A new reagent for in vivo structure probing of RNA G and U residues that improves RNA structure prediction alone and combined with DMS.

A key to understanding the roles of RNA in regulating gene expression is knowing their structures in vivo. One way to obtain this information is through probing structures of RNA with chemicals. To probe RNA structure directly in cells, membrane-permeable reagents that modify the Watson-Crick (WC) face of unpaired nucleotides can be used. While dimethyl sulfate (DMS) has led to substantial insight into RNA structure, it has limited nucleotide specificity in vivo, with WC face reactivity only at adenine (A) and cytosine (C) at neutral pH. The reagent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was recently shown to modify the WC face of guanine (G) and uracil (U). While useful at lower concentrations in experiments that measure chemical modifications by reverse transcription stops, at higher concentrations necessary for detection by mutational profiling (MaP), EDC treatment leads to degradation of RNA. Here we demonstrate EDC-stimulated degradation of RNA in Gram-negative and Gram-positive bacteria. In an attempt to overcome these limitations, we developed a new carbodiimide reagent, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide methiodide (ETC), which we show specifically modifies unpaired Gs and Us in vivo without substantial degradation of RNA. We establish ETC as a probe for MaP and optimize the reverse transcription conditions and computational analysis in Escherichia coli Importantly, we demonstrate the utility of ETC as a probe for improving RNA structure prediction both alone and with DMS.

Multitier regulation of the E. coli extreme acid stress response by CsrA.

CsrA is an RNA-binding protein that regulates processes critical for growth and survival, including central carbon metabolism, motility, biofilm formation, stress responses, and expression of virulence factors in pathogens. Transcriptomics studies in Escherichia coli suggested that CsrA repressed genes involved in surviving extremely acidic conditions. Here, we examine the effects of disrupting CsrA-dependent regulation on the expression of genes and circuitry for acid stress survival and demonstrate CsrA-mediated repression at multiple levels. We show that this repression is critical for managing the trade-off between growth and survival; overexpression of acid stress genes caused by csrA disruption enhances survival under extreme acidity but is detrimental for growth under mildly acidic conditions. In vitro studies confirmed that CsrA binds specifically to mRNAs of structural and regulatory genes for acid stress survival, causing translational repression. We also found that translation of the top-tier acid stress regulator, evgA, is coupled to that of a small leader peptide, evgL, which is repressed by CsrA. Unlike dedicated acid stress response genes, csrA and its sRNA antagonists, csrB and csrC, did not exhibit a substantial response to acid shock. Furthermore, disruption of CsrA regulation of acid stress genes impacted host-microbe interactions in Caenorhabditis elegans, alleviating GABA deficiencies. This study expands the known regulon of CsrA to genes of the extreme acid stress response of E. coli and highlights a new facet of the global role played by CsrA in balancing the opposing physiological demands of stress resistance with the capacity for growth and modulating host interactions.IMPORTANCETo colonize/infect the mammalian intestinal tract, bacteria must survive exposure to the extreme acidity of the stomach. E. coli does this by expressing proteins that neutralize cytoplasmic acidity and cope with molecular damage caused by low pH. Because of the metabolic cost of these processes, genes for surviving acid stress are tightly regulated. Here, we show that CsrA negatively regulates the cascade of expression responsible for the acid stress response. Increased expression of acid response genes due to csrA disruption improved survival at extremely low pH but inhibited growth under mildly acidic conditions. Our findings define a new layer of regulation in the acid stress response of E. coli and a novel physiological function for CsrA.

CsrA coordinates the expression of ribosome hibernation and anti-σ factor proteins.

