Biomedical subjects

E Gerhart H Wagner

Publications and source records attributed to E Gerhart H Wagner.

16 recordsLinked to original sources

PubMed2006

The role of RNAs in the regulation of virulence-gene expression.

Bacterial pathogens sense their environment, and in response, virulence genes are induced or repressed through spatial and temporal regulation. They are also subjected to stress conditions, which require appropriate responses. Recent research has revealed that RNAs are key regulators in pathogens. Small RNAs regulate the translation and/or stability of mRNAs that encode virulence proteins, or proteins with roles in adaptive responses, which are triggered by environmental cues and stresses. In most cases, these small RNAs act directly on target RNAs by an antisense mechanism. Other small RNAs act indirectly, by sequestration of regulatory proteins. Direct sensing of environmental signals can occur through induced structural changes in mRNAs.

Adaptation, Physiological↗

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

Lead(II) cleavage analysis of RNase P RNA in vivo.

The overall conformation of M1 RNA, the catalytic RNA subunit of RNase P in Escherichia coli, was analyzed in vivo and, in the presence of the C5 protein subunit, in vitro by lead(II) acetate probing. The partial cleavage patterns obtained are congruent with previous structure mapping performed in vitro. Most of the known major and minor cleavages in M1 RNA were supported and could be mapped onto a secondary structure model. The data obtained indicate that C5 has only minor effects on the overall structure of the RNA subunit. The similar cleavage patterns obtained in vitro and in vivo furthermore suggest that the intracellular environment does not greatly alter the overall conformation of M1 RNA within the holoenzyme complex. Moreover, our data indicate that M1 RNA in vivo is present in at least two states-the major fraction is bound to tRNA substrates and a minor fraction is substrate free. Finally, both in this and previous work we found that lead(II) probing data from in vivo experiments conducted on longer RNAs (tmRNA and M1 RNA) generally gives superior resolution compared to parallel in vitro experiments. This may reflect the absence of alternative conformers present in vitro and the more natural state of these RNAs in the cell due to proper, co-transcriptional folding pathways and possibly the presence of RNA chaperones.

Base Sequence↗

PubMed2005

Paenibacillus polymyxa invades plant roots and forms biofilms.

Paenibacillus polymyxa is a plant growth-promoting rhizobacterium with a broad host range, but so far the use of this organism as a biocontrol agent has not been very efficient. In previous work we showed that this bacterium protects Arabidopsis thaliana against pathogens and abiotic stress (S. Timmusk and E. G. H. Wagner, Mol. Plant-Microbe Interact. 12:951-959, 1999; S. Timmusk, P. van West, N. A. R. Gow, and E. G. H. Wagner, p. 1-28, in Mechanism of action of the plant growth promoting bacterium Paenibacillus polymyxa, 2003). Here, we studied colonization of plant roots by a natural isolate of P. polymyxa which had been tagged with a plasmid-borne gfp gene. Fluorescence microscopy and electron scanning microscopy indicated that the bacteria colonized predominantly the root tip, where they formed biofilms. Accumulation of bacteria was observed in the intercellular spaces outside the vascular cylinder. Systemic spreading did not occur, as indicated by the absence of bacteria in aerial tissues. Studies were performed in both a gnotobiotic system and a soil system. The fact that similar observations were made in both systems suggests that colonization by this bacterium can be studied in a more defined system. Problems associated with green fluorescent protein tagging of natural isolates and deleterious effects of the plant growth-promoting bacteria are discussed.

Arabidopsis↗

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↗

PubMed2004

A repeated GGA motif is critical for the activity and stability of the riboregulator RsmY of Pseudomonas fluorescens.

The riboregulator RsmY of Pseudomonas fluorescens strain CHA0 is an example of small regulatory RNAs belonging to the global Rsm/Csr regulatory systems controlling diverse cellular processes such as glycogen accumulation, motility, or formation of extracellular products in various bacteria. By binding multiple molecules of the small regulatory protein RsmA, RsmY relieves the negative effect of RsmA on the translation of several target genes involved in the biocontrol properties of strain CHA0. RsmY and functionally related riboregulators have repeated GGA motifs predicted to be exposed in single-stranded regions, notably in the loops of hairpins. The secondary structure of RsmY was corroborated by in vivo cleavage with lead acetate. RsmY mutants lacking three or five (out of six) of the GGA motifs showed reduced ability to derepress the expression of target genes in vivo and failed to bind the RsmA protein efficiently in vitro. The absence of GGA motifs in RsmY mutants resulted in reduced abundance of these transcripts and in a shorter half-life (< or = 6 min as compared with 27 min for wild type RsmY). These results suggest that both the interaction of RsmY with RsmA and the stability of RsmY strongly depend on the GGA repeats and that the ability of RsmY to interact with small regulatory proteins such as RsmA may protect this RNA from degradation.

Bacterial Proteins↗

PubMed2004

Disruption of the gene encoding the V-ATPase subunit A results in inhibition of normal growth and abolished sporulation in Aspergillus nidulans.

