PLoS One

Streptococcus pyogenes (Group A Streptococcus or GAS) is a Gram-positive bacterial pathogen that has shown complex modes of regulation of its virulence factors to cause diverse diseases. Bacterial small RNAs are regarded as novel widespread regulators of gene expression in response to environmental signals. Recent studies have revealed that several small RNAs (sRNAs) have an important role in S. pyogenes physiology and pathogenesis by regulating gene expression at the translational level. To search for new sRNAs in S. pyogenes, we performed a genomewide analysis through computational prediction followed by experimental verification. To overcome the limitation of low accuracy in computational prediction, we employed a combination of three different computational algorithms (sRNAPredict, eQRNA and RNAz). A total of 45 candidates were chosen based on the computational analysis, and their transcription was analyzed by reverse-transcriptase PCR and Northern blot. Through this process, we discovered 7 putative novel trans-acting sRNAs. Their abundance varied between different growth phases, suggesting that their expression is influenced by environmental or internal signals. Further, to screen target mRNAs of an sRNA, we employed differential RNA sequencing analysis. This study provides a significant resource for future study of small RNAs and their roles in physiology and pathogenesis of S. pyogenes

Identification of 88 regulatory small RNAs in the TIGR4 strain of the human pathogen Streptococcus pneumoniae

Streptococcus pneumoniae is the main etiological agent of community-acquired pneumonia and a major cause of mortality and morbidity among children and the elderly. Genome sequencing of several pneumococcal strains revealed valuable information about the potential proteins and genetic diversity of this prevalent human pathogen. However, little is known about its transcriptional regulation and its small regulatory noncoding RNAs. In this study, we performed deep sequencing of the S. pneumoniae TIGR4 strain RNome to identify small regulatory RNA candidates expressed in this pathogen. We discovered 1047 potential small RNAs including intragenic, 5'- and/or 3'-overlapping RNAs and 88 small RNAs encoded in intergenic regions. With this approach, we recovered many of the previously identified intergenic small RNAs and identified 68 novel candidates, most of which are conserved in both sequence and genomic context in other S. pneumoniae strains. We confirmed the independent expression of 17 intergenic small RNAs and predicted putative mRNA targets for six of them using bioinformatics tools. Preliminary results suggest that one of these six is a key player in the regulation of competence development. This study is the biggest catalog of small noncoding RNAs reported to date in S. pneumoniae and provides a highly complete view of the small RNA network in this pathogen.This work was supported by the Fondo de Investigación Sanitaria (FIS) (PI08/0442 and PI11/00656) and the Ministerio de Ciencia e Innovación (MICINN) (Ramón y Cajal program, RYC-2007-00179). A.J.M. was the recipient of a postdoctoral Miguel Servet contract from the Sistema Nacional de Salud funded by the FIS. During the first part of this work, M.A. was the recipient of a junior researcher contract funded by the Ramon y Cajal program from MICINN. S.N. was the recipient of a grant from CIBER Enfermedades Respiratorias (an initiative of the Instituto de Salud Carlos III).S

