Pseudomonas Aeruginosa AlgG Is a Polymer Level Alginate C5-mannuronan Epimerase

J Bacteriol. 1994 Apr;176(7):1821-30. doi: 10.1128/jb.176.7.1821-1830.1994.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alginates / metabolism*
  • Amino Acid Sequence
  • Base Sequence
  • Carbohydrate Epimerases / genetics
  • Carbohydrate Epimerases / metabolism*
  • Cell Compartmentation
  • Escherichia coli / genetics
  • Genetic Complementation Test
  • Hexuronic Acids / metabolism*
  • Isomerism
  • Molecular Sequence Data
  • Polysaccharides, Bacterial / metabolism*
  • Protein Precursors / metabolism
  • Pseudomonas aeruginosa / enzymology*
  • Recombinant Proteins / biosynthesis
  • Sequence Analysis, DNA
  • Uronic Acids / metabolism*

Substances

  • Alginates
  • Hexuronic Acids
  • Polysaccharides, Bacterial
  • Protein Precursors
  • Recombinant Proteins
  • Uronic Acids
  • guluronic acid
  • mannuronic acid
  • Carbohydrate Epimerases
  • mannuronan c-5-epimerase

Associated data

  • GENBANK/U27829