Bacterial growth rate varies due to changing physiological signals and is fundamentally dependent on protein synthesis. Consequently, cells alter their transcription and translation machinery to optimize the capacity for protein production under varying conditions and growth rates. Our findings demonstrate that the post-transcriptional regulator CsrA in Escherichia coli controls the expression of genes that participate in these processes. During exponential growth, CsrA represses the expression of proteins that alter or inhibit RNA polymerase (RNAP) and ribosome activity, including the ribosome hibernation factors RMF, RaiA, YqjD, ElaB, YgaM, and SRA, as well as the anti-σ70 factor, Rsd. Upon entry into the stationary phase, RaiA, YqjD, ElaB, and SRA expression was derepressed and that of RMF, YgaM, and Rsd was activated in the presence of CsrA. This pattern of gene expression likely supports global protein expression during active growth and helps limit protein production to a basal level when nutrients are limited. In addition, we identified genes encoding the paralogous C-tail anchored inner membrane proteins YqjD and ElaB as robust, direct targets of CsrA-mediated translational repression. These proteins bind ribosomes and mediate their localization to the inner cell membrane, impacting a variety of processes including protein expression and membrane integrity. Previous studies found that YqjD overexpression inhibits cell growth, suggesting that appropriate regulation of YqjD expression might play a key role in cell viability. CsrA-mediated regulation of yqjD and ribosome hibernation factors reveals a new role for CsrA in appropriating cellular resources for optimum growth under varying conditions.IMPORTANCEThe Csr/Rsm system (carbon storage regulator or repressor of stationary phase metabolites) is a global post-transcriptional regulatory system that coordinates and responds to environmental cues and signals, facilitating the transition between active growth and stationary phase. Another key determinant of bacterial lifestyle decisions is the management of the cellular gene expression machinery. Here, we investigate the connection between these two processes in Escherichia coli. Disrupted regulation of the transcription and translation machinery impacts many cellular functions, including gene expression, growth, fitness, and stress resistance. Elucidating the role of the Csr system in controlling the activity of RNAP and ribosomes advances our understanding of mechanisms controlling bacterial growth. A more complete understanding of these processes could lead to the improvement of therapeutic strategies for recalcitrant infections.

In vivo-like nearest neighbor parameters improve prediction of fractional RNA base-pairing in cells.

We conducted a thermodynamic analysis of RNA stability in Eco80 artificial cytoplasm, which mimics in vivo conditions, and compared it to transcriptome-wide probing of mRNA. Eco80 contains 80% of Escherichia coli metabolites, with biological concentrations of metal ions, including 2 mM free Mg2+ and 29 mM metabolite-chelated Mg2+. Fluorescence-detected binding isotherms (FDBI) were used to conduct a thermodynamic analysis of 24 RNA helices and found that these helices, which have an average stability of -12.3 kcal/mol, are less stable by ΔΔGo37 ∼1 kcal/mol. The FDBI data was used to determine a set of Watson-Crick free energy nearest neighbor parameters (NNPs), which revealed that Eco80 reduces the stability of three NNPs. This information was used to adjust the NN model using the RNAstructure package. The in vivo-like adjustments have minimal effects on the prediction of RNA secondary structures determined in vitro and in silico, but markedly improve prediction of fractional RNA base pairing in E. coli, as benchmarked with our in vivo DMS and EDC RNA chemical probing data. In summary, our thermodynamic and chemical probing analyses of RNA helices indicate that RNA secondary structures are less stable in cells than in artificially stable in vitro buffer conditions.

Robust regulation of transcription pausing in Escherichia coli by the ubiquitous elongation factor NusG.

Transcription elongation by multi-subunit RNA polymerases (RNAPs) is regulated by auxiliary factors in all organisms. NusG/Spt5 is the only universally conserved transcription elongation factor shared by all domains of life. NusG is a component of antitermination complexes controlling ribosomal RNA operons, an essential antipausing factor, and a transcription-translation coupling factor in Escherichia coli. We employed RNET-seq for genome-wide mapping of RNAP pause sites in wild-type and NusG-depleted cells. We demonstrate that NusG is a major antipausing factor that suppresses thousands of backtracked and nonbacktracked pauses across the E. coli genome. The NusG-suppressed pauses were enriched immediately downstream from the translation start codon but were also abundant elsewhere in open reading frames, small RNA genes, and antisense transcription units. This finding revealed a strong similarity of NusG to Spt5, which stimulates the elongation rate of many eukaryotic genes. We propose a model in which promoting forward translocation and/or stabilization of RNAP in the posttranslocation register by NusG results in suppression of pausing in E. coli.

