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 C5–C16. Promoters of the alkB1 and alkB2 genes, designated PalkB1 and PalkB2, respectively, were examined for activity in anhydrous bis (2-ethylhexyl) phthalate (BEHP) containing C5–C16n-alkanes. Two recombinant plasmids, pPalkB1EGFP and pPalkB2EGFP, were constructed by inserting the egfp gene downstream of PalkB1 and PalkB2, respectively and transformed into R. opacus B-4. Resting cells of R. opacus B-4 (pPalkB1EGFP) showed greater levels of EGFP fluorescence in anhydrous BEHP than in 0.85% NaCl, when C8–C16n-alkanes were supplied as an inducer. Furthermore, n-alkane inducibility of PalkB1 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.