Showing papers by "Jörg Vogel published in 2012"

Dual RNA-seq of pathogen and host

Abstract: A comprehensive understanding of host-pathogen interactions requires a knowledge of the associated gene expression changes in both the pathogen and the host. Traditional, probe-dependent approaches using microarrays or reverse transcription PCR typically require the pathogen and host cells to be physically separated before gene expression analysis. However, the development of the probe-independent RNA sequencing (RNA-seq) approach has begun to revolutionize transcriptomics. Here, we assess the feasibility of taking transcriptomics one step further by performing 'dual RNA-seq', in which gene expression changes in both the pathogen and the host are analysed simultaneously.

The transcriptional landscape and small RNAs of Salmonella enterica serovar Typhimurium

Abstract: More than 50 y of research have provided great insight into the physiology, metabolism, and molecular biology of Salmonella enterica serovar Typhimurium (S. Typhimurium), but important gaps in our knowledge remain. It is clear that a precise choreography of gene expression is required for Salmonella infection, but basic genetic information such as the global locations of transcription start sites (TSSs) has been lacking. We combined three RNA-sequencing techniques and two sequencing platforms to generate a robust picture of transcription in S. Typhimurium. Differential RNA sequencing identified 1,873 TSSs on the chromosome of S. Typhimurium SL1344 and 13% of these TSSs initiated antisense transcripts. Unique findings include the TSSs of the virulence regulators phoP, slyA, and invF. Chromatin immunoprecipitation revealed that RNA polymerase was bound to 70% of the TSSs, and two-thirds of these TSSs were associated with σ(70) (including phoP, slyA, and invF) from which we identified the -10 and -35 motifs of σ(70)-dependent S. Typhimurium gene promoters. Overall, we corrected the location of important genes and discovered 18 times more promoters than identified previously. S. Typhimurium expresses 140 small regulatory RNAs (sRNAs) at early stationary phase, including 60 newly identified sRNAs. Almost half of the experimentally verified sRNAs were found to be unique to the Salmonella genus, and <20% were found throughout the Enterobacteriaceae. This description of the transcriptional map of SL1344 advances our understanding of S. Typhimurium, arguably the most important bacterial infection model.

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

Abstract: 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.

The Primary Transcriptome of Barley Chloroplasts: Numerous Noncoding RNAs and the Dominating Role of the Plastid-Encoded RNA Polymerase

Abstract: Gene expression in plastids of higher plants is dependent on two different transcription machineries, a plastid-encoded bacterial-type RNA polymerase (PEP) and a nuclear-encoded phage-type RNA polymerase (NEP), which recognize distinct types of promoters. The division of labor between PEP and NEP during plastid development and in mature chloroplasts is unclear due to a lack of comprehensive information on promoter usage. Here, we present a thorough investigation into the distribution of PEP and NEP promoters within the plastid genome of barley (Hordeum vulgare). Using a novel differential RNA sequencing approach, which discriminates between primary and processed transcripts, we obtained a genome-wide map of transcription start sites in plastids of mature first leaves. PEP-lacking plastids of the albostrians mutant allowed for the unambiguous identification of NEP promoters. We observed that the chloroplast genome contains many more promoters than genes. According to our data, most genes (including genes coding for photosynthesis proteins) have both PEP and NEP promoters. We also detected numerous transcription start sites within operons, indicating transcriptional uncoupling of genes in polycistronic gene clusters. Moreover, we mapped many transcription start sites in intergenic regions and opposite to annotated genes, demonstrating the existence of numerous noncoding RNA candidates.

The mammalian microRNA response to bacterial infections.

Abstract: MicroRNAs are small RNAs that post-transcriptionally regulate eukaryotic gene expression. In addition to their involvement in a wide range of physiological and pathological processes, including viral infections, microRNAs are increasingly implicated in the eukaryotic response to bacterial pathogens. Recent studies have characterized changes in host microRNA expression following infection with exclusively extracellular (Helicobacter pylori) or intracellular (Salmonella enterica) Gram-negative bacteria, as well as in the response to Gram-positive (Listeria monocytogenes) and other pathogens (Mycobacterium and Francisella species). In this review, we discuss the emerging roles of microRNAs in mammalian host signaling and defense against bacterial pathogens.