The authors have previously reported on molecular responses of Aspergillus nidulans to bacterial antifungal metabolites, e.g. bafilomycins and the related concanamycins. These compounds are known inhibitors of V-ATPases and cause dramatic effects on mycelial growth and morphology. In Neurospora crassa, studies have shown that disruption of the gene encoding subunit A of the V-ATPase results in morphological changes and reduced growth similar to those observed after addition of concanamycin. This phenotype, and the fact that this mutation confers resistance to concanamycin, suggests that V-ATPase is the main (or only) target for the antibiotics. However, growth inhibition and morphology changes in, for example, A. nidulans and Penicillium roqueforti are more severe, and thus other targets are possible. In this study, the vmaA gene of A. nidulans, encoding the subunit A of V-ATPase, was disrupted by homologous recombination. The resulting vmaA1 mutant strain displayed extremely slow growth and failed to produce asexual spores. Furthermore, an altered morphology similar to that caused by addition of V-ATPase inhibitors, i.e. bafilomycin or concanamycin, was observed, indicating that V-ATPase is the main target for the antibiotics also in A. nidulans. The vmaA1 mutant was not viable at pH values above 7 and was highly sensitive to high Zn(2+) concentrations, in agreement with previous results from studies of Saccharomyces cerevisiae and N. crassa.

Aspergillus nidulans↗

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

Loop swapping in an antisense RNA/target RNA pair changes directionality of helix progression.

The binding pathway of the natural antisense RNA CopA to its target CopT proceeds through a hierarchical order of steps. It initiates by reversible loop-loop contacts followed by unidirectional helix progression into the upper stems. This involves extensive breakage of intramolecular base pairs and the subsequent formation of two intermolecular helices, B and B'. Based on the known tRNA anticodon loop structure and on results from the Sok/Hok antisense/target RNA system, it had been suggested that a U-turn (or pi-turn) in the loop of CopT might determine the directionality of helix progression. Data presented here show that the putative U-turn is one of the structural elements of antisense/target RNA pairs required to achieve fast binding kinetics. Swapping of the hypothetical U-turn structure from the target RNA to the antisense RNA retained regulatory performance in vivo and binding rates in vitro but altered the binding pathway by changing the direction in which the initiating helix was extended. In addition, our data indicate that a helical stem immediately adjacent to the target loop sequence is required to provide a scaffold for the U-turn.

Bacterial Proteins↗

PubMed2003

Characterization of phiA, a gene essential for phialide development in Aspergillus nidulans.

We have previously identified genes and proteins involved in the fungal response to the Streptomyces-produced antibiotics, bafilomycin B1 and concanamycin A, known inhibitors of V-ATPases. Using mRNA differential display we identified an Aspergillus nidulans gene with 30-fold up-regulated expression in the presence of bafilomycin. This gene, here denoted phiA, and its gene product, were further characterized by targeted gene disruption and immunohistochemistry. Phenotypically, the phiA mutation resulted in reduced growth and severely reduced sporulation. The abnormality could be traced to the phialides, which divided several times instead of forming a single flask-shaped cell. The importance of phiA for phialide and conidium development was supported by immunohistochemistry experiments that showed the protein to be mainly present in these two cell types. Attempts to relate phiA to inhibition of V-ATPases did not result in unambiguous conclusions, but suggest the possibility that changed expression of phiA is correlated with growth arrest caused by inhibited V-ATPases.

Amino Acid Sequence↗

PubMed2002

Antisense RNAs in bacteria and their genetic elements.

Antisense RNA-mediated regulation is widespread in bacteria. Most antisense RNA control systems have been found in plasmids, phages, and transposons. Fewer examples were identified in bacterial chromosomes. This chapter summarizes our current knowledge about antisense RNAs with respect to their occurrence, their biological roles, and their diverse mechanisms of action. Examples of cis- or trans-encoded antisense RNAs are discussed, and their properties compared. Most antisense RNAs are posttranscriptionally acting inhibitors of target genes, but a few examples of activator antisense RNAs are known. The implications of RNA structure on topologically and kinetically favored binding pathways are addressed, and solutions that have evolved to permit productive interactions between intricately folded RNAs are discussed. Finally, we describe how particular properties of individual antisense/target RNA systems match their respective biological roles.

Bacteria↗

PubMed2002

Antisense RNAs everywhere?

In recent years, systematic searches of both prokaryote and eukaryote genomes have identified a staggering number of small RNAs, the biological functions of which remain unknown. Small RNA-based regulators are well known from bacterial plasmids. They act on target RNAs by sequence complementarity; that is, they are antisense RNAs. Recent findings suggest that many of the novel orphan RNAs encoded by bacterial and eukaryotic chromosomes might also belong to a ubiquitous, heterogeneous class of antisense regulators of gene expression.

Base Sequence↗

PubMed2002

Lead(II) as a probe for investigating RNA structure in vivo.

