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

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Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results A Mycobacterium tuberculosis Mbox controls a conserved, small upstream ORF via a translational expression platform and rho-dependent termination of transcription Alexandre D’Halluin , Terry Kipkorir , Catherine Hubert , View ORCID Profile Kristine Arnvig doi: https://doi.org/10.1101/2025.08.04.667915 Alexandre D’Halluin 1 Structural and Molecular Biology, University College London , London WC1E 6BT U.K. 2 EGM CNRS, Université Paris-Cité, Institut de Biologie Physico-Chimique , 13 rue Pierre et Marie Curie, 75005 Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Terry Kipkorir 1 Structural and Molecular Biology, University College London , London WC1E 6BT U.K. 3 London School of Hygiene and Tropical Diseases, Department of Infection Biology , TB Centre, London WC1E 7HT U.K. Find this author on Google Scholar Find this author on PubMed Search for this author on this site Catherine Hubert 1 Structural and Molecular Biology, University College London , London WC1E 6BT U.K. Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kristine Arnvig 1 Structural and Molecular Biology, University College London , London WC1E 6BT U.K. Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Kristine Arnvig For correspondence: k.arnvig{at}ucl.ac.uk Abstract Full Text Info/History Metrics Supplementary material Preview PDF ABSTRACT 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 Mg 2+ transporters. This upregulation may be via Mg 2+ -sensing regulatory RNA such as the Bacillus subtilis ykoK Mbox riboswitch, which acts as a transcriptional “OFF-switch” under high Mg 2+ 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 Mg 2+ 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. INTRODUCTION Magnesium is required for a wide range of cellular functions in all domains of life and the most abundant divalent cation in living cells ( Smith et al ., 1998 ). In bacteria, these functions include cell wall integrity, biofilm formation, macromolecular metabolism and -function, making magnesium homeostasis essential ( Thomas & Rice, 2014 ; Subramani et al ., 2016 ; Yamagami et al ., 2021 ; Chatterjee et al ., 2024 ). This represents an extra challenge for intracellular pathogens as host immune responses include mechanisms for restricting access to magnesium in certain cellular compartments such as the phagosome (reviewed in Forbes & Gros 2001 ; Pokorzynski & Groisman 2023 ). To counteract these defence mechanisms pathogens express an array of transporters to ensure adequate Mg 2+ uptake. At least four types of magnesium channels and transporters regulate and maintain essential Mg 2+ levels in prokaryotes: CorA, CorB/C, MgtA/B and MgtE ( Franken et al ., 2022 ). Mycobacterium tuberculosis (Mtb) encodes CorA (Rv1239), MgtE (Rv0362) transporters MgtA/B however, Mtb does not encode homologues of MgtA/B, making the function of MgtC elusive ( Alix & Blanc-Potard, 2007 ). Riboregulated, i.e. RNA-based, stress-responses are widespread in bacteria, with small RNAs and riboswitches being the most prominent elements. Riboswitches are located in the 5’ leader regions of mRNAs regulating gene expression in cis ; they are composed of a highly structured ligand binding aptamer domain and an expression platform. The latter exerts gene expression control by either modulating premature termination of RNA Polymerase (RNAP) and/or restricting access of the ribosome to the Ribosome Binding Site (RBS) of the mRNA ( Salvail & Breaker, 2023 ). Nonpermissive control mechanisms may involve the formation of intrinsic terminators, unmasking of Rho-binding sites or occlusion of Shine-Dalgarno (SD) sequence of the downstream Open Reading Frame (ORF). The latter may in addition be associated with Rho-dependent termination of transcription within the ORF ( Salvail & Breaker, 2023 ). Binding of the specific ligand can either allow (“ON-switch”) or inhibit (“OFF-switch”) expression of the downstream gene ( Breaker, 2018 ; Kavita & Breaker, 2023 ; Schwenk & Arnvig, 2018 ). The genes regulated by riboswitches are often, but not always, involved in the metabolism or transport of the cognate ligand ( Kavita & Breaker, 2023 ; Roth & Breaker, 2009 ; Sherlock & Breaker, 2020 ). Riboswitch ligands range from sugars, amino acids, nucleotides and cofactors to metal ions including Mg 2+ ( Breaker 2022 ; Barrick et al ., 2004b ; Dann et al ., 2007 ; Mccown et al ., 2017 ). A magnesium-sensing riboswitch, referred to as Mbox, was first discovered in the Bacillus subtili s ykoK gene encoding a MgtE-type magnesium transporter ( Barrick et al ., 2004a ; Ramesh & Winkler, 2010 ; Townsend et al., 1995 ). The Mbox is a transcriptional ‘OFF-switch’; magnesium binding to