Identification of CoIR binding consensus and prediction of regulon of CoIRS two-component system

Background: Conserved two-component system CoIRS of Pseudomonas genus has been implicated in several unrelated phenotypes. For instance, deficiency of P. putida CoIRS system results in lowered phenol tolerance, hindrance of transposition of Tn4652 and lysis of a subpopulation of glucose-grown bacteria. In order to discover molecular mechanisms behind these phenotypes, we focused here on identification of downstream components of CoIRS signal transduction pathway. Results: First, highly similar CoIR binding sites were mapped upstream of outer membrane protein-encoding oprQ and a putative methyltransferase-encoding PP0903. These two CoIR binding sequences were used as an input in computational genome-wide screening for new potential CoIR recognition boxes upstream of different genes in P. putida. Biological relevance of a set of in silico predicted CoIR-binding sites was analysed in vivo by studying the effect of CoIR on transcription from promoters carrying these sites. This analysis disclosed seven novel genes of which six were positively and one negatively regulated by CoIR. Interestingly, all promoters tested responded more significantly to the over-expression than to the absence of CoIR suggesting that either CoIR is limiting or CoIS-activating signal is low under the conditions applied. The binding sites of CoIR in the promoters analysed were validated by gel mobility shift and/or DNase I footprinting assays. CoIR binding consensus was defined according to seven CoIR binding motifs mapped by DNase I protection assay and this consensus was used to predict minimal regulon of CoIRS system. Conclusion: Combined usage of experimental and computational approach enabled us to define the binding consensus for response regulator CoIR and to discover several new CoIR-regulated genes. For instance, genes of outer membrane lipid A 3-O-deacylase PagL and cytoplasmic membrane diacylglycerol kinase DgkA are the members of CoIR regulon. Furthermore, over 40 genes were predicted to be putatively controlled by CoIRS two-component system in P. putida. It is notable that many of CoIR-regulated genes encode membrane-related products thus confirming the previously proposed role of CoIRS system in regulation of membrane functionality.