Molecular Characterization of Two Gene Loci Required for Production of the Key Pathogenicity Factor Pectate Lyase in Pseudomonas viridiflava. C. H. Liao. Plant Science and Technology Research Unit, Eastern Regional Research Center, U.S. Department of Agriculture, Agricultural Research Service, Philadelphia, PA 19118 U.S.A. D. E. McCallus, and W. F. Fett. Plant Science and Technology Research Unit, Eastern Regional Research Center, U.S. Department of Agriculture, Agricultural Research Service, Philadelphia, PA 19118 U.S.A. Received 19. MPMI 7:391-400. Accepted 18 January 1994. This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. The American Phytopathological Society, 1994.

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 EcoRl genomic fragments. These EcoRl fragments (5.2-and 6.3-kb) appeared lo contain two distinct gene loci, designated repA and repB, which were required for production of extracellular enzymes in this bacterium. Cos-mid 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 EcoRl 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 alginatc also were identified. Cosmid clones carrying the repA (but not repB) DNA of strain PJ-08-6A were able to restore the enzyme and alginate production in Rep- mutants of strain SF312A. The repA gene is therefore required for production of not only extracellular enzymes but also exopolysaccharides in P. viridiflava.