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

• Published in: Applied and environmental microbiology Vol. 66; no. 8; pp. 3255 - 3261
• Main Authors: KLEMBA, M, JAKOBS, B, WITTICH, R.-M, PIEPER, D
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01.08.2000
• Subjects: Bacteria; Biological and medical sciences; Biotechnology; Blotting, Southern; Catechols - metabolism; Chlorobenzoates - metabolism; chlorocatechols; Cloning, Molecular; Conjugation, Genetic; Fundamental and applied biological sciences. Psychology; Genes; Genes, Bacterial; Genetic engineering; Genetic technics; Genetics and Molecular Biology; Hydrocarbons, Chlorinated - metabolism; m-Chlorobenzoic acid; Methods. Procedures. Technologies; Microbiology; Modification of gene expression level; Multigene Family; Phenotype; Plasmids - genetics; Polymerase Chain Reaction; Pseudomonas; Pseudomonas - genetics; Pseudomonas - metabolism; Pseudomonas putida; Pseudomonas putida - genetics; Pseudomonas putida - growth & development; Pseudomonas putida - metabolism; tcb gene; Pseudomonadales; Biodegradation; Recombinant microorganism; Gene cluster; Molecular integration; Chromosome; Metabolic pathway; Pollutant; Gene; Chlorine Organic compounds; Bacteria; Pseudomonadaceae; Aromatic compound; Bioremediation; Pseudomonas putida
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/AEM.66.8.3255-3261.2000

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.