Characterization of Redundant Vibrio Cholerae Riboflavin Biosynthetic Pathway Genes

Bacteria often possess elaborate riboflavin provision pathways with puzzling roles in physiology. Vibrio cholerae has a riboflavin biosynthetic pathway (RBP) with putative genetic redundancy. Particularly, this species contains a putative ribBX (originally annotated as ribBA) fusion gene on its main RBP operon in addition to monocistronic ribB and ribA genes. In this study, sequence analysis and heterologous complementation experiments were performed to evaluate the functions of RibBX, RibB and RibA of the RBP of V. cholerae. The results indicate that the RibB dihydroxy-butanone phosphate synthase function is provided by both the RibBX hybrid and the orphan RibB. RibBX homologs have been described in other members of the proteobacteria. These proteins conserve RibB activity, but the exact function of the RibX domain remains unelucidated. Here, structural comparisons of the RibB and RibX domains of RibBX homologs in proteobacteria evidenced structural variation in the RibX domain across bacteria, suggesting differential functions. Moreover, this analysis identified the carriage of one extra domain putatively involved in genetic regulation in a group of orthologs within the Neisseriaceae family. Together, the results show that in V. cholerae, RibB activity is provided by the presence of monofunctional RibB and a RibBX hybrid. Notably, the function of the RibX domains fused to RibB may vary across bacteria and some hybrids include yet extra functional domains. © © 2025 the Author(s).