation of expression of group IA capsular polysaccharides inEscherichia coli and related extracellular polysaccharides in other bacteria

Bacterial surface polysaccharides fulfill a number of important roles in cell-cell interactions, survival in natural environments, and formation of biofilms. Consequently, the mechanisms involved in regulation of extracellular polysaccharides are predicted to have a significant impact on microbial adaptation. Strains ofEscherichia coli, Klebsiella spp, andErwinia spp produce extracellular polysaccharides which share structural features. There are also similarities in the organization of genes required for synthesis of these cell surface polymers and, in some cases, the mechanism of synthesis may be related. Despite the diverse habitats of these bacteria, the systems which regulate expression of their extracellular polysaccharides appear to share components and mechanisms. Understanding these regulatory processes may lead to novel therapeutic approaches for pathogens, or for control of unwanted biofilm formation in industrial settings.     

Detection ofAspergillus andPenicillium extracellular polysaccharides (EPS) by ELISA: using antibodies raised against acid hydrolysed EPS

Species of the fungal generaAspergillus andPenicillium produce immunologically active extracellular polysaccharides (EPS) in which galactofuranose residues are immunodominant. The antigenic determinant of the EPSA. fumigatus, A. niger andP. digitatum could be removed by acid hydrolysis. Due to the hydrolysis of the EPS the immunological reaction between IgG anti-native EPS and hydrolysed EPS disappeared. Antibodies raised in rabbits against the acid hydrolysed EPS revealed new antigenic determinants that were exposed as a result of the acid hydrolysis. Immunological inhibitory experiments showed that the antibodies were no longer directed to galactofuranose residues.Enzyme Linked Immunosorbent Assay, carried out with antibodies raised against the acid hydrolysed EPS showed that the antibodies against the acid hydrolysed EPS were more species specific in comparison with the antibodies against the native EPS.     

Optimization of medium for extracellular nuclease formation from Rhizopus stolonifer

A strain of Rhizopus stolonifer produced a high activity of extracellular DNAase when grown on YPG (yeast extract peptone glucose) medium. The source of peptone had a marked effect on the enzyme yield and only one peptone (i.e. from Sarabhai M. Chemicals Ltd, India) supported enzyme production. Maximum enzyme activity (88 U/ml) was obtained after 4 days' growth under submerged conditions in YPG medium containing 100 M Mn²⁺, Co²⁺ or Mg²⁺, and glucose as the sole carbon source. The unpurified enzyme was optimally active at pH 7.5 and 45°C. It had a higher activity with sonicated and heat-denatured DNA than native DNA.     

Influence of carbohydrates and polyols on l-lactic acid production and fatty acid formation by Rhizopus arrhizus

Growth and l-lactic acid production on 24 different carbohydrates and polyols (glycerol, mannitol and sorbitol) by Rhizopus arrhizus CCM 8109 were determined. The d- but not the l-forms of xylose, fructose, galactose, mannose, glucose, cellobiose, maltose and sucrose and partially hydrolysed starch were converted to l-lactic acid. Changes in lipid formation and fatty acid composition were detected in biomass grown on the different sugars. In the presence of polyols, growth and considerable production of lipids were observed with little or no lactate production. Invertase was mainly associated with the mycelium during growth on sucrose, whereas glucoamylase and -amylase were produced extracellularly during growth on starch.The authors are with the Department of Biochemical Technology, Faculty of Chemical Technology, Slovak Polytechnical University, Radlinského 9, SK-812 37 Bratislava, Slovak Republic     

Enhancement of apparent thermostability of lipase from Rhizopus sp. by the treatment with a microbial transglutaminase

Crude lipase from Rhizopus sp. was moderately stable against heat treatment at 45 °C. However, after incubation for 1 h at 25 °C with Streptoverticillium transglutaminase (MTG), the half-life of crude lipase in the heat treatment was increased more than 10-fold compared to that of untreated one. The result can be ascribed by the MTGase-mediated crosslinking of contaminating proteins that affect the apparent thermostability of lipase in the crude sample.     

