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Transcription rates are regulated by the interactions between RNA polymerase, sigma factor, and promoter DNA sequences in bacteria. However, it remains unclear how non-canonical sequence motifs collectively control transcription rates. Here, we combined massively parallel assays, biophysics, and machine learning to develop a 346-parameter model that predicts site-specific transcription initiation rates for any σ70 promoter sequence, validated across 17396 bacterial promoters with diverse sequences. We applied the model to predict genetic context effects, design σ70 promoters with desired transcription rates, and identify undesired promoters inside engineered genetic systems. The model provides a biophysical basis for understanding gene regulation in natural genetic systems and precise transcriptional control for engineering synthetic genetic systems.One-Sentence SummaryA 346-parameter model predicted DNA’s interactions with RNA polymerase initiation complex, enabling accurate transcription rate predictions and automated promoter design in bacterial genetic systems.

The plant-specific VQ gene family participates in diverse physiological processes but little information is available on their role in leaf senescence. Here, we show that the VQ motif-containing proteins, Arabidopsis SIGMA FACTOR BINDING PROTEIN1 (SIB1) and SIB2 are negative regulators of abscisic acid (ABA)-mediated leaf senescence. Loss of SIB1 and SIB2 function resulted in increased sensitivity of ABA-induced leaf senescence. In contrast, overexpression of SIB1 significantly delayed this process. Moreover, biochemical studies revealed that SIBs interact with WRKY75 transcription factor. Loss of WRKY75 function decreased sensitivity to ABA-induced leaf senescence, while overexpression of WRKY75 significantly accelerated this process. Chromatin immunoprecipitation assays revealed that WRKY75 directly binds to the promoters of GOLDEN 2-LIKE1(GLK1) and GLK2, to repress their expression. SIBs repress the transcriptional function of WRKY75 and negatively regulate ABA-induced leaf senescence in a WRKY75-dependent manner. In contrast, WRKY75 positively modulates ABA-mediated leaf senescence in a GLK-dependent manner. In addition, SIBs inhibit WRKY75 function in ABA-mediated seed germination. These results demonstrate that SIBs can form a complex with WRKY75 to regulate ABA-mediated leaf senescence and seed germination., SIBs function as repressors of WRKY75 and regulate expression of GLKs in ABA-mediated leaf senescence and seed germination.

RpoS, an alternative sigma factor of RNA polymerase, regulates the expression of a great deal of genes involved in stationary-phase survival and stress response. To identify the function of RpoS homologue in Serratia marcescens FS14, in-frame deletion mutant of rpoS was constructed. It was found that RpoS activates the biosynthesis of prodigiosin in FS14 which is just opposite to what was observed in Serratia sp. ATCC 39006. We also demonstrated that RpoS positively regulates the prodigiosin production by activating the transcription of pig cluster in FS14, and the transcription of pig cluster is RpoS-dependent. Further study showed that the differences in the promoters of pig clusters in FS14 and 39006 lead to the different selection of the sigma factors and result in the different regulation mechanisms. The -10 element and the spacer region between -10 and -35 elements of the pig cluster in FS14 are vital for the RpoS recognition in FS14. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12088-021-00952-4.

Transcription initiation is the first step in gene expression, and is therefore strongly regulated in all domains of life. The RNA polymerase (RNAP) first associates with the initiation factorσto form a holoenzyme, which binds, bends and opens the promoter in a succession of reversible states. These states are critical for transcription regulation, but remain poorly understood. Here, we addressed the mechanism of open complex formation by monitoring its assembly/disassembly kinetics on individual consensuslacUV5promoters using high-throughput single-molecule magnetic tweezers. We probed the key protein–DNA interactions governing the open-complex formation and dissociation pathway by modulating the dynamics at different concentrations of monovalent salts and varying temperatures. Consistent with ensemble studies, we observed that RPOis a stable, slowly reversible state that is preceded by a kinetically significant open intermediate (RPI), from which the holoenzyme dissociates. A strong anion concentration and type dependence indicates that the RPOstabilization may involve sequence-independent interactions between the DNA and the holoenzyme, driven by a non-Coulombic effect consistent with the non-template DNA strand interacting withσand the RNAPβsubunit. The temperature dependence provides the energy scale of open-complex formation and further supports the existence of additional intermediates.

