BzdR, a Repressor That Controls the Anaerobic Catabolism of Benzoate in Azoarcus sp. CIB, Is the First Member of a New Subfamily of Transcriptional Regulators

Abstract

In this work, we have studied the transcriptional regulation of the bzd operon involved in the anaerobic catabolism of benzoate in the denitrifying Azoarcus sp. strain CIB. The transcription start site of the P N promoter running the expression of the bzd catabolic genes was identified. Gel retardation assays and P N ::lacZ translational fusion experiments performed both in Azoarcus sp. CIB and Escherichia coli cells have shown that bzdR encodes a specific repressor that controls the inducible expression of the adjacent bzd catabolic operon, being the first intermediate of the catabolic pathway (i.e. benzoyl-CoA, the actual inducer molecule). This is the first report of a transcriptional repressor and a CoA-derived aromatic inducer controlling gene expression in the anaerobic catabolism of aromatic compounds. DNase I footprinting experiments revealed that BzdR protected three regions (operators) at the P N promoter. The three operators contain direct repetitions of a TGCA sequence that forms part of longer palindromic structures. In agreement with the repressor role of BzdR, operator region I spans the transcription initiation site as well as the ؊10 sequence for recognition of the RNA polymerase. Primary sequence analyses of BzdR showed an unusual modular organization with an N-terminal region homologous to members of the HTH-XRE family of transcriptional regulators and a C-terminal region similar to shikimate kinases. A threedimensional model of the N-terminal and C-terminal regions of BzdR, generated by comparison with the crystal structures of the SinR regulator from Bacillus subtilis and the shikimate kinase I protein from E. coli, strongly suggests that they contain the helix-turn-helix DNA-binding motif and the benzoyl-CoA binding groove, respectively. The BzdR protein constitutes, therefore, the prototype of a new subfamily of transcriptional regulators.

Bacterial Degradation of Benzoate

“…The boxR gene from Azoarcus sp. CIB, which corresponds to orf10 in the box cluster from A. evansii (14), encodes a protein of 300 amino acids that shows an overall 47% sequence identity and a similar domain organization than the BzdR transcriptional repressor that controls the bzd genes responsible for the anaerobic degradation of benzoate in this strain (27). Thus, whereas the N-terminal region of BoxR (residues 1-93) exhibits significant similarity with transcriptional regulators of the HTH-XRE family, the C-terminal domain of BoxR (residues 133-300) presents high identity with shikimate kinases (Fig.…”

“…To label the fragment at the boxD end, the amplified DNA was digested with ScaI and EcoRI restriction enzymes, and the resulting 234-bp fragment was single endlabeled by filling in the overhanging EcoRI-digested end with [␣-32 ]dATP (6000 Ci/mmol; PerkinElmer Life Sciences) and the Klenow fragment of E. coli DNA polymerase I, as described previously (27). To label the fragment at the boxR end, the PCR amplification reaction was performed with the 3ЈFPAdhRev primer previously labeled at its 5Ј-end with phage T4 polynucleotide kinase and [␥-32 P]ATP (3000 Ci/mmol; PerkinElmer Life Sciences).…”

“…Gel Retardation Assays-The P N probe was obtained as described previously from plasmid pECOR7 (27). The boxDR probe was PCR-amplified from pSJ3P R plasmid (Table 1) by using oligonucleotides 5ЈFPAdhRev and 3ЈFPAdhRev ( Table 2).…”

“…This mixture was incubated for 20 min at 30°C, after which 3 l (0.05 unit) of DNase I (Amersham Biosciences) (prepared in 10 mM CaCl 2 , 10 mM MgCl 2 , 125 mM KCl, and 10 mM Tris-HCl, pH 7.5) was added, and the incubation was continued at 37°C for 20 s. The reaction was stopped by the addition of 180 l of a solution containing 0.4 M sodium acetate, 2.5 mM EDTA, 50 g/ml calf thymus DNA, and 0.3 g/ml glycogen. After phenol extraction, DNA fragments were analyzed as previously described (27). AϩG Maxam and Gilbert reactions (48) were carried out with the same fragments and loaded on the gels along with the footprinting samples.…”

“…Although the box pathway becomes a major route for aerobic benzoate degradation and its biochemistry and genetics have been investigated in some bacteria, thus far there is no information about the regulatory mechanism that controls the inducible expression of the box genes (14,22,24). Interestingly, a common feature of most box clusters is the presence of a boxR gene, first described in Azoarcus strains (14,25), that encodes a protein that might be a member of the BzdR subfamily of prokaryotic transcriptional regulators (26,27) and, therefore, might constitute the regulatory unit of the box cluster (Fig. 1B).…”