Alginate is a viscous extracellular polymer produced by mucoid strains of Pseudomonas aeruginosa that cause chronic pulmonary infections in patients with cystic fibrosis. Alginate is polymerized from GDP-mannuronate to a linear polymer of ,B-1-4-linked residues of D-mannuronate and its C5-epimer, L-guluronate. We previously identified a gene called algG in the alginate biosynthetic operon that is required for incorporation of L-guluronate residues into alginate. In this study, we tested the hypothesis that the product of algG is a C5-epimerase that directly converts D-mannuronate to L-guluronate. The DNA sequence of algG was determined, and an open reading frame encoding a protein (AlgG) of approximately 60 kDa was identified. The inferred amino terminus of AlgG protein contained a putative signal sequence of 35 amino acids. Expression of aIgG in Escherichia coli demonstrated both 60-kDa pre-AIgG and 55-kDa mature AlgG proteins, the latter of which was localized to the periplasm. An N-terminal analysis of AIgG showed that the signal sequence was removed in the mature form. Pulse-chase experiments in both E. coli and P. aeruginosa provided evidence for conversion of the 60-to the 55-kDa size in vivo. Expression of algG from a plasmid in an algG (i.e., polymannuronate-producing) mutant of P. aeruginosa restored production of an alginate containing Lguluronate residues. The observation that AlgG is apparently processed and exported from the cytoplasm suggested that it may act as a polymer-level mannuronan C5-epimerase. An in vitro assay for mannuronan C5 epimerization was developed wherein extracts of E. coli expressing high levels of AlgG were incubated with polymannuronate. Epimerization of D-mannuronate to L-guluronate residues in the polymer was detected enzymatically, using a L-guluronate-specific alginate lyase of Klebsieila aerogenes. Epimerization was also detected in the in vitro reaction between recombinant AlgG and poly-D-mannuronate, using high-performance anion-exchange chromatography. The epimerization reaction was detected only when acetyl groups were removed from the poly-D-mannuronate substrate, suggesting that AIgG epimerization activity in vivo may be sensitive to acetylation of the D-mannuronan residues. These results demonstrate that AlgG has polymer-level mannuronan C5-epimerase activity.Alginate is an unbranched polysaccharide produced by Pseudomonas species (10, 13), Azotobacter vinelandii (31), and several species of brown seaweed (22). Alginate is composed of D-mannuronate and its C5-epimer, L-guluronate, which are linked by 13-1-4 glycosidic bonds (13). The L-guluronate is probably derived from D-mannuronate by the action of a C5-epimerase (Fig. 1). In bacteria, alginate is modified by the addition of O-acetyl groups on some D-mannuronate residues (8, 42). The sugar residues of alginate do not show repeating subunits characteristic of other bacterial exopolysaccharides (46). Mucoid strains of Pseudomonas aeruginosa produce alginate as a capsule-like exopolysaccharide and are responsible f...
ABSTRACT. Disturbance has been shown to be an important component of the ecology of soft-bottom macrobenthic and meiobenthic marine communities. Its importance in the ecology of microbial communities was investigated by using sieving of marine sediments as a controlled disturbance. Following the disturbance, sediments were maintained in microcosms. Using a suite of biochemical measures, sieving was found to influence microbial biomass, community structure, and metabolic activity. Sieving caused an immedate decrease in microbial growth rates and a shift in metabolic status towards the synthesis of phospholipid. Microbial biomass was initially unaffected. Several hours later, growth rates increased and biomass had decreased by 75 %. Microbial biomass returned to pre-disturbance levels 8 h after sieving. Groups of phospholipid, ester-linked fatty acids, each associated with different functional groups of microorganisms, varied in their response to sieving. This result suggested that components of the microbial community differed in their reaction to this disturbance. Ambient sediments collected at the time of the construction of the microcosms were contrasted with sediments maintained in the laboratory microcosms for 5 d. Laboratory conditions significantly altered the microbial community structure and growth rates were significantly lower. Measures of metabolic status indicated that some of the microorganisms were stressed. This study demonstrates the potential significance of disturbance in the ecology of the benthic microbial community and that uncoupling sediment from the biotic and abiotic influences of the environment significantly affects the composition and activity of the microbial community.
Accurate measurement of the mass and activities of environmental microbial assemblies requires methods that differ from those of classical microbiology. Using the leaf-litter detrital microflora recovered after incubation in a semitropical estuary, biochemical assays of multiple components can be made which give reasonably similar estimates of microbial mass, using normalization values from bacterial monocultures. The assays involve recovery of adenosine triphosphate, the unique bacterial cell wall mucopeptide component muramic acid, the uniquely prokaryotic endogenous storage material poly-β-hydroxybutyrate, a series of exoesterase activities, and the extractable lipids, primarily the phospholipids and the glycolipids. If the sampling conditions are such that incorporation of radiosotopes into the microflora is possible, these assays become extraordinarily sensitive. If radioisotope usage can be coupled with the purification of particular lipids, it is possible to gain information on the structure and dynamics of the microbial population.
The role of disturbance in natural microbial systems was studied by examining sediments disturbed by feeding sting rays and enteropneust worms. Biochemical measures based on the chemical components of cells were used to follow changes in the microbial community. The responses of the microbial community to feeding by rays, a mechanical disturbance to the sediments, were timedependent. Several hours after the disturbance microbial metabolic status was shifted toward synthesis of phospholipid. Microbial growth rates were greater in disturbed sediments than in ambient sediment while microbial biomass was significantly lower in the disturbed sediments. Later, microbial biomass, growth rates and metabolic status were similar in the ambient and disturbed sediments. Ingestion and subsequent defecation of sediment by enteropneust worms also profoundly affected the microbial community. The biomass and growth rates of the microbial community were significantly lower In the fresh fecal castings compared to ambient sediment while metabolic status was shifted toward phospholipid synthesis. Six hours later, microbial growth rates were greater in the fecal castings than in ambient sediments while biomass had yet to recover. Groups of ester-Linked, phospholipid fatty acids, each associated with a functional group of microorganisms, v a r~e d in their response to disturbance, indcating components of the microbial community varied in their response to disturbance. An a posteriori comparison of the ambient sediments from the several study sites, each receiving differing amounts of wave and tidal action, showed that abiotic factors also influenced the microbial community structure and metabolic status. This study presents evidence that biotic and abiotic disturbances may be important factors controlling the structure of microbial communities in estuarine sediments.
The hydrophobic and electrostatic characteristics of bacterial cell surfaces were compared with attachment proclivity and biomass accumulation over time between wildtype Pseudomonas aeruginosa serotype O6 (possesses A and B band LPS), and three LPS-deficient mutants, viz. A28 (A + B -), R5 (A + B -), and Gt700 (A -B -). The hydrophobic character of each serotype was determined by hydrophobic interaction chromatography and salt-aggregation, and strains were ranked similarly by each method, viz. R5 A28 > Gt700 > 06. The anionic characteristics of cell-surfaces were determined by electrostatic interaction chromatography and by zeta-potential measurements, and ranked R5 > A28 Gt700 > 06. Adhesion and biofilm accumulation on stainless steel were significantly different between strains, following the order R5 > A28 >> O6>Gt700. Biofilm rankings were similar on glass, a second hydrophilic substratum. The mutant strains with a strongly hydrophobic character (R5 and A28) demonstrated a significantly greater capacity to form biofilms. These adherent mutants also appeared to have a more anionic cell surface, which may have played a role in biofilm formation on the hydrophilic substrata.
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