CsrA Positively and Directly Regulates the Expression of the pdu, pocR, and eut Genes Required for the Luminal Replication of Salmonella Typhimurium.

Enteric pathogens, such as Salmonella, have evolved to thrive in the inflamed gut. Genes located within the Salmonella pathogenicity island 1 (SPI-1) mediate the invasion of cells from the intestinal epithelium and the induction of an intestinal inflammatory response. Alternative electron acceptors become available in the inflamed gut and are utilized by Salmonella for luminal replication through the metabolism of propanediol and ethanolamine, using the enzymes encoded by the pdu and eut genes. The RNA-binding protein CsrA inhibits the expression of HilD, which is the central transcriptional regulator of the SPI-1 genes. Previous studies suggest that CsrA also regulates the expression of the pdu and eut genes, but the mechanism for this regulation is unknown. In this work, we show that CsrA positively regulates the pdu genes by binding to the pocR and pduA transcripts as well as the eut genes by binding to the eutS transcript. Furthermore, our results show that the SirA-CsrB/CsrC-CsrA regulatory cascade controls the expression of the pdu and eut genes mediated by PocR or EutR, which are the positive AraC-like transcriptional regulators for the pdu and eut genes, respectively. By oppositely regulating the expression of genes for invasion and for luminal replication, the SirA-CsrB/CsrC-CsrA regulatory cascade could be involved in the generation of two Salmonella populations that cooperate for intestinal colonization and transmission. Our study provides new insight into the regulatory mechanisms that govern Salmonella virulence. IMPORTANCE The regulatory mechanisms that control the expression of virulence genes are essential for bacteria to infect hosts. Salmonella has developed diverse regulatory mechanisms to colonize the host gut. For instance, the SirA-CsrB/CsrC-CsrA regulatory cascade controls the expression of the SPI-1 genes, which are required for this bacterium to invade intestinal epithelium cells and for the induction of an intestinal inflammatory response. In this study, we determine the mechanisms by which the SirA-CsrB/CsrC-CsrA regulatory cascade controls the expression of the pdu and eut genes, which are necessary for the replication of Salmonella in the intestinal lumen. Thus, our data, together with the results of previous reports, indicate that the SirA-CsrB/CsrC-CsrA regulatory cascade has an important role in the intestinal colonization by Salmonella.

CsrA-Mediated Translational Activation of the hmsE mRNA Enhances HmsD-Dependent C-di-GMP-Enabled Biofilm Production in Yersinia pestis.

The plague bacterium, Yersinia pestis, forms a biofilm-mediated blockage in the flea foregut that enhances its transmission by fleabite. Biofilm formation is positively controlled by cyclic di-GMP (c-di-GMP), which is synthesized by the diguanylate cyclases (DGC), HmsD and HmsT. While HmsD primarily promotes biofilm-mediated blockage of fleas, HmsT plays a more minor role in this process. HmsD is a component of the HmsCDE tripartite signaling system. HmsC and HmsE posttranslationally inhibit or activate HmsD, respectively. HmsT-dependent c-di-GMP levels and biofilm formation are positively regulated by the RNA-binding protein CsrA. In this study we determined whether CsrA positively regulates HmsD-dependent biofilm formation through interactions with the hmsE mRNA. Gel mobility shift assays determined that CsrA binds specifically to the hmsE transcript. RNase T1 footprint assays identified a single CsrA binding site and CsrA-induced structural changes in the hmsE leader region. Translational activation of the hmsE mRNA was confirmed in vivo using plasmid-encoded inducible translational fusion reporters and by HmsE protein expression studies. Furthermore, mutation of the CsrA binding site in the hmsE transcript significantly reduced HmsD-dependent biofilm formation. These results suggest that CsrA binding leads to structural changes in the hmsE mRNA that enhance its translation to enable increased HmsD-dependent biofilm formation. Given the requisite function of HmsD in biofilm-mediated flea blockage, this CsrA-dependent increase in HmsD activity underscores that complex and conditionally defined modulation of c-di-GMP synthesis within the flea gut is required for Y. pestis transmission. IMPORTANCE Mutations enhancing c-di-GMP biosynthesis drove the evolution of Y. pestis to flea-borne transmissibility. c-di-GMP-dependent biofilm-mediated blockage of the flea foregut enables regurgitative transmission of Y. pestis by fleabite. The Y. pestis diguanylate cyclases (DGC), HmsT and HmsD, which synthesize c-di-GMP, play significant roles in transmission. Several regulatory proteins involved in environmental sensing, as well as signal transduction and response regulation, tightly control DGC function. An example is CsrA, a global posttranscriptional regulator that modulates carbon metabolism and biofilm formation. CsrA integrates alternative carbon usage metabolism cues to activate c-di-GMP biosynthesis through HmsT. Here, we demonstrated that CsrA additionally activates hmsE translation to promote c-di-GMP biosynthesis through HmsD. This emphasizes that a highly evolved regulatory network controls c-di-GMP synthesis and Y. pestis transmission.