The effect of growth conditions on production and excretion of extracellular antigens by three ascomycetous yeasts

Ascomycetous yeasts produce extracellular antigens that are almost specific for the species. The antigen production by Hansenula wickerhamii and Stephanoascus ciferrii was independent of the carbon source and was proportional to the final cell density of the cultures. The same was true of chemostat cultures of Stephanoascus ciferrii, irrespective of the dilution rate and whether glucose or ammonia was the limiting nutrient. In cultures of Saccharomyces cerevisiae, however, antigen excretion mainly took place in the late exponential growth phase. Large amounts of antigen were extracted from the cell wall of Saccharomyces cerevisiae. A small amount was detected in the cytoplasm.     

Production of l-lactic acid by Rhizopus species

Of 19 Rhizopus spp. only four produced l-lactic acid in shake-flask culture. Aerobically and in the presence of a neutralizing agent, Rhizopus oryzae NRRL 395 produced the highest concentration of l-lactic acid (65 g/l) but with O₂-limited growth ethanol was produced instead.C.R. Soccol and V.I. Stonoga are with the Laboratório de Processos Biotecnológicos, Departmento de Engenharia Química da Universidade Federal do Paraná, 81531-970 Curitiba, PR, Brazil; M. Raimbault is with the Centre ORSTOM, Laboratoire de Biotecnologie, Physiologie et Métabolisme Cellulaires, 911, Avenue Agropolis, 34032 Montpellier, Cedex 1, France.     

Influence of growth conditions on production of capsular and extracellular polysaccharides byRhizobium leguminosarum

The influence of growth rate and medium composition on exopolymer production byRhizobium leguminosarum was studied. When grown in medium containing 10g/l mannitol and 1g/l glutamic acid,Rhizobium leguminosarum biovartrifolii TA-1 synthesized up to 2.0g/l of extracellular polysaccharide (EPS), and up to 1.6g/l of capsular polysaccharide (CPS). Under non-growing cell conditions in medium without glutamic acid, CPS synthesis by strain TA-1 could proceed to 2.1g/l, while EPS-production remained relatively low (0.8g/l). Maximal CPS-yield was 2.9g CPS/l medium in a medium containing 20g/l mannitol and 2g/l glutamic acid. TheEPS-deficient strain R. leguminosarum RBL5515,exo4::Tn5 was able to produce CPS to similar levels as strain TA-1, but CPS-recovery was easier because of the low viscosity of the medium and growth of the cells in pellets. With strain TA-1 in nitrogen-limited continuous cultures with a constant biomass of 500mg cell protein/l, EPS was the most abundant polysaccharide present at every dilution rate D (between 0.12 and 0.02 h^–1). The production rates were 50–100mg/g protein/h for EPS and 15–20mg/g protein/h for CPS. Only low amounts of cyclic -(1,2)-glucans were excreted (10–30 mg/l) over the entire range of growth rates.Abbreviations bv biovar - CPS capsular polysaccharide - EPS extracellular polysaccharide - HM_r high molecular mass - LM_r low molecular mass - YEMCR Yeast Extract-Mannitol-Congo Red agar     

Partial characterization of extracellular polysaccharides produced by cyanobacterium Arthrospira platensis

In this study, the physico-chemical characteristics of extracellular polysaccharides (EPS) produced by Arthrospira platensis were evaluated. Elemental analysis and a bicinchoninic acid (BCA) reaction indicated that the EPS were heteropolysaccharides that contain carbohydrate (13%) and protein (55%) moieties. Analysis of the infrared spectrum and elemental analysis revealed the presence of a sulfate group (0.5%). The UV-visible spectrum showed high UV absorption at 190∼230 nm and a shoulder at 260∼280 nm. In addition, this spectrum indicated that EPS can form aggregates with mycosporine-like amino acids and/or scytonemin. Gas chromatography analysis of the carbohydrate portion of the EPS indicated that it was composed of seven neutral sugars: galactose (14.9%), xylose (14.3%), glucose (13.2%), frucose (13.2%), rhamnose (3.7%), arabinose (1%), and mannose (0.3%) and two uronic acids, galacturonic acid (13.5%) and glucuronic acid (0.9%).     

