The differential response of the P ben promoter of P seudomonas putida mt ‐2 to BenR and XylS prevents metabolic conflicts in m ‐ xylene biodegradation

Summary P seudomonas putida   mt ‐2 encompasses two alternative and potentially conflicting routes for benzoate metabolism, one meta pathway encoded by xyl genes of the pWW 0 plasmid and mastered by the P m promoter and XylS , and one chromosomally encoded ortho pathway initiated by P ben and the BenR protein. Any cross‐activation of P ben promoter by XylS ought to cause a metabolic conflict during the degradation of m ‐xylene because 3‐methylbenzoate (3 MBz ) generated as an intermediate can be channelled through the ortho pathway and produce toxic dead‐end metabolites. The activation of P ben by XylS was revisited using both reporter technology and tiling arrays targeted to the sequences of interest around messenger RNA initiation of both P ben and P m promoters. Analysis of supersensitive luxCDABE fusions, inspection of xylX versus benA transcripts and growth tests of benR mutants indicated that the natural expression ranges of XylS under various conditions are insufficient to cause a significant cross‐regulation of P ben whether cells face endogenous or exogenous 3 MBz . This seems to stem from the nature of the operators for binding either transcriptional factor, which in the case of the P ben promoter of P . putida   mt ‐2 appear to have evolved for avoiding a strong interaction with XylS .