XRE-Type Regulator BioX Acts as a Negative Transcriptional Factor of Biotin Metabolism in Riemerella anatipestifer.

Biotin is essential for the growth and pathogenicity of microorganisms. Damage to biotin biosynthesis results in impaired bacterial growth and decreased virulence . However, the mechanisms of biotin biosynthesis in Riemerella anatipestifer remain unclear. In this study, two R. anatipestifer genes associated with biotin biosynthesis were identified. encoded a BirA protein lacking the N-terminal winged helix-turn-helix DNA binding domain, identifying it as a group I biotin protein ligase, and encoded a BioX protein, which was in the helix-turn-helix xenobiotic response element family of transcription factors. Electrophoretic mobility shift assays demonstrated that BioX bound to the promoter region of . In addition, the R. anatipestifer genes (encoding 7-keto-8-aminopelargonic acid synthase), (encoding dethiobiotin synthase), and (encoding 7,8-diaminopelargonic acid synthase) were in an operon and were regulated by BioX. Quantitative reverse transcription-PCR showed that transcription of the operon increased in the mutant Yb2ΔbioX in the presence of excessive biotin, compared with that in the wild-type strain Yb2, suggesting that BioX acted as a repressor of biotin biosynthesis. Streptavidin blot analysis showed that BirA caused biotinylation of BioX, indicating that biotinylated BioX was involved in metabolic pathways. Moreover, as determined by the median lethal dose, the virulence of Yb2ΔbioX was attenuated 500-fold compared with that of Yb2. To summarize, the genes and were identified in R. anatipestifer, and BioX was found to act as a repressor of the operon involved in the biotin biosynthesis pathway and identified as a bacterial virulence factor. Riemerella anatipestifer is a causative agent of diseases in ducks, geese, turkeys, and various other domestic and wild birds. Our study reveals that biotin synthesis of R. anatipestifer is regulated by the BioX through binding to the promoter region of the gene to inhibit transcription of the operon. Moreover, is required for R. anatipestifer pathogenicity, suggesting that BioX is a potential target for treatment of the pathogen. R. anatipestifer BioX has thus been identified as a novel negative regulator involved in biotin metabolism and associated with bacterial virulence in this study.