TOL Plasmid pWW0 Transposons Facilitate Adaptation of Pseudomonas putida for Growth on the New Carbon Source m ‐Cresol

ABSTRACT We investigated the development of a new, hybrid catabolic pathway for m ‐cresol utilization in Pseudomonas putida strain PaW1‐T, a subline of TOL plasmid pWW0‐possessing strain PaW1 carrying xyl catabolic operons for utilization of toluene and its methyl derivatives (xylenes) as carbon sources. The ability to hydroxylate m ‐cresol to methyl catechol was horizontally transferred within a broad‐host‐range plasmid expressing the phenol monooxygenase PheA. We observed that using m ‐cresol as a new carbon source required genetic rearrangements associated with elevated expression of meta ‐pathway enzymes encoded by the xyl lower operon with concomitant inactivation of the xyl upper operon. Analysis of DNA sequencing data from four m ‐cresol‐selected (Cre + ) strains revealed substantial heterogeneity in the populations of these strains and differences among individual Cre + strains. The DNA samples of Cre + strains Cre1 and Cre2 yielded sequencing reads consistent with separately existing circular forms of transposons Tn 4653 and Tn 4651 , respectively, with inversions that could inactivate the xyl upper operon. Thus, our results indicated that in addition to conventional transposition events, increasing gene dosage by forming circles of catabolic transposons Tn 4651 and Tn 4653 could also facilitate bacterial adaptation for growth on new carbon sources.