Identification and characterization of a CRP/Vfr homologue in Pseudomonas putida and the determination of its role in the expression of pca genes

In Pseudomonas putida (P. putida), aromatic hydrocarbons can be degraded into harmless compounds via the β-ketoadipate pathway, which consists of the cat branch and the pca branch. The cat branch merges with the pca branch at the level of the β-ketoadipate enol lactone, and the pca gene products subsequently complete the degradation process. PcaR is the positive activator of the pca branch. In the presence of β-ketoadipate, PcaR binds upstream to each promoter and activates the transcription of the pca genes. PcaR, however, is a negative regulator of its own transcription. Until recently, no positive regulator of the pcaR gene had been identified. By searching the sequence upstream of the pcaR promoter, a potential cyclic AMP receptor-binding protein consensus sequence (CCS) was found. Furthermore, a BLAST search of the P. putida genome revealed an open reading frame that bore significant homology to both the cyclic AMP receptor-binding protein (CRP) and the virulence factor regulator (Vfr) homologue. Preliminary investigation revealed that Vfr has a positive role in the expression of cloned pca genes of P. putida as, in the absence of Vfr, the expression of pca genes is completely undetectable. Inactivation of the Vfr-like ORF on the P. putida chromosome was undertaken using transposon mutagenesis. Investigations revealed that the absence of this “VfrH” locus had a negative effect on P. putida growth, metabolism and motility suggesting that VfrH, like its counterpart, acts as a global regulator of gene expression. VfrH was also shown to have an important role in the expression of pca genes of P. putida through its effect on pcaR expression. Furthermore, VfrH was able to complement the crp null mutant E. coli, restoring the inducible levels of β-galactosidase and tryptophanase activities. In studying the regulation of the vfrH gene itself, VfrH is also involved in the transcriptional activation of its own promoter in P. putida, likewise Vfr in P. aeruginosa . Also, aromatic compounds in the medium appear to have an effect on the expression of the vfrH gene promoter. cAMP levels were found to be higher in wild-type P. putida than in the vfrH mutant strain or the pcaR negative strain PRS3015, and aromatic compounds were also found to have an effect on this level. Overall, the research demonstrated the discovery of the global regulator CRP/VfrH in P. putida and its importance in regulation of the growth, metabolism, and motility of P. putida, as well as chemotaxis towards and the degradation of the aromatic compounds via the β-ketoadipate pathway.