A Repeat-Associated Small RNA Controls the Major Virulence Factors of Helicobacter pylori.

Many bacterial pathogens regulate their virulence genes via phase variation, whereby length-variable simple sequence repeats control the transcription or coding potential of those genes. Here, we have exploited this relationship between DNA structure and physiological function to discover a globally acting small RNA (sRNA) regulator of virulence in the gastric pathogen Helicobacter pylori. Our study reports the first sRNA whose expression is affected by a variable thymine (T) stretch in its promoter. We show the sRNA post-transcriptionally represses multiple major pathogenicity factors of H. pylori, including CagA and VacA, by base pairing to their mRNAs. We further demonstrate transcription of the sRNA is regulated by the nickel-responsive transcriptional regulator NikR (thus named NikS for nickel-regulated sRNA), thereby linking virulence factor regulation to nickel concentrations. Using in-vitro infection experiments, we demonstrate NikS affects host cell internalization and epithelial barrier disruption. Together, our results show NikS is a phase-variable, post-transcriptional global regulator of virulence properties in H. pylori.

tRNA epitranscriptome determines pathogenicity of the opportunistic pathogen Pseudomonas aeruginosa.

The success of bacterial pathogens depends on the coordinated expression of virulence determinants. Regulatory circuits that drive pathogenesis are complex, multilayered, and incompletely understood. Here, we reveal that alterations in tRNA modifications define pathogenic phenotypes in the opportunistic pathogen Pseudomonas aeruginosa. We demonstrate that the enzymatic activity of GidA leads to the introduction of a carboxymethylaminomethyl modification in selected tRNAs. Modifications at the wobble uridine base (cmnm5U34) of the anticodon drives translation of transcripts containing rare codons. Specifically, in P. aeruginosa the presence of GidA-dependent tRNA modifications modulates expression of genes encoding virulence regulators, leading to a cellular proteomic shift toward pathogenic and well-adapted physiological states. Our approach of profiling the consequences of chemical tRNA modifications is general in concept. It provides a paradigm of how environmentally driven tRNA modifications govern gene expression programs and regulate phenotypic outcomes responsible for bacterial adaption to challenging habitats prevailing in the host niche.

CRISPR RNA-Dependent Binding and Cleavage of Endogenous RNAs by the Campylobacter jejuni Cas9.

Cas9 nucleases naturally utilize CRISPR RNAs (crRNAs) to silence foreign double-stranded DNA. While recent work has shown that some Cas9 nucleases can also target RNA, RNA recognition has required nuclease modifications or accessory factors. Here, we show that the Campylobacter jejuni Cas9 (CjCas9) can bind and cleave complementary endogenous mRNAs in a crRNA-dependent manner. Approximately 100 transcripts co-immunoprecipitated with CjCas9 and generally can be subdivided through their base-pairing potential to the four crRNAs. A subset of these RNAs was cleaved around or within the predicted binding site. Mutational analyses revealed that RNA binding was crRNA and tracrRNA dependent and that target RNA cleavage required the CjCas9 HNH domain. We further observed that RNA cleavage was PAM independent, improved with greater complementarity between the crRNA and the RNA target, and was programmable in vitro. These findings suggest that C. jejuni Cas9 is a promiscuous nuclease that can coordinately target both DNA and RNA.

RiboReport - benchmarking tools for ribosome profiling-based identification of open reading frames in bacteria.

Small proteins encoded by short open reading frames (ORFs) with 50 codons or fewer are emerging as an important class of cellular macromolecules in diverse organisms. However, they often evade detection by proteomics or in silico methods. Ribosome profiling (Ribo-seq) has revealed widespread translation in genomic regions previously thought to be non-coding, driving the development of ORF detection tools using Ribo-seq data. However, only a handful of tools have been designed for bacteria, and these have not yet been systematically compared. Here, we aimed to identify tools that use Ribo-seq data to correctly determine the translational status of annotated bacterial ORFs and also discover novel translated regions with high sensitivity. To this end, we generated a large set of annotated ORFs from four diverse bacterial organisms, manually labeled for their translation status based on Ribo-seq data, which are available for future benchmarking studies. This set was used to investigate the predictive performance of seven Ribo-seq-based ORF detection tools (REPARATION_blast, DeepRibo, Ribo-TISH, PRICE, smORFer, ribotricer and SPECtre), as well as IRSOM, which uses coding potential and RNA-seq coverage only. DeepRibo and REPARATION_blast robustly predicted translated ORFs, including sORFs, with no significant difference for ORFs in close proximity to other genes versus stand-alone genes. However, no tool predicted a set of novel, experimentally verified sORFs with high sensitivity. Start codon predictions with smORFer show the value of initiation site profiling data to further improve the sensitivity of ORF prediction tools in bacteria. Overall, we find that bacterial tools perform well for sORF detection, although there is potential for improving their performance, applicability, usability and reproducibility.

