Benzylsuccinate synthase of Azoarcus sp. strain T: cloning, sequencing, transcriptional organization, and its role in anaerobic toluene and m-xylene mineralization

Abstract

Biochemical studies in Azoarcus sp. strain T have demonstrated that anaerobic oxidation of both toluene and m-xylene is initiated by addition of the aromatic hydrocarbon to fumarate, forming benzylsuccinate and 3-methyl benzylsuccinate, respectively. Partially purified benzylsuccinate synthase was previously shown to catalyze both of these addition reactions. In this study, we identified and sequenced the genes encoding benzylsuccinate synthase from Azoarcus sp. strain T and examined the role of this enzyme in both anaerobic toluene and m-xylene mineralization. Based on reverse transcription-PCR experiments and transcriptional start site mapping, we found that the structural genes encoding benzylsuccinate synthase, bssCAB, together with two additional genes, bssD and bssE, were organized in an operon in the order bssDCABE. bssD is believed to encode an activating enzyme, similar in function to pyruvate formate-lyase activase. bssE shows homology to tutH from Thauera aromatica strain T1, whose function is currently unknown. A second operon that is upstream of bssDCABE and divergently transcribed contains two genes, tdiS and tdiR. The predicted amino acid sequences show similarity to sensor kinase and response regulator proteins of prokaryotic two-component regulatory systems. A chromosomal null bssA mutant was constructed (the bssA gene encodes the alpha-subunit of benzylsuccinate synthase). This bssA null mutant strain was unable to grow under denitrifying conditions on either toluene or m-xylene, while growth on benzoate was unaffected. The growth phenotype of the DeltabssA mutant could be rescued by reintroducing bssA in trans. These results demonstrate that benzylsuccinate synthase catalyzes the first step in anaerobic mineralization of both toluene and m-xylene.

FIG. 1

Addition of toluene and m-xylene to fumarate catalyzed by benzylsuccinate synthase. R = H for toluene and R = CH₃ for m-xylene.

FIG. 2

(A) Restriction map of the tdiSR/bssDCABE chromosomal region of Azoarcus sp. strain T. The eight identified open reading frames are indicated with arrows below the map. Arrows above the map indicate the major transcriptional start sites identified. Restriction site abbreviations: B, BamHI; E, EcoRI; H, HindIII; X, XbaI. (B) Gene organization of tut and tdi/bss operons in Azoarcus sp. strain T, T. aromatica strain K172, and T. aromatica strain T1. Accession numbers are U57900 and AF036765 for T. aromatica T1 and AJ001848 for T. aromatica K172.

FIG. 3

Agarose gel electrophoresis of RT-PCR products. RT-PCR products observed using primers to amplify intergenic regions between (lanes 1 and 2) tdiS and tdiR, (3 and 4) bssD and bssC, (5 and 6) bssC and bssA, (7 and 8) bssA and bssB, (9 and 10) bssB and bssE, and (11 and 12) bssE and orf2. Odd-numbered lanes are controls without reverse transcriptase. Numbers on the left represent sizes of markers (in base pairs).

FIG. 4

Mapping of transcriptional start sites of bssDCABE and tdiSR by primer extension. (A) Sequencing and primer extension reactions with bssDpe primer. (B) Sequencing and primer extension reactions with tdiSpe primer. RNA was isolated from toluene-grown (lane 1), m-xylene-grown (lane 2), and benzoate-grown (lane 3) cells. (C) Nucleotide sequence of the tdiSR/bssDCABE promoter regions. Transcription start sites are indicated by +1, and putative −10 and −35 regions are underlined. Note that only one of the potential −10 regions of the bssDCABE promoter is underlined.

FIG. 5

Mapping of transcriptional start site of orf2 by primer extension. (A) RNA was isolated from toluene-grown cells (lane 1), m-xylene-grown cells (lane 2), and benzoate-grown cells (lane 3). A sequence ladder generated with the same primer is shown. (B) Nucleotide sequence of the orf2 promoter region, showing the start site of orf2 transcription (+1) and a potential −35 region. The stop codon for bssE is underlined.

FIG. 6

Role of bssA in anaerobic growth on (A) toluene and (B) m-xylene in Azoarcus sp. strain T. Growth of the wild-type strain on toluene or m-xylene (solid diamonds); AST2 (ΔbssA) on benzoate (open squares) and toluene or m-xylene (open triangles); and AST3 (AST2 + pGA14) on benzoate (solid triangles) and toluene or m-xylene (open circles). Note that wild-type Azoarcus sp. strain T has been observed to grow with a range of doubling times: 16 to 25 h on benzoate, 16 to 27 h on m-xylene, and 14 to 20 h on toluene (C. J. Krieger, unpublished data).