the aptamer leads to conformational changes of the RNA and the formation of an intrinsic terminator preventing ykoK expression. At low Mg 2+ concentrations, the absence of the terminator is permissive to ykoK expression which facilitates increased Mg 2+ uptake ( Ramesh & Winkler, 2010 ). Successful infection with Mtb requires its sensing of, and adaptation to, multiple micro-environments including different types of macrophages and their subcellular compartments such as phagosomes ( Chandra et al ., 2022 ; Samuels et al ., 2022 ; Sholeye et al ., 2022 ). Mtb has evolved mechanisms to either escape this organelle or to endure the hostile environment within by a range of adaptive responses (Ehrt et al ., 2018; Ernst, 2012 ; Huang, 2019 ). Riboswitches are likely to play a role in this adaptation by directly sensing host environments via specific metabolites. Several Mtb riboswitches have been predicted (Rfam RF00380) and their expression validated by RNA-seq, Term-seq, inline probing and functional assays ( Nawrocki et al ., 2015 ; Arnvig et al ., 2011 ; D’Halluin et al ., 2023 ; Kipkorir et al ., 2024a + b ). These include two predicted Mbox aptamers upstream of Rv1535 and Rv1806 ( pe20 locus ), respectively. Although the two Mtb aptamers show a high level of structural similarity to the Bacillus subtilis ykoK aptamer, published results suggests that these interact differently with divalent cations including Mg 2+ ( Bahoua et al ., 2021 ). The pe20 locus (encoding PE20, PPE31, PPE32, PPE33 Rv1810 and MgtC) is associated with magnesium homeostasis and acid stress, and PE20-PPE31 have been shown to be necessary for maintaining growth in a combination of low Mg 2+ and low pH, conditions that mimic the phagosomal environment ( Walters et al ., 2006 ; Wang et al ., 2020 ). The function of Rv1535 remains unknown. We recently mapped premature termination of transcription in Mtb at genome-scale and identified hundreds of RNA leaders with an abundance of potential new riboswitches and translated small upstream ORFs (uORFs) ( D’Halluin et al ., 2023 ). We validated predicted riboswitches and demonstrated that both Mtb Mbox leaders were associated with premature, Rho-dependent termination of transcription upstream of the annotated ORFs ( D’Halluin et al ., 2023 ). Here, we show that pe-ppe associated Mboxes are widely conserved across Mycobacteria and are co-transcribed with mgtC in Mtb. The Mtb Mbox upstream of pe20 is unusual as it combines a translational expression platform with a Rho-dependent transcription terminator. This is to our knowledge the first translational Mbox to be described. Using translational reporter fusion constructs we show that a conserved uORF located between the Mbox and pe20 is highly expressed. This peptide is highly conserved in the context of Mboxes across the Mycobacterium genus. While its function remains opaque, a paralogue of this ORF is expressed from an additional, Mbox-independent locus in Mtb, supporting the biological and regulatory importance of this peptide. RESULTS Conservation and genomic context of M. tuberculosis Mboxes Two Mbox aptamers have been identified within the Mtb H37Rv genome (Rfam RF00380). We used these sequences to predict their structures and compare these to the Mbox consensus structure from Rfam. The results, shown in Figure 1A , indicate a high degree of similarity between the two Mtb aptamers and the B. subtilis ykoK element, suggesting these are functional Mg 2+ -sensing elements as reported in the case of rv1535 by ( Bahoua et al ., 2021 ). Download figure Open in new tab Figure 1. Conservation of Mbox elements. A) Mbox aptamer structures from Bacillus subtilis ykoK and the two M. tuberculosis aptamers from rv1535 and pe20 ( rv1806 ). Structures were predicted using RNAstructure Web Serveur (Reuteur & Mathews, 2010) and drawn by extracting the bracket-dot plot to RNAcanvas (Johnson & Simons, 2023). B) Distribution of Mboxes and their associated genes in Mycobacteria . These can be split into the four types indicated, based on the aptamer and their downstream sequences. Notably, the two M. tuberculosis elements fall into different groups. Next, we investigated the conservation of the element and its context across the Mycobacterium genus. Both elements have been shown to be associated with multiple uORFs, and at least one of these is translated (u2, D’Halluin et al ., 2023 ). Based on a phylogenetic analysis of the two Mtb elements and Mboxes from other species, we identified four classes of mycobacterial Mboxes, represented by pe20 -type, manganese-type (Mn 2+ -type), rv1535 -type and mgtE -type elements, respectively; these classes are further supported by a conserved gene synteny ( Figure 1B ). The mgtE -type is the only Mbox found in the non-pathogenic M. smegmatis , and its genomic neighbourhood shows