The bacterial σ factor plays the central role in promoter recognition by RNA polymerase (RNAP). The primary σ factor, involved in transcription of housekeeping genes, was also shown to participate in the initiation of RNA synthesis and promoter escape by RNAP. In the open promoter complex, the σ finger formed by σ region 3.2 directly interacts with the template DNA strand upstream of the transcription start site. Here, we analysed the role of the σ finger in transcription initiation by four alternative σ factors in Escherichia coli, σ38, σ32, σ28 and σ24. We found that deletions of the σ finger to various extent compromise the activity of RNAP holoenzymes containing alternative σ factors, especially at low NTP concentrations. All four σs are able to utilize NADH as a noncanonical priming substrate but it has only mild effects on the efficiency of transcription initiation. The mediators of the stringent response, transcription factor DksA and the alarmone ppGpp decrease RNAP activity and promoter complex stability for all four σ factors on tested promoters. For all σs except σ38, deletions of the σ finger conversely increase the stability of promoter complexes and decrease their sensitivity to DksA and ppGpp. The result suggests that the σ finger plays a universal role in transcription initiation by alternative σ factors and sensitizes promoter complexes to the action of global transcription regulators DksA and ppGpp by modulating promoter complex stability.

Paenibacillus durus strain ATCC 35681T is a Gram-positive diazotroph that displayed capability of fixing nitrogen even in the presence of nitrate or ammonium. However, the nitrogen fixation activity was detected only at day 1 of growth when cultured in liquid nitrogen-enriched medium. The transcripts of all the nifH homologues were present throughout the 9-day study. When grown in nitrogen-depleted medium, nitrogenase activities occurred from day 1 until day 6 and the nifH transcripts were also present during the course of the study albeit at different levels. In both studies, the absence of nitrogen fixation activity regardless of the presence of the nifH transcripts raised the possibility of a post-transcriptional or post-translational regulation of the system. A putative SigA box sequence was found upstream of the transcription start site of nifB1, the first gene in the major nitrogen fixation cluster. The upstream region of nifB2 showed a promoter recognisable by SigE, a sigma factor normally involved in sporulation.

Alternate sigma factors play a major role in the survival of pathogenic bacteria such as Streptococcus pyogenes in adverse environment conditions. Stress induced sigma factors mediate gene expression under conditions of pathogenesis, dormancy and unusual environmental cues. In the present work, ComX, an alternate sigma factor from S. pyogenes has been characterized. The structures of ComX, RpoB β subunit and RpoC β’ subunit of RNA polymerase have been predicted using comparative and homology modelling respectively and validated. Attempts have been made to study RpoB-RpoC-ComX complex interactions with Double Strand (DS) and Single Strand (SS) pro moter regions. Stability of these complexes and the promoter melting mechanism have been analysed using Molecular Dynamic (MD) simulations. This study suggests that ComX, although identifies promoter analogous to the alternate sigma factor SigH of M. tuberculosis, follows a distinctive promoter flip out mechanism. Communicated by Ramaswamy H. Sarma

σB, an alternative sigma factor, is usually employed to tackle the general stress response in Staphylococcus aureus and other Gram-positive bacteria. This protein, involved in S. aureus-mediated pathogenesis, is typically blocked by RsbW, an antisigma factor having serine kinase activity. σB, a σ70-like sigma factor, harbors three conserved domains designated σB2, σB3, and σB4. To better understand the interaction between RsbW and σB or its domains, we have studied their recombinant forms, rRsbW, rσB, rσB2, rσB3, and rσB4, using different probes. The results show that none of the rσB domains, unlike rσB, showed binding to a cognate DNA in the presence of a core RNA polymerase. However, both rσB2 and rσB3, like rσB, interacted with rRsbW, and the order of their rRsbW binding affinity looks like rσB > rσB3 > rσB2. Furthermore, the reaction between rRsbW and rσB or rσB3 was exothermic and occurred spontaneously. rRsbW and rσB3 also associate with each other at a stoichiometry of 2:1, and different types of noncovalent bonds might be responsible for their interaction. A structural model of the RsbW-σB3 complex that has supported our experimental results indicated the binding of rσB3 at the putative dimeric interface of RsbW. A genetic study shows that the tentative dimer-forming region of RsbW is crucial for preserving its rσB binding ability, serine kinase activity, and dimerization ability. Additionally, a urea-induced equilibrium unfolding study indicated a notable thermodynamic stabilization of σB3 in the presence of RsbW. Possible implications of the stabilization data in drug discovery were discussed at length.