Analysis of novel small non-coding RNAs in Streptococcus pyogenes

Streptococcus pyogenes (S. pyogenes) ist einer der wichtigsten menschlichen Krankheitserreger, der ein breites Spektrum an Krankheiten verursacht, beginnend bei leichten Infektionen wie Hals- und Rachenentzündungen, Impetigo oder Scharlach, bis zu schweren invasiven Krankheiten wie das Toxische Schock Syndrom oder nekrotisierende Fasciitis, die auch lebensbedrohlich sein können. Um eine Infektion zu etablieren und dann auch aufrecht zu erhalten, besitzt S. pyogenes eine große Anzahl von Virulenzfaktoren, die sich entweder auf der Zelloberfläche befinden oder vom Bakterium in die Umgebung freigesetzt werden. Die Expression dieser Virulenzfaktoren wird einerseits durch protein-kodierte Zwei-Komponentensysteme und Transkriptionsfaktoren, aber auch durch erst kürzlich beschriebene RNA Moleküle reguliert. Von diesen RNAs, auch „small RNAs“ (sRNAs) genannt, sind in S. pyogenes bis jetzt drei bekannt, nämlich fasX, rivX und pel RNA. Das Ziel dieser Studie war die Identifizierung neuer nicht-kodierender sRNAs in S. pyogenes. Dafür wurden mittels Computeranalyse Regionen zwischen einzelnen Genen ermittelt, die für potenzielle sRNAs kodieren. Von den insgesamt 178 gefundenen Loci wurden 90 mittels Northern blot Analyse weiter untersucht. Dabei konnte die Expression von 29 sRNAs (~32%) unter normalen Wachstumsbedingungen nachgewiesen werden. Diese 29 Kandidaten konnten in 5 funktionelle Kategorien eingeteilt werden: (i) riboswitch (5), (ii) leader elements (6), (iii) funktionelle RNAs (4), (iv) T boxes (8), (v) sRNAs mit unbekannter Funktion (5) und (vi) SpyRNA014, welche bereits einmal in der Literatur als sRNA erwähnt wurde. Des Weiteren wurden die 5’ und 3’ Enden, sowie die Stabilität der sRNAs mittels Rifampicin Experimenten bestimmt. Für einige der Kandidaten wurde mittels Computeranalyse eine Vorhersage über mögliche Sekundärstrukturen erstellt und zusätzlich konnten bei sechs Kandidaten mögliche messenger RNAs (mRNAs) als Bindungspartner vorhergesagt werden. Kandidat SpyRNA049 wurde für die weitere funktionelle Analyses ausgewählt. Diese beinhaltete (i) den Beginn der Konstruktion einer Mutante, sowie (ii) die Anwendung der biochemischen Methode „StreptoTag“ um mögliche Proteine, die an SpyRNA049 binden, zu fischen. Während dieser Arbeit konnten neue sRNAs in S. pyogenes identifiziert werden, von denen die meisten in 5’ untranslatierten Regionen liegen. Aufgrund ihrer Lage im Genom und der Homologie mit bereits beschriebenen sRNAs, ist es wahrscheinlich, dass diese sRNAs eine wichtige Rolle in metabolischen oder virulenzbezogenen Prozessen spielen

Investigating the mechanism of a novel glycine-dependent riboswitch and a putative non-coding regulatory RNA in Streptococcus pyogenes

We investigated gene expression regulation by a putative glycine(Gly) riboswitch located in the 5′-UTR of a SAF protein gene in the S.pyogenes. Gly-dependency was studied using a luciferase reporter gene system. Maximal reporter gene expression happened in the presence of low Gly concentrations. RT-qPCR showed that in the presence of Gly (≥1 mM), expression of the gene was downregulated. Growth in the presence of 0.1 mM Gly led to the production of a full-length transcript. We conclude that the Gly riboswitch in S. pyogenes represses gene expression in the presence of high Gly concentrations

A Genome-Wide Analysis of Small Regulatory RNAs in the Human Pathogen Group A Streptococcus

The coordinated regulation of gene expression is essential for pathogens to infect and cause disease. A recently appreciated mechanism of regulation is that afforded by small regulatory RNA (sRNA) molecules. Here, we set out to assess the prevalence of sRNAs in the human bacterial pathogen group A Streptococcus (GAS). Genome-wide identification of candidate GAS sRNAs was performed through a tiling Affymetrix microarray approach and identified 40 candidate sRNAs within the M1T1 GAS strain MGAS2221. Together with a previous bioinformatic approach this brings the number of novel candidate sRNAs in GAS to 75, a number that approximates the number of GAS transcription factors. Transcripts were confirmed by Northern blot analysis for 16 of 32 candidate sRNAs tested, and the abundance of several of these sRNAs were shown to be temporally regulated. Six sRNAs were selected for further study and the promoter, transcriptional start site, and Rho-independent terminator identified for each. Significant variation was observed between the six sRNAs with respect to their stability during growth, and with respect to their inter- and/or intra-serotype-specific levels of abundance. To start to assess the contribution of sRNAs to gene regulation in M1T1 GAS we deleted the previously described sRNA PEL from four clinical isolates. Data from genome-wide expression microarray, quantitative RT-PCR, and Western blot analyses are consistent with PEL having no regulatory function in M1T1 GAS. The finding that candidate sRNA molecules are prevalent throughout the GAS genome provides significant impetus to the study of this fundamental gene-regulatory mechanism in an important human pathogen

Recombinase-based in vivo expression technology identifies a Streptococcus pyogenes bacteriocin important for niche adaptation in the nasopharynx