that the genes immediately downstream encode predicted metal transporters (MT) and/or associated proteins (e.g. MgtE), or proteins of unknown function. The other three branches are seen across fast-and slow-growing pathogenic Mycobacteria . The first branch, the pe20 -type is almost exclusively found upstream of multiple pe - ppe genes, which in Mtb, M. ulcerans, M. marinum and M. kansasii are followed by genes of unknown function (GUF) and MT (as MgtC was originally annotated as a magnesium transporter). The second Mn 2+ -type branch includes a cluster of manganese transporter-type downstream of the riboswitch, a constellation that is not seen in Mtb. The third rv1535 -type branch is found upstream of GUF like rv1535 but followed by a cluster of T-box/ ileS elements or various transferases. These results suggest the regulation of metal transporters by both Mboxes is well-conserved across Mycobacteria , while the the pe-ppe clusters and mosaic appearance suggest insertion events may have taken place in pathogenic / slow growing species. Rho-dependent premature termination of transcription within Mbox leaders TSS mapping and RNA-seq suggest that the rv1535 mRNA is monocistronic, while the pe20 mRNA is polycistronic spanning pe20 to mgtC ( Arnvig et al ., 2011 , Cortes et al ., 2013 , D’Halluin et al ., 2023 ) (Supplementary figure 1). Importantly, the entire polycistronic pe20 operon is upregulated during growth in low magnesium controlled by the Mbox ( Walters et al ., 2006 ). We recently mapped premature transcription termination (TTS) in Mtb genome-wide and identified two dominant TTS associated with the Mbox leaders ( D’Halluin et al ., 2023 ). TTS1062 is located ∼210 nucleotides downstream of the rv1535 TSS and 40 nucleotides downstream of the aptamer. TTS1209 is located ∼185 nucleotides downstream of the pe20 TSS and 10 nucleotides downstream of the aptamer ( Figure 2A ). Download figure Open in new tab Figure 2. Premature termination of transcription within Mtb Mbox loci . A) The two Mbox-associated genes, rv1535 and pe20 are shown with their respective leaders. TSS from ( Cortes et al ., 2013 ), Term-seq data and Transcription termination sites (TTS) from ( D’Halluin et al ., 2023 ). Distances from TSS to dominant TTS peaks and further to the start codons of have been indicated. B) Northern blot with log-phase total RNA from Mtb H37Rv and from RhoDUC ( Botella et al ., 2017 ) following depletion of Rho. Total RNA was separated on an 8% acrylamide gel, electroblotted and probed for leader sequences distinct for the two genes, approximately 180 nucleotides downstream of the TSS. The 5S RNA was probed as a loading control. In both loci , multiple smaller peaks are flanking the TTS, suggesting a degree of flexibility in the TTS. Both TTS were located a significant distance (>200 nucleotides) upstream of their annotated ORFs, revealing the premature termination of transcription within the two leader regions, and neither were associated with canonical intrinsic terminator structures. In our Mtb TTS mapping we predicted and validated Rho-dependent termination using RhoTermPredict ( Di Salvo et al ., 2019 ) and depletion of Rho using the Rho-DUC strain (Botella et al ., 2015; D’Halluin et al ., 2023 ). Two Rho-binding ( rut ) sites were predicted in each Mbox leader; one in each aptamer (T5468 and T6425) and one between aptamers and annotated ORFs (T5469 and T6426), while the mapped TTS1209 and TTS1062 are located between these ( Table 1 ; Figure 2A ). View this table: View inline View popup Download powerpoint Table 1. Location of rut sites within the M-box leaders. The calculated readthrough (RT) scores for the mapped TTS after Anhydrous Tetracyclin (ATc) induced depletion of Rho validated that transcription termination was in fact due to Rho ( D’Halluin et al ., 2023 ). To further confirm Rho-dependent, premature termination of transcription, we performed Northern Blotting on RNA from H37Rv and from Rho-depleted cultures probing for both Mboxes. The homology between the Mbox aptamers from rv1535 and pe20 made it impossible to design a 5’ probe that could distinguish between the two transcripts. To ensure that the signals were specific for either pe20 or rv1535 , we used a probe that was located 180 nucleotides into the transcripts beyond the homologous regions (supplementary figure 2) and as a result, transcripts shorter than this could not be detected. Several strong signals between 200 and 300 nucleotides roughly corresponding to the TTS mapping suggesting multiple points of premature termination of transcription within both leaders ( Figure 2B ). In H37Rv and in Rho-DUC time 0, we observed limited readthrough beyond 300 nucleotides for pe20 , while rv1535 displayed multiple larger signals primarily around 400 nucleotides consistent with the TTS pattern. Depletion