Transcriptome-wide probing reveals RNA thermometers that regulate translation of glycerol permease genes in Bacillus subtilis.

RNA structure regulates bacterial gene expression by several distinct mechanisms via environmental and cellular stimuli, one of which is temperature. While some genome-wide studies have focused on heat shock treatments and the subsequent transcriptomic changes, soil bacteria are less likely to experience such rapid and extreme temperature changes. Though RNA thermometers (RNATs) have been found in 5' untranslated leader regions (5' UTRs) of heat shock and virulence-associated genes, this RNA-controlled mechanism could regulate other genes as well. Using Structure-seq2 and the chemical probe dimethyl sulfate (DMS) at four growth temperatures ranging from 23°C to 42°C, we captured a dynamic response of the Bacillus subtilis transcriptome to temperature. Our transcriptome-wide results show RNA structural changes across all four temperatures and reveal nonmonotonic reactivity trends with increasing temperature. Then, focusing on subregions likely to contain regulatory RNAs, we examined 5' UTRs to identify large, local reactivity changes. This approach led to the discovery of RNATs that control the expression of glpF (glycerol permease) and glpT (glycerol-3-phosphate permease); expression of both genes increased with increased temperature. Results with mutant RNATs indicate that both genes are controlled at the translational level. Increased import of glycerols at high temperatures could provide thermoprotection to proteins.

Allosteric mechanism of transcription inhibition by NusG-dependent pausing of RNA polymerase.

NusG is a transcription elongation factor that stimulates transcription pausing in Gram+ bacteria including B. subtilis by sequence-specific interaction with a conserved pause-inducing -11TTNTTT-6 motif found in the non-template DNA (ntDNA) strand within the transcription bubble. To reveal the structural basis of NusG-dependent pausing, we determined a cryo-EM structure of a paused transcription complex (PTC) containing RNA polymerase (RNAP), NusG, and the TTNTTT motif in the ntDNA strand. The interaction of NusG with the ntDNA strand rearranges the transcription bubble by positioning three consecutive T residues in a cleft between NusG and the ß-lobe domain of RNAP. We revealed that the RNAP swivel module rotation (swiveling), which widens (swiveled state) and narrows (non-swiveled state) a cleft between NusG and the ß-lobe, is an intrinsic motion of RNAP and is directly linked to trigger loop (TL) folding, an essential conformational change of all cellular RNAPs for the RNA synthesis reaction. We also determined cryo-EM structures of RNAP escaping from the paused transcription state. These structures revealed the NusG-dependent pausing mechanism by which NusG-ntDNA interaction inhibits the transition from swiveled to non-swiveled states, thereby preventing TL folding and RNA synthesis allosterically. This motion is also reduced by the formation of an RNA hairpin within the RNA exit channel. Thus, the pause half-life can be modulated by the strength of the NusG-ntDNA interaction and/or the stability of the RNA hairpin. NusG residues that interact with the TTNTTT motif are widely conserved in bacteria, suggesting that NusG-dependent pausing is widespread.

Comprehensive transcription terminator atlas for Bacillus subtilis.