Bacterial sigma (σ) factor, along with RNA polymerase core enzyme, initiates gene transcription from specific promoter regions and therefore regulates clusters of genes in response to a particular situation. The extracytoplasmic function (ECF) σ factors are a class of alternative σ factors that are often associated with environmental signal transduction across the bacterial membrane, in which external signal triggers the release of active σ from the membrane-anchored anti-σ factor. Gram-positive model organism Bacillus subtilis (B. subtilis) has seven ECF σ factors: σM, σV, σX, σW, σY, σZ and σYlaC. Although all these ECF σ factors were found to be involved in B. subtilis antibiotic resistance, σW is among the most studied and considered to play a pivotal role in responding to antimicrobial stresses. σW is under tight control and remains deactivated until exposure to external stimuli, after which proteases PrsW and RasP cleave the specific anti-sigma factor-RsiW to release and activate σW. Membrane anchored protein YsdB is a negative regulator of this activation, possibly via its direct interaction with PrsW and/or RsiW. Importantly, YsdB is well conserved among Bacilli, including pathogenic bacteria like Bacillus cereus. In this study, we describe the chemical shift assignments of the cytoplasmic domain of YsdB (29-130) of B. subtilis in solution as a basis for further interaction studies and structure determination. The near-complete assignment and the solution structure that will follow could provide a further understanding in σW regulation.

A strain of Kytococcus sedentarius was isolated from a dehumidifier operating in a university lecture theatre. Genome analysis and phenotypic characterisation showed that this strain, K. sedentarius MBB13, was a moderately halotolerant aerobe with a branched aerobic electron transport chain and genes that could contribute to erythromycin resistance. The major compatible solute was glycine betaine, with ectoine and proline being deployed at higher osmolarities. Actinobacteria possess multiple WhiB-like (Wbl) regulatory proteins, and K. sedentarius MBB13 has four (WhiB1, WhiB2, WhiB3, and WhiB7). Wbls are iron-sulfur proteins that regulate gene expression through interactions with RNA polymerase sigma factors and/or other regulatory proteins. Bacterial two-hybrid analyses suggested that WhiB1 and WhiB2, but not WhiB3 and WhiB7, interact with the C-terminal domain of the major sigma factor, σA; no interaction was detected between any of the Wbl proteins and the only alternative sigma factors, σB, σH, or σJ. The interaction between σA and WhiB1 or WhiB2 was disrupted in a heterologous system under growth conditions that produce nitric oxide and the iron-sulfur clusters of the isolated WhiB1 and WhiB2 proteins reacted with nitric oxide. Thus, K. sedentarius strain exhibits the major phenotypic characteristics of the type strain and a comprehensive examination of the interactions between its four Wbl proteins and four sigma factors suggested that the Wbl proteins all operate through interaction with σA.

Chloroplast gene expression is controlled by both plastid-encoded RNA polymerase (PEP) and nuclear-encoded RNA polymerase and is crucial for chloroplast development and photosynthesis. Environmental factors such as light and temperature can influence transcription in chloroplasts. In this study, we showed that mutation in DUA1, which encodes a pentatricopeptide repeat (PPR) protein in rice (Oryza sativa), led to deficiency in chloroplast development and chlorophyll biosynthesis, impaired photosystems, and reduced expression of PEP-dependent transcripts at low temperature especially under low-light conditions. Furthermore, we demonstrated that sigma factor OsSIG1 interacted with DUA1 in vitro and in vivo. Moreover, the levels of chlorophyll and PEP-dependent gene expression were significantly decreased in the Ossig1 mutants at low-temperature and low-light conditions. Our study reveals that the PPR protein DUA1 plays an important role in regulating PEP-mediated chloroplast gene expression through interacting with OsSIG1, thus modulates chloroplast development in response to environmental signals.

