Regulatory sRNAs Research Articles

Identification of novel small RNAs and characterization of the 6S RNA of Coxiella burnetii.

Coxiella burnetii, an obligate intracellular bacterial pathogen that causes Q fever, undergoes a biphasic developmental cycle that alternates between a metabolically-active large cell variant (LCV) and a dormant small cell variant (SCV). As such, the bacterium undoubtedly employs complex modes of regulating its lifecycle, metabolism and pathogenesis. Small RNAs (sRNAs) have been shown to play important regulatory roles in controlling metabolism and virulence in several pathogenic bacteria. We hypothesize that sRNAs are involved in regulating growth and development of C. burnetii and its infection of host cells. To address the hypothesis and identify potential sRNAs, we subjected total RNA isolated from Coxiella cultured axenically and in Vero host cells to deep-sequencing. Using this approach, we identified fifteen novel C. burnetii sRNAs (CbSRs). Fourteen CbSRs were validated by Northern blotting. Most CbSRs showed differential expression, with increased levels in LCVs. Eight CbSRs were upregulated (≥2-fold) during intracellular growth as compared to growth in axenic medium. Along with the fifteen sRNAs, we also identified three sRNAs that have been previously described from other bacteria, including RNase P RNA, tmRNA and 6S RNA. The 6S regulatory sRNA of C. burnetii was found to accumulate over log phase-growth with a maximum level attained in the SCV stage. The 6S RNA-encoding gene (ssrS) was mapped to the 5′ UTR of ygfA; a highly conserved linkage in eubacteria. The predicted secondary structure of the 6S RNA possesses three highly conserved domains found in 6S RNAs of other eubacteria. We also demonstrate that Coxiella’s 6S RNA interacts with RNA polymerase (RNAP) in a specific manner. Finally, transcript levels of 6S RNA were found to be at much higher levels when Coxiella was grown in host cells relative to axenic culture, indicating a potential role in regulating the bacterium’s intracellular stress response by interacting with RNAP during transcription.

Identification of Bacteriophage-Encoded Anti-sRNAs in Pathogenic Escherichia coli

SummaryIn bacteria, Hfq is a core RNA chaperone that catalyzes the interaction of mRNAs with regulatory small RNAs (sRNAs). To determine in vivo RNA sequence requirements for Hfq interactions, and to study riboregulation in a bacterial pathogen, Hfq was UV crosslinked to RNAs in enterohemorrhagic Escherichia coli (EHEC). Hfq bound repeated trinucleotide motifs of A-R-N (A-A/G-any nucleotide) often associated with the Shine-Dalgarno translation initiation sequence in mRNAs. These motifs overlapped or were adjacent to the mRNA sequences bound by sRNAs. In consequence, sRNA-mRNA duplex formation will displace Hfq, promoting recycling. Fifty-five sRNAs were identified within bacteriophage-derived regions of the EHEC genome, including some of the most abundant Hfq-interacting sRNAs. One of these (AgvB) antagonized the function of the core genome regulatory sRNA, GcvB, by mimicking its mRNA substrate sequence. This bacteriophage-encoded “anti-sRNA” provided EHEC with a growth advantage specifically in bovine rectal mucus recovered from its primary colonization site in cattle.

Hfq protein deficiency in Escherichia coli affects ColE1-like but not λ plasmid DNA replication

