Biochemical Characterization of the Transcriptional Regulator BzdR from Azoarcus sp. CIB

Abstract

The BzdR transcriptional regulator that controls the P N promoter responsible for the anaerobic catabolism of benzoate in Azoarcus sp. CIB constitutes the prototype of a new subfamily of transcriptional regulators. Here, we provide some insights about the functional-structural relationships of the BzdR protein. Analytical ultracentrifugation studies revealed that BzdR is homodimeric in solution. An electron microscopy three-dimensional reconstruction of the BzdR dimer has been obtained, and the predicted structures of the respective N-and C-terminal domains of each BzdR monomer could be fitted into such a reconstruction. Gel retardation and ultracentrifugation experiments have shown that the binding of BzdR to its cognate promoter is cooperative. Different biochemical approaches revealed that the effector molecule benzoyl-CoA induces conformational changes in BzdR without affecting its oligomeric state. The BzdRdependent inhibition of the P N promoter and its activation in the presence of benzoyl-CoA have been established by in vitro transcription assays. The monomeric BzdR4 and BzdR5 mutant regulators revealed that dimerization of BzdR is essential for DNA binding. Remarkably, a BzdR⌬L protein lacking the linker region connecting the N-and C-terminal domains of BzdR is also dimeric and behaves as a super-repressor of the P N promoter. These data suggest that the linker region of BzdR is not essential for protein dimerization, but rather it is required to transfer the conformational changes induced by the benzoyl-CoA to the DNA binding domain leading to the release of the repressor. A model of action of the BzdR regulator has been proposed.

Bacterial Degradation of Benzoate

“…In this sense the TGCA direct repeats have been shown to be present also at the P N promoter controlling the anaerobic bzd operon in Azoarcus sp. CIB, and they were proposed to be the BzdR-recognition sites (27,50). This observation is in agreement with the fact that both BoxR and BzdR proteins contain highly similar helix-turn-helix (HTH) DNA binding domains (Fig.…”

“…2). The central region of BoxR (residues 94 -132) corresponds to the linker region of BzdR involved in the transmission of the conformational change from the C-terminal effector binding domain to the N-terminal DNA binding domain (50). Interestingly, the central regions of both regulators show the lowest amino acid sequence identity (24%) (Fig.…”

Benzoate mediates the simultaneous repression of anaerobic 4-methylbenzoate and succinate utilization in Magnetospirillum sp. strain pMbN1

“…This is supported by nonutilization of 4-methylbenzoate during phase 1, despite presence of some catabolic proteins (MbrBCA, Oah-2) (Figure 3). Expression of genes for anaerobic catabolism of aromatic acids is typically controlled by regulatory proteins recognizing corresponding aryl-CoA esters (e.g., [28][29][30][31]). Inhibition of 4-methylbenzoate uptake would prevent its intracellular presence and thus generation of 4-methylbenzoyl-CoA as putative inductor for expression of genes for 4-methylbenzoate catabolism.…”

AccR Is a Master Regulator Involved in Carbon Catabolite Repression of the Anaerobic Catabolism of Aromatic Compounds in Azoarcus sp. CIB

“…CIB bzd genes responsible for the anaerobic degradation of benzoate are clustered and consist of the P N promoter-driven bzdNOPQMSTUVWXYZA catabolic operon and the bzdR regulatory gene (20). BzdR-mediated repression of P N is alleviated by the inducer molecule benzoyl-CoA, the first intermediate of the catabolic pathway (23,24). However, activity of the P N promoter also strictly requires the AcpR transcriptional activator, an ortholog of the E. coli Fnr global regulator, which specifically drives expression of the bzd catabolic operon in an oxygen-dependent manner (25).…”

“…Azoarcus sp. CIB is able to degrade aromatic compounds both aerobically and anaerobically (20,22), and it has been used as a model organism to study the specific regulation of the benzoylCoA central pathways for the aerobic (box genes) and anaerobic (bzd genes) degradation of benzoate (20,(23)(24)(25)(26)(27).…”