of Rho led to an increase in larger transcripts for pe20 suggesting increased readthrough i.e. reduced termination. In contrast, rv1535 TTS pattern changed only marginally over the rho depletion time course, suggesting that Rho plays a greater role for pe20 regulation as compared to rv1535 . The regions downstream of the Mbox aptamers harbour multiple uORFs Ribosome profiling demonstrates that the regions between the Mbox aptamers and the two annotated open reading frames (ORF)s ( rv1535 and pe20 , respectively) are bound by ribosomes in agreement with on-going translation upstream of the annotated genes (Sawyer et al., 2021; Smith et al., 2022 ; D’Halluin et al., 2023 ). Sequence alignment of rv1535 and pe20 leaders with other pe20 -type leaders indicated several regions of conservation including near-identical Shine-Dalgarno (SD) sequences located at the end of the aptamer (SD1) and a second, highly conserved SD (SD2) further downstream. The ORF downstream of SD1 (upstream ORF1/uORF1) shows poor conservation, while the uORF downstream of SD2 (uORF2) is highly conserved (Supplementary figure 2). Moreover, we have previously shown that uORF2 from both loci is expressed ( D’Halluin et al ., 2023 ). To characterize the relationship between the pe20 Mbox and the two uORFs, we first investigated expression using translational lacZ -fusions. All constructs included the 5’ leader from the TSS and were gradually extended downstream to the end of uORF1 (Mbox- uORF1-lacZ ); the end of uORF2 (Mbox- uORF2-lacZ ), or the start codon of pe20 ORF (Mbox- pe20::lacZ ), respectively. All were fused in-frame to lacZ , expressed from a heterologous, constitutive promoter and integrated into the M. smegmatis genome in single copy ( Figure 3 ). Next, we performed β-galactosidase (β -gal) assays, which showed that Mbox- uORF2-lacZ expression was >10 fold higher than Mbox- pe20::lacZ (∼650 Miller Units compared to 60 Miller Units), while Mbox- uORF1-lacZ was only slightly higher than the background ( Figure 3B ). To validate the start codons of the two uORFs, we mutated each to non-start codons (GTG to GTC and ATG to ACG for uORF1 and uORF2 , respectively. This reduced expression significantly in both constructs, in support of the suggested translation start sites, although Mbox- uORF2-lacZ expression was still higher than Mbox- pe20::lacZ expression (Supplementary figure 3). Download figure Open in new tab Figure 3. Expression of pe20 uORFs. To ascertain expression of uORF1 and uORF2 from the pe20 operon, we made translational lacZ -fusions and measured β -galactosidase ( β gal) activity of the different constructs. Experiments were done in triplicates and differences of expression tested with a t-test (p-val<0.01). A) Genomic context of pe20 and the uORFs associated (green) including SD1 (yellow box) and SD2 (blue box); B) Schematic showing each reporter constructs (left) and their expression in Miller units (right). C) uORF1 sequence with amino acids and their codons. Start codon is shown in green and rare codons (<5/1000 frequency) are shown in red. D) Reporter constructs with full-length and truncated uORF1 sequence (left) followed by spotting assay of 10 pl of a culture at OD 600nm of 0.6, 0.06 and 0.006, indicating expression on Xgal plates and their respective β gal activities are shown on the right. As the translation initiation region (TIR) for uORF1 and uORF2 (i.e. SD1 and SD2 and their distances to the start codons) were almost identical, and we had not observed any premature TTS in the region, we reasoned that a polypeptide segment of the uORF1 led to lower expression. uORF1 contains several rare (≤5/1000) codons, i.e. TGC, CCT, TGC, TGT, TGT, AGG, AGG ( Figure 3C ). To explore this possibility, we deleted the majority of uORF1 from the Mbox- uORF1-lacZ construct except the first two codons (Mbox-GTG-GTC uORF1 -lacZ ) and measured lacZ expression. The results, shown in Figure 3D indicate that expression of this truncated uORF1 was 2.5-fold higher than that of the full-length uORF1, suggesting that the uORF1 sequence did indeed suppress expression. The pe20 Mbox operates via a translational expression platform In conjunction, the Rho-dependent premature termination of transcription, the highly conserved SDs at the end of the aptamer, located upstream of a well-expressed conserved uORF made us speculate that the pe20 Mbox operates via a translational expression platform. A functional translational expression platform requires the potential for the SD to be masked, e.g. by a pyrimidine-rich region (an αSD) that in turn can be sequestered by an ααSD under different conditions. We identified such a region approximately halfway between SD1 and SD2. This αSD and its flanking regions have the potential to pair with the entire translation initiation region (TIR) of uORF2 (shown in blue in Figure 4 ) or alternatively, with the aptamer-associated SD1 and its flanks (yellow in Figure 4 ). To explore this hypothesis further, we measured uORF2 expression after introducing mutations that could interfere with the proposed interactions. One was the abolishing the uORF1 start codon, the rationale being that this would partially unmask SD1 thereby favouring the SD1-αSD interaction, leading to an increase in uORF2- lacZ expression Download figure Open in new tab Figure 4. Model for a translational expression platform. The figure shows how the translation initiation region (TIR, blue) can be sequestered by base-pairing with the αTIR (orange), which in turn can base-pair with the ααTIR (yellow), depending on the conformation of the aptamer. Structure of the aptamer is shown on the left with part of the ααTIR shown in yellow. Similarly, deleting the αSD should also lead to higher expression of uORF2, as SD2 would no longer be sequestered. The results, shown in Figure 5A indicate a moderate (∼1.3-fold), but significant increase in expression, when the start codon of uORF1 was changed (Mbox- uORF2 G184C -lacZ ) and a larger (∼2-fold) increase in expression, when the-αSD was deleted (Mbox-ΔαSD -uORF2-lacZ ). Combining the two mutations did not result in an additive effect, suggesting they involved the same mechanism ( Figure 5A ). These results support a model in which SD1 (ααSD), αSD and SD2 interact to control the expression of uORF2- lacZ . Download figure Open in new tab Figure 5. Testing the model for a translational expression platform. A) Reporter constructs assessing the effect of uORF1 changes on uORF2 expression; changing the start codon of uORF1 to a no-start (G184C), deleting the proposed αSD, which is part of uORF1 or a combination of the two. B) Effect of gradual extension of region upstream of uORF2. Expression decreases, when αSD is included and increases again, when SD1 (ααSD) is included. C) Structures indicating how the reporter constructs relate to the model proposed in Figure 4 . Experiments were done in triplicates and differences of expression tested with a t-test (p-val<0.01). To further validate this model, we assessed the contribution of each element by gradually extending the region between uORF2 and the Mbox in uORF2- lacZ fusions ( Figure 5B ). The SD2-uORF2 construct displayed β-gal expression levels of ∼500 Miller Units, and the addition of the αSD motif reduced the β -gal expression by ∼35%. However, a further extension including the ααSD motif led to a substantial increase in uORF2 expression. This is likely due to the unmasking of SD2 and corroborates our model of a translational expression platform controlling uORF2 expression. Our results support a model in which uORF2 is controlled by a translational Mbox riboswitch combined with Rho-dependent termination of transcription. Based on sequence homology, we propose that the rv1535 Mbox likewise operates via a translational expression platform. To the best of our knowledge, these are the first examples of an Mbox translational expression platform and Rho-dependent termination of transcription. An Mbox-independent homologue of uORF2 encoded in a separate Mtb locus Considering the high conservation between uORF2 in the rv1535 and pe20 loci , we carried out deeper sequence searches and identified a third homologue of the uORF2 region including its SD downstream of the gca - gmhA - gmhB - hddA operon. This locus is associated with horizontal gene transfer ( Becq et al ., 2007 ) and the uORF2 homologue annotated as Rv0115A ( Figure 6A ). Download figure Open in new tab Figure 6. The RvO115A locus. BLAST identified RvO115A to be a homologue of Rv1805A. A) RvO115A (green) is encoded downstream of the gca operon (golden) but transcribed from its own promoter. B) Alignment of the promoter regions of gca and rvO115A show high degree of similarity, suggesting a duplication event. The blue arrow indicates hddA coding sequence upstream of rvO115A . The promoter elements, −35, extended −10 (−10e) and −10 are highlighted in grey. We identified two TSS and associated promoter motifs within this locus . The first drives the transcription of the gca-hddA operon, which terminates downstream of hddA ( D’Halluin et al ., 2023 ). The second drives the transcription of rv0115A , and potentially also a second ORF, rv0115B . The gca and rv0115A promoters have similar unusual motifs in the form of an AANCAT −10 hexamer, an extended −10 motif (TGN), a perfect −35 hexamer and in the case of cga , a Cytidine TSS ( Figure 6B ). A further alignment of the promoter regions from −120 to a few basepairs downstream of the mapped TSS, had a remarkable similarity more than 100 basepairs upstream of the TSS suggestive of a gene duplication event ( Figure 6B ). Alignment of uORF2 homologues including