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. 2018 Mar 22:9:506.
doi: 10.3389/fmicb.2018.00506. eCollection 2018.

Transcriptional Regulation of the Peripheral Pathway for the Anaerobic Catabolism of Toluene and m-Xylene in Azoarcus sp. CIB

Blas Blázquez  1 , Manuel Carmona  1 , Eduardo Díaz  1
Affiliations

Transcriptional Regulation of the Peripheral Pathway for the Anaerobic Catabolism of Toluene and m-Xylene in Azoarcus sp. CIB

Blas Blázquez et al. Front Microbiol. .

Abstract

Alkylbenzenes, such as toluene and m-xylene, are an important class of contaminant hydrocarbons that are widespread and tend to accumulate in subsurface anoxic environments. The peripheral pathway for the anaerobic oxidation of toluene in bacteria consists of an initial activation catalyzed by a benzylsuccinate synthase (encoded by bss genes), and a subsequent modified β-oxidation of benzylsuccinate to benzoyl-CoA and succinyl-CoA (encoded by bbs genes). We have shown here that the bss and bbs genes, which are located within an integrative and conjugative element, are essential for anaerobic degradation of toluene but also for m-xylene oxidation in the denitrifying beta-proteobacterium Azoarcus sp. CIB. New insights into the transcriptional organization and regulation of a complete gene cluster for anaerobic catabolism of toluene/m-xylene in a single bacterial strain are presented. The bss and bbs genes are transcriptionally coupled into two large convergent catabolic operons driven by the PbssD and PbbsA promoters, respectively, whose expression is inducible when cells grow anaerobically in toluene or m-xylene. An adjacent tdiSR operon driven by the PtdiS promoter encodes a putative two-component regulatory system. TdiR behaves as a transcriptional activator of the PbssD, PbbsA, and PtdiS promoters, being benzylsuccinate/(3-methyl)benzylsuccinate, rather than toluene/m-xylene, the inducers that may trigger the TdiS-mediated activation of TdiR. In addition to the TdiSR-based specific control, the expression of the bss and bbs genes in Azoarcus sp. CIB is under an overimposed regulation that depends on certain environmental factors, such as the presence/absence of oxygen or the availability of preferred carbon sources (catabolite repression). This work paves the way for future strategies toward the reliable assessment of microbial activity in toluene/m-xylene contaminated environments.

Keywords: Azoarcus; anaerobic degradation; biomarker; catabolite repression; m-xylene; tdiSR; toluene.