The transcriptome-wide contributions of Rho-dependent and intrinsic (Rho-independent) transcription termination mechanisms in bacteria are unclear. By sequencing released transcripts in a wild-type strain and strains containing deficiencies in NusA, NusG and/or Rho (10 strains), we produced an atlas of terminators for the model Gram-positive bacterium Bacillus subtilis. We found that NusA and NusG stimulate 77% and 19% of all intrinsic terminators, respectively, and that both proteins participate in Rho-dependent termination. We also show that Rho stimulates termination at 10% of the intrinsic terminators in vivo. We recapitulated Rho-stimulated intrinsic termination at 5 terminators in vitro and found that Rho requires the KOW domain of NusG to stimulate this process at one of these terminators. Computational analyses of our atlas using RNAstructure, MEME suite and DiffLogo, combined with in vitro transcription experiments, revealed that Rho stimulates intrinsic terminators with weak hairpins and/or U-rich tracts by remodelling the RNA upstream of the intrinsic terminator to prevent the formation of RNA structures that could otherwise compete with the terminator hairpin. We also identified 56 putative examples of 'hybrid Rho-dependent termination', wherein classical Rho-dependent termination occurs after readthrough of a Rho-stimulated intrinsic terminator.

Factor-stimulated intrinsic termination: getting by with a little help from some friends.

Transcription termination is known to occur via two mechanisms in bacteria, intrinsic termination (also frequently referred to as Rho-independent or factor-independent termination) and Rho-dependent termination. Based primarily on in vitro studies using Escherichia coli RNA polymerase, it was generally assumed that intrinsic termination and Rho-dependent termination are distinct mechanisms and that the signals required for intrinsic termination are present primarily within the nucleic acids. In this review, we detail recent findings from studies in Bacillus subtilis showing that intrinsic termination in this organism is highly stimulated by NusA, NusG, and even Rho. In NusA-stimulated intrinsic termination, NusA facilitates the formation of weak terminator hairpins and compensates for distal U-rich tract interruptions. In NusG-stimulated intrinsic termination, NusG stabilizes a sequence-dependent pause at the point of termination, which extends the time frame for RNA hairpins with weak terminal base pairs to form in either a NusA-stimulated or a NusA-independent fashion. In Rho-stimulated intrinsic termination, Rho prevents the formation of antiterminator-like RNA structures that could otherwise compete with the terminator hairpin. Combined, NusA, NusG, and Rho stimulate approximately 97% of all intrinsic terminators in B. subtilis. Thus, the general view that intrinsic termination is primarily a factor-independent process needs to be revised to account for recent findings. Moreover, the historical distinction between Rho-dependent and intrinsic termination is overly simplistic and needs to be modernized.

Toeprint Assays for Detecting RNA Structure and Protein-RNA Interactions.

Toeprint assays are primer extension inhibition assays that can detect the 3' end of an RNA secondary structure, the position of a bound RNA binding protein, as well as the position of a bound 30S ribosomal subunit or a stalled ribosome. Here we describe how this assay was used to identify an RNA hairpin that sequesters a Shine-Dalgarno sequence, how the RNA-binding protein CsrA can alter RNA structure and affect 30S ribosomal subunit binding, and how the macrolide antibiotic tylosin can induce ribosome stalling.

Expression of Bacillus subtilis ABCF antibiotic resistance factor VmlR is regulated by RNA polymerase pausing, transcription attenuation, translation attenuation and (p)ppGpp.

Since antibiotic resistance is often associated with a fitness cost, bacteria employ multi-layered regulatory mechanisms to ensure that expression of resistance factors is restricted to times of antibiotic challenge. In Bacillus subtilis, the chromosomally-encoded ABCF ATPase VmlR confers resistance to pleuromutilin, lincosamide and type A streptogramin translation inhibitors. Here we show that vmlR expression is regulated by translation attenuation and transcription attenuation mechanisms. Antibiotic-induced ribosome stalling during translation of an upstream open reading frame in the vmlR leader region prevents formation of an anti-antiterminator structure, leading to the formation of an antiterminator structure that prevents intrinsic termination. Thus, transcription in the presence of antibiotic induces vmlR expression. We also show that NusG-dependent RNA polymerase pausing in the vmlR leader prevents leaky expression in the absence of antibiotic. Furthermore, we demonstrate that induction of VmlR expression by compromised protein synthesis does not require the ability of VmlR to rescue the translational defect, as exemplified by constitutive induction of VmlR by ribosome assembly defects. Rather, the specificity of induction is determined by the antibiotic's ability to stall the ribosome on the regulatory open reading frame located within the vmlR leader. Finally, we demonstrate the involvement of (p)ppGpp-mediated signalling in antibiotic-induced VmlR expression.