Streptococcus pyogenes is a Gram-positive, human-specific bacterial pathogen with the ability to cause a wide range of diseases from strep throat to necrotizing fasciitis and toxic shock syndrome. In addition, S. pyogenes may also induce post-streptococcal sequelae including rheumatic fever, acute glomerulonephritis, and reactive arthritis. Although primarily recognized as a pathogen, S. pyogenes also colonizes the skin and throat often without causing disease, and while numerous surface adhesions are important to attach to these surfaces, additional factors important for colonization and persistence by S. pyogenes are poorly understood. In addition to host defence mechanisms, the upper respiratory tract also contains other endogenous microorganisms that compete for the same niche. In order to gain a more complete understanding as to how S. pyogenes is able to adapt within the nasopharyngeal environment, a recombinase-based in vivo expression technology (RIVET) system was developed to identify genes activated in the nasopharyngeal niche using a humanized murine model. Using RIVET, 82 unique clones were recovered revealing 22 ‘cryptic’, 9 ‘typical’, and 30 ‘antisense’ in vivo induced promoters with single inserts. One ‘typical’ promoter (Pblp) that controlled a putative class IIb bacteriocin operon was further characterized. Testing this promoter as a single clone in the RIVET system confirmed its activation in vivo. However, in vitro testing failed to activate this bacteriocin, despite continued addition of the characterized pheromone, SilCR. A blp operon deletion was out-competed by the wild-type S. pyogenes in vivo, but not in vitro. Activation of the bacteriocin in vivo provides a further explanation for the ability of S. pyogenes to occupy the nasopharyngeal niche and begin the process of colonization. This work provides a new molecular tool for the in vivo analysis of S. pyogenes, and demonstrates a novel bacteriocin system important for niche adaptation by S. pyogenes

Small RNA teg49 Is Derived from a sarA Transcript and Regulates Virulence Genes Independent of SarA in Staphylococcus aureus

Expression of virulence factors in Staphylococcus aureus is regulated by a wide range of transcriptional regulators, including proteins and small RNAs (sRNAs), at the level of transcription and/or translation. The sarA locus consists of three overlapping transcripts generated from three distinct promoters, all containing the sarA open reading frame (ORF). The 5= untranslated regions (UTRs) of these transcripts contain three separate regions 711, 409, and 146 nucleotides (nt) upstream of the sarA translation start, the functions of which remain unknown. Re- cent transcriptome-sequencing (RNA-Seq) analysis and subsequent characterization indicated that two sRNAs, teg49 and teg48, are processed and likely produced from the sarA P3 and sarA P1 transcripts of the sarA locus, respectively. In this report, we utilized a variety of sarA promoter mutants and cshA and rnc mutants to ascertain the contributions of these factors to the generation of teg49. We also defined the transcriptional regulon of teg49, including virulence genes not regulated by SarA. Phenotypically, teg49 did not impact biofilm formation or affect overall SarA expres- sion significantly. Comparative analyses of RNA-Seq data between the wild-type, teg49 mutant, and sarA mutant strains indicated that 133 genes are significantly upregulated while 97 are downregulated in a teg49 deletion mutant in a sarA- independent manner. An abscess model of skin infection indicated that the teg49 mutant exhibited a reduced bacterial load compared to the wild-type S. aureus. Overall, these results suggest that teg49 sRNA has a regulatory role in target gene regulation independent of SarA. The exact mechanism of this regulation is yet to be dissected

Identification of small RNAs in Francisella tularensis

Background: Regulation 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. Results: We 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. Conclusion: Our 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

Funktionelle Charakterisierung einer durch Säure regulierten SRNA in HelicobacterHelicobacter pyloripylori

