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The Pseudomonas fluorescens AlgG protein, but not its mannuronan C-5-epimerase activity, is needed for alginate polymer formation.

ABSTRACT: Bacterial alginates are produced as 1-4-linked beta-D-mannuronan, followed by epimerization of some of the mannuronic acid residues to alpha-L-guluronic acid. Here we report the isolation of four different epimerization-defective point mutants of the periplasmic Pseudomonas fluorescens mannuronan C-5-epimerase AlgG. All mutations affected amino acids conserved among AlgG-epimerases and were clustered in a part of the enzyme also sharing some sequence similarity to a group of secreted epimerases previously reported in Azotobacter vinelandii. An algG-deletion mutant was constructed and found to produce predominantly a dimer containing a 4-deoxy-L-erythro-hex-4-enepyranosyluronate residue at the nonreducing end and a mannuronic acid residue at the reducing end. The production of this dimer is the result of the activity of an alginate lyase, AlgL, whose in vivo activity is much more limited in the presence of AlgG. A strain expressing both an epimerase-defective (point mutation) and a wild-type epimerase was constructed and shown to produce two types of alginate molecules: one class being pure mannuronan and the other having the wild-type content of guluronic acid residues. This formation of two distinct classes of polymers in a genetically pure cell line can be explained by assuming that AlgG is part of a periplasmic protein complex.

SUBMITTER: Gimmestad M

PROVIDER: S-EPMC156231 | biostudies-literature | 2003 Jun

REPOSITORIES: biostudies-literature

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The Pseudomonas fluorescens AlgG protein, but not its mannuronan C-5-epimerase activity, is needed for alginate polymer formation.

Gimmestad Martin M Sletta Håvard H Ertesvåg Helga H Bakkevig Karianne K Jain Sumita S Suh Sang-jin SJ Skjåk-Braek Gudmund G Ellingsen Trond E TE Ohman Dennis E DE Valla Svein S

Journal of bacteriology 20030601 12

Bacterial alginates are produced as 1-4-linked beta-D-mannuronan, followed by epimerization of some of the mannuronic acid residues to alpha-L-guluronic acid. Here we report the isolation of four different epimerization-defective point mutants of the periplasmic Pseudomonas fluorescens mannuronan C-5-epimerase AlgG. All mutations affected amino acids conserved among AlgG-epimerases and were clustered in a part of the enzyme also sharing some sequence similarity to a group of secreted epimerases ...[more]

PMID: 12775688

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Project description: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.

Project description:Chronic pulmonary infection with Pseudomonas aeruginosa is a common and serious problem in patients with cystic fibrosis (CF). The P. aeruginosa isolates from these patients typically have a mucoid colony morphology due to overproduction of the exopolysaccharide alginate, which contributes to the persistence of the organisms in the CF lung. Most of the alginate biosynthetic genes are clustered in the algD operon, located at 34 min on the chromosome. Alginate biosynthesis begins with the formation of an activated monomer, GDP-mannuronate, which is known to occur via the products of the algA, algC, and algD genes. Polymannuronate forms in the periplasm, but the gene products involved in mannuronate translocation across the inner membrane and its polymerization are not known. One locus of the operon which remained uncharacterized was a new gene called algK between alg44 and algE. We sequenced algK from the mucoid CF isolate FRD1 and expressed it in Escherichia coli, which revealed a polypeptide of the predicted size (52 kDa). The sequence of AlgK showed an apparent signal peptide characteristic of a lipoprotein. AlgK-PhoA fusion proteins were constructed and shown to be active, indicating that AlgK has a periplasmic subcellular localization. To test the phenotype of an AlgK-mutant, the algK coding sequence was replaced with a nonpolar gentamicin resistance cassette to avoid polar effects on genes downstream of algK that are essential for polymer formation. The algKdelta mutant was nonmucoid, demonstrating that AlgK was required for alginate production. Also, AlgK- mutants demonstrated a small-colony phenotype on L agar, suggesting that the loss of AlgK also caused a growth defect. The mutant phenotypes were complemented by a plasmid expressing algK in trans. When the algKdelta mutation was placed in an algJ::Tn501 background, where algA was not expressed due to polar transposon effects, the growth defect was not observed. AlgK- mutants appeared to accumulate a toxic extracellular product, and we hypothesized that this could be an unpolymerized alginate precursor. High levels of low-molecular-weight uronic acid were produced by the AlgK- mutant. When AlgK- culture supernatants were subjected to dialysis, high levels of uronic acids diffused out of the dialysis sac, and no uronic acids were detectable after extensive dialysis. In contrast, the mucoid wild-type strain produced only polymerized uronic acids (i.e., alginate), whereas the algKdelta algJ::Tn501 mutant produced no uronic acids. Thus, the alginate pathway in an AlgK- mutant was blocked after transport but at a step before polymerization, suggesting that AlgK plays an important role in the polymerization of mannuronate to alginate.

Project description:Polysaccharides often are necessary components of bacterial biofilms and capsules. Production of these biopolymers constitutes a drain on key components in the central carbon metabolism, but so far little is known concerning if and how the cells divide their resources between cell growth and production of exopolysaccharides. Alginate is an industrially important linear polysaccharide synthesized from fructose 6-phosphate by several bacterial species. The aim of this study was to identify genes that are necessary for obtaining a normal level of alginate production in alginate-producing Pseudomonas fluorescens.Polysaccharide biosynthesis is costly, since it utilizes nucleotide sugars and sequesters carbon. Consequently, transcription of the genes necessary for polysaccharide biosynthesis is usually tightly regulated. In this study we used an engineered P. fluorescens SBW25 derivative where all genes encoding the proteins needed for biosynthesis of alginate from fructose 6-phosphate and export of the polymer are expressed from inducible Pm promoters. In this way we would avoid identification of genes merely involved in regulating the expression of the alginate biosynthetic genes. The engineered strain was subjected to random transposon mutagenesis and a library of about 11500 mutants was screened for strains with altered alginate production. Identified inactivated genes were mainly found to encode proteins involved in metabolic pathways related to uptake and utilization of carbon, nitrogen and phosphor sources, biosynthesis of purine and tryptophan and peptidoglycan recycling.The majority of the identified mutants resulted in diminished alginate biosynthesis while cell yield in most cases were less affected. In some cases, however, a higher final cell yield were measured. The data indicate that when the supplies of fructose 6-phosphate or GTP are diminished, less alginate is produced. This should be taken into account when bacterial strains are designed for industrial polysaccharide production.

Dataset Information

The Pseudomonas fluorescens AlgG protein, but not its mannuronan C-5-epimerase activity, is needed for alginate polymer formation.

Publications

The Pseudomonas fluorescens AlgG protein, but not its mannuronan C-5-epimerase activity, is needed for alginate polymer formation.

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