Mycobacterium tuberculosis depends on the ability to adjust to stresses encountered in a range of host environments, adjustments that require significant changes in gene expression. Small RNAs (sRNAs) play an important role as post-transcriptional regulators of prokaryotic gene expression, where they are associated with stress responses and, in the case of pathogens, adaptation to the host environment. In spite of this, the understanding of M. tuberculosis RNA biology remains limited. Here we have used a DosR-associated sRNA as an example to investigate multiple aspects of mycobacterial RNA biology that are likely to apply to other M. tuberculosis sRNAs and mRNAs. We have found that accumulation of this particular sRNA is slow but robust as cells enter stationary phase. Using reporter gene assays, we find that the sRNA core promoter is activated by DosR, and we have renamed the sRNA DrrS for DosR Regulated sRNA. Moreover, we show that DrrS is transcribed as a longer precursor, DrrS+, which is rapidly processed to the mature and highly stable DrrS. We characterise, for the first time in mycobacteria, an RNA structural determinant involved in this extraordinary stability and we show how the addition of a few nucleotides can lead to acute destabilisation. Finally, we show how this RNA element can enhance expression of a heterologous gene. Thus, the element, as well as its destabilising derivatives may be employed to post-transcriptionally regulate gene expression in mycobacteria in combination with different promoter variants. Moreover, our findings will facilitate further investigations into the severely understudied topic of mycobacterial RNA biology and into the role that regulatory RNA plays in M. tuberculosis pathogenesis.

In bacteria, nucleotide excision repair (NER) plays a major role in repairing DNA damage from a wide variety of sources. Therefore, its inhibition offers potential to develop a new antibacterial in combination with adjuvants, such as UV light. To date, only one known chemical inhibitor of NER is 2-(5-Amino-1,3,4-thiadiazol-2-ylbenzo(f)chromen-3-one) (ATBC) exists and targets Mycobacterium tuberculosis NER. To enable the design of future drugs we need to understand its mechanism of action. To determine the mechanism of action, we used in silico structure-based prediction, which identified the ATP binding pocket of E. coli UvrA as a probable target. Growth studies in E. coli showed it was non-toxic alone, but able to impair growth when combined with DNA damaging agents, and as we predicted it reduced by ~70% UvrA’s ATPase rate. Since UvrA’s ATPase activity is necessary for effective DNA binding, we used single molecule microscopy to directly observe DNA association. We measured a ~7-fold reduction in UvrA molecules binding to a single molecule of dsDNA suspended between optically trapped beads. These data provide a clear mechanism of action for ATBC, and show that targeting UvrA’s ATPase pocket is effective and ATBC provides an excellent framework for the derivation of more soluble inhibitors that can be tested computationally for activity.

Maintaining membrane integrity is of paramount importance to the survival of bacteria as the membrane is the site of multiple crucial cellular processes including energy generation, nutrient uptake and antimicrobial efflux. The DedA family of integral membrane proteins are widespread in bacteria and are associated with maintaining the integrity of the membrane. In addition, DedA proteins have been linked to resistance to multiple classes of antimicrobials in various microorganisms. Therefore, the DedA family are attractive targets for the development of new antibiotics. Despite DedA family members playing a key physiological role in many bacteria, their structure, function and physiological role remain unclear. To help illuminate the structure of the bacterial DedA proteins, we performed substituted cysteine accessibility method (SCAM) analysis on the most comprehensively characterized bacterial DedA protein, YqjA from Escherichia coli . By probing the accessibility of 15 cysteine residues across the length of YqjA using thiol reactive reagents, we mapped the topology of the protein. Using these data, we experimentally validated a structural model of YqjA generated using evolutionary covariance, which consists of an α-helical bundle with two re-entrant hairpin loops reminiscent of several secondary active transporters. In addition, our cysteine accessibility data suggest that YqjA forms an oligomer wherein the protomers are arranged in a parallel fashion. This experimentally verified model of YqjA lays the foundation for future work in understanding the function and mechanism of this interesting and important family.

The success of Mycobacterium tuberculosis relies on the ability to switch between active growth and non-replicating persistence, associated with latent TB infection. Resuscitation promoting factors (Rpfs) are essential for the transition between these states. Rpf expression is tightly regulated as these enzymes are able to degrade the cell wall, and hence potentially lethal to the bacterium itself. We have identified a regulatory element in the 5′ untranslated region (UTR) of rpfB. We demonstrate that this element is a transcriptionally regulated RNA switch/riboswitch candidate, which appears to be restricted to pathogenic mycobacteria, suggesting a role in virulence. We have used translation start site mapping to re-annotate the RpfB start codon and identified and validated a ribosome binding site that is likely to be targeted by an rpfB antisense RNA. Finally, we show that rpfB is co-transcribed with ksgA and ispE downstream. ksgA encodes a universally conserved methyltransferase involved in ribosome maturation and ispE encodes an essential kinase involved in cell wall synthesis. This arrangement implies co-regulation of resuscitation, cell wall synthesis and ribosome maturation via the RNA switch.