Unraveling the Specific Regulation of the mbd Central Pathway for the Anaerobic Degradation of 3- methylbenzoate*

The mbd cluster encodes the anaerobic degradation of 3-methylbenzoate in the β-proteobacterium Azoarcus sp. CIB. The specific transcriptional regulation circuit that controls the expression of the mbd genes was investigated. The PO, PB1, and P3R promoters responsible for the expression of the mbd genes, their cognate MbdR transcriptional repressor, as well as the MbdR operator regions (ATAC-N10-GTAT), have been characterized. The three-dimensional structure of MbdR has been solved revealing a conformation similar to that of other TetR family transcriptional regulators. The first intermediate of the catabolic pathway, i.e., 3methylbenzoyl-CoA, was shown to act as the inducer molecule. An additional MbdRdependent promoter, PA, which contributes to the expression of the CoA ligase that activates 3-methylbenzoate to 3-methylbenzoyl-CoA, was shown to be necessary for an efficient induction of the mbd genes. Our results suggest that the mbd cluster recruited a regulatory system based on the MbdR regulator and its target promoters, to evolve a distinct central catabolic pathway that is only expressed for the anaerobic degradation of aromatic compounds that generate 3methylbenzoyl-CoA as central metabolite. All these results highlight the importance of the regulatory systems in the evolution and adaptation of bacteria to the anaerobic degradation of aromatic compounds. Aromatic compounds are included among the most widespread organic compounds in nature and some of them are man-made environmental pollutants (1-4). Microorganisms play a fundamental role in the degradation of these aromatic compounds in diverse ecological niches (3, 5-8). Many habitats containing large amounts of aromatic compounds are often anoxic. In the last decades biochemical studies concerning the anaerobic degradation of aromatic compounds have been steadily accumulating, with benzoyl-CoA representing the intermediate to which most monocyclic aromatic compounds are converted (3-5, 9-12). On the contrary, the study on the specific regulatory systems controlling the expression of the gene clusters involved in the anaerobic degradation of aromatic compounds has been mainly restricted to the characterization of a few transcriptional regulators. Anaerobic benzoate degradation via benzoyl-CoA was shown to be controlled by the two-component BamVW regulatory system (13) or the BgeR regulator (14) in the obligate anaerobes Geobacter strains, and by the BadR/BadM (15, 16) and BzdR/BoxR (17-20) regulators in the facultative anaerobes Rhodopseudomonas palustris and Azoarcus strains, respectively. Moreover, a few global regulators, e.g., AadR, AcpR and AccR, that influence the anaerobic expression of the benzoyl-CoA central pathway have been reported (15, 21, 22). On the other hand, a TdiSR (TutC1B1) two-component regulatory system was described for the regulation of the bss/bbs genes encoding the peripheral pathway that converts toluene into benzoyl-CoA in denitrifying bacteria (4, 23-25). It was also reported that the regulation of the peripheral routes that funnel 4-hydroxybenzoate and pcoumarate into the benzoyl-CoA central pathway in the phototrophic R. palustris strain is accomplished by the HbaR and CouR proteins, respectively (26, 27). However, yet no specifictranscriptional regulators that control anaerobic degradation pathways other than that of benzoylCoA and some peripheral routes that converge to the later have been described so far. Azoarcus sp. CIB is a denitrifying βproteobacterium able to anaerobically degrade different aromatic compounds, including some