Low pH is the main environmental stress encountered by Helicobacter pylori in the human stomach. To ensure its survival under acidic conditions, this bacterium utilizes urease (encoded by the ureAB operon), a nickel-activated metalloenzyme, which cleaves urea into ammonia to buffer the periplasmic space. Expression of the ureAB operon is tightly regulated at the transcriptional level. Moreover, the urease activity is modulated post translationally via the activity of nickel-binding proteins such as HP1432 that act as nickel sponges to either sequester or release nickel depending on the pH. However, little is known how the levels of these nickel-binding proteins are regulated at the post-transcriptional level. Interestingly, more than 60 candidate small regulatory RNAs (sRNAs) have been identified in a differential RNA-seq approach in H. pylori strain 26695, suggesting an uncharacterized layer of post-transcriptional riboregulation in this pathogen. sRNAs control their trans- or cis- encoded targets by direct binding. Many of the characterized sRNAs are expressed in response to specific environmental cues and are ideal candidates to confer post-transcriptional regulation under different growth conditions. This study demonstrates that a small RNA termed ArsZ (Acid Responsive sRNA Z) and its target HP1432 constitute yet another level of urease regulation. In-vitro and in-vivo experiments show that ArsZ interacts with the ribosome binding site (RBS) of HP1432 mRNA, effectively repressing translation of HP1432. During acid adaptation, the acid-responsive ArsRS two-component system represses expression of ArsZ. ArsRS and ArsZ work in tandem to regulate expression of HP1432 via a coherent feedforward loop (FFL). ArsZ acts as a delay mechanism in this feedforward loop to ensure that HP1432 protein levels do not abruptly change upon transient pH drops encountered by the bacteria. ArsZ “fine-tunes” the dynamics of urease activity after pH shift presumably by altering nickel availability through post transcriptional control of HP1432 expression. Interestingly, after adaptation to acid stress, ArsZ indirectly activates the transcription of HP1432 and forms an incoherent FFL with ArsRS to regulate HP1432. This study identified a non-standard FFL in which ArsZ can participate directly or indirectly in two different network configurations depending on the state of acid stress adaptation. The importance of ArsZ in the acid response of H. pylori is further supported by bioinformatics analysis showing that the evolution of ArsZ is closely related to the emergence of modern H. pylori strains that globally infect humans. No homologs of arsZ were found in the non-pylori species of Helicobacter. Moreover, this study also demonstrates that the physiological role of a sRNA can be elucidated without the artificial overexpression of the respective sRNA, a method commonly used to characterize sRNAs. Coupled with time-course experiments, this approach allows the kinetics of ArsZ regulation to be studied under more native conditions. ArsZ is the first example of a trans-acting sRNA that regulates a nickel storage protein to modulate apo-urease maturation. These findings may have important implications in understanding the details of urease activation and hence the colonization capability of H. pylori, the only bacterial class I carcinogen to date (WHO, 1994).In der natürlichen Umgebung des menschlichen Magens ist Helicobacter pylori insbesondere niedrigen pH-Werten ausgesetzt. Um diese Bedingungen zu überleben, setzt das Bakterium das Enzym Urease ein (kodiert durch das ureAB Operon), ein Nickel-aktiviertes Metalloenzym, welches Urea zu Ammonium umsetzt um den pH-Wert des periplasmatischen Raums abzupuffern. Die Expression dieses Operons ist auf transkriptioneller Ebene streng reguliert. Zudem ist die Aktivität des Urease Enzyms auf post-translationaler Ebene moduliert. Dies geschieht durch die Aktivität von Nickel-Bindeproteinen wie HP1432, die in Abhängigkeit vom pH-Wert Nickelionen abfangen oder wieder freigeben. Allerdings ist nur sehr wenig darüber bekannt, wie diese Nickel-Bindeproteine auf post-transkriptioneller Ebene reguliert werden. Interessanterweise wurden mehr als 60 sRNA-Kandidaten (engl. small RNA für dt. kleine RNA) durch eine differentielle RNA-seq Methode im H. pylori Stamm 26695 identifiziert. Dies legt eine nicht charakterisierte Ebene post-transkriptioneller Riboregulierung in diesem Pathogen nahe. sRNAs kontrollieren ihre trans- oder cis-kodierten Zielgene durch direkte Interaktion. Viele der charakterisierten sRNAs werden als Antwort auf spezifische Umweltsignale exprimiert und stellen ideale Kandidaten für post-transkriptionelle Regulatoren unter verschiedenen Wachstumsbedingungen dar. In dieser Arbeit wird gezeigt, dass die kleine RNA ArsZ (engl. acid responsive sRNA Z für dt. säureabhängige sRNA Z) und ihr Zielgen HP1432 ein zusätzliches Level der Urease-Regulierung darstellen. In-vitro und in-vivo Experimente zeigen, dass ArsZ mit der Ribosomenbindestelle (RBS) der HP1432 mRNA interagiert, wodurch dessen Translation verhindert wird. Während der Säureanpassung verhindert das säureabhängige ArsRS Zweikomponentensystem die Expression von ArsZ. Zusammen regulieren ArsRS und ArsZ das Zielgen HP1432 in Form eines kohärenten Feed-forward-loops (FFL). ArsZ agiert hier als Verzögerungsmechanismus, um sicherzustellen, dass sich bei einem transienten Abfall des pH-Wertes das Proteinlevel von HP1432 nicht abrupt verändert. Nach pH-Änderungen vermittelt ArsZ eine Feinregulierung der Ureaseaktivität, vermutlich indem es durch die post-transkriptionelle Kontrolle der HP1432 Expression die Verfügbarkeit von Nickel verändert. Interessanterweise aktiviert ArsZ nach der Säureanpassung indirekt die Transkription von HP1432 und schließt dadurch einen inkohärenten FFL mit ArsRS zur Regulierung von HP1432. Diese Studie identifizierte einen Nicht-Standard-FFL, in dem ArsZ abhängig von dem Status der Säureadaptation in zwei verschiedenen Netzwerkkonfigurationen direkt oder indirekt agieren kann. Bioinformatorische Analysen unterstützen die Relevanz von ArsZ in der Säureantwort von H. pylori zusätzlich. Hierbei kann gezeigt werden, dass die Evolution von ArsZ mit dem Aufkommen moderner H. pylori Stämme einhergeht, die weltweit Menschen infizieren. In nicht-pylori Helicobacter Spezies konnten keine Homologe von arsZ gefunden werden. Zudem zeigt diese Studie, dass die physiologische Rolle einer sRNA ohne ihre artifizielle Überexpression aufgeklärt werden kann, eine standard-mäßige Herangehensweise zur Charakterisierung kleiner RNAs. In Kombination mit Zeitverlaufsexperimenten konnte die zeitabhängige Regulierung von Zielgenen durch ArsZ unter natürlicheren Bedingungen untersucht werden. ArsZ ist das erste Beispiel einer trans-agierenden sRNA die ein Nickel-Speicherprotein reguliert, um die Reifung der Apo-Urease zu modulieren. Diese Ergebnisse können wichtige Informationen liefern, um die Aktivierung des Urease Enzyms besser zu verstehen und um damit detailliertere Einblicke in die Kolonisierungsfähigkeit von H. pylori zu gewinnen, dem bislang einzigen bakteriellen Klasse-I-Karzinogen (WHO, 1994)

