A Genome-Wide Approach to Discovery of Small RNAs Involved in Regulation of Virulence in Vibrio cholerae

Small RNAs (sRNAs) are becoming increasingly recognized as important regulators in bacteria. To investigate the contribution of sRNA mediated regulation to virulence in Vibrio cholerae, we performed high throughput sequencing of cDNA generated from sRNA transcripts isolated from a strain ectopically expressing ToxT, the major transcriptional regulator within the virulence gene regulon. We compared this data set with ToxT binding sites determined by pulldown and deep sequencing to identify sRNA promoters directly controlled by ToxT. Analysis of the resulting transcripts with ToxT binding sites in cis revealed two sRNAs within the Vibrio Pathogenicity Island. When deletions of these sRNAs were made and the resulting strains were competed against the parental strain in the infant mouse model of V. cholerae colonization, one, TarB, displayed a variable colonization phenotype dependent on its physiological state at the time of inoculation. We identified a target of TarB as the mRNA for the secreted colonization factor, TcpF. We verified negative regulation of TcpF expression by TarB and, using point mutations that disrupted interaction between TarB and tpcF mRNA, showed that loss of this negative regulation was primarily responsible for the colonization phenotype observed in the TarB deletion mutant

Genomic SELEX for Hfq-binding RNAs identifies genomic aptamers predominantly in antisense transcripts

An unexpectedly high number of regulatory RNAs have been recently discovered that fine-tune the function of genes at all levels of expression. We employed Genomic SELEX, a method to identify protein-binding RNAs encoded in the genome, to search for further regulatory RNAs in Escherichia coli. We used the global regulator protein Hfq as bait, because it can interact with a large number of RNAs, promoting their interaction. The enriched SELEX pool was subjected to deep sequencing, and 8865 sequences were mapped to the E. coli genome. These short sequences represent genomic Hfq-aptamers and are part of potential regulatory elements within RNA molecules. The motif 5′-AAYAAYAA-3′ was enriched in the selected RNAs and confers low-nanomolar affinity to Hfq. The motif was confirmed to bind Hfq by DMS footprinting. The Hfq aptamers are 4-fold more frequent on the antisense strand of protein coding genes than on the sense strand. They were enriched opposite to translation start sites or opposite to intervening sequences between ORFs in operons. These results expand the repertoire of Hfq targets and also suggest that Hfq might regulate the expression of a large number of genes via interaction with cis-antisense RNAs

Expression of the neutral protease gene from a thermophilic Bacillus sp BT1 strain in Bacillus subtilis and its natural host: Identification of a functional promoter

The expression of the neutral protease gene (npr) from the thermophilic Bacillus sp. BT1 strain was studied in its natural host and in mesophilic Bacillus subtilis. In the thermophilic BT1 strain, the transcription of the protease gene is initiated from its own promoter, just 5' to the gene. In contrast, in heterologous B. subtilis this thermophilic npr promoter does not function, and expression of the npr gene results from transcription originating upstream of an adjacent gene, open reading frame X (ORF X). A functional promoter was identified 5' to ORF X that is required for efficient expression of the npr gene in Bacillus subtilis as verified by primer extension, reverse transcription-PCR, and 5' rapid amplification of cDNA ends experiments. These data suggest that transcriptional signals used in thermophilic Bacillus sp. BT1 strain are different from those used in B. subtilis

Transcriptional profiling of Bordetella pertussis reveals requirement of RNA chaperone Hfq for Type III secretion system functionality

Bordetella pertussis, the causative agent of human whooping cough (pertussis) produces a complex array of virulence factors in order to establish efficient infection in the host. The RNA chaperone Hfq and small regulatory RNAs are key players in posttranscriptional regulation in bacteria and have been shown to play an essential role in virulence of a broad spectrum of bacterial pathogens. This study represents the first attempt to characterize the Hfq regulon of the human pathogen B. pertussis under laboratory conditions as well as upon passage in the host and indicates that loss of Hfq has a profound effect on gene expression in B. pertussis. Comparative transcriptional profiling revealed that Hfq is required for expression of several virulence factors in B. pertussis cells including the Type III secretion system (T3SS). In striking contrast to the wt strain, T3SS did not become operational in the hfq mutant passaged either through mice or macrophages thereby proving that Hfq is required for the functionality of the B. pertussis T3SS. Likewise, expression of virulence factors vag8 and tcfA encoding autotransporter and tracheal colonization factor, respectively, was strongly reduced in the hfq mutant. Importantly, for the first time we demonstrate that B. pertussis T3SS can be activated upon contact with macrophage cells in vitro

