Transcriptomic profiling of the PhzIR quorum sensing, Gac- and PtrA- regulons of Pseudomonas chlororaphis PA23
Shah, Nidhi Rakesh
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The biocontrol agent Pseudomonas chlororaphis PA23 protects canola (Brassica napus) against infection by the necrotrophic fungus Sclerotinia sclerotiorum. This bacterium secretes an arsenal of antifungal (AF) compounds including pyrrolnitrin, phenazines, hydrogen cyanide together with degradative enzymes that are believed to work in concert to suppress the pathogen. Production of these compounds is governed by a complex regulatory pathway that includes the GacA/GacS two component system, the PhzI/PhzR QS system, and a novel LTTR regulator, called PtrA. The focus of this research was to elucidate the PhzRI, Gac and PtrA regulons of P. chlororaphis PA23. Transcriptomic profiling of gacA-, gacS- and ptrA-mutants, two QS-deficient strains, PA23-AHL and PA23-phzR, and the wild type was undertaken using RNA-sequencing. It was discovered that these regulators each control between 5-7% of the PA23 genome. Decreased expression of genes responsible for the biosynthesis of phenazines, pyrrolnitrin, HCN and degradative enzymes together with elevated siderophore gene expression was observed in all five strains , relative to WT expression. The largest number of differentially expressed genes fell into the COG category for unknown function (S). Comparative analysis of the QS-deficient strains revealed that the regulon of the AHL-deficient PA23-AHL was larger than that of PA23-phzR; these findings were attributed to the presence of a second uncharacterised QS system in PA23 named CsaI/CsaR. For genes under QS control, regulation may occur directly through PhzR binding to an upstream “phz box” or indirectly through control of other regulators. Only 39 genes contained putative phz boxes in the promoter region, suggesting that a large proportion of the Phz regulon is governed indirectly. Consistent with this notion, several transcriptional regulators including CsaR, the H-NS type regulator MvaT and sigma factors RpoS, RpoN and RpoD showed differential expression in one or both of the QS-deficient strains. The GacS and GacA transcriptomes shared a surprisingly low degree (42.8%) of overlap, which is unexpected since they comprise a two-component signal transduction system . Interestingly, there was a marked overlap (37.8%) between the PtrA and Gac regulons. The phenotype of a ptrA mutant closely resembles that of the gac-deficient strains and plasmid-borne gacS is able to partially complement a ptrA mutant. Thus, we were somewhat surprised to discover no change in expression of either gacS or gacA in the ptrA mutant background. At present, more questions than answers remain regarding the mechanisms underlying PtrA control.