Characterization of algG encoding C5-epimerase in the alginate biosynthetic gene cluster of Pseudomonas fluorescens

The organization of the alginate gene cluster in Pseudomonas fluorescens wa s characterized. A bank of genomic DNA from P. fluorescens was mobilized to a strain of Pseudomonas aeruginosa with a transposon insertion (algJ::Tn50 1) in the alginate biosynthetic operon that rendered it non-mucoid. Phenoty pic complementation in this heterologous host was observed, and a complemen ting clone containing 32 kb of P. fluorescens DNA was obtained. Southern hy bridization 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 m utant that produced alginate as polymannuronate due to its C5-epimerase def ect, complementation was observed and the alginate from the recombinant str ain contained L-guluronate as determined by proton nuclear magnetic resonan ce spectroscopy. A sequence analysis of the P. fluorescens DNA containing a lgG revealed sequences similar to P. aeruginosa algG that were also flanked by algE- and algX-like sequences. The predicted AlgG amino acid sequence o f P. fluorescens was 67% identical (80% similar) to P. aeruginosa AlgG and 60% identical (76% similar) to Azotobacter vinelandii AlgG. As in P. aerugi nosa, AlgG from P. fluorescens appeared to have a signal sequence that woul d 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 we re defective in alginate production, suggesting a potential role for this p rotein in polymer formation. (C) 2001 Elsevier Science B.V. All rights rese rved.