In this communication, we describe a simple and reliable method for RNA structure determination in vivo, using the divalent ion, lead(II), as a structural probe. Lead(II) is known to cleave RNA within single-stranded regions, loops, and bulges, whereas cleavages in double-stranded regions are weaker or absent. Because the ion easily entered bacterial cells, Escherichia coli cultures were treated by addition of 50-100 mM lead(II) acetate for 3-7 min, resulting in partial cleavage of RNA in vivo. Cleavage positions were mapped by reverse transcription analysis of total extracted RNA. Three RNAs were analyzed: tmRNA, CopT (the target of the antisense RNA CopA), and the leader region of the ompF mRNA. All three RNAs had previously been analyzed in vitro, and secondary structure models were available. The results presented here show that lead(II) cleavages in vivo yield detailed structural information for these RNAs, which was in good agreement with the models proposed based on in vitro work. These data illustrate the potential of lead(II) as a sequence-independent RNA structure probe for use in living cells.

Base Sequence↗

PubMed2002

An antisense RNA-mediated transcriptional attenuation mechanism functions in Escherichia coli.

Antisense RNA-mediated transcriptional attenuation is a regulatory mechanism operating in the replication control of two groups of plasmids in gram-positive bacteria, the pT181 group and the inc18 family, represented by pIP501. In contrast, this control mechanism has so far not been identified in gram-negative bacteria or their plasmids. In this work we asked whether such a mechanism can be supported by Escherichia coli. The core replication control regions of plasmids pT181 and pIP501 were transferred into this heterologous host. In vivo lacZ reporter gene assays showed that the antisense RNAs of these plasmids can inhibit lacZ expression and that most of this effect can be accounted for by reduced mRNA readthrough. Northern analyses confirmed that the ratio of attenuated to readthrough target RNA was increased in the presence of the cognate antisense RNA, as expected for this mechanism. Similarly, both antisense RNAs induced premature termination of their cognate target RNAs in an E. coli in vitro transcription system, whereas the noncognate antisense RNAs had no effect. Thus, this report shows that antisense RNA-mediated transcriptional attenuation is supported by at least one gram-negative host, although the data indicate that inhibitory efficiencies are lower than those for, e.g., Bacillus subtilis. Possible explanations for the apparent absence of this control mode in plasmids of gram-negative bacteria are discussed.

Blotting, Northern↗

PubMed1999

Changes in Aspergillus nidulans gene expression induced by bafilomycin, a Streptomyces-produced antibiotic.

In natural environments bacteria and filamentous fungi often compete for the same resources. Consequently, production of antibiotic secondary metabolites and defence mechanisms against these compounds have evolved in these organisms. An experimental model has been developed to study the response in fungi exposed to one such antibiotic. The filamentous fungus Aspergillus nidulans was treated with bafilomycin B1, a Streptomyces-produced antibiotic which reduces radial growth rate and induces morphological changes in fungi. mRNA differential display was used to study changes in fungal gene expression. For five genes, changes in abundance of the corresponding mRNAs, directly or indirectly caused by bafilomycin, were observed. Of these, three were up-regulated and two repressed. With four of these the change in mRNA abundance measured ranged from 10- to 60-fold. However, for one gene the mRNA was only detected after bafilomycin treatment. One of the down-regulated mRNAs encodes ASPND1, a glycoprotein that belongs to a known family of antigens identified in aspergilloma patients. One up-regulated mRNA shows sequence similarities, at the amino acid level, with a cell-wall protein of Saccharomyces cerevisiae. The remaining three genes were also cloned and sequenced; their sequences do not correspond to known genes in A. nidulans, and no similarities with published nucleotide or protein sequences in other organisms were found. These results indicate the feasibility of using mRNA differential display to study interactions between bacteria and filamentous fungi.

Amino Acid Sequence↗

PubMed1998

Degradation pathway of CopA, the antisense RNA that controls replication of plasmid R1.

RNA decay in bacteria is carried out by a number of enzymes that participate in the coordinated degradation of their substrates. Endo- and exonucleolytic cleavages as well as polyadenylation are generally involved in determining the half-life of RNAs. Small, untranslated antisense RNAs are suitable model systems to study decay. A study of the pathway of degradation of CopA, the copy number regulator RNA of plasmid R1, is reported here. Strains carrying mutations in the genes encoding RNase E, polynucleotide phosphorylase (PNPase), RNase II and poly(A) polymerase I (PcnB/PAP I)--alone or in combination--were used to investigate degradation patterns and relative half-lives of CopA. The results obtained suggest that RNase E initiates CopA decay. Both PNPase and RNase II can degrade the major 3'-cleavage product generated by RNase E. This exonucleolytic degradation is aided by PcnB, which may imply a requirement for A-tailing. RNase II can partially protect CopA's 3'-end from PNPase-dependent degradation. Other RNases are probably involved in decay, since in rnb/pnp double mutants, decay still occurs, albeit at a reduced rate. Experiments using purified RNase E identified cleavage sites in CopA in the vicinity of, but not identical to, those mapped in vivo, suggesting that the cleavage site specificity of this RNase is modulated by additional proteins in the cell. A model of CopA decay is presented and discussed.

Bacterial Proteins↗