Balanced cell division is secured by two different regulatory sites in OxyS RNA.

The hydrogen peroxide-induced small RNA OxyS has been proposed to originate from the 3' UTR of a peroxide mRNA. Unexpectedly, phylogenetic OxyS targetome predictions indicate that most OxyS targets belong to the category of "cell cycle," including cell division and cell elongation. Previously, we reported that Escherichia coli OxyS inhibits cell division by repressing expression of the essential transcription termination factor nusG, thereby leading to the expression of the KilR protein, which interferes with the function of the major cell division protein, FtsZ. By interfering with cell division, OxyS brings about cell-cycle arrest, thus allowing DNA damage repair. Cell division and cell elongation are opposing functions to the extent that inhibition of cell division requires a parallel inhibition of cell elongation for the cells to survive. In this study, we report that in addition to cell division, OxyS inhibits mepS, which encodes an essential peptidoglycan endopeptidase that is responsible for cell elongation. Our study indicates that cell-cycle arrest and balancing between cell division and cell elongation are important and conserved functions of the oxidative stress-induced sRNA OxyS.

Improved RNA stability estimation indicates that transcriptional interference is frequent in diverse bacteria.

We used stochastic simulations and experimental data from E. coli, K. aerogenes, Synechococcus PCC 7002 and Synechocystis PCC 6803 to provide evidence that transcriptional interference via the collision mechanism is likely a prevalent mechanism for bacterial gene regulation. Rifampicin time-series data can be used to globally monitor and quantify collision between sense and antisense transcription-complexes. Our findings also highlight that transcriptional events, such as differential RNA decay, partial termination, and internal transcriptional start sites often deviate from gene annotations. Consequently, within a single gene annotation, there exist transcript segments with varying half-lives and transcriptional properties. To address these complexities, we introduce 'rifi', an R-package that analyzes transcriptomic data from rifampicin time series. 'rifi' employs a dynamic programming-based segmentation approach to identify individual transcripts, enabling accurate assessment of RNA stability and detection of diverse transcriptional events.

DIGGER-Bac: prediction of seed regions for high-fidelity construction of synthetic small RNAs in bacteria.

Synthetic small RNAs (sRNAs) are gaining increasing attention in the field of synthetic biology and bioengineering for efficient post-transcriptional regulation of gene expression. However, the optimal design of synthetic sRNAs is challenging because alterations may impair functions or off-target effects can arise. Here, we introduce DIGGER-Bac, a toolbox for Design and Identification of seed regions for Golden Gate assembly and Expression of synthetic sRNAs in Bacteria. The SEEDling tool predicts optimal sRNA seed regions in combination with user-defined sRNA scaffolds for efficient regulation of specified mRNA targets. Results are passed on to the G-GArden tool, which assists with primer design for high-fidelity Golden Gate assembly of the desired synthetic sRNA constructs.

An Easy-to-Use Plasmid Toolset for Efficient Generation and Benchmarking of Synthetic Small RNAs in Bacteria.

Synthetic biology approaches life from the perspective of an engineer. Standardized and de novo design of genetic parts to subsequently build reproducible and controllable modules, for example, for circuit design, is a key element. To achieve this, natural systems and elements often serve as a blueprint for researchers. Regulation of protein abundance is controlled at DNA, mRNA, and protein levels. Many tools for the activation or repression of transcription or the destabilization of proteins are available, but easy-to-handle minimal regulatory elements on the mRNA level are preferable when translation needs to be modulated. Regulatory RNAs contribute considerably to regulatory networks in all domains of life. In particular, bacteria use small regulatory RNAs (sRNAs) to regulate mRNA translation. Slowly, sRNAs are attracting the interest of using them for broad applications in synthetic biology. Here, we promote a "plug and play" plasmid toolset to quickly and efficiently create synthetic sRNAs to study sRNA biology or their application in bacteria. We propose a simple benchmarking assay by targeting the acrA gene of Escherichia coli and rendering cells sensitive toward the ß-lactam antibiotic oxacillin. We further highlight that it may be necessary to test multiple seed regions and sRNA scaffolds to achieve the desired regulatory effect. The described plasmid toolset allows quick construction and testing of various synthetic sRNAs based on the user's needs.