Superfolder GFP reporters validate diverse new mRNA targets of the classic porin regulator, MicF RNA

Abstract: MicF is a textbook example of a small regulatory RNA (sRNA) that acts on a trans-encoded target mRNA through imperfect base pairing. Discovery of MicF as a post-transcriptional repressor of the major Escherichia coli porin OmpF established the paradigm for a meanwhile common mechanism of translational inhibition, through antisense sequestration of a ribosome binding site. However, whether MicF regulates additional genes has remained unknown for almost three decades. Here, we have harnessed the new superfolder variant of GFP for reporter-gene fusions to validate newly predicted targets of MicF in Salmonella. We show that the conserved 5' end of MicF acts by seed pairing to repress the mRNAs of global transcriptional regulator Lrp, and periplasmic protein YahO, while a second targeting region is also required to regulate the mRNA of the lipid A-modifying enzyme LpxR. Interestingly, MicF targets lpxR at both the ribosome binding site and deep within the coding sequence. MicF binding in the coding sequence of lpxR decreases mRNA stability through exacerbating the use of a native RNase E site proximal to the short MicF-lpxR duplex. Altogether, this study assigns the classic MicF sRNA to the growing class of Hfq-associated regulators that use diverse mechanisms to impact multiple loci.

Global regulatory functions of the Staphylococcus aureus endoribonuclease III in gene expression.

Abstract: RNA turnover plays an important role in both virulence and adaptation to stress in the Gram-positive human pathogen Staphylococcus aureus. However, the molecular players and mechanisms involved in these processes are poorly understood. Here, we explored the functions of S. aureus endoribonuclease III (RNase III), a member of the ubiquitous family of double-strand-specific endoribonucleases. To define genomic transcripts that are bound and processed by RNase III, we performed deep sequencing on cDNA libraries generated from RNAs that were co-immunoprecipitated with wild-type RNase III or two different cleavage-defective mutant variants in vivo. Several newly identified RNase III targets were validated by independent experimental methods. We identified various classes of structured RNAs as RNase III substrates and demonstrated that this enzyme is involved in the maturation of rRNAs and tRNAs, regulates the turnover of mRNAs and non-coding RNAs, and autoregulates its synthesis by cleaving within the coding region of its own mRNA. Moreover, we identified a positive effect of RNase III on protein synthesis based on novel mechanisms. RNase III–mediated cleavage in the 5′ untranslated region (5′UTR) enhanced the stability and translation of cspA mRNA, which encodes the major cold-shock protein. Furthermore, RNase III cleaved overlapping 5′UTRs of divergently transcribed genes to generate leaderless mRNAs, which constitutes a novel way to co-regulate neighboring genes. In agreement with recent findings, low abundance antisense RNAs covering 44% of the annotated genes were captured by co-immunoprecipitation with RNase III mutant proteins. Thus, in addition to gene regulation, RNase III is associated with RNA quality control of pervasive transcription. Overall, this study illustrates the complexity of post-transcriptional regulation mediated by RNase III.

A conserved RpoS-dependent small RNA controls the synthesis of major porin OmpD

Abstract: A remarkable feature of many small non-coding RNAs (sRNAs) of Escherichia coli and Salmonella is their accumulation in the stationary phase of bacterial growth. Several stress response regulators and sigma factors have been reported to direct the transcription of stationary phase-specific sRNAs, but a widely conserved sRNA gene that is controlled by the major stationary phase and stress sigma factor, σS (RpoS), has remained elusive. We have studied in Salmonella the conserved SdsR sRNA, previously known as RyeB, one of the most abundant stationary phase-specific sRNAs in E. coli. Alignments of the sdsR promoter region and genetic analysis strongly suggest that this sRNA gene is selectively transcribed by σS. We show that SdsR down-regulates the synthesis of the major Salmonella porin OmpD by Hfq-dependent base pairing; SdsR thus represents the fourth sRNA to regulate this major outer membrane porin. Similar to the InvR, MicC and RybB sRNAs, SdsR recognizes the ompD mRNA in the coding sequence, suggesting that this mRNA may be primarily targeted downstream of the start codon. The SdsR-binding site in ompD was localized by 3′-RACE, an experimental approach that promises to be of use in predicting other sRNA–target interactions in bacteria.

Genome-wide transcriptome analysis of the plant pathogen Xanthomonas identifies sRNAs with putative virulence functions

Abstract: The Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) is an important model to elucidate the mechanisms involved in the interaction with the host. To gain insight into the transcriptome of the Xcv strain 85–10, we took a differential RNA sequencing (dRNA-seq) approach. Using a novel method to automatically generate comprehensive transcription start site (TSS) maps we report 1421 putative TSSs in the Xcv genome. Genes in Xcv exhibit a poorly conserved −10 promoter element and no consensus Shine-Dalgarno sequence. Moreover, 14% of all mRNAs are leaderless and 13% of them have unusually long 5′-UTRs. Northern blot analyses confirmed 16 intergenic small RNAs and seven cis-encoded antisense RNAs in Xcv. Expression of eight intergenic transcripts was controlled by HrpG and HrpX, key regulators of the Xcv type III secretion system. More detailed characterization identified sX12 as a small RNA that controls virulence of Xcv by affecting the interaction of the pathogen and its host plants. The transcriptional landscape of Xcv is unexpectedly complex, featuring abundant antisense transcripts, alternative TSSs and clade-specific small RNAs.