Small RNAs that target G-rich sequences are generated by diverse biogenesis pathways in Epsilonproteobacteria.

Bacterial small RNAs (sRNAs) are widespread post-transcriptional regulators that control bacterial stress responses and virulence. Nevertheless, little is known about how they arise and evolve. Homologs can be difficult to identify beyond the strain level using sequence-based approaches, and similar functionalities can arise by convergent evolution. Here, we found that the virulence-associated CJnc190 sRNA of the foodborne pathogen Campylobacter jejuni resembles the RepG sRNA from the gastric pathogen Helicobacter pylori. However, while both sRNAs bind G-rich sites in their target mRNAs using a C/U-rich loop, they largely differ in their biogenesis. RepG is transcribed from a stand-alone gene and does not require processing, whereas CJnc190 is transcribed from two promoters as precursors that are processed by RNase III and also has a cis-encoded antagonist, CJnc180. By comparing CJnc190 homologs in diverse Campylobacter species, we show that RNase III-dependent processing of CJnc190 appears to be a conserved feature even outside of C. jejuni. We also demonstrate the CJnc180 antisense partner is expressed in C. coli, yet here might be derived from the 3'UTR (untranslated region) of an upstream flagella-related gene. Our analysis of G-tract targeting sRNAs in Epsilonproteobacteria demonstrates that similar sRNAs can have markedly different biogenesis pathways.

The Rsm (Csr) post-transcriptional regulatory pathway coordinately controls multiple CRISPR-Cas immune systems.

CRISPR-Cas systems provide bacteria with adaptive immunity against phages and plasmids; however, pathways regulating their activity are not well defined. We recently developed a high-throughput genome-wide method (SorTn-seq) and used this to uncover CRISPR-Cas regulators. Here, we demonstrate that the widespread Rsm/Csr pathway regulates the expression of multiple CRISPR-Cas systems in Serratia (type I-E, I-F and III-A). The main pathway component, RsmA (CsrA), is an RNA-binding post-transcriptional regulator of carbon utilisation, virulence and motility. RsmA binds cas mRNAs and suppresses type I and III CRISPR-Cas interference in addition to adaptation by type I systems. Coregulation of CRISPR-Cas and flagella by the Rsm pathway allows modulation of adaptive immunity when changes in receptor availability would alter susceptibility to flagella-tropic phages. Furthermore, we show that Rsm controls CRISPR-Cas in other genera, suggesting conservation of this regulatory strategy. Finally, we identify genes encoding RsmA homologues in phages, which have the potential to manipulate the physiology of host bacteria and might provide an anti-CRISPR activity.

Identifying Small Open Reading Frames in Prokaryotes with Ribosome Profiling.

Small proteins encoded by open reading frames (ORFs) shorter than 50 codons (small ORFs [sORFs]) are often overlooked by annotation engines and are difficult to characterize using traditional biochemical techniques. Ribosome profiling has tremendous potential to empirically improve the annotations of prokaryotic genomes. Recent improvements in ribosome profiling methods for bacterial model organisms have revealed many new sORFs in well-characterized genomes. Antibiotics that trap ribosomes just after initiation have played a key role in these developments by allowing the unambiguous identification of the start codons (and, hence, the reading frame) for novel ORFs. Here, we describe these new methods and highlight critical controls and considerations for adapting ribosome profiling to different prokaryotic species.

HRIBO: high-throughput analysis of bacterial ribosome profiling data.

MOTIVATION: Ribosome profiling (Ribo-seq) is a powerful approach based on deep sequencing of cDNA libraries generated from ribosome-protected RNA fragments to explore the translatome of a cell, and is especially useful for the detection of small proteins (50-100 amino acids) that are recalcitrant to many standard biochemical and in silico approaches. While pipelines are available to analyze Ribo-seq data, none are designed explicitly for the automatic processing and analysis of data from bacteria, nor are they focused on the discovery of unannotated open reading frames (ORFs). RESULTS: We present HRIBO (High-throughput annotation by Ribo-seq), a workflow to enable reproducible and high-throughput analysis of bacterial Ribo-seq data. The workflow performs all required pre-processing and quality control steps. Importantly, HRIBO outputs annotation-independent ORF predictions based on two complementary bacteria-focused tools, and integrates them with additional feature information and expression values. This facilitates the rapid and high-confidence discovery of novel ORFs and their prioritization for functional characterization. AVAILABILITY AND IMPLEMENTATION: HRIBO is a free and open source project available under the GPL-3 license at: https://github.com/RickGelhausen/HRIBO.

