Anaerobic toluene catabolism of Thauera aromatica: the bbs operon codes for enzymes of beta oxidation of the intermediate benzylsuccinate

Abstract

The pathway of anaerobic toluene oxidation to benzoyl coenzyme A (benzoyl-CoA) consists of an initial reaction catalyzed by benzylsuccinate synthase, a glycyl radical enzyme adding the methyl group of toluene to the double bond of a fumarate cosubstrate, and a subsequent beta-oxidation pathway of benzylsuccinate. Benzylsuccinate synthase has been studied in some detail, whereas the enzymes participating in beta oxidation of benzylsuccinate are unknown. We have investigated these enzymes by analyzing substrate-induced proteins in toluene-grown cells. Toluene-induced proteins were identified and N-terminally sequenced. Nine of these proteins are encoded by an 8.5-kb operon consisting of bbs (beta-oxidation of benzylsuccinate) genes whose products are apparently involved in the beta-oxidation pathway of benzylsuccinate. Two of the genes, bbsE and bbsF, code for the subunits of a succinyl-CoA:benzylsuccinate CoA-transferase whose activity was previously detected in toluene-grown Thauera aromatica. The bbsG gene codes for a specific benzylsuccinyl-CoA dehydrogenase, as confirmed by overexpression of the gene in Escherichia coli and detection of enzyme activity. The further enzymes of the pathway are probably encoded by bbsH (enoyl-CoA hydratase), bbsCD (3-hydroxyacyl-CoA dehydrogenase), and bbsB (3-oxoacyl-CoA thiolase). The operon contains two additional genes, bbsA and bbsI, for which no obvious function could be derived. The bbs operon is expressed only in toluene-grown cells and is regulated at the transcriptional level. Promoter mapping revealed a transcription start site upstream of the bbsA gene. This represents the first known promoter site in Thauera spp.

FIG. 1

Proposed pathway of anaerobic toluene oxidation to benzoyl-CoA. The enzymes involved are indicated by their gene names: (1) benzylsuccinate synthase, BssABC; (2) succinyl-CoA:benzylsuccinate CoA-transferase, BbsEF; (3) benzylsuccinyl-CoA dehydrogenase, BbsG; (4) phenylitaconyl-CoA hydratase, BbsH; (5) 3-hydroxyacyl-CoA dehydrogenase, BbsCD; (6) benzoylsuccinyl-CoA thiolase, BbsB; (7) succinate dehydrogenase, Sdh.

FIG. 2

Analysis of toluene-induced proteins. (A) SDS-PAGE of extracts of benzoate-grown (lane 2) and toluene-grown (lane 3) cells of T. aromatica. Molecular masses of marker proteins (lane 1) are given in the left margin; the 2D gels are aligned relative to the migration positions of these markers. Arrows point to toluene-induced proteins. (B) 2D gel of a cell extract of toluene-grown cells. Boxes indicate the positions of toluene-induced proteins. One box (4+) contains two induced proteins: the larger of these was identified as BbsH; the smaller one may be BbsC, but it could not be verified because of a blocked N terminus. (C) 2D gel of a cell extract of benzoate-grown cells. The positions of toluene-induced proteins from panel B are indicated by boxes. Numbers indicate the migration positions of the identified proteins: (1) benzylsuccinate synthase subunit BssA; (2a and 2b) BbsEF; (3) BbsG; (4) BbsH; (5a, b) BbsCD; (6) BbsB, (7) BbsA, and (8) BbsI. The correlation of the induced protein (5a) with BbsC was based on the results of T7 expression experiments. A toluene-induced protein that does not correlate to a bbs gene product and was not located on the 2D gels is labeled with X.

FIG. 3

Organization of the bbs operon. The sequenced fragment is indicated by the locations of open reading frames. The bbs genes are shown as hatched boxes; flanking open reading frames are shown as open boxes. Restriction sites for EcoRI (E) and HindIII (H) are indicated. The mapped transcription start site of the bbs operon is indicated by an arrow. Relevant subclones for expression in the T7 promoter-polymerase system are indicated as arrows below the genes; the arrows show orientations of the fragments with respect to the T7 promoter.

FIG. 4

Expression of the bbs genes by the T7 promoter-polymerase system. The autoradiogram is of a 12.5% polyacrylamide gel in which SDS lysates from equal amounts of cells were separated. Migration positions of nonlabeled standard proteins are indicated. Lanes contained lysates of E. coli K38 containing plasmid pGP1-2 and various T7 promoter vectors. Lane 1, vector pT7-4, containing the gene for β-lactamase in the direction of the T7 promoter. Lane 2, plasmid pH3 (bbsABC′). The aberrant size of the bbsC′ gene product is caused by a gene fusion of the truncated bbsC gene with the vector portion, which results in a predicted product of 29.5 kDa. Lane 3, plasmid pE1 (bbsC). Lane 4, plasmid pDH (bbsDE). Lane 5, plasmid pE3 (bbsFG). The bbsF gene product is probably not synthesized from this plasmid; the labeled protein is identical to the product synthesized from a plasmid containing only the bbsG gene. Lane 6, plasmid pHH (bbsH). Lane 7, control experiment with a plasmid containing bssA and a part of bssB in inverse orientation.

FIG. 5

Promoter mapping of the bbs operon. (A) Fluorograms of primer extension assays with RNA prepared from cells grown on toluene (Tol) and benzoate (Bz). (B) Fluorogram of a DNA sequencing assay with the same primer. (C) Indication of the transcription start and possible promoter site of the bbs operon. The mapped start point is labeled by an open box; possible promoter boxes similar to standard −10 and −35 elements are underlined, and the ribosome binding site of the bbsA gene is shown in italics. The location of the primer used for the experiment is given by an arrow below the sequence.