Biomedical subjects

Jörg Vogel

Publications and source records attributed to Jörg Vogel.

10 recordsLinked to original sources

PubMed2006

The RNA chaperone Hfq is essential for the virulence of Salmonella typhimurium.

The RNA chaperone, Hfq, plays a diverse role in bacterial physiology beyond its original role as a host factor required for replication of Qbeta RNA bacteriophage. In this study, we show that Hfq is involved in the expression and secretion of virulence factors in the facultative intracellular pathogen, Salmonella typhimurium. A Salmonella hfq deletion strain is highly attenuated in mice after both oral and intraperitoneal infection, and shows a severe defect in invasion of epithelial cells and a growth defect in both epithelial cells and macrophages in vitro. Surprisingly, we find that these phenotypes are largely independent of the previously reported requirement of Hfq for expression of the stationary phase sigma factor, RpoS. Our results implicate Hfq as a key regulator of multiple aspects of virulence including regulation of motility and outer membrane protein (OmpD) expression in addition to invasion and intracellular growth. These pleiotropic effects are suggested to involve a network of regulatory small non-coding RNAs, placing Hfq at the centre of post-transcriptional regulation of virulence gene expression in Salmonella. In addition, the hfq mutation appears to cause a chronic activation of the RpoE-mediated envelope stress response which is likely due to a misregulation of membrane protein expression.

Bacterial Outer Membrane Proteins↗

PubMed2006

Small non-coding RNAs and the bacterial outer membrane.

Recent systematic genome searches revealed that bacteria encode a tremendous number of small non-coding RNAs (sRNAs). Whereas most of these molecules remain of unknown function, it has become increasingly clear that many of them will act to modulate gene expression at the post-transcriptional level. Where studied in more detail, sRNAs have often been found to control the expression of outer membrane proteins (OMPs). Enterobacteria such as Escherichia coli and Salmonella are now known to encode at least eight OMP-regulating sRNAs (InvR, MicA, MicC, MicF, OmrAB, RseX and RybB). These sRNAs exert their functions under a variety of growth and stress conditions, including the sigmaE-mediated envelope stress response. An sRNA-OMP network is emerging in which some sRNAs act specifically on a single omp mRNA, whereas others control multiple omp mRNA targets.

Bacterial Outer Membrane Proteins↗

PubMed2006

Experimental approaches to identify non-coding RNAs.

Cellular RNAs that do not function as messenger RNAs (mRNAs), transfer RNAs (tRNAs) or ribosomal RNAs (rRNAs) comprise a diverse class of molecules that are commonly referred to as non-protein-coding RNAs (ncRNAs). These molecules have been known for quite a while, but their importance was not fully appreciated until recent genome-wide searches discovered thousands of these molecules and their genes in a variety of model organisms. Some of these screens were based on biocomputational prediction of ncRNA candidates within entire genomes of model organisms. Alternatively, direct biochemical isolation of expressed ncRNAs from cells, tissues or entire organisms has been shown to be a powerful approach to identify ncRNAs both at the level of individual molecules and at a global scale. In this review, we will survey several such wet-lab strategies, i.e. direct sequencing of ncRNAs, shotgun cloning of small-sized ncRNAs (cDNA libraries), microarray analysis and genomic SELEX to identify novel ncRNAs, and discuss the advantages and limits of these approaches.

Gene Expression Profiling↗

PubMed2005

Hfq-dependent regulation of OmpA synthesis is mediated by an antisense RNA.

This paper shows that the small RNA MicA (previously SraD) is an antisense regulator of ompA in Escherichia coli. MicA accumulates upon entry into stationary phase and down-regulates the level of ompA mRNA. Regulation of ompA (outer membrane protein A), previously attributed to Hfq/mRNA binding, is lost upon deletion of the micA gene, whereas overexpression of MicA inhibits the synthesis of OmpA. In vitro, MicA binds to the ompA mRNA leader. Enzymatic and chemical probing was used to map the structures of MicA, the ompA mRNA leader, and the complex formed upon binding. MicA binding generates a footprint across the ompA Shine-Dalgarno sequence, consistent with a 12 + 4 base-pair interaction, which is additionally supported by the effect of mutations in vivo and by bioinformatics analysis of enterobacterial micA/ompA homolog sequences. MicA is conserved in many enterobacteria, as is its ompA target site. In vitro toeprinting confirmed that binding of MicA specifically interferes with ribosome binding. We propose that MicA, when present at high levels, blocks ribosome binding at the ompA translation start site, which-in line with previous work-secondarily facilitates RNase E cleavage and subsequent mRNA decay. MicA requires the presence of the Hfq protein, although the mechanistic basis for this remains unclear.