Analysis of mRNA Decay Intermediates in Bacillus subtilis 3' Exoribonuclease and RNA Helicase Mutant Strains.

The Bacillus subtilis genome encodes four 3' exoribonucleases: polynucleotide phosphorylase (PNPase), RNase R, RNase PH, and YhaM. Previous work showed that PNPase, encoded by the pnpA gene, is the major 3' exonuclease involved in mRNA turnover; in a pnpA deletion strain, numerous mRNA decay intermediates accumulate. Whether B. subtilis mRNA decay occurs in the context of a degradosome complex is controversial. In this study, global mapping of mRNA decay intermediate 3' ends within coding sequences was performed in strains that were either deleted for or had an inactivating point mutation in the pnpA gene. The patterns of 3'-end accumulation in these strains were highly similar, which may have implications for the role of a degradosome in mRNA decay. A comparison with mapped 3' ends in a strain lacking CshA, the major RNA helicase, indicated that many mRNAs require both PNPase and CshA for efficient decay. Transcriptome sequencing (RNA-seq) analysis of strains lacking RNase R suggested that this enzyme did not play a major role in mRNA turnover in the wild-type strain. Strains were constructed that contained only one of the four known 3' exoribonucleases. When RNase R was the only 3' exonuclease present, it was able to degrade a model mRNA efficiently, showing processive decay even through a strong stem-loop structure that inhibits PNPase processivity. Strains containing only RNase PH or only YhaM were also insensitive to this RNA secondary structure, suggesting the existence of another, as-yet-unidentified, 3' exoribonuclease. IMPORTANCE The ability to rapidly change bacterial gene expression programs in response to environmental conditions is highly dependent on the efficient turnover of mRNA. While much is known about the regulation of gene expression at the transcriptional and translational levels, much less is known about the intermediate step of mRNA decay. Here, we mapped the 3' ends of mRNA decay intermediates in strains that were missing the major 3' exoribonuclease PNPase or the RNA helicase CshA. We also assessed the roles of three other B. subtilis 3' exonucleases in the mRNA decay process. The data confirm the primary role of PNPase in mRNA turnover and suggest the involvement of one or more unknown RNases.

Transcriptome-Wide Effects of NusA on RNA Polymerase Pausing in Bacillus subtilis.

Transcription elongation is a highly processive process that is punctuated by RNA polymerase (RNAP) pausing. Long-lived pauses can provide time for diverse regulatory events to occur, which play important roles in modulating gene expression. Transcription elongation factors can dramatically affect RNAP pausing in vitro. The genome-wide role of such factors in pausing in vivo has been examined only for NusG in Bacillus subtilis. NusA is another transcription elongation factor known to stimulate pausing of B. subtilis and Escherichia coli RNAP in vitro. Here, we present the first in vivo study to identify the genome-wide role of NusA in RNAP pausing. Using native elongation transcript sequencing followed by RNase digestion (RNET-seq), we analyzed factor-dependent RNAP pausing in B. subtilis and found that NusA has a relatively minor role in RNAP pausing compared to NusG. We demonstrate that NusA has both stimulating and suppressing effects on pausing in vivo. Based on our thresholding criteria on in vivo data, NusA stimulates pausing at 129 pause peaks in 93 different genes or 5' untranslated regions (5' UTRs). Putative pause hairpins were identified for 87 (67%) of the 129 NusA-stimulated pause peaks, suggesting that RNA hairpins are a common component of NusA-stimulated pause signals. However, a consensus sequence was not identified as a NusA-stimulated pause motif. We further demonstrate that NusA stimulates pausing in vitro at some of the pause sites identified in vivo. IMPORTANCE NusA is an essential transcription elongation factor that was assumed to play a major role in RNAP pausing. NusA stimulates pausing in vitro; however, the essential nature of NusA had prevented an assessment of its role in pausing in vivo. Using a NusA depletion strain and RNET-seq, we identified a similar number of NusA-stimulated and NusA-suppressed pause peaks throughout the genome. NusA-stimulated pausing was confirmed at several sites in vitro. However, NusA did not always stimulate pausing at sites identified in vivo, while in other instances NusA stimulated pausing at sites not observed in vivo. We found that NusA has only a minor role in stimulating RNAP pausing in B. subtilis.

