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Riboswitches are metabolite-binding RNA regulators that modulate gene expression at the levels of transcription and translation. One of the hallmarks of riboswitch regulation is that they undergo structural changes upon metabolite binding. While a lot of effort has been put to characterize how the metabolite is recognized by the riboswitch, there is still relatively little information regarding how ligand sensing is performed within a transcriptional context. Here, we study the ligand-dependent cotranscriptional folding of the FMN-sensing ribB riboswitch of Escherichia coli Using RNase H assays to study nascent ribB riboswitch transcripts, DNA probes targeting the P1 and sequestering stems indicate that FMN binding leads to the protection of these regions from RNase H cleavage, consistent with the riboswitch inhibiting translation initiation when bound to FMN. Our results show that ligand sensing is strongly affected by the position of elongating RNA polymerase, which is defining an FMN-binding transcriptional window that is bordered in its 3' extremity by a transcriptional pause site. Also, using successively overlapping DNA probes targeting a subdomain of the riboswitch, our data suggest the presence of a previously unsuspected helical region involving the 3' strand of the P1 stem. Our results show that this helical region is conserved across bacterial species, thus suggesting that this predicted structure, the anti*-P1 stem, is involved in the FMN-free conformation of the ribB riboswitch. Overall, our study further demonstrates that intricate folding strategies may be used by riboswitches to perform metabolite sensing during the transcriptional process., (© 2024 Eschbach et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Modifications at the wobble position (position 34) of tRNA facilitate interactions that enable or stabilize non-Watson-Crick base pairs. In bacterial tRNA, 5-hydroxyuridine (ho 5 U) derivatives xo 5 U [x: methyl (mo 5 U), carboxymethyl (cmo 5 U), and methoxycarbonylmethyl (mcmo 5 U)] present at the wobble positions of tRNAs are responsible for the recognition of NYN codon families. These modifications of U34 allow base-pairing not only with A and G but also with U, and in some cases, C. mo 5 U was originally found in Gram-positive bacteria, and cmo 5 U and mcmo 5 U were found in Gram-negative bacteria. tRNAs of Mycoplasma species, mitochondria, and chloroplasts adopt four-way decoding in which unmodified U34 recognizes codons ending in A, G, C, and U. Lactobacillus casei , Gram-positive bacteria, and lactic acid bacteria lack the modification enzyme genes for xo 5 U biosynthesis. Nevertheless, L. casei has only one type of tRNA Val with the anticodon UAC [tRNA Val (UAC)]. However, the genome of L. casei encodes an undetermined tRNA (tRNA Und ) gene, and the sequence corresponding to the anticodon region is GAC. Here, we confirm that U34 in L. casei tRNA Val is unmodified and that there is no tRNA Und expression in the cells. In addition, in vitro transcribed tRNA Und was not aminoacylated by L. casei valyl-tRNA synthetase, suggesting that tRNA Und is not able to accept valine, even if expressed in cells. Correspondingly, native tRNA Val (UAC) with unmodified U34 bound to all four valine codons in the ribosome A site. This suggests that L. casei tRNA Val decodes all valine codons by four-way decoding, similarly to tRNAs from Mycoplasma species, mitochondria, and chloroplasts., (© 2024 Sugita et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Transcription elongation is one of the most important processes in the cell. During RNA polymerase elongation, the folding of nascent transcripts plays crucial roles in the genetic decision. Bacterial riboswitches are prime examples of RNA regulators that control gene expression by altering their structure upon metabolite sensing. It was previously revealed that the thiamin pyrophosphate-sensing tbpA riboswitch in Escherichia coli cotranscriptionally adopts three main structures leading to metabolite sensing. Here, using single-molecule FRET, we characterize the transition in which the first nascent structure, a 5' stem-loop, is unfolded during transcription elongation to form the ligand-binding competent structure. Our results suggest that the structural transition occurs in a relatively abrupt manner, i.e., within a 1-2 nucleotide window. Furthermore, a highly dynamic structural exchange is observed, indicating that riboswitch transcripts perform rapid sampling of nascent co-occurring structures. We also observe that the presence of the RNAP stabilizes the 5' stem-loop along the elongation process, consistent with RNAP interacting with the 5' stem-loop. Our study emphasizes the role of early folding stem-loop structures in the cotranscriptional formation of complex RNA molecules involved in genetic regulation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The functions of non-coding small RNAs (sRNAs) within the human microbiome remain largely unexplored. In this Cell Host & Microbe issue, El Mouali et al. identify Segatella RNA colonization factor (SrcF), a sRNA from a prevalent gut bacterium Segatella copri. SrcF promotes colonization of S. copri by regulating bacterial degradation of complex dietary carbohydrates., Competing Interests: Declaration of interests M.S.D. works as a consultant and an advisory board member at Theralution GmbH, Germany., (Copyright © 2024 Elsevier Inc. All rights reserved.)

There has been recent interest in trying to understand the connection between transfer RNA (tRNA) posttranscriptional modifications and changes in-cellular environmental conditions. Here, we report on the identification of the modified nucleoside 5-methylcytidine (m 5 C) in Escherichia coli tRNAs. This modification was determined to be present at position 49 of tRNA Tyr-QUA-II. Moreover, m 5 C levels in this tRNA are significantly elevated under high reactive oxygen specieis (ROS) conditions in E. coli cells. We identified the known ribosomal RNA methyltransferase rsmF as the enzyme responsible for m 5 C synthesis in tRNA and enzyme transcript levels are responsive to elevated levels of ROS in the cell. We further find that changes in m 5 C levels in this tRNA are not specific to Fenton-like reaction conditions elevating ROS, but heat shock can also induce increased modification of tRNA Tyr-QUA-II. Altogether, this work illustrates how cells adapt to changing environmental conditions through variations in tRNA modification profiles., Competing Interests: Competing interests statement:The authors declare no competing interest.

Fe-S clusters are essential cofactors involved in many reactions across all domains of life. Their biogenesis in Escherichia coli and other enterobacteria involves two machineries: Isc and Suf. Under conditions where cells operate with the Suf system, such as during oxidative stress or iron limitation, the entry of aminoglycosides is reduced, leading to resistance to these antibiotics. The transition between Isc and Suf machineries is controlled by the transcriptional regulator IscR. Here, we found that two small regulatory RNAs (sRNAs), FnrS and OxyS, control iscR expression by base pairing to the 5'-UTR of the iscR mRNA. These sRNAs act in opposite ways and in opposite conditions: FnrS, expressed in anaerobiosis, represses the expression of iscR while OxyS, expressed during oxidative stress, activates it. Using an E. coli strain experiencing protracted oxidative stress, we further demonstrate that iscR expression is rapidly and significantly enhanced in the presence of OxyS. Consequently, we further show that OxyS induces resistance to aminoglycosides during oxidative stress through regulation of Fe-S cluster biogenesis, revealing a major role for this sRNA., Competing Interests: Competing interests statement:The authors declare no competing interest.