Structural flexibility of RNA as molecular basis for Hfq chaperone function

In enteric bacteria, many small regulatory RNAs (sRNAs) associate with the RNA chaperone host factor Q (Hfq) and often require the protein for regulation of target mRNAs. Previous studies suggested that the hexameric Escherichia coli Hfq (Hfq(Ec)) binds sRNAs on the proximal site, whereas the distal site has been implicated in Hfq-mRNA interactions. Employing a combination of small angle X-ray scattering, nuclear magnetic resonance and biochemical approaches, we report the structural analysis of a 1:1 complex of Hfq(Ec) with a 34-nt-long subsequence of a natural substrate sRNA, DsrA (DsrA(34)). This sRNA is involved in post-transcriptional regulation of the E. coli rpoS mRNA encoding the stationary phase sigma factor RpoS. The molecular envelopes of Hfq(Ec) in complex with DsrA(34) revealed an overall asymmetric shape of the complex in solution with the protein maintaining its doughnut-like structure, whereas the extended DsrA(34) is flexible and displays an ensemble of different spatial arrangements. These results are discussed in terms of a model, wherein the structural flexibility of RNA ligands bound to Hfq stochastically facilitates base pairing and provides the foundation for the RNA chaperone function inherent to Hfq

Requirement of the CsdA DEAD-box helicase for low temperature riboregulation of rpoS mRNA

The ribosome binding site of Escherichia coli rpoS mRNA, encoding the stationary sigma-factor RpoS, is sequestered by an inhibitory stem-loop structure (iss). Translational activation of rpoS mRNA at low temperature and during exponential growth includes Hfq-facilitated duplex formation between rpoS and the small regulatory RNA DsrA as well as a concomitant re-direction of RNase III cleavage in the 5′-untranslated region of rpoS upon DsrA·rpoS annealing. In this way, DsrA-mediated regulation does not only activate rpoS translation by disrupting the inhibitory secondary structure but also stabilizes the rpoS transcript. Although minor structural changes by Hfq have been observed in rpoS mRNA, a prevailing question concerns unfolding of the iss in rpoS at low growth temperature. Here, we have identified the DEAD-box helicase CsdA as an ancillary factor required for low temperature activation of RpoS synthesis by DsrA. The lack of RpoS synthesis observed in the csdA mutant strain at low growth temperature could be attributed to a lack of duplex formation between rpoS and DsrA, showing that at low temperature the sole action of Hfq is not sufficient to permit DsrA·rpoS annealing. An interactome study has previously indicated an association between Hfq and CsdA. However, immunological assays did not reveal a physical interaction between Hfq and CsdA. These findings add to a model, wherein Hfq binds upstream of the rpoS iss and presents DsrA in a conformation receptive to annealing. Melting of the iss by CsdA may then permit DsrA·rpoS duplex formation, and consequently rpoS translation

Structural insights into the dynamics and function of the C-terminus of the E. coli RNA chaperone Hfq

The hexameric Escherichia coli RNA chaperone Hfq (Hfq(Ec)) is involved in riboregulation of target mRNAs by small trans-encoded RNAs. Hfq proteins of different bacteria comprise an evolutionarily conserved core, whereas the C-terminus is variable in length. Although the structure of the conserved core has been elucidated for several Hfq proteins, no structural information has yet been obtained for the C-terminus. Using bioinformatics, nuclear magnetic resonance spectroscopy, synchrotron radiation circular dichroism (SRCD) spectroscopy and small angle X-ray scattering we provide for the first time insights into the conformation and dynamic properties of the C-terminal extension of Hfq(Ec). These studies indicate that the C-termini are flexible and extend laterally away from the hexameric core, displaying in this way features typical of intrinsically disordered proteins that facilitate intermolecular interactions. We identified a minimal, intrinsically disordered region of the C-terminus supporting the interactions with longer RNA fragments. This minimal region together with rest of the C-terminal extension provides a flexible moiety capable of tethering long and structurally diverse RNA molecules. Furthermore, SRCD spectroscopy supported the hypothesis that RNA fragments exceeding a certain length interact with the C-termini of Hfq(Ec)