Quorum sensing signaling studies in Pseudomonas syringae pv. actinidae

Quorum sensing (QS) is an intercellular communication system in bacteria linking cell density to gene expression via the production and detection of signal molecules. In Gram-negative bacteria, N-acyl homoserine lactones (AHLs) signas are most commonly used being produced by an AHL synthase belonging to the LuxI-protein family and a transcriptional regulator belonging to the LuxR family. QS gene regulation plays important roles in many bacterial plant pathogens and it is therefore of interest to determine the type and role of QS signaling in Pseudomonas syringae pv. actinidiae (PSA). It was verified that PSA does not produce AHLs and that a prototypical complete LuxI/R QS system is absent. PSA however possesses three putative LuxR solos (LuxRs devoid of a cognate LuxI synthase) which we designated as PsaR1, PsaR2 and PsaR3. PsaR1 and PsaR3 could be involved in responding to exogenous AHLs produced by neighbouring bacteria whereas PsaR2 belongs to the subfamily of LuxR solos from plant associated bacteria (PAB) that binds and responds to yet unknown plant signal molecules. In silico modelling has highlighted that PsaR1 and PsaR3 possess the molecular determinants for AHL binding, whereas PsaR2 does not and has selectivity towards plant signals. Mutation in all the three luxR solos showed reduction of in planta survival having additive effects if more that one solo was inactivated in double mutants. These LuxRs solos are therefore important for in planta growth/survival of PSA. It is believed that most bacteria undergo QS by producing and responding to an endogenous signal; for this reason we postulate that PSA most likely possesses a complete QS system. In order to find and characterize this putative system we performed a genetic screen for PSA Tn5 transposon mutants which were altered in phenotypes that are typically regulated by QS. After screening 40,000 mutants, we identified 70 Tn5 mutants which were altered in production of a secreted enzyme and/or exopolysaccharide. Mapping all these mutants revealed that the transposon was inserted in metabolism, structural, transport, bacterial movement and regulatory genes; initial analysis of all of these loci does not indicate that any of them could be a known or novel QS system (however transposon insertions in the GacA/GacS and AlgZ/AlgR regulators, which are known to regulate QS in P. aeruginosa, have been isolated). The data of the three LuxRs solos and of the genetic screen aimed to identify QS signaling system in PSA will be presented and discussed.