The toxin-antitoxin (TA) system regulates many physiological processes in free-living bacteria. One such TA system in Escherichia coli comprises an RNA toxin SdsR and an antitoxin RyeA. An overabundance of SdsR is toxic to the cells. RyeA normalizes SdsR abundance and helps the cells to adapt to altered conditions. The current study showed that a novel small RNA (sRNA) regulator GcvB directly interacts with RyeA to maintain its abundance in the cells under normal or low pH conditions. The deletion of the gcvB allele in the E. coli chromosome resulted in a ∼3-fold decrease in intrabacterial RyeA accumulation. An ectopic expression of GcvB in ΔgcvB strain reinstated RyeA abundance to its normal level. Induction of GcvB in the cells upon exposure to low pH resulted in a simultaneous increase in intracellular RyeA. While GcvB increases RyeA abundance in the cells, SdsR accumulation is divergently regulated by GcvB. The absence of the gcvB gene in E. coli leads to upregulation of SdsR and vice versa. The GcvB-mediated decrease of SdsR accumulation stems from the increased RyeA-driven normalization of SdsR. This study delineates a novel mechanism for the regulation of the expression of an RNA toxin SdsR by another sRNA regulator GcvB through a feed-forward control., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In many bacteria, the essential factors Rho and NusG mediate termination of synthesis of nascent transcripts (including antisense RNAs) that are not being simultaneously translated. It has been proposed that in Rho's absence toxic RNA-DNA hybrids (R-loops) may be generated from nascent untranslated transcripts, and genome-wide mapping studies in Escherichia coli have identified putative loci of R-loop formation from more than 100 endogenous antisense transcripts that are synthesized only in a Rho-deficient strain. Here we provide evidence that engineered expression in wild-type E. coli of several such individual antisense regions on a plasmid or the chromosome generates R-loops that, in an RNase H-modulated manner, serve to disrupt genome integrity. Rho inhibition was associated with increased prevalence of antisense R-loops also in Xanthomonas oryzae pv. oryzae and Caulobacter crescentus. Our results confirm the essential role of Rho in several bacterial genera for prevention of toxic R-loops from pervasive yet cryptic endogenous antisense transcripts. Engineered antisense R-looped regions may be useful for studies on both site-specific impediments to bacterial chromosomal replication and the mechanisms of their resolution., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

All known bacterial tRNAs adopt the canonical cloverleaf 2D and L-shaped 3D structures. We aimed to explore whether alternative tRNA structures could be introduced in bacterial translation. To this end, we crafted a vitamin-based genetic system to evolve Escherichia coli toward activity of structurally non-canonical tRNAs. The system reliably couples (escape frequency <10-12) growth with the activities of a novel orthogonal histidine suppressor tRNA (HisTUAC) and of the cognate ARS (HisS) via suppression of a GTA valine codon in the mRNA of an enzyme in thiamine biosynthesis (ThiN). Suppression results in the introduction of an essential histidine and thereby confers thiamine prototrophy. We then replaced HisTUAC in the system with non-canonical suppressor tRNAs and selected for growth. A strain evolved to utilize mini HisT, a tRNA lacking the D-arm, and we identified the responsible mutation in an RNase gene (pnp) involved in tRNA degradation. This indicated that HisS, the ribosome, and EF-Tu accept mini HisT ab initio, which we confirmed genetically and through in vitro translation experiments. Our results reveal a previously unknown flexibility of the bacterial translation machinery for the accepted fold of the adaptor of the genetic code and demonstrate the power of the vitamin-based suppression system., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

mRNA degradation is a central process that affects all gene expression levels, though it remains challenging to predict the stability of a mRNA from its sequence, due to the many coupled interactions that control degradation rate. Here, we carried out massively parallel kinetic decay measurements on over 50,000 bacterial mRNAs, using a learn-by-design approach to develop and validate a predictive sequence-to-function model of mRNA stability. mRNAs were designed to systematically vary translation rates, secondary structures, sequence compositions, G-quadruplexes, i-motifs, and RppH activity, resulting in mRNA half-lives from about 20 seconds to 20 minutes. We combined biophysical models and machine learning to develop steady-state and kinetic decay models of mRNA stability with high accuracy and generalizability, utilizing transcription rate models to identify mRNA isoforms and translation rate models to calculate ribosome protection. Overall, the developed model quantifies the key interactions that collectively control mRNA stability in bacterial operons and predicts how changing mRNA sequence alters mRNA stability, which is important when studying and engineering bacterial genetic systems., (© 2024. The Author(s).)

TLR8 senses single-stranded RNA (ssRNA) fragments, processed via cleavage by ribonuclease (RNase) T2 and RNase A family members. Processing by these RNases releases uridines and purine-terminated residues resulting in TLR8 activation. Monocytes show high expression of RNase 6, yet this RNase has not been analyzed for its physiological contribution to the recognition of bacterial RNA by TLR8. Here, we show a role for RNase 6 in TLR8 activation. BLaER1 cells, transdifferentiated into monocyte-like cells, as well as primary monocytes deficient for RNASE6 show a dampened TLR8-dependent response upon stimulation with isolated bacterial RNA (bRNA) and also upon infection with live bacteria. Pretreatment of bacterial RNA with recombinant RNase 6 generates fragments that induce TLR8 stimulation in RNase 6 knockout cells. 2'O-RNA methyl modification, when introduced at the first uridine in the UA dinucleotide, impairs processing by RNase 6 and dampens TLR8 stimulation. In summary, our data show that RNase 6 processes bacterial RNA and generates uridine-terminated breakdown products that activate TLR8., (© 2024. The Author(s).)

About 30% of all bacterial proteins execute their function outside of the cytosol and must be inserted into or translocated across the cytoplasmic membrane. This requires efficient targeting systems that recognize N-terminal signal sequences in client proteins and deliver them to protein transport complexes in the membrane. While the importance of these protein transport machineries for the spatial organization of the bacterial cell is well documented in multiple studies, the contribution of mRNA targeting and localized translation to protein transport is only beginning to emerge. mRNAs can exhibit diverse subcellular localizations in the bacterial cell and can accumulate at sites where new protein is required. This is frequently observed for mRNAs encoding membrane proteins, but the physiological importance of membrane enrichment of mRNAs and the consequences it has for the insertion of the encoded protein have not been explored in detail. Here, we briefly highlight some basic concepts of signal sequence-based protein targeting and describe in more detail strategies that enable the monitoring of mRNA localization in bacterial cells and potential mechanisms that route mRNAs to particular positions within the cell. Finally, we summarize some recent developments that demonstrate that mRNA targeting and localized translation can sustain membrane protein insertion under stress conditions when the protein-targeting machinery is compromised. Thus, mRNA targeting likely acts as a back-up strategy and complements the canonical signal sequence-based protein targeting., (© 2024 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