Hfq is a nucleic acid-binding protein involved in controlling several aspects of RNA metabolism. It achieves this regulatory function by modulating the translational activity and stability of different mRNAs, generally via interactions with stress-related small regulatory sRNAs. However, besides its role in the coordination of translation of bacterial mRNA, Hfq is also a nucleoid-associated DNA-binding protein. Motivated by the above property of Hfq, we investigated if hfq gene mutation has implications for the regulation of DNA replication. Efficiency of ColE1-like (pMB1- and p15A replicons) and bacteriophage λ-derived plasmids’ replication has been investigated in wild-type strain and otherwise isogenic hfq mutant of Escherichia coli. Significant differences in plasmid amount and kinetics of plasmid DNA synthesis were observed between the two tested bacterial hosts for ColE1-like replicons, but not for λ plasmid. Furthermore, ColE1-like plasmids replicated more efficiently in wild-type cells than in the hfq mutant in the early exponential phase of growth, but less efficiently in late exponential and early stationary phases. Hfq levels in the wild-type host, estimated by Western-blotting, were increased at the latter phases relative to the former one. Moreover, effects of the hfq mutation on ColE1-like plasmid replication were impaired in the absence of the rom gene, coding for a protein enhancing RNA I–RNA II interactions during the control of the replication initiation. These results are discussed in the light of a potential mechanism by which Hfq protein may influence replication of some, but not all, replicons in E. coli.

Small RNAs in the Vibrionaceae: an ocean still to be explored

In bacteria, the discovery of noncoding small RNAs (sRNAs) as modulators of gene expression in response to environmental signals has brought new insights into bacterial gene regulation, including control of pathogenicity. The Vibrionaceae constitute a family of marine bacteria of which many are responsible for infections affecting not only humans, such as Vibrio cholerae but also fish and marine invertebrates, representing the major cause of mortality in farmed marine species. They are able to colonize many habitats, existing as planktonic forms, in biofilms or associated with various hosts. This high adaptability is linked to their capacity to generate genetic diversity, in part through lateral gene transfer, but also by varying gene expression control. In the recent years, several major studies have illustrated the importance of small regulatory sRNAs in the Vibrionaceae for the control of pathogenicity and adaptation to environment and nutrient sources such as chitin, especially in V. cholerae and Vibrio harveyi. The existence of a complex regulatory network controlled by quorum sensing has been demonstrated in which sRNAs play central roles. This review covers major advances made in the discovery and elucidation of functions of Vibrionaceae sRNAs within the last 10 years.

Identification of novel sRNAs in mycobacterial species.

Bacterial small RNAs (sRNAs) are short transcripts that typically do not encode proteins and often act as regulators of gene expression through a variety of mechanisms. Regulatory sRNAs have been identified in many species, including Mycobacterium tuberculosis, the causative agent of tuberculosis. Here, we use a computational algorithm to predict sRNA candidates in the mycobacterial species M. smegmatis and M. bovis BCG and confirmed the expression of many sRNAs using Northern blotting. Thus, we have identified 17 and 23 novel sRNAs in M. smegmatis and M. bovis BCG, respectively. We have also applied a high-throughput technique (Deep-RACE) to map the 5′ and 3′ ends of many of these sRNAs and identified potential regulators of sRNAs by analysis of existing ChIP-seq datasets. The sRNAs identified in this work likely contribute to the unique biology of mycobacteria.

Physiological Consequences of Multiple-Target Regulation by the Small RNA SgrS in Escherichia coli

Cells use complex mechanisms to regulate glucose transport and metabolism to achieve optimal energy and biomass production while avoiding accumulation of toxic metabolites. Glucose transport and glycolytic metabolism carry the risk of the buildup of phosphosugars, which can inhibit growth at high concentrations. Many enteric bacteria cope with phosphosugar accumulation and associated stress (i.e., sugar-phosphate stress) by producing a small RNA (sRNA) regulator, SgrS, which decreases phosphosugar accumulation in part by repressing translation of sugar transporter mRNAs (ptsG and manXYZ) and enhancing translation of a sugar phosphatase mRNA (yigL). Despite a molecular understanding of individual target regulation by SgrS, previously little was known about how coordinated regulation of these multiple targets contributes to the rescue of cell growth during sugar-phosphate stress. This study examines how SgrS regulation of different targets impacts growth under different nutritional conditions when sugar-phosphate stress is induced. The severity of stress-associated growth inhibition depended on nutrient availability. Stress in nutrient-rich media necessitated SgrS regulation of only sugar transporter mRNAs (ptsG or manXYZ). However, repression of transporter mRNAs was insufficient for growth rescue during stress in nutrient-poor media; here SgrS regulation of the phosphatase (yigL) and as-yet-undefined targets also contributed to growth rescue. The results of this study imply that regulation of only a subset of an sRNA's targets may be important in a given environment. Further, the results suggest that SgrS and perhaps other sRNAs are flexible regulators that modulate expression of multigene regulons to allow cells to adapt to an array of stress conditions.

