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...
The extracellular polysaccharide alginate has been widely associated with chronic Pseudomonas aeruginosa infections in the cystic fibrosis lung. However, it is clear that alginate biosynthesis is a more widespread phenomenon. Alginate plays a key role as a virulence factor of plant-pathogenic pseudomonads, in the formation of biofilms and with the encystment process of Azotobacter spp.
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