Cloning and Characterization of Four Genes of Rhizobium leguminosarum bv. trifolii Involved in Exopolysaccharide Production and Nodulation

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Abstract
Four different genes of Rhizobium leguminosarum bv. trifolii strain RBL5599 involved in exopolysaccharide (EPS) production were identified by complementation of Tn5-induced EPS-deficient mutants (Exo mutants) with a cosmid bank. On one cosmid pssA was located, which was found to be almost identical to the pss4 gene from R. leguminosarum bv. viciae VF39 and highly homologous to a family of glycosyl transferases. Two pssA mutants, exo2 and exo4, were characterized and found to produce 19 and 1% of the wild-type amount of EPS, respectively. The three other genes were found to be closely linked on a different complementing cosmid. pssC revealed similarity to exoM and exoW of R. meliloti, both encoding glucosyl transferases involved in the synthesis of succinoglycan. A mutation in this gene (mutant exo50) did reduce EPS synthesis to 27% of the wild-type amount. We found an operon closely linked to pssC, consisting of two overlapping genes, pssD and pssE, that is essential for EPS production. Homology of pssD and pssE was found with cps14F and cps14G of Streptococcus pneumoniae, respectively: two genes responsible for the second step in capsule polysaccharide synthesis. Furthermore, pssD and pssE were homologous to the 5' and 3' parts, respectively, of spsK of Sphingomonas S88, which encodes a putative glycosyl transferase. Structural analysis of EPS produced by Exo mutants exo2, exo4, and exo50 showed it to be identical to that of the parental strain RBL5599, with the exception of acetyl groups esterified to one of the glucose residues being absent.