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Figures

FIGURE 1
FIGURE 1
Proposed toluene and m-xylene anaerobic peripheral degradation pathway in Azoarcus sp. CIB: (A) Scheme of the proposed peripheral pathway of anaerobic degradation of toluene and m-xylene. The enzymes are indicated following the color code indicated in (B). Toluene and m-xylene derivatives are shown in black and red color, respectively. Intermediates 1 correspond to benzylsuccinyl-CoA (black) and (3-methyl)benzylsuccinyl-CoA (red). Intermediates 2 correspond to phenylitaconyl-CoA (black) and (3-methyl)phenylitaconyl-CoA (red). Intermediates 3 correspond to 2-[hydroxyphenyl]-succinyl-CoA (black) and 2-[hydroxyphenyl(methyl)]-succinyl-CoA (red). Intermediates 4 correspond to benzoylsuccinyl-CoA (black) and (3-methyl)benzoylsuccinyl-CoA (red). Modified from Carmona et al. (2009). (B) Scheme of the gene cluster encoding the anaerobic peripheral pathway of toluene and m-xylene in Azoarcus sp. CIB. Genes are represented by arrows and their predicted function is annotated as follows: black, regulatory genes; dark blue, genes encoding the (3-methyl)benzylsuccinate synthase (BSS); yellow, gene encoding a putative BSS chaperone; orange, gene encoding the (3-methyl)phenylitaconyl-CoA hydratase; red, gene encoding the (3-methyl)benzylsuccinyl-CoA dehydrogenase; brown, genes encoding the succinyl-CoA:(3-methyl)benzylsuccinate CoA transferase; green, genes encoding a 2-[hydroxyphenyl(methyl)]-succinyl-CoA dehydrogenase; light blue, genes encoding the (3-methyl)benzoylsuccinyl-CoA thiolase; white, genes of unknown function. A truncated IS21 transposase istA gene (ΔistA) is shown by a gray arrow. The gray rectangle indicates the mbd cluster responsible for the 3-methylbenzoyl-CoA central pathway. Genes that have been inactivated in this work and avoid the use of toluene and m-xylene as sole carbon and energy source by the corresponding mutant strains (Azoarcus sp. CIBdtidR, Azoarcus sp. CIBdtidS, Azoarcus sp. CIBdbssD, Azoarcus sp. CIBdbssF, Azoarcus sp. CIBdbbsB) are indicated with a red star. The gene that has been inactivated and does not avoid the use of toluene and m-xylene as sole carbon and energy source in the corresponding mutant strain (Azoarcus sp. CIBdbssJ) is indicated with a green star.
FIGURE 2
FIGURE 2
Scheme of the genetic organization of the tdi-bss-bbs cluster in different bacteria. The tdi-bss-bss clusters from Azoarcus sp. CIB, Azoarcus toluclasticus MF63 (Acc. No. NZ_ARJX00000000.1), Azoarcus sp. strain T (Ac. No. AY032676), T. aromatica T1 (Ac. No. U57900 and AF113168), Thauera sp. strain DNT-1 (Ac. No. AB066263), T. aromatica K172 (Ac. No. AJ001848 and AF173961), “Aromatoleum aromaticum” EbN1 (Ac. No. NC_006513), Herminiimonas sp. CN (Ac. No. AVCC01000000), Magnetospirillum sp. strain TS-6 (Ac. No. AB167725), Geobacter metallireducens GS-15 (Ac. No. NC_007517) and Desulfobacula toluolica Tol2 (Ac. No. NC_018645) are represented here. Genes are represented by arrows following the color code indicated in Figure 1: black, regulatory genes; gray, putative regulatory genes of an aerobic toluene degradation pathway; dark blue, genes encoding the (3-methyl)benzylsuccinate synthase (BSS); yellow, genes encoding a putative BSS chaperone; orange, genes encoding the (3-methyl)phenylitaconyl-CoA hydratase; red, genes encoding the (3-methyl)benzylsuccinyl-CoA dehydrogenase and its predicted associated electron-transfer system; brown, genes encoding the succinyl-CoA:(3-methyl)benzylsuccinate CoA transferase; green, genes encoding a 2-[hydroxyphenyl(methyl)]-succinyl-CoA dehydrogenase; light blue, genes encoding the (3-methyl)benzoylsuccinyl-CoA thiolase; light green, genes encoding a putative toluene transport system; violet, gene encoding a putative succinate-dehydrogenase flavoprotein; white, genes of unknown function. Insertion sequences are indicated by gray rectangles. The white rectangle next to the bbs operon in Azoarcus sp. CIB represents orfB (AzCIB_4527), orfC (AzCIB_4528), orfD (AzCIB_4529) and orfE (AzCIB_4530) genes, that are also present in A. toluclasticus MF63. In the bss-bbs cluster of Azoarcus sp. CIB, the DNA fragment harboring the bssDCABEFG genes and whose average GC content is 58% is indicated by a disontinuous line flanked by circles. The DNA fragment containing the rest of the bss (bssIJKL) and bbs genes, whose average GC content is 65%, is indicated by a discontinuous line flanked by diamonds.
FIGURE 3
FIGURE 3
The expression of the bss, bbs and tdi genes is inducible in Azoarcus sp. CIB. Agarose gel electrophoresis of the RT-PCR products. Gene expression was monitored by RT-PCR as described in Section “Materials and Methods” with the primer pairs bssA5new/bssA3new, bbsA331.3/bbsA5new and tdiRint5/tdiSF.3 (Supplementary Table S1) that amplify a bssA gene fragment (A,D), a bbsA gene fragment (B,E), or the tdiS-tdiR intergenic region (C), respectively. (A–C) Azoarcus sp. CIB cells were grown anaerobically until mid-exponential phase by using 0.2% succinate (S), 250 mM of toluene (T) or 250 mM m-xylene (X) as sole carbon source. (D,E) Azoarcus sp. CIB cells were grown under anaerobic (–O2) or aerobic (+O2) conditions using toluene as only carbon source. Lanes M, molecular size markers (HaeIII-digested ΦX174 DNA); numbers indicate the sizes of the markers (in bp).
FIGURE 4
FIGURE 4