RNA 5'-modification with NAD+/NADH (oxidized/reduced nicotinamide adenine dinucleotide) has been found in bacteria, eukaryotes and viruses. 5'-NAD is incorporated into RNA by RNA polymerases (RNAPs) during the initiation of synthesis. It is unknown (i) which factors and physiological conditions permit substantial NAD incorporation into RNA in vivo and (ii) how 5'-NAD impacts gene expression and the fate of RNA in bacteria. Here we show in Escherichia coli that RNA NADylation is stimulated by low cellular concentration of the competing substrate ATP, and by weakening ATP contacts with RNAP active site. Additionally, RNA NADylation may be influenced by DNA supercoiling. RNA NADylation does not interfere with posttranscriptional RNA processing by major ribonuclease RNase E. It does not impact the base-pairing between RNAI, the repressor of plasmid replication, and its antisense target, RNAII. Leaderless NADylated model mRNA cI-lacZ is recognized by the 70S ribosome and is translated with the same efficiency as triphosphorylated cI-lacZ mRNA. Translation exposes the 5'-NAD of this mRNA to de-capping by NudC enzyme. We suggest that NADylated mRNAs are rapidly degraded, consistent with their low abundance in published datasets. Furthermore, we observed that ppGpp inhibits NudC de-capping activity, contributing to the growth phase-dependency of NADylated RNA levels., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The increase in antibiotic resistance in bacteria has prompted the efforts in developing new alternative strategies for pathogenic bacteria. We explored the feasibility of targeting Vibrio cholerae by neutralizing bacterial cellular processes rather than outright killing the pathogen. We investigated the efficacy of delivering engineered regulatory small RNAs (sRNAs) to modulate gene expression through DNA conjugation. As a proof of concept, we engineered several sRNAs targeting the type VI secretion system (T6SS), several of which were able to successfully knockdown the T6SS activity at different degrees. Using the same strategy, we modulated exopolysaccharide production and motility. Lastly, we delivered an sRNA targeting T6SS into V. cholerae via conjugation and observed a rapid knockdown of the T6SS activity. Coupling conjugation with engineered sRNAs represents a novel way of modulating gene expression in V. cholerae opening the door for the development of novel prophylactic and therapeutic applications., Importance: Given the prevalence of antibiotic resistance, there is an increasing need to develop alternative approaches to managing pathogenic bacteria. In this work, we explore the feasibility of modulating the expression of various cellular systems in Vibrio cholerae using engineered regulatory sRNAs delivered into cells via DNA conjugation. These sRNAs are based on regulatory sRNAs found in V. cholerae and exploit its native regulatory machinery. By delivering these sRNAs conjugatively along with a real-time marker for DNA transfer, we found that complete knockdown of a targeted cellular system could be achieved within one cell division cycle after sRNA gene delivery. These results indicate that conjugative delivery of engineered regulatory sRNAs is a rapid and robust way of precisely targeting V. cholerae ., Competing Interests: The authors declare no conflict of interest.

Tandem SAM-II/SAM-V riboswitch belongs to a class of riboswitches found in the marine bacterium ' Candidatus Pelagibacter ubique'. Previous studies have demonstrated that these riboswitches have the potential for digital modulation of gene expression at both the transcriptional and translational levels. In this study, we investigate the conformational changes in the tandem SAM-II/SAM-V riboswitch binding to S-adenosylmethionine (SAM) using selective 2'-hydroxyl acylation analyzed by the primer extension (SHAPE) assay, small-angle X-ray scattering (SAXS), and oligos depressing probing. Our findings reveal that the linker between SAM-II/SAM-V aptamers blocks the SAM response of the SAM-II domain. This result proposes a new mechanism for gene expression regulation, where the ligand-binding functions of tandem riboswitches can be selectively masked or released through a linker.

Riboswitches regulate gene expression by modulating their structure upon metabolite binding. These RNA orchestrate several layers of regulation to achieve genetic control. Although Escherichia coli riboswitches modulate translation initiation, several cases have been reported where riboswitches also modulate mRNA levels. Here, we characterize the regulation mechanisms of the thiamin pyrophosphate (TPP) tbpA riboswitch in E. coli. Our results indicate that the tbpA riboswitch modulates both levels of translation and transcription and that TPP sensing is achieved more efficiently cotranscriptionally than post-transcriptionally. The preference for cotranscriptional binding is also observed when monitoring the TPP-dependent inhibition of translation initiation. Using single-molecule approaches, we observe that the aptamer domain freely fluctuates between two main structures involved in TPP recognition. Our results suggest that translation initiation is controlled through the ligand-dependent stabilization of the riboswitch structure. This study demonstrates that riboswitch cotranscriptional sensing is the primary determinant in controlling translation and mRNA levels., (© 2024. The Author(s).)

Bacterial regulatory RNAs (sRNAs) are important players to control gene expression. In Staphylococcus aureus , SprC is an antivirulent trans -acting sRNA known to base-pair with the major autolysin atl mRNA, preventing its translation. Using MS2-affinity purification coupled with RNA sequencing, we looked for its sRNA-RNA interactome and identified 14 novel mRNA targets. In vitro biochemical investigations revealed that SprC binds two of them, czrB and deoD, and uses a single accessible region to regulate its targets, including Atl translation. Unlike Atl regulation, the characterization of the SprC- czrB interaction pinpointed a destabilization of the czrAB cotranscript, leading to a decrease of the mRNA level that impaired CzrB zinc efflux pump expression. On a physiological standpoint, we showed that SprC expression is detrimental to combat against zinc toxicity. In addition, phagocyctosis assays revealed a significant, but moderate, increase of czrB mRNA levels in a sprC -deleted mutant, indicating a functional link between SprC and czrB upon internalization in macrophages, and suggesting a role in resistance to both oxidative and zinc bursts. Altogether, our data uncover a novel pathway in which SprC is implicated, highlighting the multiple strategies used by S. aureus to balance virulence using an RNA regulator., (© 2024 Raynaud et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

SreA is one of seven candidate S -adenosyl methionine (SAM) class I riboswitches identified in Listeria monocytogenes , a saprophyte and opportunistic foodborne pathogen. SreA precedes genes encoding a methionine ATP-binding cassette (ABC) transporter, which imports methionine and is presumed to regulate transcription of its downstream genes in a SAM-dependent manner. The proposed role of SreA in controlling the transcription of genes encoding an ABC transporter complex may have important implications for how the bacteria senses and responds to the availability of the metabolite SAM in the diverse environments in which L. monocytogenes persists. Here we validate SreA as a functional SAM-I riboswitch through ligand binding studies, structure characterization, and transcription termination assays. We determined that SreA has both a structure and SAM binding properties similar to those of other well-characterized SAM-I riboswitches. Despite the apparent structural similarities to previously described SAM-I riboswitches, SreA induces transcription termination in response to comparatively lower (nanomolar) ligand concentrations. Furthermore, SreA is a leaky riboswitch that permits some transcription of the downstream gene even in the presence of millimolar SAM, suggesting that L. monocytogenes may "dampen" the expression of genes for methionine import but likely does not turn them "OFF".