Genome-Wide Identification of Regulatory RNAs in the Human Pathogen Clostridium difficile

Clostridium difficile is an emergent pathogen, and the most common cause of nosocomial diarrhea. In an effort to understand the role of small noncoding RNAs (sRNAs) in C. difficile physiology and pathogenesis, we used an in silico approach to identify 511 sRNA candidates in both intergenic and coding regions. In parallel, RNA–seq and differential 5′-end RNA–seq were used for global identification of C. difficile sRNAs and their transcriptional start sites at three different growth conditions (exponential growth phase, stationary phase, and starvation). This global experimental approach identified 251 putative regulatory sRNAs including 94 potential trans riboregulators located in intergenic regions, 91 cis-antisense RNAs, and 66 riboswitches. Expression of 35 sRNAs was confirmed by gene-specific experimental approaches. Some sRNAs, including an antisense RNA that may be involved in control of C. difficile autolytic activity, showed growth phase-dependent expression profiles. Expression of each of 16 predicted c-di-GMP-responsive riboswitches was observed, and experimental evidence for their regulatory role in coordinated control of motility and biofilm formation was obtained. Finally, we detected abundant sRNAs encoded by multiple C. difficile CRISPR loci. These RNAs may be important for C. difficile survival in bacteriophage-rich gut communities. Altogether, this first experimental genome-wide identification of C. difficile sRNAs provides a firm basis for future RNome characterization and identification of molecular mechanisms of sRNA–based regulation of gene expression in this emergent enteropathogen.

Small Regulatory RNAs in the Control of Motility and Biofilm Formation in E. coli and Salmonella

Biofilm formation in Escherichia coli and other enteric bacteria involves the inverse regulation of the synthesis of flagella and biofilm matrix components such as amyloid curli fibres, cellulose, colanic acid and poly-N-acetylglucosamine (PGA). Physiologically, these processes reflect the transition from growth to stationary phase. At the molecular level, they are tightly controlled by various sigma factors competing for RNA polymerase, a series of transcription factors acting in hierarchical regulatory cascades and several nucleotide messengers, including cyclic-di-GMP. In addition, a surprisingly large number of small regulatory RNAs (sRNAs) have been shown to directly or indirectly modulate motility and/or biofilm formation. This review aims at giving an overview of these sRNA regulators and their impact in biofilm formation in E. coli and Salmonella. Special emphasis will be put on sRNAs, that have known targets such as the mRNAs of the flagellar master regulator FlhDC, the stationary phase sigma factor σS (RpoS) and the key biofilm regulator CsgD that have recently been shown to act as major hubs for regulation by multiple sRNAs.

Quantitative Proteomic Analysis of the Hfq-Regulon in Sinorhizobium meliloti 2011

Riboregulation stands for RNA-based control of gene expression. In bacteria, small non-coding RNAs (sRNAs) are a major class of riboregulatory elements, most of which act at the post-transcriptional level by base-pairing target mRNA genes. The RNA chaperone Hfq facilitates antisense interactions between target mRNAs and regulatory sRNAs, thus influencing mRNA stability and/or translation rate. In the α-proteobacterium Sinorhizobium meliloti strain 2011, the identification and detection of multiple sRNAs genes and the broadly pleitropic phenotype associated to the absence of a functional Hfq protein both support the existence of riboregulatory circuits controlling gene expression to ensure the fitness of this bacterium in both free living and symbiotic conditions. In order to identify target mRNAs subject to Hfq-dependent riboregulation, we have compared the proteome of an hfq mutant and the wild type S. meliloti by quantitative proteomics following protein labelling with 15N. Among 2139 univocally identified proteins, a total of 195 proteins showed a differential abundance between the Hfq mutant and the wild type strain; 65 proteins accumulated ≥2-fold whereas 130 were downregulated (≤0.5-fold) in the absence of Hfq. This profound proteomic impact implies a major role for Hfq on regulation of diverse physiological processes in S. meliloti, from transport of small molecules to homeostasis of iron and nitrogen. Changes in the cellular levels of proteins involved in transport of nucleotides, peptides and amino acids, and in iron homeostasis, were confirmed with phenotypic assays. These results represent the first quantitative proteomic analysis in S. meliloti. The comparative analysis of the hfq mutant proteome allowed identification of novel strongly Hfq-regulated genes in S. meliloti.

