Molecular characterization of two gene loci required for production of the key pathogenicity factor pectate lyase in Pseudomonas viridiflava.

Four pleiotropic mutants of Pseudomonas viridiflava strain PJ-08-6A that were deficient in production of both pectate lyase (Pel) and protease (Prt) were isolated following transposon mutagenesis. Unlike secretion-defective (Out-) mutants, these four showed no accumulation of enzymes within the cells. Southern hybridization analysis revealed that each mutant had Tn5 inserted in one of two EcoRI genomic fragments. These EcoRI fragments (5.2- and 6.3-kb) appeared to contain two distinct gene loci, designated repA and repB, which were required for production of extracellular enzymes in this bacterium. Cosmid clones carrying the functional repA and repB DNA fragments were identified in a genomic library of strain PJ-08-6A. After analysis of repA+ plasmids by restriction mapping and marker-exchange mutagenesis, the repA gene was located in a joint region between the 1.8-kb EcoRI-HindIII and 2.8-kb EcoRI fragments cloned. Nucleotide sequence analysis of the repA region revealed the presence of an open reading frame consisting of 2,790 bases. The RepA protein predicted from the DNA sequence showed 93% similarity in amino acid sequence to the LemA protein of P. syringae pv. syringae, which was previously identified as a member of a two-component global regulatory system. A plasmid carrying the lemA gene of P. syringae pv. syringae was capable of complementing the RepA- mutation in P. viridiflava. The functions of the repA and lemA genes thus appear to be similar and interchangeable. Mutants of P. viridiflava strain SF312A deficient in production of Pel, Prt, and the exopolysaccharide alginate also were identified.(ABSTRACT TRUNCATED AT 250 WORDS)