BadR and BadM Proteins Transcriptionally Regulate Two Operons Needed for Anaerobic Benzoate Degradation by Rhodopseudomonas palustris

Abstract

The bacterium Rhodopseudomonas palustris grows with the aromatic acid benzoate and the alicyclic acid cyclohexanecarboxylate (CHC) as sole carbon sources. The enzymatic steps in an oxygen-independent pathway for CHC degradation have been elucidated, but it was unknown how the CHC operon (badHI aliAB badK) encoding the enzymes for CHC degradation was regulated. aliA and aliB encode enzymes for the conversion of CHC to cyclohex-1-enecarboxyl-coenzyme A (CHene-CoA). At this point, the pathway for CHC degradation merges with the pathway for anaerobic benzoate degradation, as CHene-CoA is an intermediate in both degradation pathways. Three enzymes, encoded by badK, badH, and badI, prepare and cleave the alicyclic ring of CHene-CoA to yield pimelyl-CoA. Here, we show that the MarR transcription factor family member, BadR, represses transcription of the CHC operon by binding near the transcription start site of badH. 2-Ketocyclohexane-1-carboxyl-CoA, an intermediate of CHC and benzoate degradation, interacts with BadR to abrogate repression. We also present evidence that the transcription factor BadM binds to the promoter of the badDEFGAB (Bad) operon for the anaerobic conversion of benzoate to CHene-CoA to repress its expression. Contrary to previous reports, BadR does not appear to control expression of the Bad operon. These data enhance our view of the transcriptional regulation of anaerobic benzoate degradation by R. palustris.A licyclic compounds occur in nature as functional groups on plant secondary products, components of crude oil and polyketide antibiotics (1). Cyclohexanecarboxylate (CHC) is the most structurally simple of these and is of special interest in the context of the overall metabolism of the phototrophic bacterium Rhodopseudomonas palustris, because its degradation pathway shares reactions in common with anaerobic benzoate degradation. The anaerobic benzoate degradation pathway is the main conduit for aromatic ring reduction and cleavage in R. palustris and other anaerobes. Structurally diverse aromatic compounds, including monomeric constituents of lignin, a major plant polymer, are processed by bacteria through peripheral degradation pathways to form benzoate or benzoyl coenzyme A (benzoylCoA) (2-4). In R. palustris, the benzoate degradation pathway involves a dearomatization of benzoyl-CoA by the oxygen-sensitive enzyme benzoyl-CoA reductase followed by a ␤-oxidation of the reduced product, cyclohex-1-enecarboxyl-CoA (CHeneCoA). This culminates in a ring cleavage that generates pimelylCoA (Fig. 1A) (5, 6). Pimelyl-CoA is further degraded to three acetyl-CoAs and one CO 2 (7). The ␤-oxidation segment of anaerobic benzoate degradation is catalyzed by three oxygen-insensitive enzymes: BadK, BadH, and BadI (Fig. 1A) (6, 8, 9). These are encoded by an operon, which also includes aliA and aliB, two genes that allow R. palustris to metabolize exogenously supplied CHC. aliA encodes CHC-CoA ligase, and aliB encodes a dehydrogenase, which catalyzes the conversion of CHC-CoA to CHeneCoA (10,11,12). The ba...

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