Adenosine-to-inosine (A-to-I) RNA editing plays an important role in the post-transcriptional regulation of eukaryotic cell physiology. However, our understanding of the occurrence, function and regulation of A-to-I editing in bacteria remains limited. Bacterial mRNA editing is catalysed by the deaminase TadA, which was originally described to modify a single tRNA in Escherichia coli. Intriguingly, several bacterial species appear to perform A-to-I editing on more than one tRNA. Here, we provide evidence that in the human pathogen Streptococcus pyogenes, tRNA editing has expanded to an additional tRNA substrate. Using RNA sequencing, we identified more than 27 editing sites in the transcriptome of S. pyogenes SF370 and demonstrate that the adaptation of S. pyogenes TadA to a second tRNA substrate has also diversified the sequence context and recoding scope of mRNA editing. Based on the observation that editing is dynamically regulated in response to several infection-relevant stimuli, such as oxidative stress, we further investigated the underlying determinants of editing dynamics and identified mRNA stability as a key modulator of A-to-I editing. Overall, our findings reveal the presence and diversification of A-to-I editing in S. pyogenes and provide novel insights into the plasticity of the editome and its regulation in bacteria., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Co-transcriptional assembly is an integral feature of the formation of RNA-protein complexes that mediate translation. For ribosome synthesis, prior studies have indicated that the strict order of transcription of rRNA domains may not be obligatory during bacterial ribosome biogenesis, since a series of circularly permuted rRNAs are viable. In this work, we report the structural insights into assembly of the bacterial ribosome large subunit (LSU) based on cryo-EM density maps of intermediates that accumulate during in vitro ribosome synthesis using a set of circularly permuted (CiPer) rRNAs. The observed ensemble of 23 resolved ribosome large subunit intermediates reveals conserved assembly routes with an underlying hierarchy among cooperative assembly blocks. There are intricate interdependencies for the formation of key structural rRNA helices revealed from the circular permutation of rRNA. While the order of domain synthesis is not obligatory, the order of domain association does appear to proceed with a particular order, likely due to the strong evolutionary pressure on efficient ribosome synthesis. This work reinforces the robustness of the known assembly hierarchy of the bacterial large ribosomal subunit and offers a coherent view of how efficient assembly of CiPer rRNAs can be understood in that context., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Acinetobacter baumannii is an opportunistic Gram-negative pathogen that infects critically ill patients. The emergence of antimicrobial resistant A. baumannii has exacerbated the need to characterize environmental adaptation, antibiotic resistance and pathogenicity and their genetic regulators to inform intervention strategies. Critical to adaptation to changing environments in bacteria are small regulatory RNAs (sRNAs), however, the role that sRNAs play in the biology of A. baumannii is poorly understood. To assess the regulatory function of sRNAs and to uncover their RNA interaction partners, we employed an RNA proximity ligation and sequencing method (Hi-GRIL-seq) in three different environmental conditions. Forty sRNAs were ligated to sRNA-RNA chimeric sequencing reads, suggesting that sRNA-mediated gene regulation is pervasive in A. baumannii. In-depth characterization uncovered the sRNA Aar to be a post-transcriptional regulator of four mRNA targets including the transcript encoding outer membrane protein CarO. Aar initiates base-pairing with these mRNAs using a conserved seed region of nine nucleotides, sequestering the ribosome binding sites and inhibiting translation. Aar is differentially expressed in multiple stress conditions suggesting a role in fine-tuning translation of the Aar-target molecules. Our study provides mechanistic insights into sRNA-mediated gene regulation in A. baumannii and represents a valuable resource for future RNA-centric research endeavours., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Staphylococcus aureus is a major contributor to bacterial-associated mortality, owing to its exceptional adaptability across diverse environments. Iron is vital to most organisms but can be toxic in excess. To manage its intracellular iron, S. aureus , like many pathogens, employs intricate systems. We have recently identified IsrR as a key regulatory RNA induced during iron starvation. Its role is to reduce the synthesis of non-essential iron-containing proteins under iron-depleted conditions. In this study, we unveil IsrR's regulatory action on MiaB, an enzyme responsible for methylthio group addition to specific sites on transfer RNAs (tRNAs). We use predictive tools and reporter fusion assays to demonstrate IsrR's binding to the Shine-Dalgarno sequence of miaB RNA, thereby impeding its translation. The effectiveness of IsrR hinges on the integrity of a specific C-rich region. As MiaB is non-essential and has iron-sulfur clusters, IsrR induction spares iron by downregulating miaB . This may improve S. aureus fitness and aid in navigating the host's nutritional immune defenses.IMPORTANCEIn many biotopes, including those found within an infected host, bacteria confront the challenge of iron deficiency. They employ various strategies to adapt to this scarcity of nutrients, one of which involves regulating iron-containing proteins through the action of small regulatory RNAs. Our study shows how IsrR, a small RNA from S. aureus , prevents the production of MiaB, a tRNA-modifying enzyme containing iron-sulfur clusters. With this illustration, we propose a new substrate for an iron-sparing small RNA, which, when downregulated, should reduce the need for iron and save it to essential functions., Competing Interests: The authors declare no conflict of interest.

Riboswitches are 5' RNA regulatory elements that are capable of binding to various ligands, such as small metabolites, ions and tRNAs, leading to conformational changes and affecting gene transcription or translation. They are widespread in bacteria and frequently control genes that are essential for the survival or virulence of major pathogens. As a result, they represent promising targets for the development of new antimicrobial treatments. Clostridioides difficile , a leading cause of antibiotic-associated nosocomial diarrhoea in adults, possesses numerous riboswitches in its genome. Accumulating knowledge of riboswitch-based regulatory mechanisms provides insights into the potential therapeutic targets for treating C. difficile infections. This review offers an in-depth examination of the current state of knowledge regarding riboswitch-mediated regulation in C. difficile , highlighting their importance in bacterial adaptability and pathogenicity. Particular attention is given to the ligand specificity and function of known riboswitches in this bacterium. The review also discusses the recent progress that has been made in the development of riboswitch-targeting compounds as potential treatments for C. difficile infections. Future research directions are proposed, emphasizing the need for detailed structural and functional analyses of riboswitches to fully harness their regulatory capabilities for developing new antimicrobial strategies.

Bacteria of the genus Pseudomonas are ubiquitous on Earth due to their great metabolic versatility and adaptation to fluctuating environments and different hosts. Some groups are important animal/human and plant pathogens, whereas others are studied for their biotechnological applications, including bioremediation, biological control of phytopathogens and plant growth promotion. Notably, their adaptability is mediated by various signal transduction systems, with the post-transcriptional Gac-Rsm cascade playing a key role. This pervasive Pseudomonas pathway controls major transitions at the population level, such as motile/sessile lifestyle, primary/secondary metabolism or replicative/infective behaviour. A hallmark of the Gac-Rsm cascade is the participation of small, regulatory, non-coding RNAs of the Rsm clan. These RNAs are synthetised in response to cell-density-dependent autoinducer signals channelled through the GacS/GacA two-component system, and they counteract, by molecular mimicry, the translational control that RNA-binding proteins of the RsmA family exert over hundreds of mRNAs. Rsm RNAs have been investigated in a few Pseudomonas model species, evidencing the presence of a variable number and families of genes depending on the taxonomic clade. However, the global picture of the distribution of these riboregulators at the genus level was unknown until now. We have undertaken a comprehensive survey and annotation of the vast array of gene sequences encoding members of the Rsm RNA clan in 245 complete genomes that cover 28 phylogenomic clades across the entire genus. The properties of the different families of rsm genes, their phylogenetic radiation, as well as the features of their promoters and adjacent regions, are discussed. The novel insights presented in our manuscript will significantly boost research on the biology of these prevalent RNAs in understudied species of the genus Pseudomonas and closely related genera., (© 2024 John Wiley & Sons Ltd.)