The ancestral SgrS RNA discriminates horizontally acquired Salmonella mRNAs through a single G-U wobble pair

Abstract: SgrS RNA is a model for the large class of Hfq-associated small RNAs that act to posttranscriptionally regulate bacterial mRNAs. The function of SgrS is well-characterized in nonpathogenic Escherichia coli, where it was originally shown to counteract glucose-phosphate stress by acting as a repressor of the ptsG mRNA, which encodes the major glucose transporter. We have discovered additional SgrS targets in Salmonella Typhimurium, a pathogen related to E. coli that recently acquired one-quarter of all genes by horizontal gene transfer. We show that the conserved short seed region of SgrS that recognizes ptsG was recruited to target the Salmonella-specific sopD mRNA of a secreted virulence protein. The SgrS–sopD interaction is exceptionally selective; we find that sopD2 mRNA, whose gene arose from sopD duplication during Salmonella evolution, is deaf to SgrS because of a nonproductive G-U pair in the potential SgrS-sopD2 RNA duplex vs. G-C in SgrS-sopD. In other words, SgrS discriminates the two virulence factor mRNAs at the level of a single hydrogen bond. Our study suggests that bacterial pathogens use their large suites of conserved Hfq-associated regulators to integrate horizontally acquired genes into existing posttranscriptional networks, just as conserved transcription factors are recruited to tame foreign genes at the DNA level. The results graphically illustrate the importance of the seed regions of bacterial small RNAs to select new targets with high fidelity and suggest that target predictions must consider all or none decisions by individual seed nucleotides.

The csgD mRNA as a hub for signal integration via multiple small RNAs

Abstract: The transcription factor CsgD governing the production of curli fimbriae and cellulose is a key player in the complex regulatory circuit that decides whether Escherichia coli form biofilms. The csgD gene itself is tightly controlled at the level of transcription by a large array of DNA-binding proteins, but what happens after transcription is less understood. In this issue of Molecular Microbiology, Jorgensen et al. (2012), Mika et al. (2012) and Thomason et al. (2012) report on small RNAs (McaS, RprA and GcvB) that together with the RNA-chaperone Hfq regulate the mRNAs of csgD and other biofilm genes, and illustrate the burgeoning concept that the 5' region of bacterial mRNA serves as a hub for sRNA-mediated signal integration at the post-transcriptional level.

RelA protein stimulates the activity of RyhB small RNA by acting on RNA-binding protein Hfq

Abstract: The conserved RNA-binding protein Hfq and its associated small regulatory RNAs (sRNAs) are increasingly recognized as the players of a large network of posttranscriptional control of gene expression in Gram-negative bacteria The role of Hfq in this network is to facilitate base pairing between sRNAs and their trans-encoded target mRNAs Although the number of known sRNA–mRNA interactions has grown steadily, cellular factors that influence Hfq, the mediator of these interactions, have remained unknown We report that RelA, a protein long known as the central regulator of the bacterial-stringent response, acts on Hfq and thereby affects the physiological activity of RyhB sRNA as a regulator of iron homeostasis RyhB requires RelA in vivo to arrest growth during iron depletion and to down-regulate a subset of its target mRNAs (fdoG, nuoA, and sodA), whereas the sodB and sdhC targets are barely affected by RelA In vitro studies with recombinant proteins show that RelA enhances multimerization of Hfq monomers and stimulates Hfq binding of RyhB and other sRNAs Hfq from polysomes extracted from wild-type cells binds RyhB in vitro, whereas Hfq from polysomes of a relA mutant strain shows no binding We propose that, by increasing the level of the hexameric form of Hfq, RelA enables binding of RNAs whose affinity for Hfq is low Our results suggest that, under specific conditions and/or environments, Hfq concentrations are limiting for RNA binding, which thereby provides an opportunity for cellular proteins such as RelA to impact sRNA-mediated responses by modulating the activity of Hfq