Identification of novel growth phase- and media-dependent small non-coding RNAs in Streptococcus pyogenes M49 using intergenic tiling arrays

BackgroundSmall non-coding RNAs (sRNAs) have attracted attention as a new class of gene regulators in both eukaryotes and bacteria. Genome-wide screening methods have been successfully applied in Gram-negative bacteria to identify sRNA regulators. Many sRNAs are well characterized, including their target mRNAs and mode of action. In comparison, little is known about sRNAs in Gram-positive pathogens. In this study, we identified novel sRNAs in the exclusively human pathogen Streptococcus pyogenes M49 (Group A Streptococcus, GAS M49), employing a whole genome intergenic tiling array approach. GAS is an important pathogen that causes diseases ranging from mild superficial infections of the skin and mucous membranes of the naso-pharynx, to severe toxic and invasive diseases.ResultsWe identified 55 putative sRNAs in GAS M49 that were expressed during growth. Of these, 42 were novel. Some of the newly-identified sRNAs belonged to one of the common non-coding RNA families described in the Rfam database. Comparison of the results of our screen with the outcome of two recently published bioinformatics tools showed a low level of overlap between putative sRNA genes. Previously, 40 potential sRNAs have been reported to be expressed in a GAS M1T1 serotype, as detected by a whole genome intergenic tiling array approach. Our screen detected 12 putative sRNA genes that were expressed in both strains. Twenty sRNA candidates appeared to be regulated in a medium-dependent fashion, while eight sRNA genes were regulated throughout growth in chemically defined medium. Expression of candidate genes was verified by reverse transcriptase-qPCR. For a subset of sRNAs, the transcriptional start was determined by 5′ rapid amplification of cDNA ends-PCR (RACE-PCR) analysis.ConclusionsIn accord with the results of previous studies, we found little overlap between different screening methods, which underlines the fact that a comprehensive analysis of sRNAs expressed by a given organism requires the complementary use of different methods and the investigation of several environmental conditions. Despite a high conservation of sRNA genes within streptococci, the expression of sRNAs appears to be strain specific.

Genome‐wide identification of novel small RNAs in Pseudomonas aeruginosa

Bacterial small regulatory RNAs (sRNAs) function in post-transcriptional control of gene expression and control a variety of processes including metabolic reactions, stress responses and pathogenesis in response to environmental signals. A variety of approaches have been used previously to identify 44 sRNAs in the opportunistic human pathogen Pseudomonas aeruginosa. In this work, RNA sequencing (RNA-seq) is used to identify novel transcripts in P.aeruginosa involving a combination of three different sequencing libraries. Almost all known sRNAs and over 500 novel intergenic sRNAs are identified with this approach. Although the use of three libraries increased the number of novel transcripts identified, there were significant differences in the subset of transcripts detected in each library, underscoring the importance of library preparation strategy and relative sRNA abundance for successful sRNA detection. Nearly 90% of the novel sRNAs have no orthologous bacterial sequences outside of P.aeruginosa, supporting a limited degree of sequence conservation and rapid evolution of sRNAs at the species level. We anticipate that the data will be useful for the study of regulatory sRNAs in bacteria and that the approach described here may be applied to identify sRNAs in any bacterium under different growth and stress conditions.