Riboswitches are specialized RNA structures that orchestrate gene expression in response to sensing specific metabolite or ion ligands, mostly in bacteria. Upon ligand binding, these conformationally dynamic RNA motifs undergo structural changes that control critical gene expression processes such as transcription termination and translation initiation, thereby enabling cellular homeostasis and adaptation. Because RNA folds rapidly and co-transcriptionally, riboswitches make use of the low complexity of RNA sequences to adopt alternative, transient conformations on the heels of the transcribing RNA polymerase (RNAP), resulting in kinetic partitioning that defines the regulatory outcome. This review summarizes single molecule microscopy evidence that has begun to unveil a sophisticated network of dynamic, kinetically balanced interactions between riboswitch architecture and the gene expression machinery that, together, integrate diverse cellular signals., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Nils G. Walter reports financial support was provided by National Institutes of Health. Nils G. Walter reports financial support was provided by National Science Foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Antibiotic resistance is a critical global health concern, causing millions of prolonged bacterial infections every year and straining our healthcare systems. Novel antibiotic strategies are essential to combating this health crisis and bacterial non-coding RNAs are promising targets for new antibiotics. In particular, a class of bacterial non-coding RNAs called riboswitches has attracted significant interest as antibiotic targets. Riboswitches reside in the 5'-untranslated region of an mRNA transcript and tune gene expression levels in cis by binding to a small-molecule ligand. Riboswitches often control expression of essential genes for bacterial survival, making riboswitch inhibitors an exciting prospect for new antibacterials. Synthetic ligand mimics have predominated the search for new riboswitch inhibitors, which are designed based on static structures of a riboswitch's ligand-sensing aptamer domain or identified by screening a small-molecule library. However, many small-molecule inhibitors that bind an isolated riboswitch aptamer domain with high affinity in vitro lack potency in vivo . Importantly, riboswitches fold and respond to the ligand during active transcription in vivo . This co-transcriptional folding is often not considered during inhibitor design, and may explain the discrepancy between a low K d in vitro and poor inhibition in vivo . In this review, we cover advances in riboswitch co-transcriptional folding and illustrate how intermediate structures can be targeted by antisense oligonucleotides-an exciting new strategy for riboswitch inhibitor design.

Processing of RNA is a key regulatory mechanism for all living systems. Escherichia coli protein YicC belongs to the well-conserved YicC family and has been identified as a novel ribonuclease. Here, we report a 2.8-Å-resolution crystal structure of the E. coli YicC apo protein and a 3.2-Å-cryo-EM structure of YicC bound to an RNA substrate. The apo YicC forms a dimer of trimers with a large open channel. In the RNA-bound form, the top trimer of YicC rotates nearly 70° and closes the RNA substrate inside the cavity to form a clamshell-pearl conformation that resembles no other known RNases. The structural information combined with mass spectrometry and biochemical data identified cleavage on the upstream side of an RNA hairpin. Mutagenesis studies demonstrated that the previously uncharacterized domain, DUF1732, is critical in both RNA binding and catalysis. These studies shed light on the mechanism of the previously unexplored YicC RNase family., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The study of small, regulatory RNAs (sRNA) that act by base-pairing with target RNAs in bacteria has been steadily advancing, particularly with the availability of more and more transcriptome and RNA-RNA interactome datasets. While the characterization of multiple sRNAs has helped to elucidate their mechanisms of action, these studies also are providing insights into protein function, control of metabolic flux, and connections between metabolic pathways as we will discuss here. In describing several examples of the metabolic insights gained, we will summarize the different types of base-pairing sRNAs including mRNA-derived sRNAs, sponge RNAs, RNA mimics, and dual-function RNAs as well as suggest how information about sRNAs could be exploited in the future., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024. Published by Elsevier Ltd.)

Binding of the bacterial Rho helicase to nascent transcripts triggers Rho-dependent transcription termination (RDTT) in response to cellular signals that modulate mRNA structure and accessibility of Rho utilization (Rut) sites. Despite the impact of temperature on RNA structure, RDTT was never linked to the bacterial response to temperature shifts. We show that Rho is a central player in the cold-shock response (CSR), challenging the current view that CSR is primarily a posttranscriptional program. We identify Rut sites in 5'-untranslated regions of key CSR genes/operons (cspA, cspB, cspG, and nsrR-rnr-yjfHI) that trigger premature RDTT at 37°C but not at 15°C. High concentrations of RNA chaperone CspA or nucleotide changes in the cspA mRNA leader reduce RDTT efficiency, revealing how RNA restructuring directs Rho to activate CSR genes during the cold shock and to silence them during cold acclimation. These findings establish a paradigm for how RNA thermosensors can modulate gene expression., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Escherichia coli possesses four initiator tRNA (i-tRNA) genes, three of which are present together as metZWV and the fourth one as metY . In E. coli B, all four genes ( metZWV and metY ) encode i-tRNA fMet1 , in which the G at position 46 is modified to m 7 G46 by TrmB (m 7 G methyltransferase). However, in E. coli K, because of a single-nucleotide polymorphism, metY encodes a variant, i-tRNA fMet2 , having an A in place of m 7 G46. We generated E. coli strains to explore the importance of this polymorphism in i-tRNAs. The strains were sustained either on metY A46 ( metY of E. coli K origin encoding i-tRNA fMet2 ) or its derivative metY G46 (encoding i-tRNA fMet1 ) in single (chromosomal) or plasmid-borne copies. We show that the strains sustained on i-tRNA fMet1 have a growth fitness advantage over those sustained on i-tRNA fMet2 . The growth fitness advantages are more pronounced for the strains sustained on i-tRNA fMet1 in nutrient-rich media than in nutrient-poor media. The growth fitness of the strains correlates well with the relative stabilities of the i-tRNAs in vivo . Furthermore, the atomistic molecular dynamics simulations support the higher stability of i-tRNA fMet1 than that of i-tRNA fMet2 . The stability of i-tRNA fMet1 remains unaffected upon the deletion of TrmB. These studies highlight how metY G46 and metY A46 alleles might influence the growth fitness of E. coli under certain nutrient-limiting conditions., Importance: Escherichia coli harbors four initiator tRNA (i-tRNA) genes: three of these at metZWV and the fourth one at metY loci. In E. coli B, all four genes encode i-tRNA fMet1 . In E. coli K, because of a single-nucleotide polymorphism, metY encodes a variant, i-tRNA fMet2 , having an A in place of G at position 46 of i-tRNA sequence in metY. We show that G46 confers stability to i-tRNA fMet1 . The strains sustained on i-tRNA fMet1 have a growth fitness advantage over those sustained on i-tRNA fMet2 . Strains harboring metY G46 (B mimic) or metY A46 (K mimic) show that while in the nutrient-rich media, the K mimic is outcompeted rapidly; in the nutrient-poor medium, the K mimic is outcompeted less rapidly., Competing Interests: The authors declare no conflict of interest.

Streptococcus pneumoniae (pneumococcus) causes a wide range of important human infectious diseases, including pneumonia, pneumonia-derived sepsis, otitis media, and meningitis. Pneumococcus produces numerous secreted proteins that are critical for normal physiology and pathogenesis. The membrane targeting and translocation of these secreted proteins are partly mediated by the signal recognition particle (SRP) complex, which consists of 4.5S small cytoplasmic RNA (ScRNA), and the Ffh, and FtsY proteins. Here, we report that pneumococcal ∆ scRNA , ∆ ffh, and ∆ ftsY mutants were significantly impaired in competence induction, competence pili production, exogenous DNA uptake, and genetic transformation. Also, the ∆ scRNA mutant was significantly attenuated in the mouse models of bacteremia and pneumonia. Interestingly, unlike the ∆ scRNA , both ∆ ffh and ∆ ftsY mutants had growth defects on Todd-Hewitt Agar, which were alleviated by the provision of free amino acids or serum. Differences in nutritional requirements between ∆ ffh and ∆ ftsY vs ∆ scRNA suggest that Ffh and FtsY may be partially functional in the absence of ScRNA. Finally, the insertase YidC2, which could functionally rescue some SRP mutations in other streptococcal species, was not essential for pneumococcal genetic transformation. Collectively, these results indicate that ScRNA is crucial for the successful development of genetic competence and virulence in pneumococcus., Importance: Streptococcus pneumoniae (pneumococcus) causes multiple important infectious diseases in humans. The signal recognition particle (SRP) complex, which comprised 4.5S small cytoplasmic RNA (ScRNA), and the Ffh and FtsY proteins, mediates membrane targeting and translocation of secreted proteins in all organisms. However, the role of SRP and ScRNA has not been characterized during the induction of the competence system for genetic transformation and virulence in pneumococcus. By using a combination of genetic, biochemical, proteomic, and imaging approaches, we demonstrated that the SRP complex plays a significant role in membrane targeting of competence system-regulated effectors important for genetic transformation, virulence during bacteremia and pneumonia infections, and nutritional acquisition., Competing Interests: The authors declare no conflict of interest.