Expression of the Cyanobacterial FoF1 ATP Synthase Regulator AtpΘ Depends on Small DNA-Binding Proteins and Differential mRNA Stability.

FoF1 ATP synthases produce ATP, the universal biological energy source. ATP synthase complexes on cyanobacterial thylakoid membranes use proton gradients generated either by photosynthesis or respiration. AtpΘ is an ATP synthase regulator in cyanobacteria which is encoded by the gene atpT. AtpΘ prevents the hydrolysis of ATP (reverse reaction) that otherwise would occur under unfavorable conditions. In the cyanobacterium Synechocystis sp. PCC 6803, AtpΘ is expressed maximum in darkness but at very low levels under optimum phototrophic growth conditions or in the presence of glucose. DNA coimmunoprecipitation experiments followed by mass spectrometry identified the binding of the two transcriptional regulators cyAbrB1 and cyAbrB2 to the promoter and the histone-like protein HU to the 5'UTR of atpT. Analyses of nucleotide substitutions in the promoter and GFP reporter assays identified a functionally relevant sequence motif resembling the HLR1 element bound by the RpaB transcription factor. Electrophoretic mobility shift assays confirmed interaction of cyAbrB1, cyAbrB2, and RpaB with the promoter DNA. However, overall the effect of transcriptional regulation was comparatively low. In contrast, atpT transcript stabilities differed dramatically, half-lives were 1.6 min in the light, 33 min in the dark and substantial changes were observed if glucose or DCMU were added. These findings show that transcriptional control of atpT involves nucleoid-associated DNA-binding proteins, positive regulation through RpaB, while the major effect on the condition-dependent regulation of atpT expression is mediated by controlling mRNA stability, which is related to the cellular redox and energy status. IMPORTANCE FoF1 ATP synthases produce ATP, the universal biological energy source. Under unfavorable conditions, ATP synthases can operate in a futile reverse reaction, pumping protons while ATP is used up. Cyanobacteria perform plant-like photosynthesis, but they cannot use the same mechanism as plant chloroplasts to inhibit ATP synthases during the night because respiratory and photosynthetic complexes are both located in the same membrane system. AtpΘ is a small protein encoded by the gene atpT in cyanobacteria that can prevent the ATP synthase reverse reaction (ATPase activity). Here we found that three transcription factors contribute to the regulation of atpT expression. However, the control of mRNA stability was identified as the major regulatory process governing atpT expression. Thus, it is the interplay between transcriptional and posttranscriptional regulation that position the AtpΘ-based regulatory mechanism within the context of the cellular redox and energy balance.

Integrative analysis of the salt stress response in cyanobacteria.

Microorganisms evolved specific acclimation strategies to thrive in environments of high or fluctuating salinities. Here, salt acclimation in the model cyanobacterium Synechocystis sp. PCC 6803 was analyzed by integrating transcriptomic, proteomic and metabolomic data. A dynamic reorganization of the transcriptome occurred during the first hours after salt shock, e.g. involving the upregulation of genes to activate compatible solute biochemistry balancing osmotic pressure. The massive accumulation of glucosylglycerol then had a measurable impact on the overall carbon and nitrogen metabolism. In addition, we observed the coordinated induction of putative regulatory RNAs and of several proteins known for their involvement in other stress responses. Overall, salt-induced changes in the proteome and transcriptome showed good correlations, especially among the stably up-regulated proteins and their transcripts. We define an extended salt stimulon comprising proteins directly or indirectly related to compatible solute metabolism, ion and water movements, and a distinct set of regulatory RNAs involved in post-transcriptional regulation. Our comprehensive data set provides the basis for engineering cyanobacterial salt tolerance and to further understand its regulation.

NsiR3, a nitrogen stress-inducible small RNA, regulates proline oxidase expression in the cyanobacterium Nostoc sp. PCC 7120.

