Bacterial degradation of benzoate: cross-regulation between aerobic and anaerobic pathways

Abstract

We have studied for the first time the transcriptional regulatory circuit that controls the expression of the box genes encoding the aerobic hybrid pathway used to assimilate benzoate via coenzyme A (CoA) derivatives in bacteria. The promoters responsible for the expression of the box cluster in the β-proteobacterium Azoarcus sp., their cognate transcriptional repressor, the BoxR protein, and the inducer molecule (benzoyl-CoA) have been characterized. The BoxR protein shows a significant sequence identity to the BzdR transcriptional repressor that controls the bzd genes involved in the anaerobic degradation of benzoate. Because the boxR gene is present in all box clusters so far identified in bacteria, the BoxR/benzoyl-CoA regulatory system appears to be a widespread strategy to control this aerobic hybrid pathway. Interestingly, the paralogous BoxR and BzdR regulators act synergistically to control the expression of the box and bzd genes. This cross-regulation between anaerobic and aerobic pathways for the catabolism of aromatic compounds has never been shown before, and it may reflect a biological strategy to increase the cell fitness in organisms that survive in environments subject to changing oxygen concentrations.

FIGURE 1.

Major biochemical strategies for benzoate degradation and genetic arrangements of the box clusters in proteobacteria. A, schemes of the first biochemical steps of the classical aerobic (black box) and anaerobic (gray box) benzoate degradation pathways and that of the aerobic hybrid box pathway (white box) are shown. The activation and the dearomatization/ring-cleavage steps are indicated by white and striped arrows, respectively. The lower pathway that funnels the dehydroadipyl-CoA semialdehyde into the central metabolism is represented by a dotted arrow. OX, benzoate dioxygenase; DH, benzoate dihydrodiol dehydrogenase; DOX, catechol dioxygenase (ortho (intradiol) or meta (extradiol) cleavage). The Box enzymes responsible for the activation and dearomatization/ring-cleavage reactions are indicated: BclA, benzoate-CoA ligase; BoxA, NADPH-dependent reductase; BoxB, benzoyl-CoA 2,3-epoxidase; BoxC, 2,3-epoxybenzoyl-CoA dihydrolase. B, schemes of the major arrangements of the functional modules within the box clusters from proteobacteria are shown. The activation (C1, bclA gene), dearomatization/ring-cleavage (C2, boxABC genes), and lower pathway (C3) catabolic modules are shown by white, striped, and dotted arrows, respectively. The putative regulatory module (boxR gene) is shown by black arrows. It should be noted that the gene composition and arrangement within the C3 module can differ even among strains of the same genus. Type 1 box clusters are present in many β-proteobacteria, e.g. strains of the Azoarcus/Aromatoleum, Burkholderia, Bordetella, Polaromonas, Rhodoferax, Delftia, Variovorax, Leptothrix, Verminephrobacter, Comamonas, Achromobacter, Ralstonia, Cupriavidus genera. Type 2 box clusters are present in some α-proteobacteria where the C1 module can be associated, e.g. strains of the Silicibacter and Jannaschia genera, or not, e.g. Magnetospirillum sp. AMB-1, to the box cluster. Although types 1 and 2 usually contain the boxA gene within the C2 module, in some strains this gene is missing. Type 3 box clusters are present in other α-proteobacteria where the C3 module contains only one gene and the C2 module lacks the boxA gene, e.g. strains of the Rhodopseudomonas and Bradyrhizobium genera, or in some β-proteobacteria, e.g. Thauera aromatica, where the C3 module is not found between the C1 and C2 modules. In the genome of some δ-proteobacteria, e.g. Sorangium cellulosum, there is a boxR gene divergently oriented to the C2 module (boxBC genes).

FIGURE 2.

Amino acid sequence comparison between BzdR and BoxR proteins from Azoarcus sp. CIB. The amino acid sequences of BzdR (AAQ08805) and BoxR (CCD33120) were aligned using the multiple sequence alignment program ClustalW. The amino acid residues of each sequence are numbered at the right. Amino acids are indicated by their standard one-letter code. Dark gray shows identical residues in the two sequences, whereas light gray indicates functional similarity between residues. The α-helices and β-strands predicted for the BzdR (top) and BoxR (bottom) proteins are also drawn. The N terminus, linker, and C-terminal regions of both proteins are indicated at the top. The predicted helix-turn-helix (HTH) motif for DNA binding is marked within the N-terminal region.

FIGURE 3.