A three-dimensional intestinal tissue model reveals factors and small regulatory RNAs important for colonization with Campylobacter jejuni.

The Gram-negative Epsilonproteobacterium Campylobacter jejuni is currently the most prevalent bacterial foodborne pathogen. Like for many other human pathogens, infection studies with C. jejuni mainly employ artificial animal or cell culture models that can be limited in their ability to reflect the in-vivo environment within the human host. Here, we report the development and application of a human three-dimensional (3D) infection model based on tissue engineering to study host-pathogen interactions. Our intestinal 3D tissue model is built on a decellularized extracellular matrix scaffold, which is reseeded with human Caco-2 cells. Dynamic culture conditions enable the formation of a polarized mucosal epithelial barrier reminiscent of the 3D microarchitecture of the human small intestine. Infection with C. jejuni demonstrates that the 3D tissue model can reveal isolate-dependent colonization and barrier disruption phenotypes accompanied by perturbed localization of cell-cell junctions. Pathogenesis-related phenotypes of C. jejuni mutant strains in the 3D model deviated from those obtained with 2D-monolayers, but recapitulated phenotypes previously observed in animal models. Moreover, we demonstrate the involvement of a small regulatory RNA pair, CJnc180/190, during infections and observe different phenotypes of CJnc180/190 mutant strains in 2D vs. 3D infection models. Hereby, the CJnc190 sRNA exerts its pathogenic influence, at least in part, via repression of PtmG, which is involved in flagellin modification. Our results suggest that the Caco-2 cell-based 3D tissue model is a valuable and biologically relevant tool between in-vitro and in-vivo infection models to study virulence of C. jejuni and other gastrointestinal pathogens.

The small non-coding RNA RsaE influences extracellular matrix composition in Staphylococcus epidermidis biofilm communities.

RsaE is a conserved small regulatory RNA (sRNA) which was previously reported to represent a riboregulator of central carbon flow and other metabolic pathways in Staphylococcus aureus and Bacillus subtilis. Here we show that RsaE contributes to extracellular (e)DNA release and biofilm-matrix switching towards polysaccharide intercellular adhesin (PIA) production in a hypervariable Staphylococcus epidermidis isolate. Transcriptome analysis through differential RNA sequencing (dRNA-seq) in combination with confocal laser scanning microscopy (CLSM) and reporter gene fusions demonstrate that S. epidermidis protein- and PIA-biofilm matrix producers differ with respect to RsaE and metabolic gene expression. RsaE is spatiotemporally expressed within S. epidermidis PIA-mediated biofilms, and its overexpression triggers a PIA biofilm phenotype as well as eDNA release in an S. epidermidis protein biofilm matrix-producing strain background. dRNA-seq and Northern blot analyses revealed RsaE to exist as a major full-length 100-nt transcript and a minor processed species lacking approximately 20 nucleotides at the 5'-end. RsaE processing results in expansion of the mRNA target spectrum. Thus, full-length RsaE interacts with S. epidermidis antiholin-encoding lrgA mRNA, facilitating bacterial lysis and eDNA release. Processed RsaE, however, interacts with the 5'-UTR of icaR and sucCD mRNAs, encoding the icaADBC biofilm operon repressor IcaR and succinyl-CoA synthetase of the tricarboxylic acid (TCA) cycle, respectively. RsaE augments PIA-mediated biofilm matrix production, most likely through activation of icaADBC operon expression via repression of icaR as well as by TCA cycle inhibition and re-programming of staphylococcal central carbon metabolism towards PIA precursor synthesis. Additionally, RsaE supports biofilm formation by mediating the release of eDNA as stabilizing biofilm matrix component. As RsaE itself is heterogeneously expressed within biofilms, we consider this sRNA to function as a factor favoring phenotypic heterogeneity and supporting division of labor in S. epidermidis biofilm communities.

A non-coding RNA from the intercellular adhesion (ica) locus of Staphylococcus epidermidis controls polysaccharide intercellular adhesion (PIA)-mediated biofilm formation.