NsiR3 (nitrogen stress-inducible RNA 3) is a small noncoding RNA strongly conserved in heterocyst-forming cyanobacteria. In Nostoc sp. PCC 7120, transcription of NsiR3 is induced by nitrogen starvation and depends on the global nitrogen regulator NtcA. A conserved NtcA-binding site is centered around position -42.5 with respect to the transcription start site of NsiR3 homologs, and NtcA binds in vitro to a DNA fragment containing this sequence. In the absence of combined nitrogen, NsiR3 expression is induced in all cells along the Nostoc filament but much more strongly in heterocysts, differentiated cells devoted to nitrogen fixation. Co-expression analysis of transcriptomic data obtained from microarrays hybridized with RNA obtained from Nostoc wild-type or mutant strains grown in the presence of ammonium or in the absence of combined nitrogen revealed that the expression profile of gene putA (proline oxidase) correlates negatively with that of NsiR3. Using a heterologous system in Escherichia coli, we show that NsiR3 binds to the 5'-UTR of putA mRNA, resulting in reduced expression of a reporter gene. Overexpression of NsiR3 in Nostoc resulted in strong reduction of putA mRNA accumulation, further supporting the negative regulation of putA by NsiR3. The higher expression of NsiR3 in heterocysts versus vegetative cells of the N2 -fixing filament could contribute to the previously described absence of putA mRNA and of the catabolic pathway to produce glutamate from arginine via proline specifically in heterocysts. Post-transcriptional regulation by NsiR3 represents an indirect NtcA-operated regulatory mechanism of putA expression. DATABASE: Microarray data are available in GEO database under accession numbers GSE120377 and GSE150191.

Bioinformatic prediction reveals posttranscriptional regulation of the chromosomal replication initiator gene dnaA by the attenuator sRNA rnTrpL in Escherichia coli.

DnaA is the initiator protein of chromosome replication, but the regulation of its homoeostasis in enterobacteria is not well understood. The DnaA level remains stable at different growth rates, suggesting a link between metabolism and dnaA expression. In a bioinformatic prediction, which we made to unravel targets of the sRNA rnTrpL in Enterobacteriaceae, the dnaA mRNA was the most conserved target candidate. The sRNA rnTrpL is derived from the transcription attenuator of the tryptophan biosynthesis operon. In Escherichia coli, its level is higher in minimal than in rich medium due to derepressed transcription without external tryptophan supply. Overexpression and deletion of the rnTrpL gene decreased and increased, respectively, the levels of dnaA mRNA. The decrease of the dnaA mRNA level upon rnTrpL overproduction was dependent on hfq and rne. Base pairing between rnTrpL and dnaA mRNA in vivo was validated. In minimal medium, the oriC level was increased in the ΔtrpL mutant, in line with the expected DnaA overproduction and increased initiation of chromosome replication. In line with this, chromosomal rnTrpL mutation abolishing the interaction with dnaA increased both the dnaA mRNA and the oriC level. Moreover, upon addition of tryptophan to minimal medium cultures, the oriC level in the wild type was increased. Thus, rnTrpL is a base-pairing sRNA that posttranscriptionally regulates dnaA in E. coli. Furthermore, our data suggest that rnTrpL contributes to the DnaA homoeostasis in dependence on the nutrient availability, which is represented by the tryptophan level in the cell.

GLASSgo in Galaxy: high-throughput, reproducible and easy-to-integrate prediction of sRNA homologs.

MOTIVATION: The correct prediction of bacterial sRNA homologs is a prerequisite for many downstream analyses based on comparative genomics, but it is frequently challenging due to the short length and distinct heterogeneity of such homologs. GLobal Automatic Small RNA Search go (GLASSgo) is an efficient tool for the prediction of sRNA homologs from a single input query. To make the algorithm available to a broader community, we offer a Docker container along with a free-access web service. For non-computer scientists, the web service provides a user-friendly interface. However, capabilities were lacking so far for batch processing, version control and direct interaction with compatible software applications as a workflow management system can provide. RESULTS: Here, we present GLASSgo 1.5.2, an updated version that is fully incorporated into the workflow management system Galaxy. The improved version contains a new feature for extracting the upstream regions, allowing the search for conserved promoter elements. Additionally, it supports the use of accession numbers instead of the outdated GI numbers, which widens the applicability of the tool. AVAILABILITY AND IMPLEMENTATION: GLASSgo is available at https://github.com/lotts/GLASSgo/ under the MIT license and is accompanied by instruction and application data. Furthermore, it can be installed into any Galaxy instance using the Galaxy ToolShed.

The power of cooperation: Experimental and computational approaches in the functional characterization of bacterial sRNAs.

