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

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 the ir 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-seq 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 16 retroactivity between a pre-existing metabolic network and a new biochemical pathway implanted through gene transfer. Custom Agilent tiling microarrays containing relevant regions of pWW0 plasmid were hybridized with two RNA samples each: altered and control (two channel-format). Four biological replicates were hybridized independently for each comparison.