HIERARCHICAL AUTOINDUCTION IN RALSTONIA-SOLANACEARUM - CONTROL OF ACYL-HOMOSERINE LACTONE PRODUCTION BY A NOVEL AUTOREGULATORY SYSTEM RESPONSIVE TO 3-HYDROXYPALMITIC ACID METHYL-ESTER

Bacteria employ autoinduction systems to sense the onset of appropriat e cell density for expression of developmental genes. In many gram-neg ative bacteria, autoinduction involves the production of and response to diffusible acylated-homoserine lactones (acyl-HSLs) and is mediated by members of the LuxR and LuxI families. Ralstonia (Pseudomonas) sol anacearum, a phytopathogenic bacterium that appears to autoregulate it s virulence genes, produces compounds that promote expression of sever al heterologous acyl-HSL-responsive reporter gene constructs. High-pre ssure liquid chromatography of highly concentrated ethyl acetate extra cts revealed that culture supernatants of strain AW1 contained two com pounds with retention times similar to N-hexanoyl- and N-octanoyl-HSL. To investigate the role of these acyl-HSLs in R. solanacearum virulen ce gene expression, transposon mutants that were deficient for inducin g an acyl-HSL-responsive reporter in Agrobacterium tumefaciens were ge nerated. Three loci involved in normal acyl-HSL production were identi fied, one of which was shown to contain the divergently transcribed so lR and solI genes, the luxR and luxI homologs, respectively. A 4.1-kb fragment containing solR and solI enabled all of the mutants (regardle ss of the locus inactivated) and a naturally acyl-HSL-defective strain of R. solanacearum to produce acyl-HSLs. Inactivation of soil abolish ed production of all detectable acyl-HSLs but affected neither the exp ression of virulence genes in culture nor the ability to wilt tomato p lants. AW1 has a functional autoinduction system, because (i) expressi on of soil required SolR and acyl-HSL and (ii) expression of a gene li nked to solR and solI, designated aidA, was acyl-HSL dependent. Becaus e AidA has no homologs in the protein databases, its discovery provide d no clues as to the role of acyl-HSLs in R. solanacearum gene regulat ion. However, expression of solR and solI required the global LysR-typ e virulence regulator PhcA, and both solR and solI exhibited a cell de nsity-associated pattern of expression similar to other PhcA-regulated genes. The acyl-HSL-dependent autoinduction system in R. solanacearum is part of a more complex autoregulatory hierarchy, since the transcr iptional activity of PhcA is itself controlled by a novel autoregulato ry system that responds to 3-hydroxypalmitic acid methyl ester.