High-Resolution Transcriptome Maps Reveal Strain-Specific Regulatory Features of Multiple Campylobacter jejuni Isolates

Campylobacter jejuni is currently the leading cause of bacterial gastroenteritis in humans. Comparison of multiple Campylobacter strains revealed a high genetic and phenotypic diversity. However, little is known about differences in transcriptome organization, gene expression, and small RNA (sRNA) repertoires. Here we present the first comparative primary transcriptome analysis based on the differential RNA–seq (dRNA–seq) of four C. jejuni isolates. Our approach includes a novel, generic method for the automated annotation of transcriptional start sites (TSS), which allowed us to provide genome-wide promoter maps in the analyzed strains. These global TSS maps are refined through the integration of a SuperGenome approach that allows for a comparative TSS annotation by mapping RNA–seq data of multiple strains into a common coordinate system derived from a whole-genome alignment. Considering the steadily increasing amount of RNA–seq studies, our automated TSS annotation will not only facilitate transcriptome annotation for a wider range of pro- and eukaryotes but can also be adapted for the analysis among different growth or stress conditions. Our comparative dRNA–seq analysis revealed conservation of most TSS, but also single-nucleotide-polymorphisms (SNP) in promoter regions, which lead to strain-specific transcriptional output. Furthermore, we identified strain-specific sRNA repertoires that could contribute to differential gene regulation among strains. In addition, we identified a novel minimal CRISPR-system in Campylobacter of the type-II CRISPR subtype, which relies on the host factor RNase III and a trans-encoded sRNA for maturation of crRNAs. This minimal system of Campylobacter, which seems active in only some strains, employs a unique maturation pathway, since the crRNAs are transcribed from individual promoters in the upstream repeats and thereby minimize the requirements for the maturation machinery. Overall, our study provides new insights into strain-specific transcriptome organization and sRNAs, and reveals genes that could modulate phenotypic variation among strains despite high conservation at the DNA level