Riboswitch­mediated regulation of the Mycobacterium tuberculosis resuscitation promoting factor, rpfB

Mycobacterium tuberculosis, the causative agent of tuberculosis, results in over 1.5 million deaths every year. An estimated third of the world’s population may harbour a latent infection with risk of reactivation under permissive conditions. M. tuberculosis expresses a number of homologous resuscitation promoting factors (Rpfs) believed to facilitate resuscitation from dormancy. However, little is known about the regulation of Rpf expression. This study identifies a putative novel riboswitch in the 5’UTR of rpfB, which appears to be conserved in pathogenic mycobacteria, but not in non-pathogenic species. Northern blot analysis of M. tuberculosis RNA reveals short terminated transcripts and longer read-through transcripts suggesting transcription attenuation mediated by a transcriptional riboswitch. This has been further supported by 3’RACE, reporter gene assays and in vitro transcription. Findings indicate that rpfB is co-transcribed with the universally conserved rRNA methyltransferase ksgA, implicating the riboswitch in ksgA expression regulation, thereby linking resuscitation with ribosome maturation. Moreover, through translation start site mapping the RpfB start codon has been re-annotated and a ribosome binding site has been validated. This ribosome binding site is likely targeted by an rpfB antisense RNA produced from an antisense promoter that may also serve to regulate rpfB expression either by RNA polymerase clashing, sense antisense interaction, mRNA processing or all of the above. Expression of the rpfB locus has been investigated under multiple physiological stresses and throughout biofilm formation. Collectively, findings support the presence of a novel rpfB riboswitch transcriptional ‘on-switch’ capable of regulating expression through transcription attenuation. Understanding the stimuli and regulation of Rpf gene expression will improve understanding of their role in disease and resuscitation