RNA structures are fundamentally important for RNA function. Dynamic, condition-dependent structural changes are able to modulate gene expression as shown for riboswitches and RNA thermometers. By parallel analysis of RNA structures, we mapped the RNA structurome of Yersinia pseudotuberculosis at three different temperatures. This human pathogen is exquisitely responsive to host body temperature (37°C), which induces a major metabolic transition. Our analysis profiles the structure of more than 1,750 RNAs at 25°C, 37°C, and 42°C. Average mRNAs tend to be unstructured around the ribosome binding site. We searched for 5′-UTRs that are folded at low temperature and identified novel thermoresponsive RNA structures from diverse gene categories. The regulatory potential of 16 candidates was validated. In summary, we present a dynamic bacterial RNA structurome and find that the expression of virulence-relevant functions in Y. pseudotuberculosis and reprogramming of its metabolism in response to temperature is associated with a restructuring of numerous mRNAs.RNA structure | RNA thermometer | temperature | virulence | translational control R NA structures play a pivotal role in the function of noncoding RNAs (ncRNAs) comprised of rRNAs, tRNAs, and small regulatory RNAs (sRNAs) and in the expression of proteincoding mRNAs. Structured segments affect the entire RNA life cycle from transcription, maturation, and translation to degradation and determine the specificity of interactions with other RNAs, proteins, or ligands. Although some RNAs, such as ribozymes and rRNAs, adopt rather stable secondary and tertiary structures, many regulatory RNA elements are dynamic and undergo structural rearrangements in the physiological temperature range. Well-known examples are metabolite-sensing riboswitches (1) and temperaturesensing RNA thermometers (RNATs) (2). Bacterial RNATs are usually located in the 5′-UTR or the intercistronic region (ICR) of an mRNA and differentially control translation of the downstream ORF in response to temperature (3). Typically, an RNAT folds into a structure that occludes the ribosome binding site (RBS). A temperature upshift liberates the RBS and allows the ribosome to bind and initiate translation. Structurally diverse RNATs are located upstream of many bacterial heat shock and virulence genes. Thermosensitive RNA structures playing a role in infection and host adaptation processes have been documented in Listeria monocytogenes (4), Yersinia pestis (5), Yersinia pseudotuberculosis (6), Leptospira interrogans (7), Shigella dysenteriae (8), Neisseria meningitidis (9), Pseudomonas aeruginosa (10), and Vibrio cholerae (11).In contrast to ligand-binding riboswitches, RNATs show poor, if any, conservation in sequence and structure. This diversity has hampered the in silico identification of novel RNATs, but recently developed genome-wide RNA structure-probing approaches offer new opportunities (12). Global structure probing maps the structures of the entire pool of expressed RNA molecules and provides a sna...

Photosynthetic organisms need defense systems against photooxidative stress caused by the generation of highly reactive singlet oxygen ( 1 O 2 ). Here we show that the alternative sigma factor RpoH II is required for the expression of important defense factors and that deletion of rpoH II leads to increased sensitivity against exposure to 1 O 2 and methylglyoxal in Rhodobacter sphaeroides. The gene encoding RpoH II is controlled by RpoE, and thereby a sigma factor cascade is constituted. We provide the first in vivo study that identifies genes controlled by an RpoH II -type sigma factor, which is widely distributed in the Alphaproteobacteria. RpoH IIdependent genes encode oxidative-stress defense systems, including proteins for the degradation of methylglyoxal, detoxification of peroxides, 1 O 2 scavenging, and redox and iron homeostasis. Our experiments indicate that glutathione (GSH)-dependent mechanisms are involved in the defense against photooxidative stress in photosynthetic bacteria. Therefore, we conclude that systems pivotal for the organism's defense against photooxidative stress are strongly dependent on GSH and are specifically recognized by RpoH II in R. sphaeroides.

One hallmark of pathogenic yersiniae is their ability to rapidly adjust their life-style and pathogenesis upon host entry. In order to capture the range, magnitude and complexity of the underlying gene control mechanisms we used comparative RNA-seq-based transcriptomic profiling of the enteric pathogen Y. pseudotuberculosis under environmental and infection-relevant conditions. We identified 1151 individual transcription start sites, multiple riboswitch-like RNA elements, and a global set of antisense RNAs and previously unrecognized trans-acting RNAs. Taking advantage of these data, we revealed a temperature-induced and growth phase-dependent reprogramming of a large set of catabolic/energy production genes and uncovered the existence of a thermo-regulated ‘acetate switch’, which appear to prime the bacteria for growth in the digestive tract. To elucidate the regulatory architecture linking nutritional status to virulence we also refined the CRP regulon. We identified a massive remodelling of the CRP-controlled network in response to temperature and discovered CRP as a transcriptional master regulator of numerous conserved and newly identified non-coding RNAs which participate in this process. This finding highlights a novel level of complexity of the regulatory network in which the concerted action of transcriptional regulators and multiple non-coding RNAs under control of CRP adjusts the control of Yersinia fitness and virulence to the requirements of their environmental and virulent life-styles.

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Pathogenic bacteria need to rapidly adjust their virulence and fitness program to prevent eradication by the host. So far, underlying adaptation processes that drive pathogenesis have mostly been studied in vitro, neglecting the true complexity of host-induced stimuli acting on the invading pathogen. In this study, we developed an unbiased experimental approach that allows simultaneous monitoring of genome-wide infection-linked transcriptional alterations of the host and colonizing extracellular pathogens. Using this tool for Yersinia pseudotuberculosis-infected lymphatic tissues, we revealed numerous alterations of host transcripts associated with inflammatory and acute-phase responses, coagulative activities, and transition metal ion sequestration, highlighting that the immune response is dominated by infiltrating neutrophils and elicits a mixed T H 17/T H 1 response. In consequence, the pathogen's response is mainly directed to prevent phagocytic attacks. Yersinia up-regulates the gene and expression dose of the antiphagocytic type III secretion system (T3SS) and induces functions counteracting neutrophil-induced ion deprivation, radical stress, and nutritional restraints. Several conserved bacterial riboregulators were identified that impacted this response. The strongest influence on virulence was found for the loss of the carbon storage regulator (Csr) system, which is shown to be essential for the up-regulation of the T3SS on host cell contact. In summary, our established approach provides a powerful tool for the discovery of infection-specific stimuli, induced host and pathogen responses, and underlying regulatory processes.tissue dual RNA-seq | host-pathogen interaction | host-adapted metabolism | noncoding RNAs | Yersinia