Trans-acting small regulatory RNAs (sRNAs) are key players in the regulation of gene expression in bacteria. There are hundreds of different sRNAs in a typical bacterium, which in contrast to eukaryotic microRNAs are more heterogeneous in length, sequence composition, and secondary structure. The vast majority of sRNAs function post-transcriptionally by binding to other RNAs (mRNAs, sRNAs) through rather short regions of imperfect sequence complementarity. Besides, every single sRNA may interact with dozens of different target RNAs and impact gene expression either negatively or positively. These facts contributed to the view that the entirety of the regulatory targets of a given sRNA, its targetome, is challenging to identify. However, recent developments show that a more comprehensive sRNAs targetome can be achieved through the combination of experimental and computational approaches. Here, we give a short introduction into these methods followed by a description of two sRNAs, RyhB, and RsaA, to illustrate the particular strengths and weaknesses of these approaches in more details. RyhB is an sRNA involved in iron homeostasis in Enterobacteriaceae, while RsaA is a modulator of virulence in Staphylococcus aureus. Using such a combined strategy, a better appreciation of the sRNA-dependent regulatory networks is now attainable.

Depletion of the FtsH1/3 Proteolytic Complex Suppresses the Nutrient Stress Response in the Cyanobacterium Synechocystis sp strain PCC 6803.

The membrane-embedded FtsH proteases found in bacteria, chloroplasts, and mitochondria are involved in diverse cellular processes including protein quality control and regulation. The genome of the model cyanobacterium Synechocystis sp PCC 6803 encodes four FtsH homologs designated FtsH1 to FtsH4. The FtsH3 homolog is present in two hetero-oligomeric complexes: FtsH2/3, which is responsible for photosystem II quality control, and the essential FtsH1/3 complex, which helps maintain Fe homeostasis by regulating the level of the transcription factor Fur. To gain a more comprehensive insight into the physiological roles of FtsH hetero-complexes, we performed genome-wide expression profiling and global proteomic analyses of Synechocystis mutants conditionally depleted of FtsH3 or FtsH1 grown under various nutrient conditions. We show that the lack of FtsH1/3 leads to a drastic reduction in the transcriptional response to nutrient stress of not only Fur but also the Pho, NdhR, and NtcA regulons. In addition, this effect is accompanied by the accumulation of the respective transcription factors. Thus, the FtsH1/3 complex is of critical importance for acclimation to iron, phosphate, carbon, and nitrogen starvation in Synechocystis.plantcell;31/12/2912/FX1F1fx1.

Inactivation of the RNA helicase CrhR impacts a specific subset of the transcriptome in the cyanobacterium Synechocystis sp. PCC 6803.

DEAD-box RNA-helicases catalyze the reorganization of structured RNAs and the formation of RNP complexes. The cyanobacterium Synechocystis sp. PCC 6803 encodes a single DEAD-box RNA helicase, CrhR (Slr0083), whose expression is regulated by abiotic stresses that alter the redox potential of the photosynthetic electron transport chain, including temperature downshift. Despite its proposed effect on RNA metabolism and its known relevance in cold-stress adaptation, the reported impact of a CrhR knockout on the cold adaption of the transcriptome only identified eight affected genes. Here, we utilized a custom designed microarray to assess the impact of the absence of CrhR RNA helicase activity on the transcriptome, independent of cold stress. CrhR truncation impacts an RNA subset comprising ~10% of the ncRNA and also ~10% of the mRNA transcripts. While equal numbers of mRNAs showed increased as well as decreased abundance, more than 90% of the ncRNAs showed enhanced expression in the absence of CrhR, indicative of a negative effect on ncRNA transcription or stability. We further tested the effect of CrhR on the stability of strongly responding RNAs that identify examples of post-transcriptional and transcriptional regulation. The data suggest that CrhR impacts multiple aspects of RNA metabolism in Synechocystis.

Elements of the heterocyst-specific transcriptome unravelled by co-expression analysis in Nostoc sp. PCC 7120.

