High-resolution analysis of the m-xylene/toluene biodegradation subtranscriptome of Pseudomonas putida mt-2

Pseudomonas putida mt-2 metabolizes m-xylene and other aromatic compounds through the enzymes encoded by the xyl operons of the TOL plasmid pWW0 along with other chromosomally encoded activities. Tiling arrays of densely overlapping oligonucleotides were designed to cover every gene involved in this process, allowing dissection of operon structures and exposing the interplay of plasmid and chromosomal functions. All xyl sequences were transcribed in response to aromatic substrates and the 3′-termini of both upper and lower mRNA operons extended beyond their coding regions, i.e. the 3′-end of the lower operon mRNA penetrated into the convergent xylS regulatory gene. Furthermore, xylR mRNA for the master m-xylene responsive regulator of the system was decreased by aromatic substrates, while the cognate upper operon mRNA was evenly stable throughout its full length. RNA sequencing confirmed these data at a single nucleotide level and refined the formerly misannotated xylL sequence. The chromosomal ortho route for degradation of benzoate (the ben, cat clusters and some pca genes) was activated by this aromatic, but not by the TOL substrates, toluene or m-xylene. We advocate this scenario as a testbed of natural retroactivity between a pre-existing metabolic network and a new biochemical pathway implanted through gene transfer.