Biology subjects

D'Halluin, A.

Publications and source records attributed to D'Halluin, A..

5 recordsLinked to original sources

bioRxiv2025

A Mycobacterium tuberculosis Mbox controls a conserved, small upstream ORF via a translational expression platform and rho-dependent termination of transcription

Magnesium is vital for bacterial survival, and its homeostasis is tightly regulated. Intracellular pathogens like Mycobacterium tuberculosis (Mtb) often face host-mediated magnesium limitation, which can be counteracted by upregulating the expression of Mg2+ transporters. This upregulation may be via Mg2+-sensing regulatory RNA such as the Bacillus subtilis ykoK Mbox riboswitch, which acts as a transcriptional "OFF-switch" under high Mg2+ conditions. Mtb encodes two Mbox elements with strong similarity to the ykoK Mbox. In the current study, we characterize the Mbox encoded upstream of the Mtb pe20 operon, which is required for growth in low Mg2+ combined with low pH. We show that this switch operates via a translational expression platform and Rho-dependent transcription termination, which is the first such case reported for an Mbox. Moreover, we show that the switch directly controls a small ORF (uORF2) encoded upstream of pe20. We have annotated this highly expressed and highly conserved uORF as rv1805A, but its role remains unclear. Interestingly, a homologous gene exists outside the Mbox-regulated context, suggesting functional importance beyond magnesium stress. Overall, this study uncovers a dual mechanism of riboswitch regulation in Mtb, combining translational control with Rho-mediated transcription termination. These findings expand our understanding of RNA-based gene regulation in mycobacteria, with implications for pathogenesis and stress adaptation.

molecular biology↗

bioRxiv2025

The endoribonuclease Rae1 from Bacillus subtilis cleaves mRNA upstream of stalled ribosomes

The ribosome-associated endoribonuclease 1 (Rae1) cleaves mRNAs in a translation-dependent manner. Here, we identify a new Rae1 target, the fliY mRNA, which is cleaved by Rae1 in the absence of the elongation factor P (EF-P). The Rae1 site was mapped 12 nucleotides upstream of the second proline codon of an SPP stalling motif in fliY. Remarkably, Rae1 cleavages also occur 12 nucleotides upstream of the stop codon within two validated Rae1 mRNA targets, bmrX and spyA (S1025). Shifting the stop codon relative to the Rae1 cutting site abolished Rae1 sensitivity of bmrX and spyA mRNAs. We show that ribosome pausing occurs at the spyA stop codon, confirming its crucial role, and positioning the Rae1 cleavage at the tail end of the stalled ribosome, rather than in the A-site as previously proposed. These findings reveal a compelling novel mechanism by which Rae1 mediates mRNA cleavage in coordination with immobile ribosomes.

molecular biology↗

bioRxiv2024

The RpfB switch is a novel B12-sensing riboswitch regulating (non-replicating) persistence in Mycobacterium tuberculosis.

Riboswitches are metabolite-sensing RNA elements that control a wide range of genes in bacteria. Most riboswitches identified to date are broadly conserved and control genes that are directly involved in the transport or biosynthesis of their cognate ligands. However, a minority of switches are restricted to a few species and in addition may bind less obvious ligands. One such switch controls the expression of the Mycobacterium tuberculosis rpfB operon, which is critical for resuscitation of dormant bacteria, ribosome maturation and reactivation of latent tuberculosis infection. The switch is restricted to pathogenic mycobacteria and until now, its ligand was unknown. However, in the current study, we identify the ligand as cobalamin or vitamin B12. Using in-line probing, we show that vitamin B12 binds directly to the riboswitch RNA, and we predict a structure based on the cleavage pattern. Moreover, we show that B12 suppresses the expression of an rpfB-lacZ reporter fusion and crucially, that B12 suppresses resuscitation of M. tuberculosis from a state of non-replicating persistence. These findings demonstrate a pivotal role of crosstalk between a host-derived metabolite and a pathogen riboswitch in controlling M. tuberculosis persistence with potential for improved interventions.

molecular biology↗

bioRxiv2023

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.

molecular biology↗

bioRxiv2022

Term-seq reveals an abundance of conditional, Rho-dependent termination in Mycobacterium tuberculosis.

Little is known about the decisions behind transcription elongation versus termination in the human pathogen Mycobacterium tuberculosis. By applying Term-seq to M. tuberculosis 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 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. tuberculosis 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. tuberculosis adaptation to the host environment offering novel potential points of intervention.

microbiology↗