Sulfation of extracellular polysaccharides of red microalgae: preparation, characterization and properties

Polysaccharides are natural polymers with a variety of properties that may be translated into significant commercial applications. A program of chemical modifications of the extracellular polysaccharides of red microalgae, such as Porphyridium sp. and Rhodella reticulata, has been undertaken by our group in order to tailor new properties and hence to broaden the spectrum of potential applications. These algal biopolymers are anionic in nature due to the presence of uronic acids (about 10%) and sulfate half esters (about 7%). In the current study, the sulfate content of these biopolymers was increased to 35-40% by means of sulfation agents such as pyridine SO(3), DMF.SO(3) and ClSO(3)H. Reaction conditions were optimized in a model system based on potato starch as the model polysaccharide (type of reagent, temperature and time of reaction). After work-up procedures, the highest sulfate content was obtained by sulfation of the polysaccharide of Porphyridium sp. with a mixture of ClSO(3)H and pyridine at 70 degrees C for 1 h. The sulfated products were characterized by chemical and rheological analyses, IR spectroscopy, and GPC-HPLC chromatography. "Oversulfated" polymers (having sulfate contents exceeding 20%) with high molecular weights were found to inhibit mammalian cell growth when used at certain concentrations; for example, over 80% inhibition was obtained when oversulfated polymers at a concentration of 200 microg/ml were tested on T-cell lymphoma line 24-1. These preliminary results indicate that the modified polysaccharides do indeed exhibit potential therapeutic properties.     

Studies on the production of extracellular protease by Alcaligenes faecalis

Alcaligenes faecalis produced extracellular protease when incubated in media containing protein substrates. Enzyme production was found to be influenced by various culture conditions. Enzyme production was growth-associated, expressed linearity with growth and reached a maximum at the end of the growth phase. Carbohydrates and inorganic nitrogen sources could not be utilized by the bacterium for its growth, and organic nitrogen appeared to be a primary determinant in protease production. Enzyme production reached its maximum level of 171.2 U/ml when the culture was incubated at 30 °C at pH 8.0. Ca²⁺ and Mg²⁺ enhanced the enzyme production. The crude enzyme powder was stable at high alkaline pH and stable upto 6 months at the storage temperature of 0–4 °C. This revised version was published online in July 2006 with corrections to the Cover Date.     

Formation of fermentation products and extracellular protease during anaerobic growth of Bacillus licheniformis in chemostat and batch-culture

For a relaxed (rel-), protease producing (A-type) and a stringent (rel+), not-protease producing (B-type) variant of Bacillus licheniformis we determined fermentation patterns and products, growth parameters and alkaline protease-production (if any) in anaerobic, glucose-grown chemostats and batch-cultures. Glucose is dissimilated via glycolysis and oxidative pentose phosphate pathway simultaneously; the relative share of these two routes depends on growth phase (in batch) and specific growth rate (in chemostat). Predominant products are lactate, glycerol and acetaldehyde for A-type batches and acetaldehyde, ethanol, acetate and lactate for B-type batches. Both types show a considerable acetaldehyde production. In chemostat cultures, the fermentation products resemble those in batch-culture. From the anaerobic batches and chemostats, we conclude that the A-type (with low ATP-yield) will have a YATPmax of probably 12.9 g/mol and the B-type (with high ATP-yield) a YATPmax of about 10.1 g/mol. For batch-cultures, both types have about the same, high Yglucose (12 g/mol). So, the slow-growing A-type has a relatively high efficiency of anaerobic growth (i.e. an efficient use of ATP) and the fast-growing B-type a relatively low efficiency of anaerobic growth. In aerobic batch-cultures, we found 48, respectively 41% glucose-carbon conversion into mainly glycerol and pyruvate, respectively acetate as overflow metabolites in the A- and B-type. In both aerobic and anaerobic batch-cultures of the A-type, protease is produced predominantly in the logarithmic and early stationary phase, while a low but steady production is maintained in the stationary phase. Protease production occurs via de novo synthesis; up to 10% of the total protease in a culture is present in a cell-associated form. Although anaerobic protease production (expressed as protease per amount of biomass) is much higher than for aerobic conditions, specific rates of production are in the same range as for aerobic conditions while, most important, the substrate costs of anaerobic production are very much higher than for aerobic conditions.     

