EVIDENCE FOR INVOLVEMENT OF A VOLATILE EXTRACELLULAR FACTOR IN PSEUDOMONAS-SOLANACEARUM VIRULENCE GENE-EXPRESSION

A complex regulatory network controls virulence genes of Pseudomonas solanacearum. Analysis of the transposon-generated mutant AW1-83 suggests that a new locus, designated phcB, may play a role in this network. AW1-83 (phcB83) produced at least 30-fold less than the wild type of extracellular polysaccharide (EPS I, encoded in part by eps) and at least seven extracellular proteins, but these traits were fully restored in response to one or more extracellular factors (EF) released by wild-type P. solanacearum. Presence of EF increased transcription of a genomic eps::lacZ fusion in a phcB83 background more than 50-fold, restoring wild-type expression. The EF made by P. solanacearum was present in both the aqueous and the vapor phases. Millimolar levels of methanol (but not larger alcohols) and micromolar levels of C14- to C18-fatty acid methyl esters (but not larger or smaller methyl esters) also restored nearly wild-type expression of eps::lacZ in a phcB83 background. The methoxy group was essential for this increase, since neither free fatty acids nor the ethyl or propyl esters were active. Growth with the C16-methyl ester restored normal production of EPS I and extracellular proteins by AW1-83. The wild-type phcB locus was subcloned on a 4-kb fragment and delimited to less than 2 kb by transposon inactivation and complementation studies. Genomic phcB::Tn3HoHo1 mutations appeared to eliminate EF production but did not uniformly reduce production of EPS I and extracellular proteins. Site-specific recombination of the phcB83 allele into the genome of five other P. solanacearum strains revealed that they have a structurally and functionally conserved phcB locus. Although all 80 wild-type strains of P. solanacearum tested made some EF, out of seven genera of bacteria tested, only Agrobacterium produced an EF-like activity that stimulated visible EPS production by AW1-83. Our results suggest that the EF may be an extracellular signal molecule in P. solanacearum that is different from the acyl-homoserine lactone signal compounds produced by Vibrio fischeri and other Gram-negative bacteria.