Roles of Ring-Hydroxylating Dioxygenases in Styrene and Benzene Catabolism inRhodococcus jostiiRHA1

Abstract

Proteomics and targeted gene disruption were used to investigate the catabolism of benzene, styrene, biphenyl, and ethylbenzene in Rhodococcus jostii RHA1, a well-studied soil bacterium whose potent polychlorinated biphenyl (PCB)-transforming properties are partly due to the presence of the related Bph and Etb pathways. Of 151 identified proteins, 22 Bph/Etb proteins were among the most abundant in biphenyl-, ethylbenzene-, benzene-, and styrene-grown cells. Cells grown on biphenyl, ethylbenzene, or benzene contained both Bph and Etb enzymes and at least two sets of lower Bph pathway enzymes. By contrast, styrene-grown cells contained no Etb enzymes and only one set of lower Bph pathway enzymes. Gene disruption established that biphenyl dioxygenase (BPDO) was essential for growth of RHA1 on benzene or styrene but that ethylbenzene dioxygenase (EBDO) was not required for growth on any of the tested substrates. Moreover, whole-cell assays of the ⌬bphAa and etbAa1::cmrA etbAa2::aphII mutants demonstrated that while both dioxygenases preferentially transformed biphenyl, only BPDO transformed styrene. Deletion of pcaL of the ␤-ketoadipate pathway disrupted growth on benzene but not other substrates. Thus, styrene and benzene are degraded via meta-and ortho-cleavage, respectively. Finally, catalases were more abundant during growth on nonpolar aromatic compounds than on aromatic acids. This suggests that the relaxed specificities of BPDO and EBDO that enable RHA1 to grow on a range of compounds come at the cost of increased uncoupling during the latter's initial transformation. The stress response may augment RHA1's ability to degrade PCBs and other pollutants that induce similar uncoupling.Rhodococci are mycolic acid-producing actinomycetes that degrade a wide variety of organic compounds (13). These catabolic capabilities are of interest for a range of bioremediation and biocatalytic applications (53). To better understand the physiology and metabolism of this important genus, we have conducted genomic studies of Rhodococcus jostii RHA1 (formerly Rhodococcus sp. strain RHA1, the species was recently identified by A. L. Jones and M. Goodfellow [personal communication]), a strain that possesses an exceptional ability to aerobically degrade polychlorinated biphenyls (PCBs) (47). Annotation of the 9.7-Mb RHA1 genome (31; http://www .rhodococcus.ca) predicted over 200 oxygenases and 30 pathways involved in the catabolism of aromatic compounds. Transcriptomic and proteomic studies have revealed a number of unique features of RHA1 metabolism, including a novel nitrile hydratase (37) and multiple steroid-degrading pathways (54).RHA1 transforms PCBs using the homologous Bph and Etb pathways (19), so named for their presumed respective specificities for biphenyl (47) and ethylbenzene (16, 57), respectively. The pathways utilize a meta-cleavage strategy in which vicinal dihydroxylation of an aromatic ring enables oxygenolytic extradiol (or meta) ring opening (Fig.

Biphenyl and ethylbenzene dioxygenases of Rhodococcus jostii RHA1 transform PBDEs

“…1), and their catalytic components (BphAa/BphAb and EtbAa/EtbAb) share 37% amino acid sequence identity. Furthermore, there are two copies of the etbAa and etbAb genes, which differ by a single nucleotide substitution (Patrauchan et al, 2008). While BPDO and EBDO each transform various aromatic compounds, including benzene, biphenyl, ethylbenzene, and naphthalene, EBDO prefers larger substrates, such as naphthalene, consistent with its phylogeny, while BPDO prefers substrates containing a single aromatic nucleus (Iwasaki et al, 2007;Masai et al, 1997).…”

Styrene oxide isomerase of Sphingopyxis sp. Kp5.2

“…It cannot be ruled out completely that in some cases the activity determination of the SOI may have been inhibited by uncommonly high PAD activity, but the low abundance of SOIs amongst the soil bacteria tested indicated that alternative or modified pathways for styrene degradation may have been dominant. A few studies have revealed the possibility of styrene degradation via alternative routes (Patrauchan et al, 2008;Toda & Itoh, 2012;Warhurst et al, 1994).…”

Vanillin Catabolism in Rhodococcus jostii RHA1

“…These results are in good agreement with the bioinformatic and transcriptomic analyses indicating that ro04165 and ro04163 Targeted gene deletion. To further validate the annotation of the vanillin degradation genes, vdh (ro02986) and vanA (ro04165) were disrupted by targeted mutagenesis using a sacB counterselection system as previously described (11,14). The oligonucleotides used in constructing the mutants are listed in Table S1 in the supplemental material, and the plasmids generated in the process are listed in Table S2 in the supplemental material.…”