Mouse skin passage of a Streptococcus pyogenes Tn917 mutant of sagA/pel restores virulence, beta-hemolysis and sagA/pel expression without altering the position or sequence of the transposon

BACKGROUND: Streptolysin S (SLS), the oxygen-stable hemolysin of Streptococcus pyogenes, has recently been shown to be encoded by the sagA/pel gene. Mutants lacking expression of this gene were less virulent in a dermonecrotic mouse infection model. Inactivation of the sagA/pel gene affect the expression of a variety of virulence factors in addition to the hemolysin. Insertion of a Tn917 transposon into the promoter region of the sagA/pel gene of S. pyogenes isolate CS101 eliminated expression of SLS, as well as decreased expression of the streptococcal pyrogenic exotoxin B, streptokinase and M protein. RESULTS: In this study a mouse skin air sac model was utilized to analyze the effect of biological pressures on expression of SLS and other sagA/pel regulated gene products. The insertion delayed the lethal effect of S. pyogenes in a mouse skin infection model. Despite this, bacteria could be cultured from the kidneys 72 hours post infection. These kidney-recovered isolates were β-hemolytic despite the transposon being present in its original location and had equivalent virulence to the wild type isolate when re-injected into naive mice. Northern blot analysis of the kidney-recovered isolates confirmed that transcription of sagA/pel was restored; however the expression of all sagA/pel regulated genes was not restored to wild type levels. CONCLUSIONS: These results show that biological pressure present in the mouse can select for variants with altered expression of key virulence factor genes in S. pyogenes

Relationships Between Levels of Mindfulness and Subjective Well-Being in Undergraduate Students

College is often a period of unique demands and increased autonomy. The heightened stress associated with this period can contribute to mental health difficulties and maladaptive behaviors in a subset of students. The goal of the current study was to examine the role of mindfulness (i.e., awareness of, and appreciation for, the present moment and experiences) in experiencing negative emotions and lifestyle behaviors in 23 male and 19 female undergraduate students. Participants were randomly assigned to a mindfulness meditation condition or a cognitive activity control condition. Results suggest that higher levels of mindfulness are associated with better lifestyle habits, lower levels of stress, and reduced negative affect. Results also suggest mindfulness meditation is effective for individuals reporting lower initial levels of mindfulness. The Five Facet Mindfulness Questionnaire-Short Form (FFMQ-SF; Baer et al., 2006) was used to examine specific facets of mindfulness. Notably, scores from the facet of Observing were predictive of environmental concern, social concern, and safety concern while scores from the Describing facet were predictive of a greater sense of purpose. The results of this study provide preliminary evidence of the effectiveness of mindfulness practices on students’ subjective well-being

Translational activation of rpoS mRNA by the non-coding RNA DsrA and Hfq does not require ribosome binding

At low temperature, translational activation of rpoS mRNA, encoding the stationary phase sigma-factor, σS, involves the small regulatory RNA (sRNA) DsrA and the RNA chaperone Hfq. The Hfq-mediated DsrA-rpoS interaction relieves an intramolecular secondary structure that impedes ribosome access to the rpoS ribosome binding site. In addition, DsrA/rpoS duplex formation creates an RNase III cleavage site within the duplex. Previous biochemical studies suggested that DsrA and Hfq associate with the 30S ribosomal subunit protein S1, which implied a role for the ribosome in sRNA-mediated post-transcriptional regulation. Here, we show by ribosome profiling that Hfq partitions with the cytoplasmic fraction rather than with 30S subunits. Besides, by employing immunological techniques, no evidence for a physical interaction between Hfq and S1 was obtained. Similarly, in vitro studies did not reveal a direct interaction between DsrA and S1. By employing a ribosome binding deficient rpoS mRNA, and by using the RNase III clevage in the DsrA/rpoS duplex as a diagnostic marker, we provide in vivo evidence that the Hfq-mediated DsrA/rpoS interaction, and consequently the structural changes in rpoS mRNA precede ribosome binding. These data suggest a simple mechanistic model in which translational activation by DsrA provides a translationally competent rpoS mRNA to which 30S subunits can readily bind

