The Sponge RNAs of bacteria - How to find them and their role in regulating the post-transcriptional network

In bacteria small regulatory RNAs (sRNAs) interact with their mRNA targets through non-consecutive base-pairing. The loose base-pairing specificity allows sRNAs to regulate large numbers of genes, either affecting the stability and/or the translation of mRNAs. Mechanisms enabling post-transcriptional regulation of the sRNAs themselves have also been described involving so-called sponge RNAs. Sponge RNAs modulate free sRNA levels in the cell through RNA-RNA interactions that sequester ("soak up") the sRNA and/or promote degradation of the target sRNA or the sponge RNA-sRNA complex. The development of complex RNA sequencing strategies for the detection of RNA-RNA interactions has enabled identification of several sponge RNAs, as well as previously known regulatory RNAs able to act as both regulators and sponges. This review highlights techniques that have enabled the identification of these sponge RNAs, the origins of sponge RNAs and the mechanisms by which they function in the post-transcriptional network.</p

The reduction in small ribosomal subunit abundance in ethanol-stressed cells of Bacillus subtilis is mediated by a SigB-dependent antisense RNA

One of the best-characterized general stress responses in bacteria is the sigma(B)-mediated stress response of the Gram-positive soil bacterium Bacillus subtilis. The sigma(B) regulon contains approximately 200 protein-encoding genes and 136 putative regulatory RNAs. One of these sigma(B)-dependent RNAs, named S1136-S1134, was recently mapped as being transcribed from the S1136 promoter on the opposite strand of the essential rpsD gene, which encodes the ribosomal primary-binding protein S4. Accordingly, S1136-S1134 transcription results in an rpsD-overlapping antisense RNA (asRNA). Upon exposure of B. subtilis to ethanol, the S1136 promoter was found to be induced, while rpsD transcription was downregulated. By quantitative PCR, we show that the activation of transcription from the S1136 promoter is directly responsible for the downregulation of rpsD upon ethanol exposure. We also show that this downregulation of rpsD leads to a reduced level of the small (30S) ribosomal subunit upon ethanol stress. The activation of the S1136 promoter thus represents the first example of antisense transcription-mediated regulation in the general stress response of B. subtilis and implicates the reduction of ribosomal protein abundance as a new aspect in the sigma(B)-dependent stress response. We propose that the observed reduction in the level of the small ribosomal subunit, which contains the ribosome-decoding center, may protect B. subtilis cells against misreading and spurious translation of possibly toxic aberrant peptides under conditions of ethanol stress. (C) 2015 Elsevier B.V. All rights reserved.</p

Homogeneity and heterogeneity in amylase production by Bacillus subtilis under different growth conditions

Background Bacillus subtilis is an important cell factory for the biotechnological industry due to its ability to secrete commercially relevant proteins in large amounts directly into the growth medium. However, hyper-secretion of proteins, such as α-amylases, leads to induction of the secretion stress-responsive CssR-CssS regulatory system, resulting in up-regulation of the HtrA and HtrB proteases. These proteases degrade misfolded proteins secreted via the Sec pathway, resulting in a loss of product. The aim of this study was to investigate the secretion stress response in B. subtilis 168 cells overproducing the industrially relevant α-amylase AmyM from Geobacillus stearothermophilus, which was expressed from the strong promoter P(amyQ)-M. Results Here we show that activity of the htrB promoter as induced by overproduction of AmyM was “noisy”, which is indicative for heterogeneous activation of the secretion stress pathway. Plasmids were constructed to allow real-time analysis of P(amyQ)-M promoter activity and AmyM production by, respectively, transcriptional and out-of-frame translationally coupled fusions with gfpmut3. Our results show the emergence of distinct sub-populations of high- and low-level AmyM-producing cells, reflecting heterogeneity in the activity of P(amyQ)-M. This most likely explains the heterogeneous secretion stress response. Importantly, more homogenous cell populations with regard to P(amyQ)-M activity were observed for the B. subtilis mutant strain 168degUhy32, and the wild-type strain 168 under optimized growth conditions. Conclusion Expression heterogeneity of secretory proteins in B. subtilis can be suppressed by degU mutation and optimized growth conditions. Further, the out-of-frame translational fusion of a gene for a secreted target protein and gfp represents a versatile tool for real-time monitoring of protein production and opens novel avenues for Bacillus production strain improvement

Regulatory RNAs in Bacillus subtilis:a Gram-Positive Perspective on Bacterial RNA-Mediated Regulation of Gene Expression