The boxR gene encodes a transcriptional repressor of the box genes. A, agarose gel electrophoresis of RT-PCR products is shown. Total RNA was isolated from Azoarcus sp. CIB (CIBwt) or Azoarcus sp. CIBdboxR (CIBdboxR) cells grown in alanine (0.4%)-containing MC medium in the presence (lanes 1 and 5) or in the absence (lanes 3 and 7) of 1 mm benzoate (Bz). RT-PCRs were performed as indicated under “Experimental Procedures” with the primer pair 5′pboxdQ/3′pboxdQ (Table 2) that amplifies a 153-bp fragment of the boxD gene (arrow). Lanes 2, 4, 6, and 8, control reactions in which reverse transcriptase was omitted from the reaction mixture. Lane M, molecular size markers (HaeIII-digested ΦX174 DNA). Numbers on the right represent the sizes of the markers (in base pairs). B, shown is β-galactosidase activity of E. coli CC118 cells grown aerobically in glycerol-containing minimal medium and harboring plasmids pSJ3P_D (P_D::lacZ) (white bars) or pSJ3P_X (P_X::lacZ) (black bars) and the pCK01BoxR (BoxR) or the control plasmid pCK01 (−). Values for β-galactosidase activity (in Miller units) were determined as indicated under “Experimental Procedures.” Each value is the average from three separate experiments; error bars indicate S.D.

FIGURE 4.

Identification of the transcription start site in the P_D and P_X promoters. Total RNA was isolated from E. coli CC118 cells bearing the lacZ translational fusion plasmid pSJ3P_D (P_D::lacZ) or pSJ3P_X (P_X::lacZ) as described under “Experimental Procedures.” The size of the extended product (lanes P_D and P_X) was determined by comparison with the DNA sequence ladder (lanes A, T, C, and G) of the P_D (A) and P_X (B) regions. Primer extension and sequencing reactions were performed with primers 5′boxDext (A) and Lac57 (B), respectively, as described under “Experimental Procedures.” An expanded view of the nucleotides surrounding the transcription initiation site (asterisk) in the noncoding strand is shown. C, shown is an expanded view of the boxD-boxR intergenic region. The nucleotide sequence between the translation initiation codons (italics) of the divergent boxD (GTG) and boxR (ATG) genes is shown. The transcription initiation site (+1) of the boxD and boxR genes is indicated with white letters. The ribosome-binding site (RBS) and the inferred −10 and −35 regions of each promoter are underlined. The BoxR-mediated protection of the intergenic region from digestion by DNase I is shown with boldface letters. The TGC(A) sequences within the protected region are underlined with broken lines, and some of them form part of palindromic structures (convergent arrows).

FIGURE 5.

Interaction of the BoxR protein with boxD-boxR intergenic region. Gel retardation analyses of BoxR binding to the boxD-boxR intergenic region were performed as indicated under “Experimental Procedures.” A, lane 1, free boxDR probe; lane 2, retardation assay containing 1000 ng of E. coli M15 (pREP4, pQE32) control cell extract; lanes 3–5 show retardation assays containing 50, 100, and 200 ng, respectively, of E. coli M15 (pREP4, pQE32-His₆BoxR) cell extract harboring the His₆-BoxR protein (it represents about 12% of the total protein). B, lane 1, free boxDR probe; lanes 2–5, show retardation assays containing 200 ng of E. coli M15 (pREP4, pQE32-His₆BoxR) cell extract and 0, 0.5, 1.0, and 2.0 mm benzoyl-CoA, respectively; lanes 6 and 7 show retardation assays containing 200 ng of E. coli M15 (pREP4, pQE32-His₆BoxR) cell extract and 2.0 mm benzoate or phenylacetyl-CoA, respectively. C, lane 1, free boxDR probe; lanes 2–7 show retardation assays containing 200 ng of E. coli M15 (pREP4, pQE32-His₆BoxR) cell extract and 0, 5, 25, 50, 75, and 150 ng of boxDR unlabeled probe, respectively. The boxDR probe and the boxDR-BoxR complex are indicated by the arrows.

FIGURE 6.

