Expression of Rhodococcus opacus alkB genes in anhydrous organic solvents.
Rhodococcus opacus B-4 is a benzene-tolerant bacterium which was isolated from a gasoline-contaminated soil sample. We previously demonstrated that this organism was able to survive and exhibit biocatalytic activity in anhydrous organic solvents for at least 5 d. In the present study, we cloned the alkB1 and alkB2 genes encoding alkane hydroxylases from R. opacus B-4. Heterologous expression of the alkB1 and alkB2 genes in Escherichia coli JM109 showed that they encode functional alkane hydroxylases with a substrate range of C(5)-C(16). Promoters of the alkB1 and alkB2 genes, designated P(alkB1) and P(alkB2), respectively, were examined for activity in anhydrous bis (2-ethylhexyl) phthalate (BEHP) containing C(5)-C(16)n-alkanes. Two recombinant plasmids, pP(alkB1)EGFP and pP(alkB2)EGFP, were constructed by inserting the egfp gene downstream of P(alkB1) and P(alkB2), respectively and transformed into R. opacus B-4. Resting cells of R. opacus B-4 (pP(alkB1)EGFP) showed greater levels of EGFP fluorescence in anhydrous BEHP than in 0.85% NaCl, when C(8)-C(16)n-alkanes were supplied as an inducer. Furthermore, n-alkane inducibility of P(alkB1) activity in anhydrous BEHP was noticeably different from that in 0.85% NaCl. This paper presents the first evidence that bacteria can express their genes in essentially anhydrous organic solvents.
Sameshima
Yuka
Y
Department of Biotechnology, Graduate School of Engineering, Suita, Osaka, Japan. yuka_sameshima@bio.eng.osaka-u.ac.jp
Honda
Kohsuke
K
Kato
Junichi
J
Omasa
Takeshi
T
Ohtake
Hisao
H
D001483:Base Sequence; D003001:Cloning, Molecular; D003470:Culture Media; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics*; D005453:Fluorescence; D005798:Genes, Bacterial; D006899:Mixed Function Oxygenases / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012240:Rhodococcus / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D012997:Solvents / Q000737:chemistry*
D003470:Culture Media; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D012997:Solvents; D006899:Mixed Function Oxygenases; C466427:AlkB protein, E coli