rv0115A across mycobacterial species reveals a well-conserved N-terminal domain, including a universally conserved Proline residue ( Figure 7 ). This peptide is specific for Mycobacteria, which indicates that uORF2 peptides and their homologues have functions uniquely associated with this genus. Based on this finding, we suggest renaming uORF2 from the pe20 operon rv1805A . Download figure Open in new tab Figure 7. Conservation of uORF2 within Mycobacteria. Alignment of Mbox associated uORF2 extracted from Figure 1B and Mtb Rv0115A peptides showed high conservation of several residues mainly at the N-terminal sequence, including 100% conservation of a proline at position 7 in most peptides. Consensus sequence and amino acid conservation were assessed using Chimera ( Meng et al ., 2006 ). No evident role for Rv1805A in biofilm formation during magnesium stress Realising the ubiquitous presence of Rv1805A homologues, we sought to find a role for this peptide. PE20 and PPE31 are necessary for Mtb growth in conditions of low Mg 2+ combined with low pH ( Wang et al ., 2020 ). To probe a potential role of uORF2 in this process, we exploited the fact that magnesium is required for Mycobacterium biofilm formation ( Chatterjee et al ., 2024 ) and leveraged the trick that Mycobacterium smegmatis , a closely related species, has no homolog of pe20 locus. In agreement with literature, the growth and biofilm formation of M. smegmatis were compromised in low Mg 2+ , and that this phenotype was exacerbated at acidic pH values ( Figure 8 ). We tested whether the expression of pe20-ppe31 or rv1805A-pe20-ppe31 might rescue this phenotype by transforming M. smegmatis with plasmids expressing the cognate genes. The results in figure 8 indicate no visible difference between strains expressing pe20-ppe31 with or without rv1805A or rv0115A ; further investigations are required to identify a role of this peptide and its homologues in mycobacterial biology. Download figure Open in new tab Figure 8. Biofilm formation in M. smegmatis during Mg2+-depletion and acid stress. Cultures of Mycobacterium smegmatis were grown to mid-log phase, washed in Mg2+-free medium, resuspended in 1 mL of indicated medium at OD 0.01. Plates were sealed in plastic bags and left for static incubation at 37°C_ _ for a week. Plates shown are representative of three independent experiments. DISCUSSION In the current study we have discovered a novel complex magnesium-controlled riboregulatory system which controls pe20 gene expression in Mtb. Our results show that premature termination occurring in the 5’ leader of pe20 (and rv1535 ) relies on Rho-dependent termination of transcription ( D’Halluin et al ., 2023 ). Moreover, the pe20 Mbox contributes a translational expression platform, where the translation initiation region including the SD of the first gene in the operon can be sequestered by an anti Shine-Dalgarno motif (α SD). This is also, to the best of our knowledge, the first example of a translationally controlled Mbox. This type of control is consistent with the scarcity of intrinsic terminators in Mtb, and it echoes the finding that a mycobacterial T-box is the only known T-box with a translational expression platform ( Sherwood et al ., 2018 ). Finally, we identified a highly conserved uORF ( rv1805A ), which is the primary regulated ORF within the pe20 operon and, based on homology, likely also in the rv1535 operon. The pe20 operon is suppressed in the presence of high Mg 2+ like the Mbox controlled ykoK gene in B. subtilis ( Walters et al ., 2006 ; Ramesh & Winkler, 2010 ). pe20 and ppe31 are critical for magnesium uptake in low-pH/low-magnesium conditions suggesting that the gene products form (part of) a magnesium transporter ( Feng e t al ., 2021 ; Wang et al ., 2020 ). We propose that the Mbox– rv1805A module acts as the key regulatory gate, enabling expression of the magnesium-responsive PE/PPE transporter complex only under specific environmental conditions, such as low Mg 2+ and acidic pH. The structure of the pe20 operon, including the presence of mgtC raises questions about its ancestry. Given what is known about pe-ppe gene expansion ( Fishbein et al ., 2015 ) and what we have observed in other riboswitch-controlled pe/ppe loci (i . e. the Cbl-ppe2-cobQ locus , and the PE-containing uORF recently identified downstream of the Mtb glycine riboswitch ( D’Halluin et al ., 2023 ; Kipkorir et al ., 2024), it is tempting to speculate that an early pe (− ppe ) element invaded the current pe20 locus and subsequently expanded whereby Rv1805A became the first gene in this operon. A recent study suggests that the rv1535 Mbox, and by extension likely also the pe20 Mbox associates with other divalent cations in addition to Mg 2+ ( Bahoua et al ., 2021 ). Regardless of the identity of the cognate