Biological activity and regulation of cyclic lipopeptide production in Pseudomonas corrugata and Pseudomonas mediterranea

Pseudomonas corrugata and P. mediterranea, are two closely related bacteria both causal agents of tomato pith necrosis and recently evaluated as biocontrol agents and producers of industrially-promising biomolecules. The interest of our work focused on cyclic lipopeptides (CLPs) that are surface active molecules with antibacterial, antifungal and cytotoxic properties. P. corrugata is known to produce two kinds of cyclic lipopeptides, corpeptins and cormycin A. Investigations on P. corrugata strain CFBP5454 quorum sensing (QS) system helped to postulate its role on virulence mechanism mediated by CLPs during detrimental interaction with tomato plants. On the contrary no information was available on P. mediterranea/tomato molecular interaction. Both species are able to induce on tomato undistinguishable symptoms thus we thoughts that they likely share common virulence mechanisms. Starting from these premises we demonstrated that P. mediterranea CFBP 5447 harbours one system designated PmeI/R that is highly homologous to the PcoI/R system of P. corrugata and that virulence on tomatoes is affected by inactivation of QS genes in both species although with some differences. A LuxR regulator RfiA encoded by rfiA cotranscribed both with pcoI and pmeI was also demonstrated to be pivotal for virulence. The MALDI-TOF MS spectra of bacterial cell-free culture filtrates showed that P. corrugata CFBP5454 produces both corpeptins and cormycin and for the first time these CLPs were also found in a P. mediterranea strain which spectrum also showed a putatively unidentified bioactive compound. Mutational analysis revealed that P. mediterranea CFBP 5447 PmeI- and PmeR-, P. corrugata CFBP 5454 PcoR- and both RfiA-, were impaired in CLP production but also showed a drastic reduction of virulence when inoculated in tomato plants demonstrating their pivotal role. These results also evidence that the QS is involved in CLP production and all the correlated phenotypes (colony morphology, biosurfactant activity) via the RfiA transcriptional regulator. In the P. corrugata CFBP 5454 cosmid insert sequence from which the AHL QS genes were identified we describes three genes crpC, crpD, crpE. Bioinformatics and mutational analysis of crpC and crpDE demonstrated they are involved in the production and secretion of corpeptins since the derivative mutant strain cell-free culture filtrates showed only the peaks of cormycin in their MALDI-TOF MS spectra. Moreover, pathogenicity tests revealed that these long chain CLPs are important for P. corrugata virulence. Using real-time PCR we also ascertained that the regulators PcoR and RfiA are involved in corpeptins production/secretion. P. corrugata CFBP 5454 and P. mediterranea CFBP5447 produced in vitro diffusible compounds with antimicrobial properties which inhibited the growth of plant pathogenic fungi and bacteria, as well as antifungal volatile compounds. Antagonistic activity of QS mutants didn t different from that of their respective parent strains whereas antifungal activity was completely abolished in the rfiA- mutants. Since both the pcoR- (and pmeR-) and rfiA- mutants did not produce CLPs the production of one or more additional antimicrobial compound is suggested. In vivo experiment showed the good biocontrol activity of P. corrugata CFBP 5454 but also that the QS and rfiA mutants are still able to reduce the disease incidence in the in vivo investigated pathosystems. P. corrugata CFBP 5454 draft genome sequence helped in identifying a biosynthetic cluster hcnABC coding for the enzyme HCN synthase involved in this volatile compound biosynthesis. P. corrugata hcnA and hcnC knockout mutants failed to produce HCN and showed a reduced antimicrobial activity against Botrytis cinerea. Cyanogenesis seems to be not regulated either by AHL-QS or by RfiA because mutants still retained the ability to produce HCN.