The TetR Family Repressor HpaR Negatively Regulates the Catabolism of 5-Hydroxypicolinic Acid in Alcaligenes faecalis JQ135 by Binding to Two Unique DNA Sequences in the Promoter of Operon.

5-Hydroxypicolinic acid (5HPA), an important natural pyridine derivative, is microbially degraded in the environment. Previously, a gene cluster, , responsible for 5HPA degradation, was identified in Alcaligenes faecalis JQ135. However, the transcription regulation mechanism of the cluster is still unknown. In this study, the transcription start site and promoter of the operon was identified. Quantitative reverse transcription-PCR and promoter activity analysis indicated that the transcription of the operon was negatively regulated by a TetR family regulator, HpaR, whereas the transcription of itself was not regulated by HpaR. Electrophoretic mobility shift assay and DNase I footprinting revealed that HpaR bound to two DNA sequences, covering the -35 region and -10 region, respectively, in the promoter region of the operon. Interestingly, the two binding sequences are partially palindromic, with 3 to 4 mismatches and are complementary to each other. 5HPA acted as a ligand of HpaR, preventing HpaR from binding to promoter region and derepressing the transcription of the operon. The study revealed that HpaR binds to two unique complementary sequences of the promoter of the operon to negatively regulate the catabolism of 5HPA. This study revealed that the transcription of the operon was negatively regulated by a TetR family regulator, HpaR. The binding of HpaR to the promoter of the operon has the following unique features: (i) HpaR has two independent binding sites in the promoter of the operon, covering -35 region and -10 region, respectively; (ii) the palindrome sequences of the two binding sites are complementary to each other; and (iii) both of the binding sites include a 10-nucleotide partial palindrome sequence with 3 to 4 mismatches. This study provides new insights into the binding features of the TetR family regulator with DNA sequences.