RP4::Mu3A-mediated in vivo cloning and transfer of a chlorobiphenyl catabolic pathway

Abstract

Chromosomal DNA fragments encoding the ability to utilize biphenyl as sole carbon source (Bph+) were mobilized by means of plasmid RP4::Mu3A from strain JB1 (tentatively identified as Burkholderia sp.) to Alcaligenes eutrophus CH34 at a frequency of 10(-3) per transferred plasmid. The mobilized DNA integrated into the recipient chromosome or was recovered as catabolic prime plasmids. Three Bph+ prime plasmids were transferred from A. eutrophus to Escherichia coli and back to A. eutrophus without modification of the phenotype. The transferred Bph+ DNA segments allowed metabolism of biphenyl, 2-, 3- and 4-chlorobiphenyl, and diphenylmethane. Genes involved in biphenyl degradation were identified on the prime plasmids by DNA-DNA hybridization and by gene cloning. Bph+ prime plasmids were transferred to Burkholderia cepacia, Pseudomonas aeruginosa, Comamonas testosteroni and A. eutrophus and the catabolic genes were expressed in those hosts. Transfer of the plasmid to the 3-chlorobenzoate-degrading bacterium Pseudomonas sp. B13 allowed the recipient to mineralize 3-chlorobiphenyl. Other catabolic prime plasmids were obtained from JB1 by selection on m-hydroxybenzoate and tyrosine as carbon sources. 16S rRNA sequence data demonstrated that the in vivo transfer of bph was achieved between bacteria belonging to two different branches of the beta-Proteobacteria.