In bacteria, rapid adaptation to changing environmental conditions depends on the interplay between housekeeping and alternative σ factors, responsible for transcription of specific regulons by RNA polymerase (RNAP). In comparison with alternative σ factors, primary σs contain poorly conserved region 1.1, whose functions in transcription are only partially understood. We found that a single mutation in region 1.1 in Escherichia coli σ70 rewires transcription regulation during cell growth resulting in profound phenotypic changes. Despite its destabilizing effect on promoter complexes, this mutation increases the activity of rRNA promoters and also decreases RNAP sensitivity to the major regulator of stringent response DksA. Using total RNA sequencing combined with single-cell analysis of gene expression we showed that changes in region 1.1 disrupt the balance between the "greed" and "fear" strategies thus making the cells more susceptible to environmental threats and antibiotics. Our results reveal an unexpected role of σ region 1.1 in growth-dependent transcription regulation and suggest that changes in this region may facilitate rapid switching of RNAP properties in evolving bacterial populations.

Synthetic methylotrophy aims to engineer methane and methanol utilization pathways in platform hosts like Escherichia coli for industrial bioprocessing of natural gas and biogas. While recent attempts to engineer synthetic methylotrophs have proved successful, autonomous methylotrophy, i.e. the ability to utilize methane or methanol as sole carbon and energy substrates, has not yet been realized. Here, we address an important limitation of autonomous methylotrophy in E. coli: the inability of the organism to synthesize several amino acids when grown on methanol. By activating the stringent/stress response via ppGpp overproduction, or DksA and RpoS overexpression, we demonstrate improved biosynthesis of proteinogenic amino acids via endogenous upregulation of amino acid synthesis pathway genes. Thus, we were able to achieve biosynthesis of several limiting amino acids from methanol-derived carbon, in contrast to the control methylotrophic E. coli strain. This study addresses a key limitation currently preventing autonomous methylotrophy in E. coli and possibly other synthetic methylotrophs and provides insight as to how this limitation can be alleviated via stringent/stress response activation.

Bacterial RNA polymerase is a potent target for antibiotics, which utilize a plethora of different modes of action, some of which are still not fully understood. Ureidothiophene (Urd) was found in a screen of a library of chemical compounds for ability to inhibit bacterial transcription. The mechanism of Urd action is not known. Here, we show that Urd inhibits transcription at the early stage of closed complex formation by blocking interaction of RNA polymerase with the promoter –10 element, while not affecting interactions with –35 element or steps of transcription after promoter closed complex formation. We show that mutation in the region 1.2 of initiation factor σ decreases sensitivity to Urd. The results suggest that Urd may directly target σ region 1.2, which allosterically controls the recognition of –10 element by σ region 2. Alternatively, Urd may block conformational changes of the holoenzyme required for engagement with –10 promoter element, although by a mechanism distinct from that of antibiotic fidaxomycin (lipiarmycin). The results suggest a new mode of transcription inhibition involving the regulatory domain of σ subunit, and potentially pinpoint a novel target for development of new antibacterials.

The Lyme disease spirochete Borrelia burgdorferi exhibits dramatic changes in gene expression as it transits between its tick vector and vertebrate host. A major hurdle to understanding the mechanisms underlying gene regulation in B. burgdorferi has been the lack of a functional assay to test how gene regulatory proteins and sigma factors interact with RNA polymerase to direct transcription. To gain mechanistic insight into transcriptional control in B. burgdorferi, and address sigma factor function and specificity, we developed an in vitro transcription assay using the B. burgdorferi RNA polymerase holoenzyme. We established reaction conditions for maximal RNA polymerase activity by optimizing pH, temperature, and the requirement for divalent metals. Using this assay system, we analyzed the promoter specificity of the housekeeping sigma factor RpoD to promoters encoding previously identified RpoD consensus sequences in B. burgdorferi. Collectively, this study established an in vitro transcription assay that revealed RpoD-dependent promoter selectivity by RNA polymerase and the requirement of specific metal cofactors for maximal RNA polymerase activity. The establishment of this functional assay will facilitate molecular and biochemical studies on how gene regulatory proteins and sigma factors exert control of gene expression in B. burgdorferi required for the completion of its enzootic cycle.