ligand, our results suggest an ability to alternate between two structures: a non-permissive (ligand-bound) structure that sequesters SD1, allowing αSD/ αTIR to pair with SD2/TIR thereby preventing translation of uORF2/Rv1805A. This could in turn lead Rho-dependent termination of transcription, which will affect the entire operon ( Hao et al ., 2021 ; Molodtsov et al ., 2023 ). We note, however, that according to Term-seq results, the primary TTS is located upstream of rv1805A , suggesting that Rho-dependent termination does not depend on translation of this uORF. An alternative explanation of our results could therefore be that the pyrimidine-rich region that we have annotated as α SD, might act as a Rho-binding ( rut ) site that would be masked by translation of uORF1. Deleting this region increased expression 2.5-fold, likely due to reduced termination of transcription or by unmasking of SD2. The marginal increase in the expression of uORF2 in the context of an untranslated uORF1 (Mbox- rv1805A G184C -lacZ , Figure 5 ) and the conservation of the ααSD-αSD interaction, suggests a functional interaction. The two models are not mutually exclusive, and further experiments will eludidate the structural and mechanistic basis underlying the riboregulation.. What is the function of Rv1805A? Given its high conservation and position upstream of pe20 , we hypothesize that Rv1805A acts as a regulatory peptide modulating the activity or assembly of the PE20–PPE31 complex. Alternatively, it may serve as a structural component of a magnesium-responsive transporter. Conservation between Rv1805A, the Rv1535 uORF2 (Rv1535A) and Rv0115A suggests important roles for these peptides and future work will focus on identifying interaction partners of Rv1805A and assessing its impact on magnesium uptake and stress responses. In conclusion, our findings reveal a previously unrecognized mode of riboswitch control in Mtb, where a translational Mbox integrates with Rho-dependent termination to regulate a conserved uORF. This multilayered modus operandi underscores the sophistication of RNA-based regulation in Mtb stress adaptation. MATERIAL AND METHODS Strains and cultures Strains used in this study are listed in Supplementary Table 1. M. tuberculosis H37Rv and M. smegmatis MC 2 155 were cultured on solid media Middlebrook agar 7H11 supplemented with 10% OADC (Sigma), 0.5% Glycerol and 50 µg/ml hygromycin if appropriate. Liquid cultures were done in Middlebrook 7H9 supplemented with 10% ADC (Sigma), 0.5% Glycerol, 0.05% Tween 80 and 50 µg/ml hygromycin where appropriate. Cultures were harvested at an OD 600nm ∼0.6 for mid-log phase. Mtb RhoDUC strain, a gift obtained from Pr. Dirk Schnappinger, was grown as previously described with 50 µg/ml hygromycin, 20 µg/ml Kanamycin and 50 µg/ml zeocin ( Botella et al ., 2017 ; D’Halluin et al ., 2023 ). When the cultures reached an OD 600nm ∼0.6, depletion of Rho was induced using 500 ng/ml of anhydrotetracycline. Cells were harvested after 0, 1.5, 3 and 4.5 hours. Escherichia coli DH5α was used for cloning the lacZ fusion reporters and were cultured on solid LB 1.5% agar supplemented with 50 µM of 5-bromo-4-chloro-3-indolyl-β;-D-galactopyranoside (X-gal) or in liquid LB supplemented with 250 µg/ml Hygromycin. Plasmids constructions and primers Plasmids and primers used in this study are listed in Supplementary Table 1 and 2. pIRATE plasmids, describe in D’Halluin et al ., 2023 , were used for lacZ translational fusion reporters and for Beta-galactosidase assay. Reporters were constructed using Gibson assemblies with oligos (Sigma) or geneBlocks (IDT) listed in Table 3 between HindIII and NcoI sites. Start codons point mutations and deletion of the αSD sequence were generated using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs). Plasmids were cloned in E. coli DH5α, extracted and sequenced by Sanger sequencing. Plasmids were transformed into M. smegmatis by electroporation and selected on Middlebrook 7H11 agar plates containing 50 µg/ml Hygromycin. RNA extraction and Northern Blotting M. tuberculosis H37Rv were stopped using 37.5% of cold ice and centrifuge 10min at 5000 rpm 4°C. Total RNA were extracted as previously described using the FastRNA Pro Blue kit (MP Biomedicals) according to the manufacturer’s protocol ( Arnvig et al ., 2011 ; D’Halluin et al ., 2023 ). RNA concentration and purity was assessed using the Nanodrop 2000 (ThermoFisher), residual genomic DNA removed using Turbo DNase (ThermoFisher) and RNA integrity assessed with 2100 Bioanalyzer (Agilent). 