Polysaccharide intercellular adhesin (PIA)-associated biofilm formation is mediated by the intercellular adhesin (ica) locus and represents a major pathomechanism of Staphylococcus epidermidis. Here, we report on a novel long non-coding (nc)RNA, named IcaZ, which is approximately 400 nucleotides in size. icaZ is located downstream of the ica repressor gene icaR and partially overlaps with the icaR 3' UTR. icaZ exclusively exists in ica-positive S. epidermidis, but not in S. aureus or other staphylococci. Inactivation of the gene completely abolishes PIA production. IcaZ is transcribed as a primary transcript from its own promoter during early- and mid-exponential growth and its transcription is induced by low temperature, ethanol and salt stress. IcaZ targets the icaR 5' UTR and hampers icaR mRNA translation, which alleviates repression of icaADBC operon transcription and results in PIA production. Interestingly, other than in S. aureus, posttranscriptional control of icaR mRNA in S. epidermidis does not involve icaR mRNA 5'/3' UTR base pairing. This suggests major structural and functional differences in icaADBC operon regulation between the two species that also involve the recruitment of ncRNAs. Together, the IcaZ ncRNA represents an unprecedented novel species-specific player involved in the control of PIA production in NBSP S. epidermidis.

Identification of the RNA Pyrophosphohydrolase RppH of Helicobacter pylori and Global Analysis of Its RNA Targets.

RNA degradation is crucial for regulating gene expression in all organisms. Like the decapping of eukaryotic mRNAs, the conversion of the 5'-terminal triphosphate of bacterial transcripts to a monophosphate can trigger RNA decay by exposing the transcript to attack by 5'-monophosphate-dependent ribonucleases. In both biological realms, this deprotection step is catalyzed by members of the Nudix hydrolase family. The genome of the gastric pathogen Helicobacter pylori, a Gram-negative epsilonproteobacterium, encodes two proteins resembling Nudix enzymes. Here we present evidence that one of them, HP1228 (renamed HpRppH), is an RNA pyrophosphohydrolase that triggers RNA degradation in H. pylori, whereas the other, HP0507, lacks such activity. In vitro, HpRppH converts RNA 5'-triphosphates and diphosphates to monophosphates. It requires at least two unpaired nucleotides at the 5' end of its substrates and prefers three or more but has only modest sequence preferences. The influence of HpRppH on RNA degradation in vivo was examined by using RNA-seq to search the H. pylori transcriptome for RNAs whose 5'-phosphorylation state and cellular concentration are governed by this enzyme. Analysis of cDNA libraries specific for transcripts bearing a 5'-triphosphate and/or monophosphate revealed at least 63 potential HpRppH targets. These included mRNAs and sRNAs, several of which were validated individually by half-life measurements and quantification of their 5'-terminal phosphorylation state in wild-type and mutant cells. These findings demonstrate an important role for RppH in post-transcriptional gene regulation in pathogenic Epsilonproteobacteria and suggest a possible basis for the phenotypes of H. pylori mutants lacking this enzyme.

Differential RNA-seq of Vibrio cholerae identifies the VqmR small RNA as a regulator of biofilm formation.

Quorum sensing (QS) is a process of cell-to-cell communication that enables bacteria to transition between individual and collective lifestyles. QS controls virulence and biofilm formation in Vibrio cholerae, the causative agent of cholera disease. Differential RNA sequencing (RNA-seq) of wild-type V. cholerae and a locked low-cell-density QS-mutant strain identified 7,240 transcriptional start sites with ∼ 47% initiated in the antisense direction. A total of 107 of the transcripts do not appear to encode proteins, suggesting they specify regulatory RNAs. We focused on one such transcript that we name VqmR. vqmR is located upstream of the vqmA gene encoding a DNA-binding transcription factor. Mutagenesis and microarray analyses demonstrate that VqmA activates vqmR transcription, that vqmR encodes a regulatory RNA, and VqmR directly controls at least eight mRNA targets including the rtx (repeats in toxin) toxin genes and the vpsT transcriptional regulator of biofilm production. We show that VqmR inhibits biofilm formation through repression of vpsT. Together, these data provide to our knowledege the first global annotation of the transcriptional start sites in V. cholerae and highlight the importance of posttranscriptional regulation for collective behaviors in this human pathogen.

CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.

