Genome-wide annotation of transcript boundaries using bacterial Rend-seq datasets.

Accurate annotation to single-nucleotide resolution of the transcribed regions in genomes is key to optimally analyse RNA-seq data, understand regulatory events and for the design of experiments. However, currently most genome annotations provided by GenBank generally lack information about untranslated regions. Additionally, information regarding genomic locations of non-coding RNAs, such as sRNAs, or anti-sense RNAs is frequently missing. To provide such information, diverse RNA-seq technologies, such as Rend-seq, have been developed and applied to many bacterial species. However, incorporating this vast amount of information into annotation files has been limited and is bioinformatically challenging, resulting in UTRs and other non-coding elements being overlooked or misrepresented. To overcome this problem, we present pyRAP (python Rend-seq Annotation Pipeline), a software package that analyses Rend-seq datasets to accurately resolve transcript boundaries genome-wide. We report the use of pyRAP to find novel transcripts, transcript isoforms, and RNase-dependent sRNA processing events. In Bacillus subtilis we uncovered 63 novel transcripts and provide genomic coordinates with single-nucleotide resolution for 2218 5'UTRs, 1864 3'UTRs and 161 non-coding RNAs. In Escherichia coli, we report 117 novel transcripts, 2429 5'UTRs, 1619 3'UTRs and 91 non-coding RNAs, and in Staphylococcus aureus, 16 novel transcripts, 664 5'UTRs, 696 3'UTRs, and 81 non-coding RNAs. Finally, we use pyRAP to produce updated annotation files for B. subtilis 168, E. coli K-12 MG1655, and S. aureus 8325 for use in the wider microbial genomics research community.

Identification of an RNA sponge that controls the RoxS riboregulator of central metabolism in Bacillus subtilis.

sRNAs are a taxonomically-restricted but transcriptomically-abundant class of post-transcriptional regulators. While of major importance for adaption to the environment, we currently lack global-scale methodology enabling target identification, especially in species without known RNA hub proteins (e.g. Hfq). Using psoralen RNA cross-linking and Illumina-sequencing we identify RNA-RNA interacting pairs in vivo in Bacillus subtilis, resolving previously well-described interactants. Although sRNA-sRNA pairings are rare (compared with sRNA-mRNA), we identify a robust example involving the conserved sRNA RoxS and an unstudied sRNA RosA (Regulator of sRNA A). We show RosA to be the first confirmed RNA sponge described in a Gram-positive bacterium. RosA interacts with at least two sRNAs, RoxS and FsrA. The RosA/RoxS interaction not only affects the levels of RoxS but also its processing and regulatory activity. We also found that the transcription of RosA is repressed by CcpA, the key regulator of carbon-metabolism in B. subtilis. Since RoxS is already known to be transcriptionally controlled by malate via the transcriptional repressor Rex, its post-transcriptional regulation by CcpA via RosA places RoxS in a key position to control central metabolism in response to varying carbon sources.

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.

Differential expression of a prophage-encoded glycocin and its immunity protein suggests a mutualistic strategy of a phage and its host.

Sublancin 168 is a highly potent and stable antimicrobial peptide secreted by the Gram-positive bacterium Bacillus subtilis. Production of sublancin gives B. subtilis a major competitive growth advantage over a range of other bacteria thriving in the same ecological niches, the soil and plant rhizosphere. B. subtilis protects itself against sublancin by producing the cognate immunity protein SunI. Previous studies have shown that both the sunA gene for sublancin and the sunI immunity gene are encoded by the prophage SPß. The sunA gene is under control of several transcriptional regulators. Here we describe the mechanisms by which sunA is heterogeneously expressed within a population, while the sunI gene encoding the immunity protein is homogeneously expressed. The key determinants in heterogeneous sunA expression are the transcriptional regulators Spo0A, AbrB and Rok. Interestingly, these regulators have only a minor influence on sunI expression and they have no effect on the homogeneous expression of sunI within a population of growing cells. Altogether, our findings imply that the homogeneous expression of sunI allows even cells that are not producing sublancin to protect themselves at all times from the active sublancin produced at high levels by their isogenic neighbors. This suggests a mutualistic evolutionary strategy entertained by the SPß prophage and its Bacillus host, ensuring both stable prophage maintenance and a maximal competitive advantage for the host at minimal costs.

