Biology subjects

Guillier, M.

Publications and source records attributed to Guillier, M..

8 recordsLinked to original sources

bioRxiv2026

Studying the regulons of OmrA and OmrB paralogous small RNAs reveals targets involved in central carbon metabolism and lipogenesis

Small regulatory RNAs (sRNAs) are key players in bacterial adaptation to stress. They often occupy central positions in regulatory networks and control the expression of multiple targets. In a striking example of this, the enterobacterial OmrA and OmrB paralogous sRNAs are known to regulate about ten different targets, with extensive data suggesting the regulon is in fact much larger. Here we performed transcriptome and proteome analyses and identified more than fifteen new targets of Escherichia coli OmrA and OmrB. We validated several, including genes involved in central carbon metabolism and fatty acid synthesis, among which ppc, actP and fabA. Consistent with a role in carbon metabolism, overproducing OmrA or OmrB inhibited growth on glucose minimal medium. The analysis of suppressor mutants shows that this is due to a decreased carbon flux through the TCA cycle. Incorporating other datasets such as RIL-seq, we generated a multi-omics-based prediction of target candidates. Together, our results show that OmrA/B base-pair to various regions of their mRNA targets, and therefore likely act through diverse regulatory mechanisms. Hence, this work extends the OmrA and OmrB regulons, establishes an unsuspected connection with carbon usage, and shows the benefits of combining global analyses to investigate sRNA regulons.

Molecular Biology↗

bioRxiv2026

A novel peptide modulator of a two-component system revealed by the specific activation of a small RNA in Enterobacteriaceae

Small regulatory RNAs (sRNAs) are major post-transcriptional regulators in bacteria and, together with transcriptional regulators such as the two-component systems (TCSs), participate in the rapid adaptation of these microorganisms to changing environments. Several examples of paralogous sRNAs with overlapping functions have been reported, that could in theory integrate different environmental cues. Consistent with this idea, we have identified the acid-responsive RstB-RstA two-component system, important for virulence of multiple bacterial species, as a specific multicopy activator of the Escherichia coli OmrB sRNA, but not of the paralogous sRNA OmrA. Further characterization of this regulation unexpectedly revealed the asr-ydgU operon, itself a target of RstB-RstA, as a dual modulator of this TCS via two opposite effects. First, the 27 aminoacids YdgU small protein exerts a negative feedback by directly interacting with RstB and, second, Asr in contrast mediates a positive feedback on RstB-RstA activity via a not completely elucidated mechanism. These results provide a new example of retro-control of a TCS, here RstB-RstA, by one of its direct targets. They further highlight the major role of small proteins in controlling TCS activity and ydgU was thus renamed samT, for Small Acid-responsive Modulator of the RstB-RstA TCS.

microbiology↗

bioRxiv2025

30S-seq redefines the bacterial Ribosome Binding Site

The translation initiation step is rate limiting for the efficiency of gene expression in all organisms. However, the mechanism of ribosome recruitment to mRNA start sites strikingly differs between eukaryotes and prokaryotes. The eukaryotic small (40S) ribosomal subunit binds 5 end caps and scans for the start codon while the bacterial small (30S) subunit directly binds to the Shine-Dalgarno (SD) motif close to the initiation site. Pioneer studies have shown rare 30S loading events further upstream within 5 untranslated regions (5UTRs), at ribosome standby sites1-3. Together with the frequent occurrence of long bacterial mRNA 5UTRs and degenerated SD sequences, this indicates that the 30S subunit might bind upstream of the SD more commonly than currently thought. We therefore developed 30S-seq to map 30S-mRNA interactions in a bacterial transcriptome (Escherichia coli), inspired by translation complex profile sequencing (TCP-seq) previously used in eukaryotes4,5. Our results provide new and unsuspected insights into the behaviour of 30S and 70S complexes during the canonical translation initiation process. Notably, 30S subunits are recruited upstream of the start codon, primed to receive the SD released by the departing 70S ribosome. Remarkably, we also find hundreds of non-canonical 30S binding sites within mRNA 5UTRs, sometimes over 100 nucleotides upstream of the start region. We validated several of these upstream ribosome binding sites, and demonstrated their strong impact on gene expression. Thus, even in bacteria, ribosomes frequently bind mRNAs outside of the start region to initiate translation, challenging the classic ribosome binding site model.

molecular biology↗

bioRxiv2025

EASY-CRISPR: a toolbox for high-throughput single-step custom genetic editing in bacteria

Targeted gene editing can be achieved using CRISPR/Cas9-assisted recombineering. However, high-efficiency editing requires careful optimization for each locus to be modified which can be tedious and time-consuming. In this work, we developed a simple, fast and cheap method for the Editing and Assembly of SYnthetic operons using CRISPR/Cas9-assisted recombineering (EASY-CRISPR) in Escherichia coli. Highly efficient editing of the different constitutive elements of the operons can be achieved by using a set of optimized guide RNAs and single- or double-stranded DNA repair templates carrying relatively short homology arms. This facilitates the construction of multiple genetic tools, including mutant libraries or reporter genes. EASY-CRISPR is also highly modular, as we provide alternative and complementary versions of the operon inserted in three loci which can be edited iteratively and easily combined. As a proof of concept, we report the construction of several fusions with reporter genes confirming known post-transcriptional regulation mechanisms and the construction of saturated and unbiased mutant libraries. In summary, the EASY-CRISPR system provides a flexible genomic expression platform that can be used both for the understanding of biological processes and as a tool for bioengineering applications.

microbiology↗

bioRxiv2024

Control of iron acquisition by multiple small RNAs unravels a new role for transcriptional terminator loops in gene regulation