Riboswitches are structured RNA elements that regulate gene expression upon binding to small molecule ligands. Understanding the mechanisms by which small molecules impact riboswitch activity is key to developing potent, selective ligands for these and other RNA targets. We report the structure-informed design of chemically diverse synthetic ligands for PreQ 1 riboswitches. Multiple X-ray co-crystal structures of synthetic ligands with the Thermoanaerobacter tengcongensis (Tte)-PreQ 1 riboswitch confirm a common binding site with the cognate ligand, despite considerable chemical differences among the ligands. Structure probing assays demonstrate that one ligand causes conformational changes similar to PreQ 1 in six structurally and mechanistically diverse PreQ 1 riboswitch aptamers. Single-molecule force spectroscopy is used to demonstrate differential modes of riboswitch stabilization by the ligands. Binding of the natural ligand brings about the formation of a persistent, folded pseudoknot structure, whereas a synthetic ligand decreases the rate of unfolding through a kinetic mechanism. Single round transcription termination assays show the biochemical activity of the ligands, while a GFP reporter system reveals compound activity in regulating gene expression in live cells without toxicity. Taken together, this study reveals that diverse small molecules can impact gene expression in live cells by altering conformational changes in RNA structures through distinct mechanisms., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

T-box riboswitches are widespread bacterial regulatory noncoding RNAs that directly interact with tRNAs and switch conformations to regulate the transcription or translation of genes related to amino acid metabolism. Recent studies in Bacilli have revealed the core mechanisms of T-boxes that enable multivalent, specific recognition of both the identity and aminoacylation status of the tRNA substrates. However, in-depth knowledge on a vast number of T-boxes in other bacterial species remains scarce, although a remarkable structural diversity, particularly among pathogens, is apparent. In the present study, analysis of T-boxes that control the transcription of glycyl-tRNA synthetases from four prominent human pathogens revealed significant structural idiosyncrasies. Nonetheless, these diverse T-boxes maintain functional T-box:tRNA Gly interactions both in vitro and in vivo. Probing analysis not only validated recent structural observations, but also expanded our knowledge on the substantial diversities among T-boxes and suggest interesting distinctions from the canonical Bacilli T-boxes. Surprisingly, some glycyl T-boxes seem to redirect the T-box trajectory in the absence of recognizable K-turns or contain Stem II modules that are generally absent in glycyl T-boxes. These results consolidate the notion of a lineage-specific diversification and elaboration of the T-box mechanism and corroborate the potential of T-boxes as promising species-specific RNA targets for next-generation antibacterial compounds., (© 2024 Giarimoglou et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Ribosomal RNAs are transcribed as part of larger precursor molecules. In Escherichia coli , complementary RNA segments flank each rRNA and form long leader-trailer (LT) helices, which are crucial for subunit biogenesis in the cell. A previous study of 15 representative species suggested that most but not all prokaryotes contain LT helices. Here, we use a combination of in silico folding and covariation methods to identify and characterize LT helices in 4464 bacterial and 260 archaeal organisms. Our results suggest that LT helices are present in all phyla, including Deinococcota, which had previously been suspected to lack LT helices. In very few organisms, our pipeline failed to detect LT helices for both 16S and 23S rRNA. However, a closer case-by-case look revealed that LT helices are indeed present but escaped initial detection. Over 3600 secondary structure models, many well supported by nucleotide covariation, were generated. These structures show a high degree of diversity. Yet, all exhibit extensive base-pairing between the leader and trailer strands, in line with a common and essential function., (© 2024 Gemler et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Pneumolysin (Ply) of Streptococcus pneumoniae (pneumococcus) at relatively high and low levels facilitates pneumococcal invasion into the lung and brain, respectively; however, the regulatory mechanisms of Ply expression are poorly understood. Here, we find that a small RNA plyT, processed from the 3'UTR of the ply operon, is expressed higher in anaerobically- than in statically-cultured pneumococcus D39. Using bioinformatic, biochemical and genetic approaches, we reveal that PlyT inhibits Ply synthesis and hemolytic activities by pairing with an RBS-embedded intergenic region of the ply operon. The RNA-binding protein SPD&#95;1558 facilitates the pairing. Importantly, PlyT inhibition of Ply synthesis is stronger in anaerobic culture and leads to lower Ply abundance. Deletion of plyT decreases the number of pneumococci in the infected mouse brain and reduces the virulence, demonstrating that PlyT-regulated lower Ply in oxygen-void microenvironments, such as the blood, is important for pneumococcus to cross the blood-brain barrier and invade the brain. PlyT-mediated repression of Ply synthesis at anoxic niches is also verified in pneumococcal serotype 4 and 14 strains; moreover, the ply operon with a 3'UTR-embedded plyT, and the pairing sequences of IGR and plyT are highly conserved among pneumococcal strains, implying PlyT-regulated Ply synthesis might be widely employed by pneumococcus., (© 2024. The Author(s).)

Vital organismal processes, including development, differentiation and adaptation, involve altered gene expression. Although expression is frequently controlled at the transcriptional stage, various regulation mechanisms operate at downstream levels. Here, we leverage the photoreceptor NmPAL to optogenetically induce RNA refolding and the translation of bacterial mRNAs. Blue-light-triggered NmPAL binding disrupts a cis-repressed mRNA state, thereby relieves obstruction of translation initiation, and upregulates gene expression. Iterative probing and optimization of the circuit, dubbed riboptoregulator, enhanced induction to 30-fold. Given action at the mRNA level, the riboptoregulator can differentially regulate individual structural genes within polycistronic operons. Moreover, it is orthogonal to and can be wed with other gene-regulatory circuits for nuanced and more stringent gene-expression control. We thus advance the pAurora2 circuit that combines transcriptional and translational mechanisms to optogenetically increase bacterial gene expression by >1000-fold. The riboptoregulator strategy stands to upgrade numerous regulatory circuits and widely applies to expression control in microbial biotechnology, synthetic biology and materials science., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Pseudouridine (Ψ) is an ubiquitous RNA modification, present in the tRNAs and rRNAs of species across all domains of life. Conserved pseudouridine synthases modify the mRNAs of diverse eukaryotes, but the modification has yet to be identified in bacterial mRNAs. Here, we report the discovery of pseudouridines in mRNA from E. coli. By testing the mRNA modification capacity of all 11 known pseudouridine synthases, we identify RluA as the predominant mRNA-modifying enzyme. RluA, a known tRNA and 23S rRNA pseudouridine synthase, modifies at least 31 of the 44 high-confidence sites we identified in E. coli mRNAs. Using RNA structure probing data to inform secondary structures, we show that the target sites of RluA occur in a common sequence and structural motif comprised of a ΨURAA sequence located in the loop of a short hairpin. This recognition element is shared with previously identified target sites of RluA in tRNAs and rRNA. Overall, our work identifies pseudouridine in key mRNAs and suggests the capacity of Ψ to regulate the transcripts that contain it., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Schaening-Burgos et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In this issue of Structure, Elghondakly et al. 1 present the crystal structure of Thermoanaerobacter pseudethanolicus antiterminator LoaP, a member of a ubiquitous family of NusG transcription factors, bound to its target, a dfn RNA hairpin. LoaP uses RNA as a recognition determinant, which is unique among NusG paralogs and makes unusual contacts in the major groove of the RNA., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