Non-coding RNAs in marine Synechococcus and their regulation under environmentally relevant stress conditions

Regulatory small RNAs (sRNAs) have crucial roles in the adaptive responses of bacteria to changes in the environment. Thus far, potential regulatory RNAs have been studied mainly in marine picocyanobacteria in genetically intractable Prochlorococcus, rendering their molecular analysis difficult. Synechococcus sp. WH7803 is a model cyanobacterium, representative of the picocyanobacteria from the mesotrophic areas of the ocean. Similar to the closely related Prochlorococcus it possesses a relatively streamlined genome and a small number of genes, but is genetically tractable. Here, a comparative genome analysis was performed for this and four additional marine Synechococcus to identify the suite of possible sRNAs and other RNA elements. Based on the prediction and on complementary microarray profiling, we have identified several known as well as 32 novel sRNAs. Some sRNAs overlap adjacent coding regions, for instance for the central photosynthetic gene psbA. Several of these novel sRNAs responded specifically to environmentally relevant stress conditions. Among them are six sRNAs changing their accumulation level under cold stress, six responding to high light and two to iron limitation. Target predictions suggested genes encoding components of the light-harvesting apparatus as targets of sRNAs originating from genomic islands and that one of the iron-regulated sRNAs might be a functional homolog of RyhB. These data suggest that marine Synechococcus mount adaptive responses to these different stresses involving regulatory sRNAs.

RNA mimicry, a decoy for regulatory proteins

Small non-coding RNA molecules (sRNA) are key regulators participating in complex networks, which adapt metabolism in response to environmental changes. In this issue of Molecular Microbiology, and in a related paper in Proc. Natl. Acad. Sci. USA, Moreno et al. (2011) and Sonnleitner et al. (2009) report on novel sRNAs, which act as decoys to inhibit the activity of the master post-transcriptional regulatory protein Crc. Crc is a key protein involved in carbon catabolite repression that optimizes metabolism improving the adaptation of the bacteria to their diverse habitats. Crc is a novel RNA-binding protein that regulates translation of multiple target mRNAs. Two regulatory sRNAs in Pseudomonas putida mimic the natural mRNA targets of Crc and counteract the action of Crc by sequestrating the protein when catabolite repression is absent. Crc trapping by a sRNA is a mechanism reminiscent to the regulation of the repressor of secondary metabolites (RsmA) in Pseudomonas, and highlights the suitability of RNA-dependent regulation to rapidly adjust cell growth in response to environmental changes.

Dual-function RNA regulators in bacteria

The importance of small RNA (sRNA) regulators has been recognized across all domains of life. In bacteria, sRNAs typically control the expression of virulence and stress response genes via antisense base pairing with mRNA targets. Originally dubbed “non-coding RNAs,” a number of bacterial antisense sRNAs have been found to encode functional proteins. Although very few of these dual-function sRNAs have been characterized, they have been found in both gram-negative and gram-positive organisms. Among the few known examples, the functions and mechanisms of regulation by dual-function sRNAs are variable. Some dual-function sRNAs depend on the RNA chaperone Hfq for base pairing-dependent regulation (riboregulation); this feature appears so far exclusive to gram-negative bacterial sRNAs. Other variations can be found in the spatial organization of the coding region with respect to the riboregulation determinants. How the functions of encoded proteins relate to riboregulation is for the most part not understood. However, in one case it appears that there is physiological redundancy between protein and riboregulation functions. This mini-review focuses on the two best-studied bacterial dual-function sRNAs: RNAIII from Staphylococcus aureus and SgrS from Escherichia coli and includes a discussion of what is known about the structure, function and physiological roles of these sRNAs as well as what questions remain outstanding.

