Expression of the meta-cleavage pathway operon of the TOL plasmid of Pseudomonas putida in the phototrophic bacterium Rhodobacter sphaeroides

Abstract

Plasmid pPL392, a pBR322 derivative containing a 16.4 kb fragment harboring the whole meta-cleavage pathway operon genes of the TOL plasmid pWWO as well as its regulatory gene xylS, was inserted into the HindIII site of the broad-host-range plasmid pRK404 to yield the plasmid pUA58. This pUA58 plasmid was mobilized into the phototrophic bacteria Rhodobacter sphaeroides using the mobilizing plasmid pRK2013. Meta-cleavage pathway genes, measured by the catechol 2,3-dioxygenase (C230) enzyme activity, were constitutively expressed at a high level in the R. sphaeroides (pUA58) strain, since the inducing compound m-toluate did not increase the level of the C230 enzyme. This behaviour of R. sphaeroides (pUA58) strain was not due to any modification of the pUA58 plasmid in this bacterium, because synthesis of the C230 enzyme in both E. coli and P. putida cells containing this plasmid were inducible by m-toluate. Nevertheless, and despite the high level of expression of meta-cleavage genes, R. sphaeroides (pUA58) cells were unable to grow using m-toluate as the sole carbon source. By subcloning some of the whole meta-cleavage operon genes, it has been shown that the growth inability of R. sphaeroides (pUA58) using m-toluate must be attributed to an inhibitory effect of one of the intermediate metabolites of the m-toluate degradation over the R. sphaeroides cells.