Transcriptomic Approaches for Studying Quorum Sensing in Vibrio cholerae

Transcriptome analysis using RNA-sequencing (RNA-seq) has now become the standard approach to determine the transcriptional output of an organism. Various modifications to this technology have been developed over the years, usually aiming to improve the annotation of transcript borders, or to identify novel classes of RNAs, such as small regulatory RNAs (sRNAs) and antisense transcripts. RNA-seq has also led to the identification of dozens of new sRNAs in the major human pathogen, Vibrio cholerae. Several of these sRNAs function in the context of a cell-to-cell communication process, called quorum sensing (QS). QS is key for pathogenicity and biofilm formation of V. cholerae and the sRNAs involved typically act by base pairing with multiple target mRNAs to control gene expression at the posttranscriptional level. In this chapter, we describe the use of RNA-seq technologies for the discovery and characterization of regulatory RNAs in V. cholerae and discuss their relevance to QS and collective functions, such as biofilm formation. We further outline possible methods for the identification and validation of sRNA target genes, which can provide crucial information as to the physiological roles of an sRNA

Neutron-Star-Merger Equation of State

In this work, we discuss the dense matter equation of state (EOS) for the extreme range of conditions encountered in neutron stars and their mergers. The calculation of the properties of such an EOS involves modeling different degrees of freedom (such as nuclei, nucleons, hyperons, and quarks), taking into account different symmetries, and including finite density and temperature effects in a thermodynamically consistent manner. We begin by addressing subnuclear matter consisting of nucleons and a small admixture of light nuclei in the context of the excluded volume approach. We then turn our attention to supranuclear homogeneous matter as described by the Chiral Mean Field (CMF) formalism. Finally, we present results from realistic neutron-star-merger simulations performed using the CMF model that predict signatures for deconfinement to quark matter in gravitational wave signals.Comment: Contribution to the Special Issue "Compact Stars in the QCD Phase Diagram and in the Multi-Messenger Era of Astronomy" dedicated to the conference: Compact Stars in the QCD Phase Diagram VI

Cross-Regulation between Bacteria and Phages at a Posttranscriptional Level.

The study of bacteriophages (phages) and prophages has provided key insights into almost every cellular process as well as led to the discovery of unexpected new mechanisms and the development of valuable tools. This is exemplified for RNA-based regulation. For instance, the characterization and exploitation of the anti-phage CRISPR systems is revolutionizing molecular biology. Phage-encoded proteins such as the RNA binding MS2 protein, which is broadly used to isolate tagged RNAs, also have been developed as valuable tools. Hfq, the RNA chaperone protein central to the function of many base pairing small RNAs (sRNAs), was first characterized as a bacterial host factor required for Qβ phage replication. The ongoing studies of RNAs are continuing to reveal regulatory connections between infecting phages, prophages and bacteria and to provide novel insights. There are bacterial and prophage sRNAs that regulate prophage genes, which impact bacterial virulence as well as bacterial cell killing. Conversely, phage- and prophage-encoded sRNAs modulate the expression of bacterial genes modifying metabolism. An interesting subcategory of the prophage-encoded sRNAs are sponge RNAs that inhibit the activities of bacterial-encoded sRNAs. Phages also affect post-transcriptional regulation in bacteria through proteins that inhibit or alter the activities of key bacterial proteins involved in posttranscriptional regulation. However, what is most exciting about phage and prophage research, given the millions of phage-encoded genes that have not yet been characterized, is the vast potential for discovering new RNA regulators and novel mechanisms and for gaining insight into the evolution of regulatory RNAs

Nucleic Acids Res.

A remarkable feature of many small non-coding RNAs (sRNAs) of Escherichia coli and Salmonella is their accumulation in the stationary phase of bacterial growth. Several stress response regulators and sigma factors have been reported to direct the transcription of stationary phase-specific sRNAs, but a widely conserved sRNA gene that is controlled by the major stationary phase and stress sigma factor, Sigma(S) (RpoS), has remained elusive. We have studied in Salmonella the conserved SdsR sRNA, previously known as RyeB, one of the most abundant stationary phase-specific sRNAs in E. coli. Alignments of the sdsR promoter region and genetic analysis strongly suggest that this sRNA gene is selectively transcribed by Sigma(S). We show that SdsR down-regulates the synthesis of the major Salmonella porin OmpD by Hfq-dependent base pairing; SdsR thus represents the fourth sRNA to regulate this major outer membrane porin. Similar to the InvR, MicC and RybB sRNAs, SdsR recognizes the ompD mRNA in the coding sequence, suggesting that this mRNA may be primarily targeted downstream of the start codon. The SdsR-binding site in ompD was localized by 3'-RACE, an experimental approach that promises to be of use in predicting other sRNA-target interactions in bacteria

Small RNA-based feedforward loop with AND-gate logic regulates extrachromosomal DNA transfer in Salmonella

Horizontal gene transfer via plasmid conjugation is a major driving force in microbial evolution but constitutes a complex process that requires synchronization with the physiological state of the host bacteria. Although several host transcription factors are known to regulate plasmid-borne transfer genes, RNA-based regulatory circuits for host-plasmid communication remain unknown. We describe a posttranscriptional mechanism whereby the Hfq-dependent small RNA, RprA, inhibits transfer of pSLT, the virulence plasmid of Salmonella enterica. RprA employs two separate seed-pairing domains to activate the mRNAs of both the sigma-factor σS and the RicI protein, a previously uncharacterized membrane protein here shown to inhibit conjugation. Transcription of ricI requires σS and, together, RprA and σS orchestrate a coherent feedforward loop with AND-gate logic to tightly control the activation of RicI synthesis. RicI interacts with the conjugation apparatus protein TraV and limits plasmid transfer under membrane-damaging conditions. To our knowledge, this study reports the first small RNA-controlled feedforward loop relying on posttranscriptional activation of two independent targets and an unexpected role of the conserved RprA small RNA in controlling extrachromosomal DNA transfer.Bildung und Forschung Project eBio:RNAsys BIO2013-44220-RMinisterio de Economía y Competitividad CSD2008-00013Junta de Andalucía CVI-587

Gene autoregulation by 3' UTR-derived bacterial small RNAs

Negative feedback regulation, that is the ability of a gene to repress its own synthesis, is the most abundant regulatory motif known to biology. Frequently reported for transcriptional regulators, negative feedback control relies on binding of a transcription factor to its own promoter. Here, we report a novel mechanism for gene autoregulation in bacteria relying on small regulatory RNA (sRNA) and the major endoribonuclease, RNase E. TIER-seq analysis (transiently-inactivating-an-endoribonuclease-followed-by-RNA-seq) revealed similar to 25,000 RNase E-dependent cleavage sites in Vibrio cholerae, several of which resulted in the accumulation of stable sRNAs. Focusing on two examples, OppZ and CarZ, we discovered that these sRNAs are processed from the 3' untranslated region (3' UTR) of the oppABCDF and carAB operons, respectively, and basepair with their own transcripts to inhibit translation. For OppZ, this process also triggers Rho-dependent transcription termination. Our data show that sRNAs from 3' UTRs serve as autoregulatory elements allowing negative feedback control at the post-transcriptional level