Bacterial Outer Membrane Proteins↗

PubMed2005

Identification of cyanobacterial non-coding RNAs by comparative genome analysis.

BACKGROUND: Whole genome sequencing of marine cyanobacteria has revealed an unprecedented degree of genomic variation and streamlining. With a size of 1.66 megabase-pairs, Prochlorococcus sp. MED4 has the most compact of these genomes and it is enigmatic how the few identified regulatory proteins efficiently sustain the lifestyle of an ecologically successful marine microorganism. Small non-coding RNAs (ncRNAs) control a plethora of processes in eukaryotes as well as in bacteria; however, systematic searches for ncRNAs are still lacking for most eubacterial phyla outside the enterobacteria. RESULTS: Based on a computational prediction we show the presence of several ncRNAs (cyanobacterial functional RNA or Yfr) in several different cyanobacteria of the Prochlorococcus-Synechococcus lineage. Some ncRNA genes are present only in two or three of the four strains investigated, whereas the RNAs Yfr2 through Yfr5 are structurally highly related and are encoded by a rapidly evolving gene family as their genes exist in different copy numbers and at different sites in the four investigated genomes. One ncRNA, Yfr7, is present in at least seven other cyanobacteria. In addition, control elements for several ribosomal operons were predicted as well as riboswitches for thiamine pyrophosphate and cobalamin. CONCLUSION: This is the first genome-wide and systematic screen for ncRNAs in cyanobacteria. Several ncRNAs were both computationally predicted and their presence was biochemically verified. These RNAs may have regulatory functions and each shows a distinct phylogenetic distribution. Our approach can be applied to any group of microorganisms for which more than one total genome sequence is available for comparative analysis.

Base Sequence↗

PubMed2005

How to find small non-coding RNAs in bacteria.

Small non-coding RNAs (sRNAs) have attracted considerable attention as an emerging class of gene expression regulators. In bacteria, a few regulatory RNA molecules have long been known, but the extent of their role in the cell was not fully appreciated until the recent discovery of hundreds of potential sRNA genes in the bacterium Escherichia coli. Orthologs of these E. coli sRNA genes, as well as unrelated sRNAs, were also found in other bacteria. Here we review the disparate experimental approaches used over the years to identify sRNA molecules and their genes in prokaryotes. These include genome-wide searches based on the biocomputational prediction of non-coding RNA genes, global detection of non-coding transcripts using microarrays, and shotgun cloning of small RNAs (RNomics). Other sRNAs were found by either co-purification with RNA-binding proteins, such as Hfq or CsrA/RsmA, or classical cloning of abundant small RNAs after size fractionation in polyacrylamide gels. In addition, bacterial genetics offers powerful tools that aid in the search for sRNAs that may play a critical role in the regulatory circuit of interest, for example, the response to stress or the adaptation to a change in nutrient availability. Many of the techniques discussed here have also been successfully applied to the discovery of eukaryotic and archaeal sRNAs.

Cloning, Molecular↗

PubMed2004

The small RNA IstR inhibits synthesis of an SOS-induced toxic peptide.

More than 60 small RNAs (sRNA) have been identified in E. coli. The functions of the majority of these sRNAs are still unclear. For the few sRNAs characterized, expression and functional studies indicate that they act under stress conditions. Here, we describe a novel E. coli chromosome locus that is part of the SOS response to DNA damage. This locus encodes two sRNAs, IstR-1 and IstR-2, and a toxic peptide, TisB, encoded by tisAB mRNA. Transcription of tisAB and istR-2 is SOS regulated, whereas IstR-1 is present throughout growth. IstR-1 inhibits toxicity by base-pairing to a short region in the tisAB mRNA. This antisense interaction entails RNase III-dependent cleavage, thereby inactivating the mRNA for translation. In the absence of the SOS response, IstR-1 is present in high excess over its target. However, SOS induction leads to depletion of the IstR-1 pool, concomitant with accumulation of tisAB mRNA. Under such conditions, TisB exerts its toxic effect, slowing down growth. We propose that the inhibitory sRNA prevents inadvertent TisB synthesis during normal growth and, possibly, also limits SOS-induced toxicity. Our study adds the SOS regulon to the growing list of global regulatory circuits controlled by sRNA genes.

