title: Development of a genetic transformation system for benzene-tolerant Rhodococcus opacus strains.
abstract: 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.
authors.affiliation: Department of Molecular Biotechnology, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8530, Japan.
authors.forename: Kyung-Su
authors.initials: KS
authors.lastname: Na
authors.forename: Kan
authors.initials: K
authors.lastname: Nagayasu
authors.forename: Akio
authors.initials: A
authors.lastname: Kuroda
authors.forename: Noboru
authors.initials: N
authors.lastname: Takiguchi
authors.forename: Tsukasa
authors.initials: T
authors.lastname: Ikeda
authors.forename: Hisao
authors.initials: H
authors.lastname: Ohtake
authors.forename: Junichi
authors.initials: J
authors.lastname: Kato
mesh_terms: D001554:Benzene / Q000494:pharmacology*; D004265:DNA Helicases / Q000235:genetics* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D024881:Drug Resistance, Bacterial / Q000235:genetics; D018274:Electroporation / Q000379:methods; D024861:Genetic Enhancement / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism; D012240:Rhodococcus / Q000145:classification / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D013045:Species Specificity; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism*; D014169:Transformation, Bacterial / Q000502:physiology*
chemical_list: D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D004265:DNA Helicases; D001554:Benzene