CsrA regulation via binding to the base-pairing small RNA Spot 42.

The carbon storage regulator system and base-pairing small RNAs (sRNAs) represent two predominant modes of bacterial post-transcriptional regulation, which globally influence gene expression. Binding of CsrA protein to the 5' UTR or initial mRNA coding sequences can affect translation, RNA stability, and/or transcript elongation. Base-pairing sRNAs also regulate these processes, often requiring assistance from the RNA chaperone Hfq. Transcriptomics studies in Escherichia coli have identified many new CsrA targets, including Spot 42 and other base-pairing sRNAs. Spot 42 synthesis is repressed by cAMP-CRP, induced by the presence of glucose, and Spot 42 post-transcriptionally represses operons that facilitate metabolism of nonpreferred carbon sources. CsrA activity is also increased by glucose via effects on CsrA sRNA antagonists, CsrB/C. Here, we elucidate a mechanism wherein CsrA binds to and protects Spot 42 sRNA from RNase E-mediated cleavage. This protection leads to enhanced repression of srlA by Spot 42, a gene required for sorbitol uptake. A second, independent mechanism by which CsrA represses srlA is by binding to and inhibiting translation of srlM mRNA, encoding a transcriptional activator of srlA. Our findings demonstrate a novel means of regulation, by CsrA binding to a sRNA, and indicate that such interactions can help to shape complex bacterial regulatory circuitry.

An incoherent feedforward loop formed by SirA/BarA, HilE and HilD is involved in controlling the growth cost of virulence factor expression by Salmonella Typhimurium.

An intricate regulatory network controls the expression of Salmonella virulence genes. The transcriptional regulator HilD plays a central role in this network by controlling the expression of tens of genes mainly required for intestinal colonization. Accordingly, the expression/activity of HilD is highly regulated by multiple factors, such as the SirA/BarA two-component system and the Hcp-like protein HilE. SirA/BarA positively regulates translation of hilD mRNA through a regulatory cascade involving the small RNAs CsrB and CsrC, and the RNA-binding protein CsrA, whereas HilE inhibits HilD activity by protein-protein interaction. In this study, we show that SirA/BarA also positively regulates translation of hilE mRNA through the same mentioned regulatory cascade. Thus, our results reveal a paradoxical regulation exerted by SirA/BarA-Csr on HilD, which involves simultaneous opposite effects, direct positive control and indirect negative control through HilE. This kind of regulation is called an incoherent type-1 feedforward loop (I1-FFL), which is a motif present in certain regulatory networks and represents a complex biological problem to decipher. Interestingly, our results, together with those from a previous study, indicate that HilE, the repressor component of the I1-FFL reported here (I1-FFLSirA/BarA-HilE-HilD), is required to reduce the growth cost imposed by the expression of the genes regulated by HilD. Moreover, we and others found that HilE is necessary for successful intestinal colonization by Salmonella. Thus, these findings support that I1-FFLSirA/BarA-HilE-HilD cooperates to control the precise amount and activity of HilD, for an appropriate balance between the growth cost and the virulence benefit generated by the expression of the genes induced by this regulator. I1-FFLSirA/BarA-HilE-HilD represents a complex regulatory I1-FFL that involves multiple regulators acting at distinct levels of gene expression, as well as showing different connections to the rest of the regulatory network governing Salmonella virulence.