Amino Acid Sequence↗

PubMed2003

RNomics in Escherichia coli detects new sRNA species and indicates parallel transcriptional output in bacteria.

Recent bioinformatics-aided searches have identified many new small RNAs (sRNAs) in the intergenic regions of the bacterium Escherichia coli. Here, a shot-gun cloning approach (RNomics) was used to generate cDNA libraries of small sized RNAs. Besides many of the known sRNAs, we found new species that were not predicted previously. The present work brings the number of sRNAs in E.coli to 62. Experimental transcription start site mapping showed that some sRNAs were encoded from independent genes, while others were processed from mRNA leaders or trailers, indicative of a parallel transcriptional output generating sRNAs co-expressed with mRNAs. Two of these RNAs (SroA and SroG) consist of known (THI and RFN) riboswitch elements. We also show that two recently identified sRNAs (RyeB and SraC/RyeA) interact, resulting in RNase III-dependent cleavage. To the best of our knowledge, this represents the first case of two non-coding RNAs interacting by a putative antisense mechanism. In addition, intracellular metabolic stabilities of sRNAs were determined, including ones from previous screens. The wide range of half-lives (<2 to >32 min) indicates that sRNAs cannot generally be assumed to be metabolically stable. The experimental characterization of sRNAs analyzed here suggests that the definition of an sRNA is more complex than previously assumed.

Blotting, Northern↗

PubMed2003

Experimental and computational analysis of transcriptional start sites in the cyanobacterium Prochlorococcus MED4.

In contrast to certain model eubacteria, little is known as to where transcription is initiated in the genomes of cyanobacteria, which are largely distinct from other prokaryotes. In this work, 25 transcription start sites (TSS) of 21 different genes of Prochlorococcus sp. MED4 were determined experimentally. The data suggest more than one TSS for the genes ftsZ, petH, psbD and ntcA. In contrast, the rbcL-rbcS operon encoding ribulose 1,5-bisphosphate carboxylase/oxygenase lacks a detectable promoter and is co-transcribed with the upstream located gene ccmK. The entire set of experimental data was used in a genome-wide scan for putative TSS in Prochlorococcus. A -10 element could be defined, whereas at the -35 position there was no element common to all investigated sequences. However, splitting the data set into sub-classes revealed different types of putative -35 boxes. Only one of them resembled the consensus sequence TTGACA recognized by the vegetative sigma factor (sigma70) of enterobacteria. Using a scoring matrix of the -10 element, more than 3000 TSS were predicted, about 40% of which were estimated to be functional. This is the first systematic study of transcription initiation sites in a cyanobacterium.

Base Sequence↗

PubMed2002

Lariat formation and a hydrolytic pathway in plant chloroplast group II intron splicing.

Lariat formation has been studied intensively only with a few self-splicing group II introns, and little is known about how the numerous diverse introns in plant organelles are excised. Several of these introns have branch-points that are not a single bulge but are adjoined by A:A, A:C, A:G and G:G pairs. Using a highly sensitive in vivo approach, we demonstrate that all but one of the barley chloroplast introns splice via the common pathway that produces a branched product. RNA editing does not improve domain 5 and 6 structures of these introns. The conserved branch-point in tobacco rpl16 is chosen even if an adjacent unpaired adenosine is available, suggesting that spatial arrangements in domain 6 determine correct branch-point selection. Lariats were not detected for the chloroplast trnV intron, which lacks an unpaired adenosine in domain 6. Instead, this intron is released as linear molecules that undergo further polyadenylation. trnV, which is conserved throughout plant evolution, constitutes the first example of naturally occurring hydrolytic group II intron splicing in vivo.

Chloroplasts↗