LoaP is a member of the universal NusG protein family. Previously, we reported that unlike other characterized homologs, LoaP binds RNA sequence-specifically, recognizing a stem-loop in the 5'-untranslated region of operons it regulates. To elucidate how this NusG homolog acquired this ability, we now determined the co-crystal structure of Thermoanaerobacter pseudethanolicus LoaP bound to its cognate 26-nucleotide dfn RNA element. Our structure reveals that the LoaP C-terminal KOW domain recognizes the helical portion of the RNA by docking into a broadened major groove, while a protruding β-hairpin of the N-terminal NusG-like domain binds the UNCG tetraloop capping the stem-loop. Major-groove RNA recognition is unusual and is made possible by conserved features of the dfn hairpin. Superposition with structures of other NusG proteins implies that LoaP can bind concurrently to the dfn RNA and the transcription elongation complex, suggesting a new level of co-transcriptional regulation by proteins of this conserved family., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

Synonymous codons were originally viewed as interchangeable, with no phenotypic consequences. However, substantial evidence has now demonstrated that synonymous substitutions can perturb a variety of gene expression and protein homeostasis mechanisms, including translational efficiency, translational fidelity, and cotranslational folding of the encoded protein. To date, most studies of synonymous codon-derived perturbations have focused on effects within a single gene. Here, we show that synonymous codon substitutions made far within the coding sequence of Escherichia coli plasmid-encoded chloramphenicol acetyltransferase ( cat ) can significantly increase expression of the divergent upstream tetracycline resistance gene, tetR . In four out of nine synonymously recoded cat sequences tested, expression of the upstream tetR gene was significantly elevated due to transcription of a long antisense RNA (asRNA) originating from a transcription start site within cat . Surprisingly, transcription of this asRNA readily bypassed the native tet transcriptional repression mechanism. Even more surprisingly, accumulation of the TetR protein correlated with the level of asRNA, rather than total tetR RNA. These effects of synonymous codon substitutions on transcription and translation of a neighboring gene suggest that synonymous codon usage in bacteria may be under selection to both preserve the amino acid sequence of the encoded gene and avoid DNA sequence elements that can significantly perturb expression of neighboring genes. Avoiding such sequences may be especially important in plasmids and prokaryotic genomes, where genes and regulatory elements are often densely packed. Similar considerations may apply to the design of genetic circuits for synthetic biology applications., Competing Interests: Competing interests statement:The authors declare no competing interest.

Among the two Y RNAs in Deinococcus radiodurans, the functional properties of Yrn2 are still not known. Yrn2 although consists of a long stem-loop for Rsr binding, differs from Yrn1 in the effector binding site. An initial study on Yrn2 delineated it to be a UV-induced noncoding RNA. Apart from that Yrn2 has scarcely been investigated. In the current study, we identified Yrn2 as an γ-radiation induced Y RNA, which is also induced upon H 2 O 2 and mitomycin treatment. Ectopically expressed Yrn2 appeared to be nontoxic to the cell growth. An overabundance of Yrn2 was found to ameliorate cell survival under oxidative stress through the detoxification of intracellular reactive oxygen species with a subsequent decrease in total protein carbonylation. A significant accumulation of intracellular Mn(II) with unaltered Fe(II) and Zn(II) with detected while Yrn2 is overabundant in the cells. This study identified the role of a novel Yrn2 under oxidative stress in D. radiodurans., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Bacteria reside in constantly changing environments and require rapid and precise adjustments of gene expression to ensure survival. Small regulatory RNAs (sRNAs) are a crucial element that bacteria utilize to achieve this. sRNAs are short RNA molecules that modulate gene expression usually through base-pairing interactions with target RNAs, primarily mRNAs. These interactions can lead to either negative outcomes such as mRNA degradation or translational repression or positive outcomes such as mRNA stabilization or translation enhancement. In recent years, high-throughput approaches such as RIL-seq (RNA interaction by ligation and sequencing) revolutionized the sRNA field by enabling the identification of sRNA targets on a global scale, unveiling intricate sRNA-RNA networks. In this review, we discuss the insights gained from investigating sRNA-RNA networks in well-studied bacterial species as well as in understudied bacterial species. Having a complete understanding of sRNA-mediated regulation is critical for the development of new strategies for controlling bacterial growth and combating bacterial infections., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Pseudomonas aeruginosa, an opportunistic bacterial pathogen of public health concern, is known for its metabolic versatility, adaptability in harsh environment, and pathogenic aggressiveness. P. aeruginosa relies on various regulatory networks modulated by small non-coding RNAs, which in turn influence different physiological traits such as metabolism, stress response, and pathogenesis. In this study, srbA sRNA has been shown to play a diverse role in regulating cellular metabolism and the production of different virulence factors in P. aeruginosa. srbA was found to control the TCA cycle, a key regulatory pathway for cellular metabolism and energy production, by regulating three main enzymes: citrate synthase (gltA), isocitrate dehydrogenase (icd), and α-ketoglutarate dehydrogenase E1 subunit (sucA) at both the transcriptional and translational levels. By modulating the TCA cycle, srbA could help the bacteria to adapt nutritional stress by lowering energy consumption. Additionally, srbA has been found to differentially regulate production of various virulence factors such as rhamnolipid, elastase, LasA protease, and pyocyanin under both nutrient-rich and nutrient-limiting conditions. It could also influence motilities in P. aeruginosa, linked to biofilm formation and pathogenicity. Thus, srbA might hold a promise in the research area for identifying virulence pathways and developing novel therapeutic targets to combat the global pathogenic threat of P. aeruginosa., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The type VI secretion system (T6SS) is essential for Gram-negative bacteria to antagonize a wide variety of prokaryotic and eukaryotic competitors and thus gain survival advantages. Two sets of T6SS have been found in Vibrio fluvialis , namely VflT6SS1 and VflT6SS2, among which VflT6SS2 is functionally expressed. The CqsA/LuxS-HapR quorum sensing (QS) system with CAI-1 and AI-2 as signal molecules can regulate VflT6SS2 by regulators LuxO and HapR, with LuxO repressing while HapR activating VflT6SS2. Quorum regulatory small RNAs (Qrr sRNAs) are Hfq-dependent trans -encoded sRNAs that control Vibrio quorum sensing. In V. fluvialis , Qrr sRNAs have not been characterized and their regulatory function is unknown. In this study, we first identified four Qrr sRNAs in V. fluvialis and demonstrated that these Qrr sRNAs are regulated by LuxO and involved in the modulation of VflT6SS2 function. On the one hand, Qrr sRNAs act on HapR, the activator of both the major and the auxiliary clusters of VflT6SS2, and then indirectly repress VflT6SS2. On the other hand, they directly repress VflT6SS2 by acting on tssB 2 and tssD 2&#95;a, the first gene of the major cluster and the highly transcriptional one among the two units of the first auxiliary cluster, respectively. Our results give insights into the role of Qrr sRNAs in CAI-1/AI-2 based QS and VflT6SS2 modulation in V . fluvialis and further enhance understandings of the network between QS and T6SS regulation in Vibrio species.

