In gram-negative bacteria, the RNA-binding protein Hfq has emerged as an important regulatory factor in a variety of physiological processes, including stress resistance and virulence. In Escherichia coli, Hfq modulates the stability or the translation of mRNAs and interacts with numerous small regulatory RNAs. Here, we studied the role of Hfq in the stress tolerance and virulence of the gram-positive food-borne human pathogen Listeria monocytogenes. We present evidence that Hfq is involved in the ability of L. monocytogenes to tolerate osmotic and ethanol stress and contributes to long-term survival under amino acid-limiting conditions. However, Hfq is not required for resistance to acid and oxidative stress. Transcription of hfq is induced under various stress conditions, including osmotic and ethanol stress and at the entry into the stationary growth phase, thus supporting the view that Hfq is important for the growth and survival of L. monocytogenes in harsh environments. The stress-inducible transcription of hfq depends on the alternative sigma factor B , which controls the expression of numerous stress-and virulence-associated genes in L. monocytogenes. Infection studies showed that Hfq contributes to pathogenesis in mice, yet plays no role in the infection of cultured cell lines. This study provides, for the first time, information on the role of Hfq in the stress tolerance and virulence of a gram-positive pathogen.

The RNA-binding protein Hfq plays important roles in bacterial physiology and is required for the activity of many small regulatory RNAs in prokaryotes. We have previously shown that Hfq contributes to stress tolerance and virulence in the Grampositive human pathogen Listeria monocytogenes. In the present study, we performed coimmunoprecipitations followed by enzymatic RNA sequencing to identify Hfq-binding RNA molecules in L. monocytogenes. The approach resulted in the discovery of three small RNAs (sRNAs). The sRNAs are conserved between Listeria species, but were not identified in other bacterial species. The initial characterization revealed a number of unique features displayed by each individual sRNA. The first sRNA is encoded from within an annotated gene in the L. monocytogenes EGD-e genome. Analogous to most regulatory sRNAs in Escherichia coli, the stability of this sRNA is highly dependent on the presence of Hfq. The second sRNA appears to be produced by a transcription attenuation mechanism, and the third sRNA is present in five copies at two different locations within the L. monocytogenes EGD-e genome. The cellular levels of the sRNAs are growth phase dependent and vary in response to growth medium. All three sRNAs are expressed when L. monocytogenes multiplies within mammalian cells. This study represents the first attempt to identify sRNAs in L. monocytogenes.

Small trans-encoded RNAs (sRNAs) modulate the translation and decay of mRNAs in bacteria. In Gram-negative species, antisense regulation by trans-encoded sRNAs relies on the Sm-like protein Hfq. In contrast to this, Hfq is dispensable for sRNA-mediated riboregulation in the Gram-positive species studied thus far. Here, we provide evidence for Hfq-dependent translational repression in the Gram-positive human pathogen Listeria monocytogenes, which is known to encode at least 50 sRNAs. We show that the Hfq-binding sRNA LhrA controls the translation and degradation of its target mRNA by an antisense mechanism, and that Hfq facilitates the binding of LhrA to its target. The work presented here provides the first experimental evidence for Hfq-dependent riboregulation in a Gram-positive bacterium. Our findings indicate that modulation of translation by trans-encoded sRNAs may occur by both Hfq-dependent and -independent mechanisms, thus adding another layer of complexity to sRNA-mediated riboregulation in Gram-positive species.

Members of the ferritin-like Dps protein family are found in a number of bacterial species, where they demonstrate the potential to bind iron, and have been implicated in tolerance to oxidative stress. In this study of the food-borne pathogen Listeria monocytogenes, the fri gene encoding a Dps homologue was deleted, and, compared to wild-type cells, it was found that the resulting mutant was less resistant to hydrogen peroxide, and demonstrated reduced survival following long-term (7–11 days) incubation in laboratory media. In view of this, it is shown that fri gene expression is controlled by the hydrogen peroxide regulator PerR, as well as the general stress sigma factor σ B. When fri mutant cells were transferred to iron-limiting conditions, growth was retarded relative to wild-type cells, indicating that Fri may be required for iron storage. This notion is supported by the observation that the L. monocytogenes genome appears not to encode other ferritin-like proteins. Given the role of Fri in resistance to oxidative stress, and growth under iron-limiting conditions, the ability of the fri mutant to infect mice was examined. When injected by the intraperitoneal route, the fri mutant demonstrated a reduced capacity to proliferate in the organs of infected mice relative to the wild-type, whereas when the bacteria were supplied intravenously this effect was mitigated. In addition, the mutant was impaired in its ability to survive and grow in J774.A1 mouse macrophage cells. Thus, the data suggest that Fri contributes to the ability of L. monocytogenes to survive in environments where oxidative stress and low iron availability may impede bacterial proliferation.

The multicopy sRNA LhrC of the intracellular pathogen Listeria monocytogenes has been shown to be induced under infection-relevant conditions, but its physiological role and mechanism of action is not understood. In an attempt to pinpoint the exact terms of LhrC expression, cell envelope stress could be defined as a specific inducer of LhrC. In this process, the two-component system LisRK was shown to be indispensable for expression of all five copies of LhrC. lapB mRNA, encoding a cell wall associated protein that was recently identified as an important virulence factor, was disclosed to be directly bound by LhrC leading to an impediment of its translation. Although LhrC binds to Hfq, it does not require the RNA chaperone for stability or lapB mRNA interaction. The mechanism of LhrC-lapB mRNA binding was shown to involve three redundant CU-rich sites and a structural rearrangement in the sRNA. This study represents an extensive depiction of a so far uncharacterized multicopy sRNA and reveals interesting new aspects concerning its regulation, virulence association and mechanism of target binding.

