title: Pseudomonas aeruginosa AlgG is a polymer level alginate C5-mannuronan epimerase.
abstract: 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 beta-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 algG in Escherichia coli demonstrated both 60-kDa pre-AlgG and 55-kDa mature AlgG proteins, the latter of which was localized to the periplasm. An N-terminal analysis of AlgG 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 inan algG (i.e., polymannuronate-producing) mutant of P. aeruginosa restored production of an alginate containing L-guluronate 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 Klebsiella 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 AlgG 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.
authors.affiliation: Department of Microbiology and Immunology, University of Tennessee, Memphis.
authors.forename: M J
authors.initials: MJ
authors.lastname: Franklin
authors.forename: C E
authors.initials: CE
authors.lastname: Chitnis
authors.forename: P
authors.initials: P
authors.lastname: Gacesa
authors.forename: A
authors.initials: A
authors.lastname: Sonesson
authors.forename: D C
authors.initials: DC
authors.lastname: White
authors.forename: D E
authors.initials: DE
authors.lastname: Ohman
mesh_terms: D000464:Alginates / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002238:Carbohydrate Epimerases / Q000235:genetics / Q000378:metabolism*; D002451:Cell Compartmentation; D004926:Escherichia coli / Q000235:genetics; D005816:Genetic Complementation Test; D006603:Hexuronic Acids / Q000378:metabolism*; D007536:Isomerism; D008969:Molecular Sequence Data; D011135:Polysaccharides, Bacterial / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D011994:Recombinant Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D014574:Uronic Acids / Q000378:metabolism*
chemical_list: D000464:Alginates; D006603:Hexuronic Acids; D011135:Polysaccharides, Bacterial; D011498:Protein Precursors; D011994:Recombinant Proteins; D014574:Uronic Acids; C007896:guluronic acid; C008324:mannuronic acid; D002238:Carbohydrate Epimerases; C086519:mannuronan c-5-epimerase