Microbiology (1996), 142,3241-3251 Printed in Great Britain Studies on the isopropylbenzene 2,3=dioxygenase and the 3-isopropylcatechol 2,3=dioxygenase genes encoded by the linear plasmid of Rhodococcus erythropolis BD2 Maria Kesseler,’ Eric R. Dabbs,’ Beate Averhoffl and Gerhard Gottschalk’ Author for correspondence: Beate Averhoff. Tel: +49 551 394041. Fax: +49 551 393793. e-mail: BAVERHO@gwdg.de 1 lnstitut fur Mikrobiologie The enzymes responsible for the degradation of isopropylbenzene (IPB) and co- der Georg-August- Universitilt, GrisebachstraOe 8, 0-37077 GBttingen, Germany 2 University of the Witwatersrand, Johannesburg, South Africa oxidation of trichloroethene (TCE) by Rhodococcus erythropolis BD2 are encoded by the linear plasmid pBD2. Fragments containing IPB catabolic genes were cloned from pBD2 and the nucleotide sequence was determined. By means of database searches and expression of the cloned genes in recombinant strains, we identified five clustered genes, ipbA7A2A3A4C, which encode the three components of the IPB 2,3=dioxygenase system, reductase,,, (ipbA4, ferredoxin,,, (ipbA3) and the two subunits of the terminal dioxygenase (ipbA 9A2), as well as the 3-isopropylcatechol (IPC) 2,3- dioxygenase (ipbC). The protein sequences deduced from the i’bA 7A2A3A4C gene cluster exhibited significant homology with the corresponding proteins of analogous degradative pathways in Gram-negative and Gram-positive bacteria, but the gene order differed from most of them. IPB 2,3=dioxygenase and 3-IPC 2,3=dioxygenase could both be expressed in Escherichia coli, but the IPB 2,3=dioxygenase activities were too low to be detected by polarographic and TCE degradative means. However, inhibitor studies with the R. erythmpolis BD2 wild-type are in accordance with the involvement of the IPB 2,3-dioxygenase in TCE oxidation. Keywords : Rhodococcus erythropolis, isopropylbenzene 2,3-dioxygenase, 3-isoprop yl- catechol2,3-dioxygenase, trichloroethene co-oxidation INTRODUCTION Multicomponent oxygenases often play a dual crucial role in the bacterial degradation of aromatic and chlorinated aliphatic compounds (Ensley, 1991). They catalyse the oxygenation of their inducer substrates and in addition oxidize chloroalkenes such as trichloroethene (TCE), a toxic groundwater contaminant with suspected carcino- genic activity which is widely distributed throughout the world (Miller & Guengerich, 1983). Since the first documentation by Nelson et al. (1986) of a pure culture capable of metabolizing TCE under aerobic conditions, a number of TCE-degrading micro-organisms have been Abbreviations: IPB, isopropylbenzene; IPC, isopropylcatechol ; Plac, lactose operon promoter; Ptac, hybrid promoter of the lactose and the tryptophan operon; TCE, trichloroethene. The GenBank accession number for the sequences reported in this paper is U24277. isolated (Ensley, 1991). Most investigations on TCE degradation have addressed Gram-negative bacteria and revealed that TCE is only biodegradable through co- metabolic transformation processes mediated by oxygen- ases which exhibit broad substrate specificities, such as monooxygenases acting on methane (Oldenhuis et al., 1989), propane (Wackett et al., 1989), isoprene (Evers et al., 1990), ammonia (Arciero et al., 1989), toluene (Whited & Gibson, 1991) or phenol (Folsom et al., 1990). By analogy with metabolism of chlorinated alkenes by mammalian cytochrome P-450 monooxygenase, bacterial oxygenases might also activate TCE and release inter- mediate reaction products, such as TCE epoxide and chloral, which produce cytotoxic effects. So far, only one dioxygenase of a Gram-negative micro-organism, the toluene dioxygenase of Psetrdomonasptrtida F1, is known to oxidize TCE (Nelson et al., 1988; Wackett & House- holder, 1989). Two representatives of Gram-positive genera, isoprene-oxidizer Rhodococct~r eythropolis strain J E77 and the propane-utilizer Mycobacteritlm vaccae, have 0002-0875 0 1996 SGM 3241