CRISPR/Cas systems constitute a widespread class of immunity systems that protect bacteria and archaea against phages and plasmids, and commonly use repeat/spacer-derived short crRNAs to silence foreign nucleic acids in a sequence-specific manner. Although the maturation of crRNAs represents a key event in CRISPR activation, the responsible endoribonucleases (CasE, Cas6, Csy4) are missing in many CRISPR/Cas subtypes. Here, differential RNA sequencing of the human pathogen Streptococcus pyogenes uncovered tracrRNA, a trans-encoded small RNA with 24-nucleotide complementarity to the repeat regions of crRNA precursor transcripts. We show that tracrRNA directs the maturation of crRNAs by the activities of the widely conserved endogenous RNase III and the CRISPR-associated Csn1 protein; all these components are essential to protect S. pyogenes against prophage-derived DNA. Our study reveals a novel pathway of small guide RNA maturation and the first example of a host factor (RNase III) required for bacterial RNA-mediated immunity against invaders.

A variable homopolymeric G-repeat defines small RNA-mediated posttranscriptional regulation of a chemotaxis receptor in Helicobacter pylori.

Phase variation of hypermutable simple sequence repeats (SSRs) is a widespread and stochastic mechanism to generate phenotypic variation within a population and thereby contributes to host adaptation of bacterial pathogens. Although several examples of SSRs that affect transcription or coding potential have been reported, we now show that a SSR also impacts small RNA-mediated posttranscriptional regulation. Based on in vitro and in vivo analyses, we demonstrate that a variable homopolymeric G-repeat in the leader of the TlpB chemotaxis receptor mRNA of the human pathogen Helicobacter pylori is directly targeted by a small RNA (sRNA), RepG (Regulator of polymeric G-repeats). Whereas RepG sRNA is highly conserved, the tlpB G-repeat length varies among diverse H. pylori strains, resulting in strain-specific RepG-mediated tlpB regulation. Based on modification of the G-repeat length within one strain, we demonstrate that the G-repeat length determines posttranscriptional regulation and can mediate both repression and activation of tlpB through RepG. In vitro translation assays show that this regulation occurs at the translational level and that RepG influences tlpB translation dependent on the G-repeat length. In contrast to the digital ON-OFF switches through frame-shift mutations within coding sequences, such modulation of posttranscriptional regulation allows for a gradual control of gene expression. This connection to sRNA-mediated posttranscriptional regulation might also apply to other genes with SSRs, which could be targeting sites of cis- or trans-encoded sRNAs, and thereby could facilitate host adaptation through sRNA-mediated fine-tuning of virulence gene expression.

Genome-wide identification of transcriptional start sites in the haloarchaeon Haloferax volcanii based on differential RNA-Seq (dRNA-Seq).

BACKGROUND: Differential RNA-Seq (dRNA-Seq) is a recently developed method of performing primary transcriptome analyses that allows for the genome-wide mapping of transcriptional start sites (TSSs) and the identification of novel transcripts. Although the transcriptomes of diverse bacterial species have been characterized by dRNA-Seq, the transcriptome analysis of archaeal species is still rather limited. Therefore, we used dRNA-Seq to characterize the primary transcriptome of the model archaeon Haloferax volcanii. RESULTS: Three independent cultures of Hfx. volcanii grown under optimal conditions to the mid-exponential growth phase were used to determine the primary transcriptome and map the 5'-ends of the transcripts. In total, 4749 potential TSSs were detected. A position weight matrix (PWM) was derived for the promoter predictions, and the results showed that 64 % of the TSSs were preceded by stringent or relaxed basal promoters. Of the identified TSSs, 1851 belonged to protein-coding genes. Thus, fewer than half (46 %) of the 4040 protein-coding genes were expressed under optimal growth conditions. Seventy-two percent of all protein-coding transcripts were leaderless, which emphasized that this pathway is the major pathway for translation initiation in haloarchaea. A total of 2898 of the TSSs belonged to potential non-coding RNAs, which accounted for an unexpectedly high fraction (61 %) of all transcripts. Most of the non-coding TSSs had not been previously described (2792) and represented novel sequences (59 % of all TSSs). A large fraction of the potential novel non-coding transcripts were cis-antisense RNAs (1244 aTSSs). A strong negative correlation between the levels of antisense transcripts and cognate sense mRNAs was found, which suggested that the negative regulation of gene expression via antisense RNAs may play an important role in haloarchaea. The other types of novel non-coding transcripts corresponded to internal transcripts overlapping with mRNAs (1153 iTSSs) and intergenic small RNA (sRNA) candidates (395 TSSs). CONCLUSION: This study provides a comprehensive map of the primary transcriptome of Hfx. volcanii grown under optimal conditions. Fewer than half of all protein-coding genes have been transcribed under these conditions. Unexpectedly, more than half of the detected TSSs belonged to several classes of non-coding RNAs. Thus, RNA-based regulation appears to play a more important role in haloarchaea than previously anticipated.

