The bacterium Listeria monocytogenes is ubiquitous in the environment and can lead to severe food-borne infections. It has recently emerged as a multifaceted model in pathogenesis. However, how this bacterium switches from a saprophyte to a pathogen is largely unknown. Here, using tiling arrays and RNAs from wild-type and mutant bacteria grown in vitro, ex vivo and in vivo, we have analysed the transcription of its entire genome. We provide the complete Listeria operon map and have uncovered far more diverse types of RNAs than expected: in addition to 50 small RNAs (<500 nucleotides), at least two of which are involved in virulence in mice, we have identified antisense RNAs covering several open-reading frames and long overlapping 5' and 3' untranslated regions. We discovered that riboswitches can act as terminators for upstream genes. When Listeria reaches the host intestinal lumen, an extensive transcriptional reshaping occurs with a SigB-mediated activation of virulence genes. In contrast, in the blood, PrfA controls transcription of virulence genes. Remarkably, several non-coding RNAs absent in the non-pathogenic species Listeria innocua exhibit the same expression patterns as the virulence genes. Together, our data unravel successive and coordinated global transcriptional changes during infection and point to previously unknown regulatory mechanisms in bacteria.

In Listeria monocytogenes, virulence genes are maximally expressed at 37 degrees C, almost silent at 30 degrees C and controlled by PrfA, a transcriptional activator whose expression is thermoregulated. Here, we show that the untranslated mRNA (UTR) preceding prfA, forms a secondary structure, which masks the ribosome binding region. Mutations predicted to destabilize this structure led to virulence gene expression and invasion of mammalian cells at 30 degrees C. Chemical probing, native gel electrophoresis, in vitro translation, and "compensatory" and "increased stability" mutations demonstrated that the UTR switches between a structure active at high temperatures, and another inactive at low temperatures. Strikingly, when the DNA corresponding to the UTR was fused to gfp in E. coli, bacteria became fluorescent at 37 degrees C, but not at 30 degrees C. This mechanism of posttranscriptional thermoregulation may have important applications.

Riboswitches are RNA elements acting in cis, controlling expression of their downstream genes through a metabolite-induced alteration of their secondary structure. Here, we demonstrate that two S-adenosylmethionine (SAM) riboswitches, SreA and SreB, can also function in trans and act as noncoding RNAs in Listeria monocytogenes. SreA and SreB control expression of the virulence regulator PrfA by binding to the 5'-untranslated region of its mRNA. Absence of the SAM riboswitches SreA and SreB increases the level of PrfA and virulence gene expression in L. monocytogenes. Thus, the impact of the SAM riboswitches on PrfA expression highlights a link between bacterial virulence and nutrient availability. Together, our results uncover an unexpected role for riboswitches and a distinct class of regulatory noncoding RNAs in bacteria.

SummaryWe discovered a new small non-coding RNA (sRNA) gene, vrrA of Vibrio cholerae O1 strain A1552. A vrrA mutant overproduces OmpA porin, and we demonstrate that the 140 nt VrrA RNA represses ompA translation by base-pairing with the 5Ј region of the mRNA. The RNA chaperone Hfq is not stringently required for VrrA action, but expression of the vrrA gene requires the membrane stress sigma factor, s E , suggesting that VrrA acts on ompA in response to periplasmic protein folding stress. We also observed that OmpA levels inversely correlated with the number of outer membrane vesicles (OMVs), and that VrrA increased OMV production comparable to loss of OmpA. VrrA is the first sRNA known to control OMV formation. Moreover, a vrrA mutant showed a fivefold increased ability to colonize the intestines of infant mice as compared with the wild type. There was increased expression of the main colonization factor of V. cholerae, the toxin co-regulated pili, in the vrrA mutant as monitored by immunoblot detection of the TcpA protein. VrrA overproduction caused a distinct reduction in the TcpA protein level. Our findings suggest that VrrA contributes to bacterial fitness in certain stressful environments, and modulates infection of the host intestinal tract.

SummarySignature-tagged mutagenesis (STM) was used to identify new genes involved in the virulence of the Gram-positive intracellular pathogen Listeria monocytogenes . One of the mutants isolated by this technique had the transposon inserted in virR, a gene encoding a putative response regulator of a twocomponent system. Deletion of virR severely decreased virulence in mice as well as invasion in cell-culture experiments. Using a transcriptomic approach, we identified 12 genes regulated by VirR, including the dlt -operon, previously reported to be important for L. monocytogenes virulence. However, a strain lacking dltA , was not as impaired in virulence as the D D D D virR strain, suggesting a role in virulence for other members of the vir regulon. Another VirR-regulated gene is homologous to mprF , which encodes a protein that modifies membrane phosphatidyl glycerol with L -lysine and that is involved in resistance to human defensins in Staphylococcus aureus . VirR thus appears to control virulence by a global regulation of surface components modifications. These modifications may affect interactions with host cells, including components of the innate immune system. Surprisingly, although controlling the same set of genes as VirR, the putative cognate histidine kinase of VirR, VirS, encoded by a gene located three genes downstream of virR, was shown not to be essential for virulence. By monitoring the activity of VirR with a GFP reporter construct, we showed that VirR can be activated independently of VirS, for example through a mechanism involving variations in the level of intracellular acetyl phosphate. In silico analysis of the VirR-regulated promoters revealed a VirR DNA-binding consensus site and specific interaction between purified VirR protein and this consensus sequence was demonstrated by gel mobility shift assays. This study identifies a second key virulence regulon in L. monocytogenes , after the prfA regulon .