Mycobacterium tuberculosis employs atypical and different classes of B12 switches to control separate operons

Vitamin B12 (B12), an essential cofactor in all domains of life, is produced de novo by only a small subset of prokaryotes, but B12-sensing riboswitches are some of the most widely distributed riboswitches in bacteria. Mycobacterium tuberculosis , the causative agent of the ongoing tuberculosis pandemic, encodes two distinct vitamin B12 riboswitches. One controls the expression of metE , encoding a B12-independent methionine synthase, while the other is located upstream of ppe2, a PE/PPE family gene whose function is still unresolved. Here, we analyse ligand sensing, secondary structure architecture, and gene expression control mechanisms of these two riboswitches. Our results provide the first evidence of direct ligand binding by metE and ppe2 riboswitches and show that the two switches exhibit different preferences for natural isoforms of B12, use distinct regulatory and structural elements, and act as translational OFF switches. Based on our results, we propose that the ppe2 switch represents a new Class IIc of B12-sensing riboswitches. Moreover, we have identified small translated open reading frames (uORFs) upstream of both metE and ppe2 , which modulate the expression of the respective downstream genes in opposite directions. Translation of the metE riboswitch uORF suppresses MetE expression, while translation of the uORF in the ppe2 switch is essential for PPE2 expression via the synthesis of a uORF-PPE2 fusion protein. In summary, our findings reveal an unexpected diversity and complexity of B12-dependent cis -regulation in M. tuberculosis , with potential implications for host-pathogen interactions. ### Competing Interest Statement The authors have declared no competing interest.