Chromosomal integration of tcb chlorocatechol degradation pathway genes as a 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 minitransposon. The minitransposon carrying the tcb gene cluster and a kanamycin resistance gene was transferred to Pseudomonas putida KT2442, and chromosomal integration was monitored by selection either for growth on 3-chlorobenzoate or for kanamycin resistance. Transconjugants able to utilize 3-chlorobenzoate 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-chlorobenzoate. Southern blot analysis revealed that many transconjugants selected directly on 3-chlorobenzoate contained multiple chromosomal copies of the tcb gene cluster, whereas those selected for kanamycin resistance possessed a single copy. Subsequent selection of kanamycin resistance-selected single-copy transconjugants for growth on 3-chlorobenzoate yielded colonies capable of utilizing this carbon source, but no amplification of the tcb gene 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 catabolic operon in P. putida thus represents a useful model system for analysis of the relationship among gene dosage, enzyme expression level, and growth on chloroaromatic substrates.
Klemba
M
M
Division of Microbiology, GBF-National Research Center for Biotechnology, Braunschweig, Germany. klembam@borcim.wustl.edu
Jakobs
B
B
Wittich
R M
RM
Pieper
D
D
D015139:Blotting, Southern; D002396:Catechols / Q000378:metabolism*; D002723:Chlorobenzoates / Q000378:metabolism; D003001:Cloning, Molecular; D003227:Conjugation, Genetic; D005798:Genes, Bacterial; D006843:Hydrocarbons, Chlorinated / Q000378:metabolism*; D005810:Multigene Family; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011549:Pseudomonas / Q000235:genetics / Q000378:metabolism; D016958:Pseudomonas putida / Q000235:genetics* / Q000254:growth & development* / Q000378:metabolism
D002396:Catechols; D002723:Chlorobenzoates; D006843:Hydrocarbons, Chlorinated; C036427:3-chlorobenzoic acid; C031137:3-chlorocatechol; C038861:4-chlorocatechol