An atlas of Hfq-bound transcripts reveals 3' UTRs as a genomic reservoir of regulatory small RNAs.

The small RNAs associated with the protein Hfq constitute one of the largest classes of post-transcriptional regulators known to date. Most previously investigated members of this class are encoded by conserved free-standing genes. Here, deep sequencing of Hfq-bound transcripts from multiple stages of growth of Salmonella typhimurium revealed a plethora of new small RNA species from within mRNA loci, including DapZ, which overlaps with the 3' region of the biosynthetic gene, dapB. Synthesis of the DapZ small RNA is independent of DapB protein synthesis, and is controlled by HilD, the master regulator of Salmonella invasion genes. DapZ carries a short G/U-rich domain similar to that of the globally acting GcvB small RNA, and uses GcvB-like seed pairing to repress translation of the major ABC transporters, DppA and OppA. This exemplifies double functional output from an mRNA locus by the production of both a protein and an Hfq-dependent trans-acting RNA. Our atlas of Hfq targets suggests that the 3' regions of mRNA genes constitute a rich reservoir that provides the Hfq network with new regulatory small RNAs.

Differential RNA-seq (dRNA-seq) for annotation of transcriptional start sites and small RNAs in Helicobacter pylori.

The global mapping of transcription boundaries is a key step in the elucidation of the full complement of transcriptional features of an organism. It facilitates the annotation of operons and untranslated regions as well as novel transcripts, including cis- and trans-encoded small RNAs (sRNAs). So called RNA sequencing (RNA-seq) based on deep sequencing of cDNAs has greatly facilitated transcript mapping with single nucleotide resolution. However, conventional RNA-seq approaches typically cannot distinguish between primary and processed transcripts. Here we describe the recently developed differential RNA-seq (dRNA-seq) approach, which facilitates the annotation of transcriptional start sites (TSS) based on deep sequencing of two differentially treated cDNA library pairs, with one library being enriched for primary transcripts. Using the human pathogen Helicobacter pylori as a model organism, we describe the application of dRNA-seq together with an automated TSS annotation approach for generation of a genome-wide TSS map in bacteria. Besides a description of transcriptome and regulatory features that can be identified by this approach, we discuss the impact of different library preparation protocols and sequencing platforms as well as manual and automated TSS annotation. Moreover, we have set up an easily accessible online browser for visualization of the H. pylori transcriptome data from this and our previous H. pylori dRNA-seq study.

The primary transcriptome of the major human pathogen Helicobacter pylori.

Genome sequencing of Helicobacter pylori has revealed the potential proteins and genetic diversity of this prevalent human pathogen, yet little is known about its transcriptional organization and noncoding RNA output. Massively parallel cDNA sequencing (RNA-seq) has been revolutionizing global transcriptomic analysis. Here, using a novel differential approach (dRNA-seq) selective for the 5' end of primary transcripts, we present a genome-wide map of H. pylori transcriptional start sites and operons. We discovered hundreds of transcriptional start sites within operons, and opposite to annotated genes, indicating that complexity of gene expression from the small H. pylori genome is increased by uncoupling of polycistrons and by genome-wide antisense transcription. We also discovered an unexpected number of approximately 60 small RNAs including the epsilon-subdivision counterpart of the regulatory 6S RNA and associated RNA products, and potential regulators of cis- and trans-encoded target messenger RNAs. Our approach establishes a paradigm for mapping and annotating the primary transcriptomes of many living species.

Small RNA binding to 5' mRNA coding region inhibits translational initiation.

Small noncoding RNAs (sRNAs) have predominantly been shown to repress bacterial mRNAs by masking the Shine-Dalgarno (SD) or AUG start codon sequence, thereby preventing 30S ribosome entry and, consequently, translation initiation. However, many recently identified sRNAs lack obvious SD and AUG complementarity, indicating that sRNA-mediated translational control could also take place at other mRNA sites. We report that Salmonella RybB sRNA represses ompN mRNA translation by pairing with the 5' coding region. Results of systematic antisense interference with 30S binding to ompN and unrelated mRNAs suggest that sRNAs can act as translational repressors by sequestering sequences within the mRNA down to the fifth codon, even without SD and AUG start codon pairing. This "five codon window" for translational control in the 5' coding region of mRNA not only has implications for sRNA target predictions but might also apply to cis-regulatory systems such as RNA thermosensors and riboswitches.