NusG is an intrinsic transcription termination factor that stimulates motility and coordinates gene expression with NusA.

NusA and NusG are transcription factors that stimulate RNA polymerase pausing in Bacillus subtilis. While NusA was known to function as an intrinsic termination factor in B. subtilis, the role of NusG in this process was unknown. To examine the individual and combinatorial roles that NusA and NusG play in intrinsic termination, Term-seq was conducted in wild type, NusA depletion, ΔnusG, and NusA depletion ΔnusG strains. We determined that NusG functions as an intrinsic termination factor that works alone and cooperatively with NusA to facilitate termination at 88% of the 1400 identified intrinsic terminators. Our results indicate that NusG stimulates a sequence-specific pause that assists in the completion of suboptimal terminator hairpins with weak terminal A-U and G-U base pairs at the bottom of the stem. Loss of NusA and NusG leads to global misregulation of gene expression and loss of NusG results in flagella and swimming motility defects.

Diverse Mechanisms and Circuitry for Global Regulation by the RNA-Binding Protein CsrA.

The carbon storage regulator (Csr) or repressor of stationary phase metabolites (Rsm) system of Gammaproteobacteria is among the most complex and best-studied posttranscriptional regulatory systems. Based on a small RNA-binding protein, CsrA and homologs, it controls metabolism, physiology, and bacterial lifestyle decisions by regulating gene expression on a vast scale. Binding of CsrA to sequences containing conserved GGA motifs in mRNAs can regulate translation, RNA stability, riboswitch function, and transcript elongation. CsrA governs the expression of dozens of transcription factors and other regulators, further expanding its influence on cellular physiology, and these factors can participate in feedback to the Csr system. Expression of csrA itself is subject to autoregulation via translational inhibition and indirect transcriptional activation. CsrA activity is controlled by small noncoding RNAs (sRNAs), CsrB and CsrC in Escherichia coli, which contain multiple high affinity CsrA binding sites that compete with those of mRNA targets. Transcription of CsrB/C is induced by certain nutrient limitations, cellular stresses, and metabolites, while these RNAs are targeted for degradation by the presence of a preferred carbon source. Consistent with these findings, CsrA tends to activate pathways and processes that are associated with robust growth and repress stationary phase metabolism and stress responses. Regulatory loops between Csr components affect the signaling dynamics of the Csr system. Recently, systems-based approaches have greatly expanded our understanding of the roles played by CsrA, while reinforcing the notion that much remains to be learned about the Csr system.

NusG-dependent RNA polymerase pausing is a frequent function of this universally conserved transcription elongation factor.

Although transcription by RNA polymerase (RNAP) is highly processive, elongation can be transiently halted by RNAP pausing. Pausing provides time for diverse regulatory events to occur such as RNA folding and regulatory factor binding. The transcription elongation factors NusA and NusG dramatically affect the frequency and duration of RNAP pausing, and hence regulation of transcription. NusG is the only transcription factor conserved in all three domains of life; its homolog in archaea and eukaryotes is Spt5. This review focuses on NusG-dependent pausing, which is a common occurrence in Bacillus subtilis. B. NusG induces pausing about once per 3 kb at a consensus TTNTTT motif in the non-template DNA strand within the paused transcription bubble. A conserved region of NusG contacts the TTNTTT motif to stabilize the paused transcription elongation complex (TEC) in multiple catalytically inactive RNAP conformations. The density of NusG-dependent pause sites is 3-fold higher in untranslated regions, suggesting that pausing could regulate the expression of hundreds of genes in B. subtilis. We describe how pausing in 5' leader regions contributes to regulating the expression of B. subtilis genes by transcription attenuation and translation control mechanisms. As opposed to the broadly accepted view that NusG is an anti-pausing factor, phylogenetic analyses suggest that NusG-dependent pausing is a widespread mechanism in bacteria. This function of NusG is consistent with the well-established role of its eukaryotic homolog Spt5 in promoter-proximal pausing. Since NusG is present in all domains of life, NusG-dependent pausing could be a conserved mechanism in all organisms.