Chromosomal integration of tcb chlorocatechol degradation pathway genes asa means of expanding the growth substrate range of bacteria to include haloaromatics

The tcbR-tcbCDEF gene cluster? coding for the chlorocatechol ortho-cleavage pathway in Pseudomonas sp, strain P51, has been cloned into a Tn5-based mi nitransposon. The minitransposon carrying the tcb gene cluster and a kanamy cin resistance gene was transferred to Pseudomonas putida KT2442, and chrom osomal integration was monitored by selection either for growth on 3-chloro benzoate or for kanamycin resistance. Transconjugants able to utilize 3-chl orobenzoate as a sole carbon source were obtained, although at a >100-fold lower frequency than kanamycin-resistant transconjugants, The vast majority of kanamycin-resistant transconjugants were not capable of growth on 3-chl orobenzoate. Southern blot analysis revealed that many transconjugants sele cted directly on 3-chlorobenzoate contained multiple chromosomal copies of the tcb gene cluster, whereas those selected for kanamycin resistance posse ssed a single copy. Subsequent selection of kanamycin resistance-selected s ingle-copy transconjugants for growth on 3-chlorobenzoate yielded colonies capable of utilizing this carbon source, but no amplification of the tcb ge ne cluster was apparent. Introduction of two copies of the tcb gene cluster without prior 3-chlorobenzoate selection resulted in transconjugants able to grow on this carbon source. Expression of the tcb chlorocatechol catabol ic operon in P. putida thus represents a useful model system for analysis o f the relationship among gene dosage, enzyme expression level, and growth o n chloroaromatic substrates.