Formation of thionates by freshwater and marine strains of sulfate-reducing bacteria

The formation of thionates (thiosulfate, trithionate and tetrahionate) during the reduction of sulfate or sulfite was studied with four marine and four freshwater strains of sulfate-reducing bacteria. Growing cultures of two strains of the freshwater species Desulfovibrio desulfuricans formed up to 400 M thiosulfate and 100 M trithionate under conditions of electron donor limitation. Tetrathionate was observed in lower concentrations of up to 30 M. Uncoupler-treated washed cells of the four freshwater strains formed thiosulfate and trithionate at low electron donor concentrations with sulfite in excess. In contrast, only one of four marine strains formed thionates. The freshwater strain Desulfobulbus propionicus transformed sulfite almost completely to thiosulfate and trithionate. The amounts produced increased with time, concentration of added sulfite and cell density. Tetrathionate was detected only occasionally and in low concentrations, and was probably formed by chemical oxidation of thiosulfate. The results confirm the diversity of the sulfite reduction pathways in sulfate-reducing bacteria, and suggest that thiosulfate and trithionate are normal by-products of sulfate reduction.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone     

Production of extracellular enzymes and aflatoxins in solid substrate fermentation with aflatoxigenic<Emphasis Type="Italic">Aspergillus</Emphasis> spp.

AflatoxigenicAspergillus flavus andAspergillus parasiticus were subjected to solid substrate fermentation process for 6 days to determine the formation of aflatoxins and production of extracellular enzymes (amyloglucosidase, cellulase, invertase and proteinase). Both organisms produced enzymes which generally increased with fermentation.Aspergillus flavus produced four enzymes whereasA. parasiticus produced three with no proteinase activity.Aspergillus parasiticus produced aflatoxins B₁, B₂ and G₁ but no G₂ andA. flavus produced aflatoxins B₁ and B₂. Invertase showed the highest activity withA. parasiticus and that corresponded with the highest total toxin produced. The enzyme activities were higher withA. parasiticus thanA. flavus although total toxins produced byA. parasiticus were lower than total toxins produced byA. flavus under the same environmental conditions.     

Oxidation of syringic acid by extracellular peroxidase of white-rot fungus,Pleurotus ostreatus

The oxidative degradation of syringic acid by the extracellular peroxidase ofPleurotus ostreatus was studied. Three products formed in the oxidation of syringic acid by the peroxidase in the presence of O₂ and H₂O₂ were identified as 2,6-dimethoxyphenol, 2,6-dimethoxy-1,4-dihydroxybenzene, and 2,6-dimethoxy-1,4-benzoquinone. A free radical was detected as the reaction intermediate of the extracellular peroxidase-catalyzed oxidation of acetosyringone. These results can be explained by mechanisms involving the production of a phenoxy radical and subsequent decarboxylation. This is the first time that 2,6-dimethoxyphenol has been identified in extracellular peroxidase-catalyzed reactions.     

Biosensor studies of the binding of extracellular matrix components with immobilized Escherichia coli O157:H7 and inhibition by polysulfated polysaccharides

Binding interactions of immobilized E. coli O157:H7 with collagen I, fibronectin, laminin and glucoaminoglycans were studied utilizing a surface plasmon resonance biosensor. A model system was developed to evaluate the inhibition of collagen-laminin binding on the E. coli sensor surface with polysulfated polysaccharides such as heparan sulfate and carrageenans. Results showed that carrageenans inhibited 71–99% while heparan sulfate inhibited 39–41% of collagen/laminin binding to E. coli sensor surface. These studies allowed a rapid assessment of compounds for carcass treatment to inhibit or detach pathogens from meat and poultry.     