SummaryIn the past few years an increasing number of small non-coding RNAs (sRNAs) in enterobacteria have been found to negatively regulate the expression of outer membrane proteins (OMPs) at the posttranscriptional level. These RNAs act under various growth and stress conditions, suggesting that one important physiological role of regulatory RNA molecules in Gram-negative bacteria is to modulate the cell surface and/or to prevent accumulation of OMPs in the envelope. Here, we extend the OMP-sRNA network by showing that the expression of the OMP YbfM is silenced by a conserved sRNA, designated MicM (also known as RybC/SroB). The regulation is strictly dependent on the RNA chaperone Hfq, and mutational analysis indicates that MicM sequesters the ribosome binding site of ybfM mRNA by an antisense mechanism. Furthermore, we provide evidence that Hfq strongly enhances the on-rate of duplex formation between MicM and its target RNA in vitro, supporting the idea that a major cellular role of the RNA chaperone is to act as a catalyst in RNA-RNA duplex formation.

Small non-coding RNAs (sRNA) have emerged as important elements of gene regulatory circuits. In enterobacteria such as Escherichia coli and Salmonella many of these sRNAs interact with the Hfq protein, an RNA chaperone similar to mammalian Sm-like proteins and act in the post-transcriptional regulation of many genes. A number of these highly conserved ribo-regulators are stringently regulated at the level of transcription and are part of major regulons that deal with the immediate response to various stress conditions, indicating that every major transcription factor may control the expression of at least one sRNA regulator. Here, we extend this view by the identification and characterization of a highly conserved, anaerobically induced small sRNA in E. coli, whose expression is strictly dependent on the anaerobic transcriptional fumarate and nitrate reductase regulator (FNR). The sRNA, named FnrS, possesses signatures of base-pairing RNAs, and we show by employing global proteomic and transcriptomic profiling that the expression of multiple genes is negatively regulated by the sRNA. Intriguingly, many of these genes encode enzymes with "aerobic" functions or enzymes linked to oxidative stress. Furthermore, in previous work most of the potential target genes have been shown to be repressed by FNR through an undetermined mechanism. Collectively, our results provide insight into the mechanism by which FNR negatively regulates genes such as sodA, sodB, cydDC, and metE, thereby demonstrating that adaptation to anaerobic growth involves the action of a small regulatory RNA.In recent years non-coding RNAs have emerged as important components of regulatory circuits both in bacteria and eukaryotes (1-3). In enteric bacteria such as Escherichia coli and Salmonella more than a hundred different sRNAs 2 have been identified, and a major class consists of trans-encoded gene regulatory RNAs that act by an antisense mechanism to activate or, more frequently, to repress translation of target mRNAs. Many of these sRNAs are conserved in related species, are made in response to changes in environmental conditions, and are required for adaptation to stress or specific growth conditions. Several common features have been uncovered. First, the sRNA genes are generally highly regulated at the transcriptional level and are frequently expressed as components of global regulatory systems. Well studied regulatory cases include OxyR regulation of OxyS RNA (oxidative stress) (4), Fur regulation of RyhB RNA (iron limitation) (5), cAMP-CRP regulation of CyaR and Spot 42 RNA (glucose limitation) (6 -9), OmpR regulation of MicF, OmrA, and OmrB RNAs (osmotic shock) (10, 11), E -mediated transcription of MicA and RybB RNAs (envelope stress) (2, 12), PhoPQ regulation of MgrR (Mg 2ϩ /Ca 2ϩ transport and virulence) (13), and LuxO control of Qrr1-4 (quorum sensing in Vibrio) (14). Furthermore, small RNA expression may be under control of a dedicated transcription factor, as exemplified by SgrR/SgrS RNA (glucose-phosphate stress) (15). Second, pairin...

It was previously shown that enhanced nisin resistance in some mutants was associated with increased expression of three genes, pbp2229, hpk1021, and lmo2487, encoding a penicillin-binding protein, a histidine kinase, and a protein of unknown function, respectively. In the present work, we determined the direct role of the three genes in nisin resistance. Interruption of pbp2229 and hpk1021 eliminated the nisin resistance phenotype. Interruption of hpk1021 additionally abolished the increase in pbp2229 expression. The results indicate that this nisin resistance mechanism is caused directly by the increase in pbp2229 expression, which in turn is brought about by the increase in hpk1021 expression. We also found a degree of cross-protection between nisin and class IIa bacteriocins and investigated possible mechanisms. The expression of virulence genes in one nisin-resistant mutant and two class IIa bacteriocin-resistant mutants of the same wild-type strain was analyzed, and each mutant consistently showed either an increase or a decrease in the expression of virulence genes (prfA-regulated as well as prfA-independent genes). Although the changes mostly were moderate, the consistency indicates that a mutant-specific change in virulence may occur concomitantly with bacteriocin resistance development.Nisin and the class IIa bacteriocins (also called pediocin-like bacteriocins) are antimicrobial peptides that are produced by lactic acid bacteria and that have the greatest potential as biopreservatives for food. One of the main target organisms in this context is Listeria monocytogenes, a food-borne pathogen that causes severe human illness as well as economic losses for the food industry.Nisin exerts its antimicrobial action by forming pores in the cytoplasmic membrane through an interaction with the peptidoglycan precursor lipid II (for a recent review, see reference 17). Enhanced nisin resistance in L. monocytogenes generally constitutes less than a 10-fold increase in the MIC. Nisin resistance in several, but not all, spontaneous mutants of L.