A novel regulatory interplay between atypical B12 riboswitches and uORF translation in Mycobacterium tuberculosis.

Vitamin B12 is an essential cofactor in all domains of life and B12-sensing riboswitches are some of the most widely distributed riboswitches. Mycobacterium tuberculosis, the causative agent of tuberculosis, harbours two B12-sensing riboswitches. One controls expression of metE, encoding a B12-independent methionine synthase, the other controls expression of ppe2 of uncertain function. Here, we analysed ligand sensing, secondary structure and gene expression control of the metE and ppe2 riboswitches. Our results provide the first evidence of B12 binding by these riboswitches and show that they exhibit different preferences for individual 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 variant of Class IIb B12-sensing riboswitches. Moreover, we have identified short translated open reading frames (uORFs) upstream of metE and ppe2, which modulate the expression of their downstream genes. Translation of the metE uORF suppresses MetE expression, while translation of the ppe2 uORF is essential for PPE2 expression. Our findings reveal an unexpected regulatory interplay between B12-sensing riboswitches and the translational machinery, highlighting a new level of cis-regulatory complexity in M. tuberculosis. Attention to such mechanisms will be critical in designing next-level intervention strategies.

Premature termination of transcription is shaped by Rho and translated uORFS in Mycobacterium tuberculosis.

Little is known about the decisions behind transcription elongation versus termination in the human pathogen Mycobacterium tuberculosis (M.TB). By applying Term-seq to M.TB we found that the majority of transcription termination is premature and associated with translated regions, i.e., within previously annotated or newly identified open reading frames. Computational predictions and Term-seq analysis, upon depletion of termination factor Rho, suggests that Rho-dependent transcription termination dominates all transcription termination sites (TTS), including those associated with regulatory 5' leaders. Moreover, our results suggest that tightly coupled translation, in the form of overlapping stop and start codons, may suppress Rho-dependent termination. This study provides detailed insights into novel M.TB cis-regulatory elements, where Rho-dependent, conditional termination of transcription and translational coupling together play major roles in gene expression control. Our findings contribute to a deeper understanding of the fundamental regulatory mechanisms that enable M.TB adaptation to the host environment offering novel potential points of intervention.

The Mycobacterium tuberculosis sRNA F6 Modifies Expression of Essential Chaperonins, GroEL2 and GroES.

Almost 140 years after the identification of Mycobacterium tuberculosis as the etiological agent of tuberculosis, important aspects of its biology remain poorly described. Little is known about the role of posttranscriptional control of gene expression and RNA biology, including the role of most of the small RNAs (sRNAs) identified to date. We have carried out a detailed investigation of the M. tuberculosis sRNA F6 and shown it to be dependent on SigF for expression and significantly induced in starvation conditions in vitro and in a mouse model of infection. Further exploration of F6 using an in vitro starvation model of infection indicates that F6 affects the expression of the essential chaperonins GroEL2 and GroES. Our results point toward a role for F6 during periods of low metabolic activity typically associated with long-term survival of M. tuberculosis in human granulomas. IMPORTANCE Control of gene expression via small regulatory RNAs (sRNAs) is poorly understood in one of the most successful pathogens, Mycobacterium tuberculosis. Here, we present an in-depth characterization of the sRNA F6, including its expression in different infection models and the differential gene expression observed upon deletion of the sRNA. Our results demonstrate that deletion of F6 leads to dysregulation of the two essential chaperonins GroEL2 and GroES and, moreover, indicate a role for F6 in the long-term survival and persistence of M. tuberculosis in the human host.

Primary transcriptome analysis reveals importance of IS elements for the shaping of the transcriptional landscape of Bordetella pertussis.

Bordetella pertussis is the causative agent of whooping cough, a respiratory disease still considered as a major public health threat and for which recent re-emergence has been observed. Constant reshuffling of Bordetella pertussis genome organization was observed during evolution. These rearrangements are essentially mediated by Insertion Sequences (IS), a mobile genetic elements present in more than 230 copies in the genome, which are supposed to be one of the driving forces enabling the pathogen to escape from vaccine-induced immunity. Here we use high-throughput sequencing approaches (RNA-seq and differential RNA-seq), to decipher Bordetella pertussis transcriptome characteristics and to evaluate the impact of IS elements on transcriptome architecture. Transcriptional organization was determined by identification of transcription start sites and revealed also a large variety of non-coding RNAs including sRNAs, leaderless mRNAs or long 3' and 5'UTR including seven riboswitches. Unusual topological organizations, such as overlapping 5'- or 3'-extremities between oppositely orientated mRNA were also unveiled. The pivotal role of IS elements in the transcriptome architecture and their effect on the transcription of neighboring genes was examined. This effect is mediated by the introduction of IS harbored promoters or by emergence of hybrid promoters. This study revealed that in addition to their impact on genome rearrangements, most of the IS also impact on the expression of their flanking genes. Furthermore, the transcripts produced by IS are strain-specific due to the strain to strain variation in IS copy number and genomic context.