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 RNA-mediated 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.

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.

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 σB-mediated stress response of the Gram-positive soil bacterium Bacillus subtilis. The σB regulon contains approximately 200 protein-encoding genes and 136 putative regulatory RNAs. One of these σB-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 σB-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.

Small regulatory RNA-induced growth rate heterogeneity of Bacillus subtilis.

Isogenic bacterial populations can consist of cells displaying heterogeneous physiological traits. Small regulatory RNAs (sRNAs) could affect this heterogeneity since they act by fine-tuning mRNA or protein levels to coordinate the appropriate cellular behavior. Here we show that the sRNA RnaC/S1022 from the Gram-positive bacterium Bacillus subtilis can suppress exponential growth by modulation of the transcriptional regulator AbrB. Specifically, the post-transcriptional abrB-RnaC/S1022 interaction allows B. subtilis to increase the cell-to-cell variation in AbrB protein levels, despite strong negative autoregulation of the abrB promoter. This behavior is consistent with existing mathematical models of sRNA action, thus suggesting that induction of protein expression noise could be a new general aspect of sRNA regulation. Importantly, we show that the sRNA-induced diversity in AbrB levels generates heterogeneity in growth rates during the exponential growth phase. Based on these findings, we hypothesize that the resulting subpopulations of fast- and slow-growing B. subtilis cells reflect a bet-hedging strategy for enhanced survival of unfavorable conditions.

The multidrug ABC transporter BmrC/BmrD of Bacillus subtilis is regulated via a ribosome-mediated transcriptional attenuation mechanism.

Expression of particular drug transporters in response to antibiotic pressure is a critical element in the development of bacterial multidrug resistance, and represents a serious concern for human health. To obtain a better understanding of underlying regulatory mechanisms, we have dissected the transcriptional activation of the ATP-binding cassette (ABC) transporter BmrC/BmrD of the Gram-positive model bacterium Bacillus subtilis. By using promoter-GFP fusions and live cell array technology, we demonstrate a temporally controlled transcriptional activation of the bmrCD genes in response to antibiotics that target protein synthesis. Intriguingly, bmrCD expression only occurs during the late-exponential and stationary growth stages, irrespective of the timing of the antibiotic challenge. We show that this is due to tight transcriptional control by the transition state regulator AbrB. Moreover, our results show that the bmrCD genes are co-transcribed with bmrB (yheJ), a small open reading frame immediately upstream of bmrC that harbors three alternative stem-loop structures. These stem-loops are apparently crucial for antibiotic-induced bmrCD transcription. Importantly, the antibiotic-induced bmrCD expression requires translation of bmrB, which implies that BmrB serves as a regulatory leader peptide. Altogether, we demonstrate for the first time that a ribosome-mediated transcriptional attenuation mechanism can control the expression of a multidrug ABC transporter.

TLM-Quant: an open-source pipeline for visualization and quantification of gene expression heterogeneity in growing microbial cells.

Gene expression heterogeneity is a key driver for microbial adaptation to fluctuating environmental conditions, cell differentiation and the evolution of species. This phenomenon has therefore enormous implications, not only for life in general, but also for biotechnological applications where unwanted subpopulations of non-producing cells can emerge in large-scale fermentations. Only time-lapse fluorescence microscopy allows real-time measurements of gene expression heterogeneity. A major limitation in the analysis of time-lapse microscopy data is the lack of fast, cost-effective, open, simple and adaptable protocols. Here we describe TLM-Quant, a semi-automatic pipeline for the analysis of time-lapse fluorescence microscopy data that enables the user to visualize and quantify gene expression heterogeneity. Importantly, our pipeline builds on the open-source packages ImageJ and R. To validate TLM-Quant, we selected three possible scenarios, namely homogeneous expression, highly 'noisy' heterogeneous expression, and bistable heterogeneous expression in the Gram-positive bacterium Bacillus subtilis. This bacterium is both a paradigm for systems-level studies on gene expression and a highly appreciated biotechnological 'cell factory'. We conclude that the temporal resolution of such analyses with TLM-Quant is only limited by the numbers of recorded images.

