Identification of ColR binding consensus and prediction of regulon of ColRS two-component system

Conserved two-component system ColRS of Pseudomonas genus has been implicated in several unrelated phenotypes. For instance, deficiency of P. putida ColRS 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 ColRS signal transduction pathway.

First, highly similar ColR binding sites were mapped upstream of outer membrane protein-encoding oprQ and a putative methyltransferase-encoding PP0903. These two ColR binding sequences were used as an input in computational genome-wide screening for new potential ColR recognition boxes upstream of different genes in P. putida. Biological relevance of a set of in silico predicted ColR-binding sites was analysed in vivo by studying the effect of ColR on transcription from promoters carrying these sites. This analysis disclosed seven novel genes of which six were positively and one negatively regulated by ColR. Interestingly, all promoters tested responded more significantly to the over-expression than to the absence of ColR suggesting that either ColR is limiting or ColS-activating signal is low under the conditions applied. The binding sites of ColR in the promoters analysed were validated by gel mobility shift and/or DNase I footprinting assays. ColR binding consensus was defined according to seven ColR binding motifs mapped by DNase I protection assay and this consensus was used to predict minimal regulon of ColRS system.

Combined usage of experimental and computational approach enabled us to define the binding consensus for response regulator ColR and to discover several new ColR-regulated genes. For instance, genes of outer membrane lipid A 3-O-deacylase PagL and cytoplasmic membrane diacylglycerol kinase DgkA are the members of ColR regulon. Furthermore, over 40 genes were predicted to be putatively controlled by ColRS two-component system in P. putida. It is notable that many of ColR-regulated genes encode membrane-related products thus confirming the previously proposed role of ColRS system in regulation of membrane functionality.