Computational approach to ensure the stability of the favorable ATP binding site in E. coli Hfq

Bacterial Hfq is a highly conserved thermostable protein of about 10 kDa. The Hfq protein was discovered in 1968 as an E. coli host factor that was essential for replication of the bacteriophage Qβ. It is now clear that Hfq has many important physiological roles. In E. coli, Hfq mutants show a multiple stress response related phenotypes. Hfq is now known to regulate the translation of two major stress transcription factors RpoS and RpoE in Enterobacteria and mediates its plieotrophic effects through several mechanisms. It interacts with regulatory sRNA and facilitates their antisense interaction with their targets. It also acts independently to modulate mRNA decay and in addition acts as a repressor of mRNA translation. Recent paper from Arluison et al. [9] provided the first evidence indicating that Hfq is an ATP-binding protein. They determined a plausible ATP-binding site in Hfq and tested Hfq's ATP-binding affinity and stoichiometry. Experimental data suggest that the ATP-binding by the Hfq–RNA complex results in its significant destabilization of the protein and the result also proves important role of Tyr25 that flanks the cleft and stabilizes the adenine portion of ATP, possibly via aromatic stacking. In our study, the ATP molecule was docked into the predicted binding cleft using GOLD docking software. The binding nature of ATP and its effect on Hfq–RNA complex was studied using molecular dynamics simulations. Importance of Tyr25 residue was monitored and revealed using mutational study on the modeled systems. Our data and the corresponding results point to one of Hfq functional structural consequences due to ATP binding and Tyr25Ala mutation.

Identification of small RNAs in Francisella tularensis

BackgroundRegulation of bacterial gene expression by small RNAs (sRNAs) have proved to be important for many biological processes. Francisella tularensis is a highly pathogenic Gram-negative bacterium that causes the disease tularaemia in humans and animals. Relatively little is known about the regulatory networks existing in this organism that allows it to survive in a wide array of environments and no sRNA regulators have been identified so far.ResultsWe have used a combination of experimental assays and in silico prediction to identify sRNAs in F. tularensis strain LVS. Using a cDNA cloning and sequencing approach we have shown that F. tularensis expresses homologues of several sRNAs that are well-conserved among diverse bacteria. We have also discovered two abundant putative sRNAs that share no sequence similarity or conserved genomic context with any previously annotated regulatory transcripts. Deletion of either of these two loci led to significant changes in the expression of several mRNAs that likely include the cognate target(s) of these sRNAs. Deletion of these sRNAs did not, however, significantly alter F. tularensis growth under various stress conditions in vitro, its replication in murine cells, or its ability to induce disease in a mouse model of F. tularensis infection. We also conducted a genome-wide in silico search for intergenic loci that suggests F. tularensis encodes several other sRNAs in addition to the sRNAs found in our experimental screen.ConclusionOur findings suggest that F. tularensis encodes a significant number of non-coding regulatory RNAs, including members of well conserved families of structural and housekeeping RNAs and other poorly conserved transcripts that may have evolved more recently to help F. tularensis deal with the unique and diverse set of environments with which it must contend.

Bacterial Small RNA Regulators: Versatile Roles and Rapidly Evolving Variations

Small RNA regulators (sRNAs) have been identified in a wide range of bacteria and found to play critical regulatory roles in many processes. The major families of sRNAs include true antisense RNAs, synthesized from the strand complementary to the mRNA they regulate, sRNAs that also act by pairing but have limited complementarity with their targets, and sRNAs that regulate proteins by binding to and affecting protein activity. The sRNAs with limited complementarity are akin to eukaryotic microRNAs in their ability to modulate the activity and stability of multiple mRNAs. In many bacterial species, the RNA chaperone Hfq is required to promote pairing between these sRNAs and their target mRNAs. Understanding the evolution of regulatory sRNAs remains a challenge; sRNA genes show evidence of duplication and horizontal transfer but also could be evolved from tRNAs, mRNAs or random transcription.