Phenol-soluble modulins, hellhounds from the staphylococcal virulence-factor pandemonium.

Phenol-soluble modulins are secreted peptides with multiple functions in Staphylococcus aureus pathogenesis and spreading. Recent studies by Otto and coworkers show that these hellhounds of the staphylococcal virulence-factor pandemonium are unleashed through an essential ABC transporter, which represents an exciting new target for stopping the spread of this important pathogen.

Is proteomics a reliable tool to probe the oxidative folding of bacterial membrane proteins?

The oxidative folding of proteins involves disulfide bond formation, which is usually catalyzed by thiol-disulfide oxidoreductases (TDORs). In bacteria, this process takes place in the cytoplasmic membrane and other extracytoplasmic compartments. While it is relatively easy to study oxidative folding of water-soluble proteins on a proteome-wide scale, this has remained a major challenge for membrane proteins due to their high hydrophobicity. Here, we have assessed whether proteomic techniques can be applied to probe the oxidative folding of membrane proteins using the Gram-positive bacterium Bacillus subtilis as a model organism. Specifically, we investigated the membrane proteome of a B. subtilis bdbCD mutant strain, which lacks the primary TDOR pair BdbC and BdbD, by gel-free mass spectrometry. In total, 18 membrane-associated proteins showed differing behavior in the bdbCD mutant and the parental strain. These included the ProA protein involved in osmoprotection. Consistent with the absence of ProA, the bdbCD mutant was found to be sensitive to osmotic shock. We hypothesize that membrane proteomics is a potentially effective approach to profile oxidative folding of bacterial membrane proteins.

Distinct roles of phenol-soluble modulins in spreading of Staphylococcus aureus on wet surfaces.

The human pathogen Staphylococcus aureus is renowned for the rapid colonization of contaminated wounds, medical implants, and food products. Nevertheless, little is known about the mechanisms that allow S. aureus to colonize the respective wet surfaces. The present studies were therefore aimed at identifying factors used by S. aureus cells to spread over wet surfaces, starting either from planktonic or biofilm-associated states. Through proteomics analyses we pinpoint phenol-soluble modulins (PSMs) as prime facilitators of the spreading process. To dissect the roles of the eight PSMs produced by S. aureus, these peptides were chemically synthesized and tested in spreading assays with different psm mutant strains. The results show that PSMα3 and PSMγ are the strongest facilitators of spreading both for planktonic cells and cells in catheter-associated biofilms. Compared to the six other PSMs of S. aureus, PSMα3 and PSMγ combine strong surfactant activities with a relatively low overall hydropathicity. Importantly, we show that PSM-mediated motility of S. aureus facilitates the rapid colonization of wet surfaces next to catheters and the colonization of fresh meat.

Definition of the σ(W) 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 σ(W) regulon of the Gram-positive bacterium Bacillus subtilis from the overlapping ECF σ(X), σ(Y), and σ(M) 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 σ(W)-regulated. Under these conditions, σ(W) exhibits a basal level of activity. Subsequently, we verified the σ(W)-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 σ(W) anti-sigma factor RsiW and subsequent activation of the σ(W)-regulon. Taken together, our studies identify 89 genes as being strictly σ(W)-regulated, including several genes for non-coding RNAs. The effects of rasP or prsW mutations on the expression of σ(W)-dependent genes were relatively mild, which implies that σ(W)-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 σ(W), but that this membrane protease also exerts other important post-transcriptional regulatory functions.

The sortase A substrates FnbpA, FnbpB, ClfA and ClfB antagonize colony spreading of Staphylococcus aureus.