Listeria pathogenicity island 1 (LIPI-1) is a genetic region containing a cluster of genes essential for virulence of the bacterial pathogen Listeria monocytogenes . Main virulence factors in LIPI-1 include long 5' untranslated regions (5'UTRs), among which is Rli51, a small RNA (sRNA) in the 5'UTR of the Zn-metalloprotease-coding mpl . So far, Rli51 function and molecular mechanisms have remained obscure. Here, we show that Rli51 exhibits a dual mechanism of regulation, functioning as a cis - and as a trans -acting sRNA. Under nutrient-rich conditions, rli51-mpl transcription is prematurely terminated, releasing a short 121-nucleotide-long sRNA. Rli51 is predicted to function as a transcription attenuator that can fold into either a terminator or a thermodynamically more stable antiterminator. We show that the sRNA Rli21/RliI binds to a single-stranded RNA loop in Rli51, which is essential to mediate premature transcription termination, suggesting that sRNA binding could stabilize the terminator fold. During intracellular infection, rli51 transcription is increased, which generates a higher abundance of the short Rli51 sRNA and allows for transcriptional read-through into mpl . Comparative intracellular bacterial transcriptomics in rli51 -null mutants and the wild-type reference strain EGD-e suggests that Rli51 upregulates iron-scavenging proteins and downregulates virulence factors from LIPI-1. MS2 affinity purification confirmed that Rli51 binds transcripts of the heme-binding protein Lmo2186 and Lmo0937 in vivo. These results prove that Rli51 functions as a trans -acting sRNA in intracellular bacteria. Our research shows a growth condition-dependent mechanism of regulation for Rli51, preventing unintended mpl transcription in extracellular bacteria and regulating genes important for virulence in intracellular bacteria.

Small RNAs (sRNAs) are major regulators of gene expression in bacteria, exerting their regulation primarily via base pairing with their target transcripts and modulating translation. Accumulating evidence suggest that sRNAs can also affect the stability of their target transcripts by altering their accessibility to endoribonucleases. Yet, the effects of sRNAs on transcript stability and the mechanisms underlying them have not been studied in wide scale. Here we employ large-scale RNA-seq-based methodologies in the model bacterium Escherichia coli to quantitatively study the functional interaction between a sRNA and an endoribonuclease in regulating gene expression, using the well-established sRNA, GcvB, and the major endoribonuclease, RNase E. Studying single and double mutants of gcvB and rne and analysing their RNA-seq results by the Double Mutant Cycle approach, we infer distinct modes of the interplay between GcvB and RNase E. Transcriptome-wide mapping of RNase E cleavage sites provides further support to the results of the RNA-seq analysis, identifying cleavage sites in targets in which the functional interaction between GcvB and RNase E is evident. Together, our results indicate that the most dominant mode of GcvB-RNase E functional interaction is GcvB enhancement of RNase E cleavage, which varies in its magnitude between different targets., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The ompD transcript, encoding an outer membrane porin in Salmonella, harbors a controlling element in its coding region that base-pairs imperfectly with a 'seed' region of the small regulatory RNA (sRNA) MicC. When tagged with the sRNA, the ompD mRNA is cleaved downstream of the pairing site by the conserved endoribonuclease RNase E, leading to transcript destruction. We observe that the sRNA-induced cleavage site is accessible to RNase E in vitro upon recruitment of ompD into the 30S translation pre-initiation complex (PIC) in the presence of the degradosome components. Evaluation of substrate accessibility suggests that the paused 30S PIC presents the mRNA for targeted recognition and degradation. Ribonuclease activity on PIC-bound ompD is critically dependent on the recruitment of RNase E into the multi-enzyme RNA degradosome, and our data suggest a process of substrate capture and handover to catalytic sites within the degradosome, in which sequential steps of seed matching and duplex remodelling contribute to cleavage efficiency. Our findings support a putative mechanism of surveillance at translation that potentially terminates gene expression efficiently and rapidly in response to signals provided by regulatory RNA., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Although RNA structures play important roles in regulating gene expression, the mechanism and function of mRNA folding in plant bacterial pathogens remain elusive. Therefore, we perform dimethyl sulfate sequencing (DMS-seq) on the Pseudomonas syringae under nutrition-rich and -deficient conditions, revealing that the mRNA structure changes substantially in the minimal medium (MM) that tunes global translation efficiency (TE), thereby inducing virulence. This process is led by the increased expression of hfq, which is directly activated by transcription regulators RpoS and CysB. The co-occurrence of Hfq and RpoS in diverse bacteria and the deep conservation of Hfq Y25 is critical for RNA-mediated regulation and implicates the wider biological importance of mRNA structure and feedback loops in the control of global gene expression., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Hypervirulent Klebsiella pneumoniae (hvKp) can infect healthy individuals, in contrast to classical strains that commonly cause nosocomial infections. The recent convergence of hypervirulence with carbapenem-resistance in K. pneumoniae can potentially create 'superbugs' that are challenging to treat. Understanding virulence regulation of hvKp is thus critical. Accumulating evidence suggest that posttranscriptional regulation by small RNAs (sRNAs) plays a role in bacterial virulence, but it has hardly been studied in K. pneumoniae. We applied RIL-seq to a prototypical clinical isolate of hvKp to unravel the Hfq-dependent RNA-RNA interaction (RRI) network. The RRI network is dominated by sRNAs, including predicted novel sRNAs, three of which we validated experimentally. We constructed a stringent subnetwork composed of RRIs that involve at least one hvKp virulence-associated gene and identified the capsule gene loci as a hub target where multiple sRNAs interact. We found that the sRNA OmrB suppressed both capsule production and hypermucoviscosity when overexpressed. Furthermore, OmrB base-pairs within kvrA coding region and partially suppresses translation of the capsule regulator KvrA. This agrees with current understanding of capsule as a major virulence and fitness factor. It emphasizes the intricate regulatory control of bacterial phenotypes by sRNAs, particularly of genes critical to bacterial physiology and virulence., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Bacterial gene expression is a complex process involving extensive regulatory mechanisms. Along with growing interests in this field, Nanopore Direct RNA Sequencing (DRS) provides a promising platform for rapid and comprehensive characterization of bacterial RNA biology. However, the DRS of bacterial RNA is currently deficient in the yield of mRNA-mapping reads and has yet to be exploited for transcriptome-wide RNA modification mapping. Here, we showed that pre-processing of bacterial total RNA (size selection followed by ribosomal RNA depletion and polyadenylation) guaranteed high throughputs of sequencing data and considerably increased the amount of mRNA reads. This way, complex transcriptome architectures were reconstructed for Escherichia coli and Staphylococcus aureus and extended the boundaries of 225 known E. coli operons and 89 defined S. aureus operons. Utilizing unmodified in vitro-transcribed (IVT) RNA libraries as a negative control, several Nanopore-based computational tools globally detected putative modification sites in the E. coli and S. aureus transcriptomes. Combined with Next-Generation Sequencing-based N6-methyladenosine (m6A) detection methods, 75 high-confidence m6A candidates were identified in the E. coli protein-coding transcripts, while none were detected in S. aureus. Altogether, we demonstrated the potential of Nanopore DRS in systematic and convenient transcriptome and epitranscriptome analysis., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)