Nitrogen is frequently limiting microbial growth in the environment. As a response, many filamentous cyanobacteria differentiate heterocysts, cells devoted to N2 fixation. Heterocyst differentiation is under the control of the master regulator HetR. Through the characterization of the HetR-dependent transcriptome in Nostoc sp. PCC 7120, we identified the new candidate genes likely involved in heterocyst differentiation. According to their maximum induction, we defined E-DIF (early in differentiation) and L-DIF (late in differentiation) genes. Most of the genes known to be involved in the critical aspects of heterocyst differentiation or function were also classified into these groups, showing the validity of the approach. Using fusions to gfp, we verified the heterocyst-specific transcription of several of the found genes, antisense transcripts and potentially trans-acting sRNAs. Through comparative sequence analysis of promoter regions, we noticed the prevalence of the previously described DIF1 motif and identified a second motif, called DIF2, in other promoters of the E-DIF cluster. Both motifs are widely conserved in heterocystous cyanobacteria. We assigned alr2522 as a third member, besides nifB and nifP, to the CnfR regulon. The elements identified here are of interest for understanding cell differentiation, engineering of biological nitrogen fixation or production of O2 -sensitive molecules in cyanobacteria.

Biocomputational Analyses and Experimental Validation Identify the Regulon Controlled by the Redox-Responsive Transcription Factor RpaB.

Oxygenic photosynthesis requires the coordination of environmental stimuli with the regulation of transcription. The transcription factor RpaB is conserved from the simplest unicellular cyanobacteria to complex eukaryotic algae, representing more than 1 billion years of evolution. To predict the RpaB-controlled regulon in the cyanobacterium Synechocystis, we analyzed the positional distribution of binding sites together with high-resolution mapping data of transcriptional start sites (TSSs). We describe more than 150 target promoters whose activity responds to fluctuating light conditions. Binding sites close to the TSS mediate repression, whereas sites centered ∼50 nt upstream mediate activation. Using complementary experimental approaches, we found that RpaB controls genes involved in photoprotection, cyclic electron flow and state transitions, photorespiration, and nirA and isiA for which we suggest cross-regulation with the transcription factors NtcA or FurA. The deep integration of RpaB with diverse photosynthetic gene functions makes it one of the most important and versatile transcriptional regulators.

The primary transcriptome of the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973.

BACKGROUND: Cyanobacteria have shown promising potential for the production of various biofuels and chemical feedstocks. Synechococcus elongatus UTEX 2973 is a fast-growing strain with pronounced tolerance to high temperatures and illumination. Hence, this strain appears to be ideal for the development of photosynthetic biotechnology. However, molecular insights on how this strain can rapidly accumulate biomass and carbohydrates under high-light and high-temperature conditions are lacking. RESULTS: Differential RNA-Sequencing (dRNA-Seq) enabled the genome-wide identification of 4808 transcription start sites (TSSs) in S. elongatus UTEX 2973 using a background reduction algorithm. High light promoted the transcription of genes associated with central metabolic pathways, whereas the highly induced small RNA (sRNA) PsrR1 likely contributed to the repression of phycobilisome genes and the accelerated glycogen accumulation rates measured under this condition. Darkness caused transcriptome remodeling with a decline in the expression of genes for carbon fixation and other major metabolic pathways and an increase in the expression of genes for glycogen catabolism and Calvin cycle inhibitor CP12. Two of the identified TSSs drive the transcription of highly abundant sRNAs in darkness. One of them is widely conserved throughout the cyanobacterial phylum. Its gene is fused to a protein-coding gene in some species, illustrating the evolutionary origin of sRNAs from an mRNA 3'-end. CONCLUSIONS: Our comprehensive set of genome-wide mapped TSSs, sRNAs and promoter activities will be valuable for projects requiring precise information about the control of transcription aimed at metabolic engineering and the elucidation of stress acclimation mechanisms in this promising strain.

Widespread Antisense Transcription in Prokaryotes.

Although bacterial genomes are usually densely protein-coding, genome-wide mapping approaches of transcriptional start sites revealed that a significant fraction of the identified promoters drive the transcription of noncoding RNAs. These can be trans-acting RNAs, mainly originating from intergenic regions and, in many studied examples, possessing regulatory functions. However, a significant fraction of these noncoding RNAs consist of natural antisense transcripts (asRNAs), which overlap other transcriptional units. Naturally occurring asRNAs were first observed to play a role in bacterial plasmid replication and in bacteriophage λ more than 30 years ago. Today's view is that asRNAs abound in all three domains of life. There are several examples of asRNAs in bacteria with clearly defined functions. Nevertheless, many asRNAs appear to result from pervasive initiation of transcription, and some data point toward global functions of such widespread transcriptional activity, explaining why the search for a specific regulatory role is sometimes futile. In this review, we give an overview about the occurrence of antisense transcription in bacteria, highlight particular examples of functionally characterized asRNAs, and discuss recent evidence pointing at global relevance in RNA processing and transcription-coupled DNA repair.

