Development of a genetic transformation system for benzene-tolerant Rhodococcus opacus strains.

Rhodococcus opacus B-4 and B-9 are tolerant to various organic solvents including benzene, toluene, ethylbenzene, xylenes and styrene, and are suitable bacterial hosts for the production of chemical products from hydrophobic substrates. A 4.4-kb endogenous plasmid (pKNR 01) was isolated from R. opacus B-4 and sequenced completely. Plasmid pKNR 01 encodes proteins that share similarity to replication proteins from the enteric bacterial and actinomycete theta-replication plasmids. A 7.4-kb chimeric plasmid, designated pKNR 01.1, was constructed by fusing XhoI-digested pKNR 01 and Escherichia coli vector pSTV 28. Plasmid pKNR 01.1 had the ability to replicate in B-4 and B-9. A protocol for transformation of B-9 by electroporation was optimized employing pKNR 01.1. Frequencies of 4.1 x 10(5) transformants per mug of plasmid DNA were obtained for B-9 cells, whereas B-4 harboring naturally occurring pKNR 01 was transformed at lower frequencies (approximately 1 x 10(4) transformants per mug of plasmid DNA). Deletion analysis of pKNR 01.1 showed that the 1.9-kb SphI-XhoI region containing the repA and rep B genes and the 0.6-kb region upstream of repA was essential for plasmid maintenance in R. opacus strains.