Translational control and target recognition by Escherichia coli small RNAs in vivo

Small non-coding RNAs (sRNAs) are an emerging class of regulators of bacterial gene expression. Most of the regulatory Escherichia coli sRNAs known to date modulate translation of trans-encoded target mRNAs. We studied the specificity of sRNA target interactions using gene fusions to green fluorescent protein (GFP) as a novel reporter of translational control by bacterial sRNAs in vivo. Target sequences were selected from both monocistronic and polycistronic mRNAs. Upon expression of the cognate sRNA (DsrA, GcvB, MicA, MicC, MicF, RprA, RyhB, SgrS and Spot42), we observed highly specific translation repression/activation of target fusions under various growth conditions. Target regulation was also tested in mutants that lacked Hfq or RNase III, or which expressed a truncated RNase E (rne701). We found that translational regulation by these sRNAs was largely independent of full-length RNase E, e.g. despite the fact that ompA fusion mRNA decay could no longer be promoted by MicA. This is the first study in which multiple well-defined E.coli sRNA target pairs have been studied in a uniform manner in vivo. We expect our GFP fusion approach to be applicable to sRNA targets of other bacteria, and also demonstrate that Vibrio RyhB sRNA represses a Vibrio sodB fusion when co-expressed in E.coli

Spectroscopic observation of RNA chaperone activities of Hfq in post-transcriptional regulation by a small non-coding RNA

Hfq protein is vital for the function of many non-coding small (s)RNAs in bacteria but the mechanism by which Hfq facilitates the function of sRNA is still debated. We developed a fluorescence resonance energy transfer assay to probe how Hfq modulates the interaction between a sRNA, DsrA, and its regulatory target mRNA, rpoS. The relevant RNA fragments were labelled so that changes in intra- and intermolecular RNA structures can be monitored in real time. Our data show that Hfq promotes the strand exchange reaction in which the internal structure of rpoS is replaced by pairing with DsrA such that the Shine-Dalgarno sequence of the mRNA becomes exposed. Hfq appears to carry out strand exchange by inducing rapid association of DsrA and a premelted rpoS and by aiding in the slow disruption of the rpoS secondary structure. Unexpectedly, Hfq also disrupts a preformed complex between rpoS and DsrA. While it may not be a frequent event in vivo, this melting activity may have implications in the reversal of sRNA-based regulation. Overall, our data suggests that Hfq not only promotes strand exchange by binding rapidly to both DsrA and rpoS but also possesses RNA chaperoning properties that facilitates dynamic RNA–RNA interactions

Posttraumatic Stress Disorder, Depression, and HIV Risk Behavior Among Ohio Army National Guard Soldiers

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/96665/1/jts21777.pd

Crystal structure of Hfq from Bacillus subtilis in complex with SELEX-derived RNA aptamer: insight into RNA-binding properties of bacterial Hfq

Bacterial Hfq is a protein that plays an important role in the regulation of genes in cooperation with sRNAs. Escherichia coli Hfq (EcHfq) has two or more sites that bind RNA(s) including U-rich and/or the poly(A) tail of mRNA. However, functional and structural information about Bacillus subtilis Hfq (BsHfq) including the RNA sequences that specifically bind to it remain unknown. Here, we describe RNA aptamers including fragment (AG)3A that are recognized by BsHfq and crystal structures of the BsHfq–(AG)3A complex at 2.2 Å resolution. Mutational and structural studies revealed that the RNA fragment binds to the distal site, one of the two binding sites on Hfq, and identified amino acid residues that are critical for sequence-specific interactions between BsHfq and (AG)3A. In particular, R32 appears to interact with G bases in (AG)3A. Poly(A) also binds to the distal site of EcHfq, but the overall RNA structure and protein–RNA interaction patterns engaged in the R32 residues of BsHfq–(AG)3A differ from those of EcHfq–poly(A). These findings provide novel insight into how the Hfq homologue recognizes RNA