Bacteria can employ widely diverse RNA molecules to regulate their gene expression. Such molecules include trans-acting small regulatory RNAs, antisense RNAs, and a variety of transcriptional attenuation mechanisms in the 5= untranslated region. Thus far, most regulatory RNA research has focused on Gram-negative bacteria, such as Escherichia coli and Salmonella. Hence, there is uncertainty about whether the resulting insights can be extrapolated directly to other bacteria, such as the Gram-positive soil bacterium Bacillus subtilis. A recent study identified 1,583 putative regulatory RNAs in B. subtilis, whose expression was assessed across 104 conditions. Here, we review the current understanding of RNA-based regulation in B. subtilis, and we categorize the newly identified putative regulatory RNAs on the basis of their conservation in other bacilli and the stability of their predicted secondary structures. Our present evaluation of the publicly available data indicates that RNAmediated gene regulation in B. subtilis mostly involves elements at the 5= ends of mRNA molecules. These can include 5= secondary structure elements and metabolite-, tRNA-, or protein-binding sites. Importantly, sense-independent segments are identified as the most conserved and structured potential regulatory RNAs in B. subtilis. Altogether, the present survey provides many leads for the identification of new regulatory RNA functions in B. subtilis

Novel regulation from novel interactions: Identification of an RNA sponge that controls the levels, processing and efficacy of the RoxS riboregulator of central metabolism in Bacillus subtilis

Small RNAs (sRNAs) are a taxonomically-restricted but transcriptomically-abundant class of post-transcriptional regulators. While potentially of importance, we know the function of few. This is in nosmall part because we lack global-scale methodology enabling target identification, this being especiallyacute in species without known RNA meeting point proteins (e.g. Hfq). We apply a combination ofpsoralen RNA cross-linking and Illumina-sequencing to identify RNA-RNA interacting pairs in vivo inBacillus subtilis, resolving previously well-described interactants. Although sRNA-sRNA pairings arerare (compared with sRNA/mRNA), we identify a robust example involving the unusually conservedsRNA (RoxS/RsaE) and an unstudied sRNA that we term Regulator of small RNA A (RosA). Thisinteraction is found in independent samples across multiple conditions. Given the possibility of a novelassociated regulatory mechanism, and the rarity of well-characterised bacterial sRNA-sRNAinteractions, we mechanistically dissect RosA and its interactants. RosA we show to be a sponge RNA,the first to be described in a Gram-positive bacterium. RosA interacts with at least two sRNAs, RoxSand FsrA. Unexpectedly, it acts differently on each. As expected of a sponge RNA, FsrA is sequesteredby RosA. The RosA/RoxS interaction is more complex affecting not only the level of RoxS but also itsprocessing and efficacy. Importantly, RosA provides the condition-dependent intermediary betweenCcpA, the key regulator of carbon metabolism, and RoxS. This not only provides evidence for a novel,and functionally important, regulatory mechanism, but in addition, provides the missing link betweentranscriptional and post-transcriptional regulation of central metabolism

Homogeneity and heterogeneity in amylase production by Bacillus subtilis under different growth conditions

__Background:__ Bacillus subtilis is an important cell factory for the biotechnological industry due to its ability to secrete commercially relevant proteins in large amounts directly into the growth medium. However, hyper-secretion of proteins, such as a-amylases, leads to induction of the secretion stress-responsive CssR-CssS regulatory system, resulting in up-regulation of the HtrA and HtrB proteases. These proteases degrade misfolded proteins secreted via the Sec pathway, resulting in a loss of product. The aim of this study was to investigate the secretion stress response in B. subtilis 168 cells overproducing the industrially relevant a-amylase AmyM from Geobacillus stearothermophilus, which was expressed from the strong promoter P(amyQ)-M. __Results:__ Here we show that activity of the htrB promoter as induced by overproduction of AmyM was "noisy", which is indicative for heterogeneous activation of the secretion stress pathway. Plasmids were constructed to allow real-time analysis of P(amyQ)-M promoter activity and AmyM production by, respectively, transcriptional and outof- frame translationally coupled fusions with gfpmut3. Our results show the emergence of distinct sub-populations of high- and low-level AmyM-producing cells, reflecting heterogeneity in the activity of P(amyQ)-M. This most likely explains the heterogeneous secretion stress response. Importantly, more homogenous cell populations with regard to P(amyQ)-M activity were observed for the B. subtilis mutant strain 168degUhy32, and the wild-type strain 168 under optimized growth conditions. __Conclusion:__ Expression heterogeneity of secretory proteins in B. subtilis can be suppressed by degU mutation and optimized growth conditions. Further, the out-of-frame translational fusion of a gene for a secreted target protein and gfp represents a versatile tool for real-time monitoring of protein production and opens novel avenues for Bacillus production strain improvement

A Qualitative Study Exploring Access to Mental Health and Substance Use Support among Individuals Experiencing Homelessness during COVID-19

People experiencing homelessness have higher rates of mental ill-health and substance use and lower access to health services compared to the general population. The COVID-19 pandemic led to changes in service delivery across health and social care services, with many adopting virtual or telephone support for service users. This paper explores the experiences of access to community-based mental health and substance use support for people experiencing homelessness during the COVID-19 pandemic. Qualitative telephone interviews were conducted with 10 women and 16 men (ages 25 to 71) who self-identified as experiencing homelessness in North East England between February and May 2021. With five individuals with lived experience, results were analysed using inductive reflexive thematic analysis. Reactive changes to support provision often led to inadvertent exclusion. Barriers to access included: physical locations, repetition of recovery stories, individual readiness, and limited availability. Participants suggested creating services reflective of need and opportunities for choice and empowerment. Community mental health and substance use support for people experiencing homelessness should ensure the support is personalised, responsive to need, inclusive, and trauma-informed. The findings of this research have important implications for mental health and substance use policy and practice for individuals who experience homelessness during a public health crisis