GLASSgo - Automated and Reliable Detection of sRNA Homologs From a Single Input Sequence.

Bacterial small RNAs (sRNAs) are important post-transcriptional regulators of gene expression. The functional and evolutionary characterization of sRNAs requires the identification of homologs, which is frequently challenging due to their heterogeneity, short length and partly, little sequence conservation. We developed the GLobal Automatic Small RNA Search go (GLASSgo) algorithm to identify sRNA homologs in complex genomic databases starting from a single sequence. GLASSgo combines an iterative BLAST strategy with pairwise identity filtering and a graph-based clustering method that utilizes RNA secondary structure information. We tested the specificity, sensitivity and runtime of GLASSgo, BLAST and the combination RNAlien/cmsearch in a typical use case scenario on 40 bacterial sRNA families. The sensitivity of the tested methods was similar, while the specificity of GLASSgo and RNAlien/cmsearch was significantly higher than that of BLAST. GLASSgo was on average ∼87 times faster than RNAlien/cmsearch, and only ∼7.5 times slower than BLAST, which shows that GLASSgo optimizes the trade-off between speed and accuracy in the task of finding sRNA homologs. GLASSgo is fully automated, whereas BLAST often recovers only parts of homologs and RNAlien/cmsearch requires extensive additional bioinformatic work to get a comprehensive set of homologs. GLASSgo is available as an easy-to-use web server to find homologous sRNAs in large databases.

Freiburg RNA tools: a central online resource for RNA-focused research and teaching.

The Freiburg RNA tools webserver is a well established online resource for RNA-focused research. It provides a unified user interface and comprehensive result visualization for efficient command line tools. The webserver includes RNA-RNA interaction prediction (IntaRNA, CopraRNA, metaMIR), sRNA homology search (GLASSgo), sequence-structure alignments (LocARNA, MARNA, CARNA, ExpaRNA), CRISPR repeat classification (CRISPRmap), sequence design (antaRNA, INFO-RNA, SECISDesign), structure aberration evaluation of point mutations (RaSE), and RNA/protein-family models visualization (CMV), and other methods. Open education resources offer interactive visualizations of RNA structure and RNA-RNA interaction prediction as well as basic and advanced sequence alignment algorithms. The services are freely available at http://rna.informatik.uni-freiburg.de.

Workflow for a Computational Analysis of an sRNA Candidate in Bacteria.

Computational methods can often facilitate the functional characterization of individual sRNAs and furthermore allow high-throughput analysis on large numbers of sRNA candidates. This chapter outlines a potential workflow for computational sRNA analyses and describes in detail methods for homolog detection, target prediction, and functional characterization based on enrichment analysis. The cyanobacterial sRNA IsaR1 is used as a specific example. All methods are available as webservers and easily accessible for nonexpert users.

OxyS small RNA induces cell cycle arrest to allow DNA damage repair.

To maintain genome integrity, organisms employ DNA damage response, the underlying principles of which are conserved from bacteria to humans. The bacterial small RNA OxyS of Escherichia coli is induced upon oxidative stress and has been implicated in protecting cells from DNA damage; however, the mechanism by which OxyS confers genome stability remained unknown. Here, we revealed an OxyS-induced molecular checkpoint relay, leading to temporary cell cycle arrest to allow damage repair. By repressing the expression of the essential transcription termination factor nusG, OxyS enables read-through transcription into a cryptic prophage encoding kilR The KilR protein interferes with the function of the major cell division protein FtsZ, thus imposing growth arrest. This transient growth inhibition facilitates DNA damage repair, enabling cellular recovery, thereby increasing viability following stress. The OxyS-mediated growth arrest represents a novel tier of defense, introducing a new regulatory concept into bacterial stress response.