Small RNAs (sRNAs) controlling gene expression by imperfect base-pairing with mRNA(s) are widespread in bacteria and regulate multiple genes, including genes involved in iron homeostasis, through a wide variety of mechanisms. We previously showed that OmrA and OmrB sRNAs repress the synthesis of the Escherichia coli FepA receptor for iron-enterobactin complexes. We now report that five additional sRNAs, namely RprA, RybB, ArrS, RseX and SdsR, that respond to different environmental cues, also repress fepA, independently of one another. While RprA follows the canonical mechanism of pairing with the translation initiation region, repression by ArrS or RseX requires a secondary structure far upstream within the long fepA 5UTR. We also demonstrate a dual action of SdsR, whose 5 end pairs with the fepA translation initiation region while its 3 end behaves like ArrS or RseX. Strikingly, mutation analysis shows a key role for the loops of these sRNAs intrinsic terminators in the regulation. Regulation furthermore depends on both the Hfq chaperone and the RNase E endonuclease. Overall, our data strongly suggest that FepA levels must be tightly controlled under a variety of conditions, and highlight the diversity of mechanisms that underly the regulation of gene expression by sRNAs in bacteria.

microbiology↗

bioRxiv2023

Riboswitch and small RNA modulate btuB translation initiation in Escherichia coli and trigger distinct mRNA regulatory mechanisms

Small RNAs (sRNAs) and riboswitches represent distinct classes of RNA regulators that control gene expression upon sensing metabolic or environmental variations. While sRNAs and riboswitches regulate gene expression by affecting mRNA and protein levels, existing studies have been limited to the characterization of each regulatory system in isolation, suggesting that sRNAs and riboswitches target distinct mRNA populations. We report that the expression of btuB in Escherichia coli, which is regulated by an adenosylcobalamin (AdoCbl) riboswitch, is also controlled by the small RNAs OmrA and, to a lesser extent, OmrB. Strikingly, we find that the riboswitch and sRNAs reduce mRNA levels through distinct pathways. Our data show that while the riboswitch triggers Rho-dependent transcription termination, sRNAs rely on the degradosome to modulate mRNA levels. Importantly, OmrA pairs with the btuB mRNA through its central region, which is not conserved in OmrB, indicating that these two sRNAs may have specific targets in addition to their common regulon. In contrast to canonical sRNA regulation, we find that OmrA repression of btuB is lost using an mRNA binding-deficient Hfq variant. Together, our study demonstrates that riboswitch and sRNAs modulate btuB expression, providing an example of cis- and trans-acting RNA-based regulatory systems maintaining cellular homeostasis.

molecular biology↗

bioRxiv2021

Regulatory interplay between RNase III and asRNAs in E. coli; the case of AsflhD and the master regulator of motility, flhDC

In order to respond to ever-changing environmental cues, bacteria have evolved resilient regulatory mechanisms controlling gene expression. At the post-transcriptional level, this is achieved by a combination of RNA-binding proteins, such as ribonucleases (RNases) and RNA chaperones, and regulatory RNAs including antisense RNAs (asRNAs). AsRNAs bound to their complementary mRNA are primary targets for the double-strand-specific endoribonuclease, RNase III. By comparing primary and processed transcripts in an rnc strain, mutated for RNase III, and its isogenic wild type strain, we detected several asRNAs. We confirmed the existence of RNase III-sensitive asRNA for crp, ompR, phoP and flhD genes, encoding master regulators of gene expression. AsflhD, the asRNA to the master regulator of motility flhDC, is slightly induced under heat-shock conditions in a sigma24 (RpoE)-dependent manner. We demonstrate that expression of AsflhD asRNA is involved in the transcriptional attenuation of flhD and thus participates in the control of the whole motility cascade. This study demonstrates that AsflhD and RNase III are additional players in the complex regulation ensuring a tight control of flagella synthesis and motility. ImportanceThe importance of asRNAs in the regulation of gene expression has long been underestimated. Here, we confirm that asRNAs can be part of layered regulatory networks since some are found opposite to genes encoding global regulators. In particular, we show how an antisense RNA (AsflhD) to the gene expressing a transcription factor serving as the primary regulator of bacterial swimming motility (FlhD4C2) is involved in the transcriptional attenuation of flhD, which in turn impacts the expression of other genes of the motility cascade. The role of AsflhD highlights the importance of discrete fine-tuning mechanisms in the control of complex regulatory networks.

microbiology↗

bioRxiv2021

Synthesis of the NarP response regulator of nitrate respiration in Escherichia coli is regulated at multiple levels by Hfq and small RNAs

Two-component systems (TCS) and small RNAs (sRNA) are widespread regulators that participate in the response and the adaptation of bacteria to their environments. They mostly act at the transcriptional and post-transcriptional levels, respectively, and can be found integrated in regulatory circuits, where TCSs control sRNAs transcription and/or sRNAs post-transcriptionally regulate TCSs synthesis. In response to nitrate and nitrite, the paralogous NarQ-NarP and NarX-NarL TCSs regulate the expression of genes involved in anaerobic respiration of these alternative electron acceptors. In addition to the previously reported repression of NarP synthesis by the SdsN137 sRNA, we show here that RprA, another Hfq-dependent sRNA, also negatively controls narP. Interestingly, the repression of narP by RprA actually relies on two independent controls. The first is via the direct pairing of the central region of RprA to the narP translation initiation region and presumably occurs at the translation initiation level. In contrast, the second control requires only the very 5 end of the narP mRNA, which is targeted, most likely indirectly, by the full-length or the shorter, processed, form of RprA. In addition, our results raise the possibility of a direct role of Hfq in narP control, further illustrating the diversity of post-transcriptional regulation mechanisms in the synthesis of TCSs.

microbiology↗