The symbiotic nature of the relationship between algae and marine bacteria is well-studied among the complex microbial interactions. The mutual profit between algae and bacteria occurs via nutrient and vitamin exchange. It is necessary to analyze the genome sequence of a bacterium to predict its symbiotic relationships. In this study, the genome of a marine bacterium, Pseudoruegeria sp. M32A2M, isolated from the south-eastern isles (GeoJe-Do) of South Korea, was sequenced and analyzed. A draft genome (91 scaffolds) of 5.5 Mb with a DNA G+C content of 62.4% was obtained. In total, 5,101 features were identified from gene annotation, and 4,927 genes were assigned to functional proteins. We also identified transcription core proteins, RNA polymerase subunits, and sigma factors. In addition, full flagella-related gene clusters involving the flagellar body, motor, regulator, and other accessory compartments were detected even though the genus Pseudoruegeria is known to comprise non-motile bacteria. Examination of annotated KEGG pathways revealed that Pseudoruegeria sp. M32A2M has the metabolic pathways for all seven vitamin Bs, including thiamin (vitamin B1), biotin (vitamin B7), and cobalamin (vitamin B12), which are necessary for symbiosis with vitamin B auxotroph algae. We also identified gene clusters for seven secondary metabolites including ectoine, homoserine lactone, beta-lactone, terpene, lasso peptide, bacteriocin, and nonribosomal proteins.

The bacterial protein WhiD belongs to the Wbl family of iron–sulfur [Fe-S] proteins present only in the actinomycetes. In Streptomyces coelicolor, it is required for the late stages of sporulation, but precisely how it functions is unknown. Here, we report results from in vitro and in vivo experiments with WhiD from Streptomyces venezuelae (SvWhiD), which differs from S. coelicolor WhiD (ScWhiD) only at the C terminus. We observed that, like ScWhiD and other Wbl proteins, SvWhiD binds a [4Fe-4S] cluster that is moderately sensitive to O2 and highly sensitive to nitric oxide (NO). However, although all previous studies have reported that Wbl proteins are monomers, we found that SvWhiD exists in a monomer–dimer equilibrium associated with its unusual C-terminal extension. Several Wbl proteins of Mycobacterium tuberculosis are known to interact with its principal sigma factor SigA. Using bacterial two-hybrid, gel filtration, and MS analyses, we demonstrate that SvWhiD interacts with domain 4 of the principal sigma factor of Streptomyces, σHrdB (σHrdB4). Using MS, we determined the dissociation constant (Kd) for the SvWhiD–σHrdB4 complex as ~0.7 μM, consistent with a relatively tight binding interaction. We found that complex formation was cluster dependent and that a reaction with NO, which was complete at 8–10 NO molecules per cluster, resulted in dissociation into the separate proteins. The SvWhiD [4Fe-4S] cluster was significantly less sensitive to reaction with O2 and NO when SvWhiD was bound to σHrdB4, consistent with protection of the cluster in the complex.

Genetic circuit-based biosensors have emerged as an effective analytical tool in synthetic biology; these biosensors can be applied to high-throughput screening of new biocatalysts and metabolic pathways. Sigma 54 (σ54)-dependent transcription factor (TF) can be a valuable component of these biosensors owing to its intrinsic silent property compared to most of the housekeeping sigma 70 (σ70) TFs. Here, we show that these unique characteristics of σ54-dependent TFs can be used to control the host cell state to be more appropriate for high-throughput screening. The acclimation of cell state was achieved by using guanosine (penta)tetraphosphate ((p)ppGpp)-related genes (relA, spoT) and nutrient conditions, to link the σ54 TF-based reporter expression with the target enzyme activity. By controlling stringent programmed responses and optimizing assay conditions, catalytically improved tyrosine phenol lyase (TPL) enzymes were successfully obtained using a σ54-dependent DmpR as the TF component, demonstrating the practical feasibility of this biosensor. This combinatorial strategy of biosensors using σ factor-dependent TFs will allow for more effective high-throughput enzyme engineering with broad applicability.