Increased adipose tissue indices of androgen catabolism and aromatization in women with metabolic dysfunction

Abstract Background Body fat distribution is a risk factor for obesity-associated comorbidities, and adipose tissue dysfunction plays a role in this association. In humans, there is a sex difference in body fat distribution, and steroid hormones are known to regulate several cellular processes within adipose tissue. Our aim was to investigate if intra-adipose steroid concentration and expression or activity of steroidogenic enzymes were associated with features of adipose tissue dysfunction in individuals with severe obesity. Methods Samples from 40 bariatric candidates (31 women, 9 men) were included in the study. Visceral (VAT) and subcutaneous adipose tissue (SAT) were collected during surgery. Adipose tissue morphology was measured by a combination of histological staining and semi-automated quantification. Following extraction, intra-adipose and plasma steroid concentrations were determined by liquid chromatography, electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Aromatase activity was estimated using product-over-substrate ratio, while AKR1C2 activity was measured directly by fluorogenic probe. Gene expression was measured by quantitative PCR. Results VAT aromatase activity was positively associated with VAT adipocyte hypertrophy (p-valueadj &lt; 0.01) and negatively with plasma HDL-cholesterol (p-valueadj &lt; 0.01), while SAT aromatase activity predicted dyslipidemia in women even after adjustment for waist circumference, age and hormonal contraceptive use. We additionally compared women with high and low visceral adiposity index (VAI) and found that VAT excess is characterized by adipose tissue dysfunction, increased androgen catabolism mirrored by increased AKR1C2 activity and higher aromatase expression and activity indices. Conclusion In women, increased androgen catabolism or aromatization is associated with visceral adiposity and adipose tissue dysfunction

Definition of the σW regulon of Bacillus subtilis in the absence of stress

Bacteria employ extracytoplasmic function (ECF) sigma factors for their responses to environmental stresses. Despite intensive research, the molecular dissection of ECF sigma factor regulons has remained a major challenge due to overlaps in the ECF sigma factor-regulated genes and the stimuli that activate the different ECF sigma factors. Here we have employed tiling arrays to single out the ECF σW regulon of the Gram-positive bacterium Bacillus subtilis from the overlapping ECF σX, σY, and σM regulons. For this purpose, we profiled the transcriptome of a B. subtilis sigW mutant under non-stress conditions to select candidate genes that are strictly σW-regulated. Under these conditions, σW exhibits a basal level of activity. Subsequently, we verified the σW-dependency of candidate genes by comparing their transcript profiles to transcriptome data obtained with the parental B. subtilis strain 168 grown under 104 different conditions, including relevant stress conditions, such as salt shock. In addition, we investigated the transcriptomes of rasP or prsW mutant strains that lack the proteases involved in the degradation of the σW anti-sigma factor RsiW and subsequent activation of the σW-regulon. Taken together, our studies identify 89 genes as being strictly σW-regulated, including several genes for non-coding RNAs. The effects of rasP or prsW mutations on the expression of σW-dependent genes were relatively mild, which implies that σW-dependent transcription under non-stress conditions is not strictly related to RasP and PrsW. Lastly, we show that the pleiotropic phenotype of rasP mutant cells, which have defects in competence development, protein secretion and membrane protein production, is not mirrored in the transcript profile of these cells. This implies that RasP is not only important for transcriptional regulation via σW, but that this membrane protease also exerts other important post-transcriptional regulatory functions

Environmental Salinity Determines the Specificity and Need for Tat-Dependent Secretion of the YwbN Protein in Bacillus subtilis

Twin-arginine protein translocation (Tat) pathways are required for transport of folded proteins across bacterial, archaeal and chloroplast membranes. Recent studies indicate that Tat has evolved into a mainstream pathway for protein secretion in certain halophilic archaea, which thrive in highly saline environments. Here, we investigated the effects of environmental salinity on Tat-dependent protein secretion by the Gram-positive soil bacterium Bacillus subtilis, which encounters widely differing salt concentrations in its natural habitats. The results show that environmental salinity determines the specificity and need for Tat-dependent secretion of the Dyp-type peroxidase YwbN in B. subtilis. Under high salinity growth conditions, at least three Tat translocase subunits, namely TatAd, TatAy and TatCy, are involved in the secretion of YwbN. Yet, a significant level of Tat-independent YwbN secretion is also observed under these conditions. When B. subtilis is grown in medium with 1% NaCl or without NaCl, the secretion of YwbN depends strictly on the previously described “minimal Tat translocase” consisting of the TatAy and TatCy subunits. Notably, in medium without NaCl, both tatAyCy and ywbN mutants display significantly reduced exponential growth rates and severe cell lysis. This is due to a critical role of secreted YwbN in the acquisition of iron under these conditions. Taken together, our findings show that environmental conditions, such as salinity, can determine the specificity and need for the secretion of a bacterial Tat substrate