Regulatory Mechanisms of Exoribonuclease PNPase and Regulatory Small RNA on T3SS of Dickeya dadantii

The type III secretion system (T3SS) is an essential virulence factor for many bacterial pathogens. Polynucleotide phosphorylase (PNPase) is one of the major exoribonucleases in bacteria and plays important roles in mRNA degradation, tRNA processing, and small RNA (sRNA) turnover. In this study, we showed that PNPase downregulates the transcription of T3SS structural and effector genes of the phytopathogenic bacterium Dickeya dadantii. This negative regulation of T3SS by PNPase occurs by repressing the expression of hrpL, encoding a master regulator of T3SS in D. dadantii. By reducing rpoN mRNA stability, PNPase downregulates the transcription of hrpL, which leads to a reduction in T3SS gene expression. Moreover, we have found that PNPase downregulates T3SS by decreasing hrpL mRNA stability. RsmB, a regulatory sRNA, enhances hrpL mRNA stability in D. dadantii. Our results suggest that PNPase decreases the amount of functional RsmB transcripts that could result in reduction of hrpL mRNA stability. In addition, bistable gene expression (differential expression of a single gene that creates two distinct subpopulations) of hrpA, hrpN, and dspE was observed in D. dadantii under in vitro conditions. Although PNPase regulates the proportion of cells in the high state and the low state of T3SS gene expression, it appears that PNPase is not the key switch that triggers the bistable expression patterns of T3SS genes.

A dual‐function sRNA from B. subtilis: SR1 acts as a peptide encoding mRNA on the gapA operon

Small non-coding RNAs (sRNAs) have been found to regulate gene expression in all three kingdoms of life. So far, relatively little is known about sRNAs from Gram-positive bacteria. SR1 is a regulatory sRNA from the Bacillus subtilis chromosome that inhibits by base-pairing translation initiation of ahrC mRNA encoding a transcriptional activator of the arginine catabolic operons. Here we present a novel target of SR1, the glycolytic gapA operon. Both microarray and Northern blot analyses show that the amount of gapA operon mRNA is significantly higher in the presence of SR1 when cells were grown in complex medium until stationary phase. Translational lacZ fusions and toeprinting analyses demonstrate that SR1 does not promote translation of gapA mRNA. By contrast, the half-life of gapA operon mRNA is strongly reduced in the sr1 knockout strain. SR1 does not act as a base-pairing sRNA on gapA operon mRNA. Instead, we demonstrate that the 39 aa peptide encoded by SR1, SR1P, is responsible for the effect of SR1 on the gapA operon. We show that SR1P binds GapA, thereby stabilizing the gapA operon mRNA by a hitherto unknown mechanism. SR1 is the first dual-function sRNA found in B. subtilis.

Translational Regulation of Gene Expression by an Anaerobically Induced Small Non-coding RNA in Escherichia coli

Small non-coding RNAs (sRNA) have emerged as important elements of gene regulatory circuits. In enterobacteria such as Escherichia coli and Salmonella many of these sRNAs interact with the Hfq protein, an RNA chaperone similar to mammalian Sm-like proteins and act in the post-transcriptional regulation of many genes. A number of these highly conserved ribo-regulators are stringently regulated at the level of transcription and are part of major regulons that deal with the immediate response to various stress conditions, indicating that every major transcription factor may control the expression of at least one sRNA regulator. Here, we extend this view by the identification and characterization of a highly conserved, anaerobically induced small sRNA in E. coli, whose expression is strictly dependent on the anaerobic transcriptional fumarate and nitrate reductase regulator (FNR). The sRNA, named FnrS, possesses signatures of base-pairing RNAs, and we show by employing global proteomic and transcriptomic profiling that the expression of multiple genes is negatively regulated by the sRNA. Intriguingly, many of these genes encode enzymes with "aerobic" functions or enzymes linked to oxidative stress. Furthermore, in previous work most of the potential target genes have been shown to be repressed by FNR through an undetermined mechanism. Collectively, our results provide insight into the mechanism by which FNR negatively regulates genes such as sodA, sodB, cydDC, and metE, thereby demonstrating that adaptation to anaerobic growth involves the action of a small regulatory RNA.