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.

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Accurately controlled termination of transcription is critical for gene expression and adaptation to changing environments. The aim of this study was to define transcription termination sites in Mycobacterium tuberculosis at nucleotide resolution. To this end, we applied Term-seq to cultures of M. tuberculosis and established that by far the most abundant mechanism of transcription termination is Rho-dependent. Moreover, we find that conditional or premature termination of transcription regulates a large proportion of genes and that this is often associated with translated upstream open reading frames (uORFs). Finally, our results show that overlapping ORFs are abundant in many cases in the absence of a recognisable Shine-Dalgarno sequence, indicating tightly coordinated/coupled translation. Our results provide a detailed image and comprehensive catalogue of novel M. tuberculosis cis-regulatory elements, where Rho-dependent conditional termination of transcription and translational coupling together play major roles in gene expression control.

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.

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 post-transcriptional 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 show it to be dependent on SigF for expression and significantly induced during in vitro starvation and in a mouse model of infection. However, we found no evidence of attenuation of a ΔF6 strain within the first 20 weeks of infection. A further exploration of F6 using in vitro models of infection suggests a role for F6 as a highly specific regulator of the heat shock repressor, HrcA. Our results point towards a role for F6 during periods of low metabolic activity similar to cold shock and associated with nutrient starvation such as that found in human granulomas in later stages of infection.

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 ofmetE, encoding a B12-independent methionine synthase, while the other is located upstream ofppe2, 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 bymetEandppe2riboswitches 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 theppe2switch represents a new Class IIc of B12-sensing riboswitches. Moreover, we have identified small translated open reading frames (uORFs) upstream of bothmetEandppe2, which modulate the expression of the respective downstream genes in opposite directions. Translation of themetEriboswitch uORF suppresses MetE expression, while translation of the uORF in theppe2switch 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 inM. tuberculosis, with potential implications for host-pathogen interactions.