Activity and thermostability of carboxymethylcellulase from Aspergillus niger is strongly influenced by noncovalently attached polysaccharides

Removal of non-covalently attached polysaccharides from carboxymethylcellulase (CMCase) of Aspergillus niger improved its activity but decreased its thermostability and protease resistance. The activation energy profile of the hydrolysis of carboxymethylcellulose (CMC) was triphasic with increasing values of 17,-55 and-562 kJ/mol for polysaccharide-free and 19, -21 and -207 kJ/mol for polysaccharide-complexed CMCase. The specificity constant (V_max/K_m) of polysaccharide-free CMCase was 1.41 compared to polysaccharide-complexed CMCase which was only 0.68. The polysaccharide free CMCase had lower thermostability (melting point = 82°C) and higher protease susceptibility compared to polysaccharide-complexed CMCase (melting point>100°C).The authors are with the National Institute for Biotechnology and Genetic Engineering (NIBGE), P.O. Box 577, Jhang Road, Faisalabad, Pakistan;     

Carbon assimilation and extracellular antigens of some yeast-like fungi

Many yeast-like fungi assimilated n-hexadecane, butylamine and putrescine as sole carbon sources. Methanol was not assimilated. This points to a physiological similarity to endomycetous, hydrocarbon-utilizing yeasts. Stephanoascus ciferrii assimilated uric acid, adenine and allantoin as sole source of carbon and nitrogen. All strains of Geotrichum candidum and many other yeast-like fungi assimilated acetoin and butan-2,3-diol. Assimilation tests for adenine, uric acid, allantoin, acetoin and butan-2,3-diol were found to be suitable for taxonomic purposes.Extracellular antigens immunologically related to those produced by Geotrichum candidum were detected in the cell-free culture liquids of several yeast-like fungi. The extracellular antigen excreted by Stephanoascus ciferrii was species-specific.     

Improvement of lipid profile and antioxidant of hypercholesterolemic albino rats by polysaccharides extracted from the green alga Ulva lactuca Linnaeus

Sulfated polysaccharides from Ulva lactuca were extracted in hot water and precipitated by ethanol then orally gavaged to rats fed on a hypercholesterolemic diet for 21 days to evaluate the antihypercholesterolemic and antioxidant actions. Atorvastatine Ca (Lipitor) was used as a reference drug. The intragastric administration of U. lactuca extract to hypercholesterolemic rats caused significant decrease of serum total lipids, triglycerides, total cholesterol, LDL-cholesterol and vLDL-cholesterol levels. Whereas, HDL-cholesterol concentration was markedly increased by 180%. Aqueous extract showed a significant ameliorative action on elevated atherogenic index, creatine kinase and lactate dehydrogenase activities of hypercholesterolemic group. Furthermore, serum activities of transaminases and alkaline phosphatase were also improved. High fat diet intake caused a highly significantly elevated serum urea, creatinine concentration. These effects were reversed by oral administration of U. lactuca extract. Sulfates polysaccharides extract of U. lactuca ameliorate hepatic enzymatic (catalase, glutathione peroxidase and superoxide dismutase), non-enzymatic (reduced glutathione & total thiol) antioxidant defenses and thiobarbituric acid reactive substances. In conclusion, the tested U. lactuca polysaccharides extract has potent hypocholesterolemic and antioxidant effects in experimentally-induced hypercholesterolemic animal model.     

Characterisation of the extracellular polysaccharides produced by isolates of the fungus Acremonium

Solid state (13)C NMR studies of the extracellular glucans from the fungi Acremonium persicinum C38 (QM107a) and Acremonium sp. strain C106 indicated a backbone of (1-->3)-beta-linked glucosyl residues with single (1-->6)-beta-linked glucosyl side branches for both glucans. Analyses of enzymatic digestion products suggested that the average branching frequency for the A. persicinum glucan (66.7% branched) was much higher than that of the Acremonium sp. strain C106 glucan (28.6% branched). The solid state (13)C NMR spectra also indicated that both glucans are amorphous polymers with no crystalline regions, and the individual chains are probably arranged as triple helices.