10 µg of total RNA were separated on a denaturing 8% acrylamide:bis-acrylamide (19:1) gel and transfer to a nylon membrane. An RNA probe was synthetized using the mirVana miRNA probe synthesis kit (Ambion) to reveal the pe20 and rv1535 Mbox transcripts and labelled with 3µM final concentration of 32 P α-UTP (3000Ci/mmol; Hartmann AnalyticGmbH). Northern blots were revealed using radiosensitive screens and visualized on a Typhoon FLA 9500 phosphoimager (GEHealthcare). Beta-galactosidase activity M. smegmatis carrying the lacZ reporter fusions were cultured at OD 600nm ∼0.6 and centrifuge 10min 5000 rpm. Pellets were washed four times in Z-buffer composed of 60mM Na 2 HPO 4 , 40mM NaH 2 PO 4 , 10mM KCl, 1mM MgSO 4 and lysed using beads with the FastPrep bio-pulveriser (MP Biomedicals). The supernatant was kept after centrifugation and the protein level assessed using a Bradford yield with the BCA kit (ThermoFisher) following the manufacturer’s recommendations. Beta-galactosidase were done using the Beta-galactosidase assay kit (ThermoFisher) following manufacturer’s protocol. Proteins were pre-incubated for 5min at 28°C before addition of ONPG. Biofilm formation M. smegmatis expressing pe20-ppe31, rv1805A-pe20-ppe31 or rv0115A+pe20-ppe31 was grown to mid-log phase, washed in Mg 2+ -free medium, resuspended in 1 mL of the indicated medium at OD 0.01 and seeded in 24-well plates. Plates were sealed in plastic bags and left for static incubation at 37°C for a week. Biofilm formation was monitored every day for a week. Folding, sequence conservation and distribution across Mycobacteria Representative genomes of several Mycobacteria were selected for sequences conservation: Mycobacterium tuberculosis H37Rv (NB_000962), Mycobacterium leprae TN (AL450380), Mycobacterium avium K10 (NZ_CP106873), Mycobacterium kansasii Kuro I (AP023343), Mycobacterium ulcerans ATCC33728 (NZ_AP017624), Mycobacterium marinum M (CP000854), Mycobacterium abscessus ATCC19977 (NC_010397), Mycobacterium haemophilum DSM 44634 (CP011883) and Mycobacterium smegmatis MC 2 155 (NZ_CP009494). The aptamer sequences of the Mboxes were extracted from RFam database (Rfam RF00380) ( Nawrocki et al ., 2015 ) and extended to the next annotated ORF. DNA and peptidic sequences were aligned using ClustalW ( Thompson et al ., 1994 ), and alignment strengthen using T-coffee ( Notredame et al ., 2000 ). The conservation of uORF2 within Mycobacteria was determined using Blast ( Altschul et al ., 1990 ) and amino acid sequences aligned using ClustalW ( Thompson et al ., 1994 ) and Chimera ( Meng et al ., 2006 ). The phylogenetic tree was generated by Clustal Omega using the sequences from the aptamer sequence to the start codon of the next in frame annotated ORF (Sievers & Higgins, 2021). Aptamer secondary structures were predicted using the RNAstructure Web Serveur for RNA Secondary Structure Prediction ( Reuter & Mathews, 2010 ). The resulting Connectivity Table (CT) file was then uploaded to RNAcanvas ( Johnson & Simon, 2023 ) for visualization and structural editing FUNDING KBA was funded by The UK Medical Research Council grants MR/S009647/1 and MR/X009211/1. TK was funded by The Newton International Fellowship grants (NIF\R1\180833 & NIF\R5A\0035), the Wellcome Institutional Strategic Support Fund grant (204841/Z/16/Z), and the Wellcome Early Career Award (225605/Z/22/Z). The views expressed are those of the author(s) and not necessarily those of the funders. The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication. AUTHOR CONTRIBUTIONS AD, TK, KBA designed the study. AD, TK, CH, KBA performed experiments. AD, TK, KBA performed data analysis and wrote the manuscript. 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Share A Mycobacterium tuberculosis Mbox controls a conserved, small upstream ORF via a translational expression platform and rho-dependent termination of transcription Alexandre D’Halluin , Terry Kipkorir , Catherine Hubert , Kristine Arnvig bioRxiv 2025.08.04.667915; doi: https://doi.org/10.1101/2025.08.04.667915 Share This Article: Copy Citation Tools A Mycobacterium tuberculosis Mbox controls a conserved, small upstream ORF via a translational expression platform and rho-dependent termination of transcription Alexandre D’Halluin , Terry Kipkorir , Catherine Hubert , Kristine Arnvig bioRxiv 2025.08.04.667915; doi: https://doi.org/10.1101/2025.08.04.667915 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Molecular Biology Subject Areas All Articles Animal Behavior and Cognition (7629) Biochemistry (17660) Bioengineering (13881) Bioinformatics (41913) Biophysics (21436) Cancer Biology (18578) Cell Biology (25482) Clinical Trials (138) Developmental Biology (13372) Ecology (19889) Epidemiology (2067) Evolutionary Biology (24302) Genetics (15599) Genomics (22483) Immunology (17728) Microbiology (40365) Molecular Biology (17163) Neuroscience (88540) Paleontology (666) Pathology (2830) Pharmacology and Toxicology (4821) Physiology (7637) Plant Biology (15136) Scientific Communication and Education (2045) Synthetic Biology (4290) Systems Biology (9818) Zoology (2269)