Small RNAs of the Bradyrhizobium/Rhodopseudomonas lineage and their analysis

Abstract: Small RNAs (sRNAs) play a pivotal role in bacterial gene regulation. However, the sRNAs of the vast majority of bacteria with sequenced genomes still remain unknown since sRNA genes are usually difficult to recognize and thus not annotated. Here, expression of seven sRNAs (BjrC2a, BjrC2b, BjrC2c, BjrC68, BjrC80, BjrC174 and BjrC1505) predicted by genome comparison of Bradyrhizobium and Rhodopseudomonas members, was verified by RNA gel blot hybridization, microarray and deep sequencing analyses of RNA from the soybean symbiont Bradyrhizobium japonicum USDA 110. BjrC2a, BjrC2b and BjrC2c belong to the RNA family RF00519, while the other sRNAs are novel. For some of the sRNAs we observed expression differences between free-living bacteria and bacteroids in root nodules. The amount of BjrC1505 was decreased in nodules. By contrast, the amount of BjrC2a, BjrC68, BjrC80, BjrC174 and the previously described 6S RNA was increased in nodules, and accumulation of truncated forms of these sRNAs was observed. Comparative genomics and deep sequencing suggest that BjrC2a is an antisense RNA regulating the expression of inositol-monophosphatase. The analyzed sRNAs show a different degree of conservation in Rhizobiales, and expression of homologs of BjrC2, BjrC68, BjrC1505, and 6S RNA was confirmed in the free-living purple bacterium Rhodopseudomonas palustris 5D.

Experimental tools to identify RNA-protein interactions in Helicobacter pylori

Abstract: Helicobacter pylori, one of the most prevalent human pathogens, used to be thought to lack small regulatory RNAs (sRNAs) which are otherwise considered abundant in all bacteria. However, our recent analysis of the primary transcriptome of H. pylori discovered an unexpectedly large number of sRNAs, and suggested that this model organism also uses riboregulation to control the expression of its genes. Nonetheless, whereas most enterobacterial sRNAs require the RNA chaperone Hfq for function, Epsilonproteobacteria including H. pylori seem to have no Hfq homologue, which prompted us to search for other auxiliary proteins in sRNA-mediated regulation. Therefore, we have developed two orthogonal methods to isolate and investigate in vivo and in vitro assembled RNA-protein complexes in H. pylori: (i) an affinity chromatography strategy based on aptamer-tagged sRNAs of interest to identify their protein binding partners; and (ii) a rapid method for chromosomal FLAG-tagging of proteins to facilitate co-immunoprecipitation of associated RNA species. Using these methods, we have identified RNA-protein interactions between the ribosomal protein S1 and various mRNAs and sRNAs of H. pylori. Moreover, both methods reported a stable RNA-protein complex between the abundant HPnc6910 sRNA and HP1334, a protein of unknown function that is encoded downstream of HPnc6910. Given that 50% of all bacteria may lack Hfq, our methods can be useful to identify RNA-protein interactions in a wider range of bacterial pathogens.

Use of aptamer tagging to identify in vivo protein binding partners of small regulatory RNAs.

Abstract: Small regulatory RNAs (sRNAs) are short, generally noncoding RNAs that act posttranscriptionally to control target gene expression. Over the past 10 years there has been a rapid expansion in the discovery and characterization of sRNAs in a diverse range of bacteria. Paradigm shifts in our understanding of the breadth of posttranscriptional control by sRNAs were achieved in a number of pioneering studies that involved immunoprecipitation of a known RNA chaperone, the near-ubiquitous Hfq, followed by sequencing to identify novel putative regulators and targets. To perform the converse experiment, we previously developed a method which uses an aptamer-tagged sRNA to allow purification of in vivo assembled RNA-protein complexes and subsequent identification of bound proteins. We successfully implemented this protocol using the Hfq-associated sRNA, InvR, tagged with a tandem repeat of the commonly used MS2-aptamer. Incorporation of the aptamer had no effect on sRNA stability or activity. InvR-MS2 could be effectively purified along with associated proteins, such as Hfq, using maltose binding protein fused to the MS2 coat protein (MBP-MS2) immobilized on an amylose column. Mass-spectroscopy was also used to identify previously uncharacterized protein partners. These results have been described previously (Said et al., Nucleic Acids Res 37:e133, 2009) and thus the figures presented here are intended solely as an illustrative guide to complement this detailed step-by-step protocol.

Hfq-associated Regulatory Small RNAs

Abstract: Regulatory RNAs in bacteria represent a diverse group of molecules that act through a variety of intricate mechanisms to modulate a broad range of physiological responses Some regulatory RNAs act to modulate protein function, often by acting as molecular mimics of other nucleic acids that are regulatory targets of the protein and thus capturing the protein in an unproductive complex (Babitzke & Romeo, 2007) The majority of regulatory RNAs characterized to date act by basepairing with target messenger RNAs (mRNAs) to modulate their stability and/or translation (Papenfort & Vogel, 2010, Waters & Storz, 2009) Base-pairing regulatory RNAs can be divided into cis- and trans-acting RNAs Well characterized groups of cis-acting regulatory RNAs include riboswitches, which are regulatory elements encoded within an mRNA that can control translation by adopting alternative structures in response to signals such as temperature or the presence of small molecules specifically bound by the riboswitch (Grundy & Henkin, 2006) Other cis-acting RNAs include antisense encoded RNAs, which are transcribed from the opposite strand within or near the target coding sequence and therefore contain perfect sequence complementarity to the target mRNA (Thomason & Storz, 2010)