Staphylococcus aureus is an important human pathogen that is renowned both for its rapid transmission within hospitals and the community, and for the formation of antibiotic resistant biofilms on medical implants. Recently, it was shown that S. aureus is able to spread over wet surfaces. This motility phenomenon is promoted by the surfactant properties of secreted phenol-soluble modulins (PSMs), which are also known to inhibit biofilm formation. The aim of the present studies was to determine whether any cell surface-associated S. aureus proteins have an impact on colony spreading. To this end, we analyzed the spreading capabilities of strains lacking non-essential components of the protein export and sorting machinery. Interestingly, our analyses reveal that the absence of sortase A (SrtA) causes a hyper-spreading phenotype. SrtA is responsible for covalent anchoring of various proteins to the staphylococcal cell wall. Accordingly, we show that the hyper-spreading phenotype of srtA mutant cells is an indirect effect that relates to the sortase substrates FnbpA, FnbpB, ClfA and ClfB. These surface-exposed staphylococcal proteins are known to promote biofilm formation, and cell-cell interactions. The hyper-spreading phenotype of srtA mutant staphylococcal cells was subsequently validated in Staphylococcus epidermidis. We conclude that cell wall-associated factors that promote a sessile lifestyle of S. aureus and S. epidermidis antagonize the colony spreading motility of these bacteria.

Global network reorganization during dynamic adaptations of Bacillus subtilis metabolism.

Adaptation of cells to environmental changes requires dynamic interactions between metabolic and regulatory networks, but studies typically address only one or a few layers of regulation. For nutritional shifts between two preferred carbon sources of Bacillus subtilis, we combined statistical and model-based data analyses of dynamic transcript, protein, and metabolite abundances and promoter activities. Adaptation to malate was rapid and primarily controlled posttranscriptionally compared with the slow, mainly transcriptionally controlled adaptation to glucose that entailed nearly half of the known transcription regulation network. Interactions across multiple levels of regulation were involved in adaptive changes that could also be achieved by controlling single genes. Our analysis suggests that global trade-offs and evolutionary constraints provide incentives to favor complex control programs.

Condition-dependent transcriptome reveals high-level regulatory architecture in Bacillus subtilis.

Bacteria adapt to environmental stimuli by adjusting their transcriptomes in a complex manner, the full potential of which has yet to be established for any individual bacterial species. Here, we report the transcriptomes of Bacillus subtilis exposed to a wide range of environmental and nutritional conditions that the organism might encounter in nature. We comprehensively mapped transcription units (TUs) and grouped 2935 promoters into regulons controlled by various RNA polymerase sigma factors, accounting for ~66% of the observed variance in transcriptional activity. This global classification of promoters and detailed description of TUs revealed that a large proportion of the detected antisense RNAs arose from potentially spurious transcription initiation by alternative sigma factors and from imperfect control of transcription termination.

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.

Requirement of the agr locus for colony spreading of Staphylococcus aureus.

The important human pathogen Staphylococcus aureus is known to spread on soft agar plates. Here, we show that colony spreading of S. aureus involves the agr quorum-sensing system. This finding can be related to the agr-dependent expression of biosurfactants, such as phenol-soluble modulins, suggesting a connection between spreading motility and virulence.

pBaSysBioII: an integrative plasmid generating gfp transcriptional fusions for high-throughput analysis of gene expression in Bacillus subtilis.

Plasmid pBaSysBioII was constructed for high-throughput analysis of gene expression in Bacillus subtilis. It is an integrative plasmid with a ligation-independent cloning (LIC) site, allowing the generation of transcriptional gfpmut3 fusions with desired promoters. Integration is by a Campbell-type event and is non-mutagenic, placing the fusion at the homologous chromosomal locus. Using phoA, murAA, gapB, ptsG and cggR promoters that are responsive to phosphate availability, growth rate and carbon source, we show that detailed profiles of promoter activity can be established, with responses to changing conditions being measurable within 1 min of the stimulus. This makes pBaSysBioII a highly versatile tool for real-time gene expression analysis in growing cells of B. subtilis.