Transcriptional organization of the tdi-bss-bbs genes: (A) Schematic representation of the tdi-bss-bbs gene cluster from Azoarcus sp. CIB. The regulatory tdi genes are indicated by black arrows; the catabolic bss and bbs genes are indicated by gray and white arrows, respectively. The PtdiS, PbssD and PbbsA promoters are represented by bent arrows. The intergenic regions whose expression was checked by RT-PCR are marked by the oligonucleotides (thin arrows) used in the analysis (1–20; see Supplementary Table S1). (B) Agarose gel electrophoresis of RT-PCR products. RT-PCRs from Azoarcus sp. CIB cells grown under denitrifying conditions on 250 mM toluene (lanes T) or 250 mM m-xylene (lanes X) were performed as described in Section “Materials and Methods.” The numbers correspond to each primer pair that amplifies each of the intergenic regions indicated in (A). Lanes M, molecular size markers (HaeIII-digested ΦX174 DNA); numbers indicate the sizes of the markers (in bp). (C) RT-PCRs from Azoarcus sp. CIB, Azoarcus sp. CIBdbssD and Azoarcus sp. CIBdbbsB cells anaerobically grown until stationary phase in 0.2% pyruvate plus 250 mM toluene (lanes T) by using the oligonucleotide pairs 11/12 and 13/14 (Supplementary Table S1). Lanes C, PCRs performed with the same primer pairs and with genomic DNA as a positive control. Lanes M, molecular size markers (HaeIII-digested ΦX174 DNA); numbers indicate the sizes of the markers (in bp).
FIGURE 5
FIGURE 5
TdiR is a transcriptional activator of the PbssD, PbbsA and PtdiS promoters. Cells of Azoarcus sp. CIB (A) or Azoarcus sp. CIBdtdiR (B) containing plasmids pBBRPbssD, pBBRPbbsA, or pBBRPtdiS that express the PbssD::lacZ, PbbsA::lacZ, or PtdiS::lacZ translational fusions, respectively, were grown anaerobically on 0.2% pyruvate (–) or 0.2% pyruvate plus toluene (+) until stationary growth phase. β-galactosidase activity values were determined as detailed in Section “Materials and Methods.” Error bars represent standard deviation of three different experiments, and asterisks mark the results that are statistically significant (unpaired t-test; ∗∗∗P-value < 0.001, ∗P-value 0.01–0.05).
FIGURE 6
FIGURE 6
Analysis of the inducers of the PbbsA promoter. Cells of Azoarcus sp. CIBdbssD (A) or Azoarcus sp. CIBdbbsB (B) containing plasmid pBBRPbbsA that expresses the PbbsA::lacZ translational fusion, were grown anaerobically until stationary growth phase in 0.2% pyruvate (–) or in 0.2% pyruvate plus toluene (T), m-xylene (mX), or benzylsuccinate (Bs). β-galactosidase activity values were determined as detailed in Section “Materials and Methods.” Error bars represent standard deviation of three different experiments, and asterisks mark the results that are statistically significant (unpaired t-test; ∗∗∗P-value < 0.001).
FIGURE 7
FIGURE 7
Phylogenetic relationships of the two-component regulatory systems involved in the regulation of aromatic hydrocarbon catabolic pathways: (A) Phylogenetic tree built from the multiple amino acid sequence alignment of the sensor histidine kinases TdiS from Azoarcus sp. CIB (ABK15651), TutC1 from T. aromatica T1 (AAD12187), Tcs2 from “A. aromaticum” sp. EbN1 (YP_158339), StyS from Pseudomonas sp. Y2 (CAA03998), TutC from P. mendocina (AAL13332), TodS from P. putida DOT-T1 (AAC45438), TmoS from P. mendocina (AAL13333), Tcs1 from A. aromaticum sp. EbN1 (YP_158337), BphS from Rhodococcus sp. RHA1 (BAC75411) and BpdS from Rhodococcus sp. M5 (AAB52543) using the program CLUSTALW and visualized with the TreeView software application. The aerobic TodS-like and anaerobic TdiS-like families are circled by red and blue lines, respectively, and a scheme of their different domain architecture and primary structure is shown at the bottom. PAS, HK, and REC correspond to the PAS sensor, autokinase and receiver domains, respectively. H and D indicate the presence of key phosphorylatable histidine and aspartic acid residues at the HK and REC domains, respectively. (B) Phylogenetic tree built from the multiple amino acid sequence alignment of the regulatory protein TdiR from Azoarcus sp. CIB (ABK15650), TutB1 from T. aromatica T1 (AAD12186), Tcr2 from “A. aromaticum” sp. EbN1 (YP_158340), TutB from T. aromatica T1 (AAD12186), StyR from Pseudomonas sp. Y2 (CAA03999), TodT from P. putida DOT-T1 (CAB43736), TmoT from P. mendocina (AAL13333), Tcr1 from A. aromaticum sp. EbN1 (YP_158338), BphT from Rhodococcus sp. RHA1 (BAC75412) and BpdT from Rhodococcus sp. M5 (AAB52544) using the program CLUSTALW and visualized with the TreeView software application. The aerobic TodT-like and anaerobic TdiR-like families are circled by red and blue lines, respectively, and a scheme of their similar domain architecture but different primary structure is shown at the bottom. REC and HTH correspond to the receiver and helix-turn-helix DNA-binding domains, respectively. D indicates the presence of key phosphorylatable aspartic acid residue at the REC domain. The bars represent one inferred amino acid substitution per ten amino acids.
FIGURE 8
FIGURE 8
Carbon catabolite control of the PbbsA, PbssD and PtdiS promoters in Azoarcus sp. CIB. Azoarcus sp. CIB cells containing plasmid pBBRPbssD (PbssD::lacZ fusion) (A), pBBRPbbsA (PbbsA::lacZ fusion) (B) or pBBRPtdiS (PtdiS::lacZ fusion) (C) were grown anaerobically in 0.2% pyruvate plus 250 mM toluene, and after 72 h they were supplemented with an additional amount of 10 mM nitrate. Culture samples were collected at mid-exponential phase (48 h) or at stationary phase (96 h). β-galactosidase activity values were determined as detailed in Section “Materials and Methods.” Error bars represent standard deviation of three different experiments, and asterisks mark the results that are statistically significant (unpaired t-test; ∗∗∗P-value < 0.001, ∗∗P-value 0.001–0.01).
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