The organization of the alginate gene cluster in Pseudomonas fluorescens was characterized. A bank of genomic DNA from P. fluorescens was mobilized to a strain of Pseudomonas aeruginosa with a transposon insertion (algJ::Tn501) in the alginate biosynthetic operon that rendered it non-mucoid. Phenotypic complementation in this heterologous host was observed, and a complementing clone containing 32 kb of P. fluorescens DNA was obtained. Southern hybridization studies showed that genes involved in alginate biosynthesis (e.g. algD, algG, and algA) were approximately in the same order and position as in P. aeruginosa. When the clone was mobilized to a P. aeruginosa algG mutant that produced alginate as polymannuronate due to its C5-epimerase defect, complementation was observed and the alginate from the recombinant strain contained L-guluronate as determined by proton nuclear magnetic resonance spectroscopy. A sequence analysis of the P. fluorescens DNA containing algG revealed sequences similar to P. aeruginosa algG that were also flanked by algE- and algX-like sequences. The predicted AlgG amino acid sequence of P. fluorescens was 67% identical (80% similar) to P. aeruginosa AlgG and 60% identical (76% similar) to Azotobacter vinelandii AlgG. As in P. aeruginosa, AlgG from P. fluorescens appeared to have a signal sequence that would localize it to the periplasm where AlgG presumably acts as a C5- epimerase at the polymer level. Non-polar algG knockout mutants of P. fluorescens were defective in alginate production, suggesting a potential role for this protein in polymer formation
biosynthesis, complementation, biosynthetic, chemistry, gene, Bacterial, genetics, growth & development, host, metabolism, phenotype, potential, role, Sequence Analysis, DNA, strain, Support, characterization, polysaccharide, analysis, Pseudomonas, Pseudomonas aeruginosa, molecular, polymer, acid, cloning, level, Molecular Sequence Data, protein, signal, cluster, gene cluster, Multigene Family, mutant, mutants, epimerase, insertion, position, production, transposon, Magnetic Resonance Spectroscopy, nuclear, nuclear magnetic resonance, nuclear magnetic resonance spectroscopy, resonance, spectroscopy, sequence, mutation, formation, Pseudomonas fluorescens, amino, clone, biotechnology, Base Sequence, heterologous, mutagenesis, operon, amino acid, recombinant, organization, alginate, Azotobacter, Azotobacter vinelandii, U.S.Gov't, Non-P.H.S., order, amino acid sequence, Sequence Alignment, Sequence Homology, P.H.S., Carbohydrate Epimerases, alga, alginate biosynthesis, alginate gene, alginate production, Alginates, Cell Division, Gene Order, Insertional, nonmucoid, periplasm, phenotypic, proton, capsula, heterologuos complementation
NCBI PubMed ID: 11707327Journal NLM ID: 7706761Publisher: Amsterdam: Elsevier
Institutions: CRIBI, Biotechnology Centre, University of Padova, Viale G. Colombo 3, 35121 Padova, Italy
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