DNase I footprinting analysis of the interaction of BoxR with the boxD-boxR intergenic region. The DNase I footprinting experiments were carried out using the boxD-boxR intergenic fragments labeled at the boxD (A) or boxR (B) ends as indicated under “Experimental Procedures.” A, lane AG shows the A + G Maxam and Gilbert sequencing reaction. Lanes 1 and 13, footprinting assays containing 1600 ng of E. coli M15 (pREP4, pQE32) control cell extract. Lanes 2–8, footprinting assays containing 25, 50, 100, 200, 400, 800, and 1600 ng of E. coli M15 (pREP4, pQE32-His₆BoxR) cell extract harboring the His₆-BoxR protein (it represents about 12% of the total protein), respectively. Lanes 9 and 10, footprinting assays containing 400 and 800 ng, respectively, of cell extracts harboring the His₆-BoxR protein and 2 mm benzoyl-CoA. Lanes 11 and 12, footprinting assays containing 400 ng of cell extract harboring the His₆-BoxR protein and 2 mm benzoate or phenylacetyl-CoA, respectively. B, lane AG shows the A + G Maxam and Gilbert sequencing reaction. Lane 1, footprinting assay containing 1600 ng of E. coli M15 (pREP4, pQE32) control cell extract. Lanes 2–8, footprinting assays containing 25, 50, 100, 200, 400, 800, and 1600 ng of E. coli M15 (pREP4, pQE32-His₆BoxR) cell extract harboring the His₆-BoxR protein, respectively. Lanes 9–11, footprinting assays containing 1600 ng of E. coli M15 (pREP4, pQE32-His₆BoxR) cell extract harboring the His₆-BoxR protein and 2 mm benzoate, phenylacetyl-CoA, and benzoyl-CoA, respectively. The protected regions are marked by brackets, and the phosphodiester bonds hypersensitive to DNase I cleavage are indicated by asterisks. The −10 and −35 boxes and the transcription initiation sites (+1) of the P_D and P_X promoters are also shown.

FIGURE 7.

Expression of the boxD, boxR, and bzdR genes under aerobic and anaerobic conditions. Total RNA was isolated from Azoarcus sp. CIB cells growing aerobically (black bars) or anaerobically (white bars) in 3 mm benzoate, and the expression of boxD, boxR, and bzdR genes was measured by real-time RT-PCR as detailed under “Experimental Procedures.” The relative expression of the genes is shown in arbitrary units. Each value is the average from three separate experiments; error bars indicate S.D.

FIGURE 8.

Cross-interaction between BoxR and P_N and between BzdR and P_D/P_X promoters. Gel retardation analyses of BoxR binding to the P_N promoter (A) and BzdR binding to the boxD-boxR intergenic region (B) and P_N promoter (C) were performed as indicated under “Experimental Procedures.” Lanes 1, free probes. Lanes 2–6 show retardation assays containing 25, 50, 100, 200, and 400 ng, respectively, of E. coli M15 (pREP4, pQE32-His₆BoxR) cell extract harboring His₆-BoxR protein (A) or E. coli M15 (pREP4, pQE32-His₆BzdR) cell extract containing His₆-BzdR protein (B and C). Lane c (A), retardation assay containing 400 ng of E. coli M15 (pREP4, pQE32) control cell extract. The P_N and boxDR probes as well as the P_N/BoxR, boxDR/BzdR and P_N/BzdR complexes are indicated by the arrows. D, E, and F, expression shown is of the P_N::lacZ, P_X::lacZ, or P_D::lacZ translational fusions, respectively. D, E. coli AFMCP_N cells, which harbor a chromosomal P_N::lacZ insertion, carrying plasmid pCK01BoxR (BoxR), pCK01BzdR (BzdR) or the control plasmid pCK01 (−) were grown anaerobically in glycerol-containing minimal medium. E and F, E. coli CC118 (pSJ3P_X) (P_X::lacZ) and E. coli CC118 (pSJ3P_D) (P_D::lacZ) cells, respectively, carrying plasmid pCK01BzdR (BzdR) or the control plasmid pCK01 (−) were grown aerobically in glycerol-containing minimal medium. Values for β-galactosidase activity (in Miller units) were determined when cultures reached mid-exponential phase as indicated under “Experimental Procedures.” Each value is the average from three separate experiments; error bars indicate S.D.

FIGURE 9.

Synergistic effect of BzdR and BoxR repressors on the activity of the P_D an P_N promoters in Azoarcus sp. CIB. Azoarcus sp. CIB (CIBwt), Azoarcus sp. CIBdboxR (CIBdboxR), Azoarcus sp. CIBdbzdR (CIBdbzdR), and Azoarcus sp. CIBdboxRbzdR (CIBdboxRbzdR) cells were grown anaerobically in 3 mm benzoate (gray bars) or 0.4% alanine (black bars) until the culture reached mid-exponential phase. Total RNA was isolated from cells, and the activity of the P_N (stripped bars) or P_D (filled bars) promoters was measured by real time RT-PCR as detailed under “Experimental Procedures.” The relative activity of the promoters is shown in arbitrary units. Each value is the average from three separate experiments; error bars indicate S.D.