It is shown that a freshly inoculated culture of the model cyanobacterium Synechocystis sp. PCC 6803 consumed almost all phosphate and 50% of nitrate within 6 days from the nutrient-rich BG-11 growth medium, indicating potential of cyanobacteria to purify wastewaters. Synechocystis sp. PCC 6803 control strain also collected nutrients efficiently from a landfill leachate wastewater KA2 (5.9–6.9 mM ammonium and 0.073–0.077 mM phosphate). Wastewaters might induce oxidative stress to microalgae, which prompted us to test growth of sigma factor inactivation strains, as ΔsigBCE and ΔsigCDE strains show superior growth in chemically induced oxidative stress. All cyanobacterial strains, including a stress-sensitive strain ΔsigBCDE, grew well in KA2 for four days, indicating that KA2 did not cause immediate oxidative stress. Completely arrested growth and bleaching of ΔsigBCDE cells after one week in KA2 wastewater point to the importance of group 2 sigma factor-mediated changes in gene expression during wastewater treatment. The growth of ΔsigBCD was arrested early in un-buffered and Hepes buffered (pH 7.5) KA2. In ΔsigBCD, all phosphate transporter genes are upregulated in standard conditions, and ΔsigBCD cells showed growth defects in low-phosphate BG-11 medium. ΔsigBCD cells removed phosphate slower from KA2 than the control strain, but phosphate supplementation of KA2 did not improve growth of ΔsigBCD. The ΔsigBCE strain showed superior growth in a laboratory-scale bioreactor in bright light and removed phosphate even slightly more efficiently than the control strain if KA2 was Hepes buffered although ΔsigBCE grew slowly in un-buffered KA2 and in low-phosphate BG-11 medium. The results indicate that engineering expression of regulatory group 2 sigma factor(s) might be useful for practical applications. Electronic supplementary material The online version of this article (10.1007/s00284-020-01973-w) contains supplementary material, which is available to authorized users.

RNA polymerases (RNAPs) carry out transcription in the three domains of life, Bacteria, Archaea, and Eukarya. Transcription initiation is highly regulated by a variety of transcription factors, whose number and subunit complexity increase during evolution. This process is regulated in Bacteria by the σ factor, while the three eukaryotic RNAPs require a complex set of transcription factors (TFs) and a TATA-binding protein (TBP). The archaeal transcription system appears to be an ancestral version of the eukaryotic RNAPII, requiring transcription factor B (TFB), TBP, and transcription factor E (TFE). The function of the bacterial sigma (σ) factor has been correlated to the roles played by the eukaryotic RNAP II and the archaeal RNAP. In addition, σ factors, TFB, and TFIIB all contain multiple DNA binding helix-turn-helix (HTH) structural motifs; although TFIIB and TFB display two HTH domains, while the bacterial σ factor spans 4 HTH motifs. The sequence similarities and structure alignments of the bacterial σ factor, eukaryotic TFIIB, and archaeal TFB evidence that these three proteins are homologs.Key Points• Transcription initiation is highly regulated by TFs.• Transcription is finely regulated in all domains of life by different sets of TFs.• Specific TFs in Bacteria, Eukarya and Archaea are homologs.

All organisms--bacteria, archaea, and eukaryotes--have a transcription initiation factor that contains a structural module that binds within the RNA polymerase (RNAP) active-center cleft and interacts with template-strand single-stranded DNA (ssDNA) in the immediate vicinity of the RNAP active center. This transcription-initiation-factor structural module pre-organizes template-strand ssDNA to engage the RNAP active center, thereby facilitating binding of initiating nucleotides and enabling transcription initiation from initiating mononucleotides. However, this transcription-initiation-factor structural module occupies the path of nascent RNA and thus presumably must be displaced before or during initial transcription. Here, we report four sets of crystal structures of bacterial initially transcribing complexes that demonstrate, and define details of, stepwise, RNA-extension-driven displacement of the “σ finger” of the bacterial transcription initiation factor σ. The structures reveal that--for both the primary σ factor and extracytoplasmic (ECF) σ factors, and for both 5’-triphosphate RNA and 5’-hydroxy RNA--the “σ finger” is displaced in stepwise fashion, progressively folding back upon itself, driven by collision with the RNA 5’-end, upon extension of nascent RNA from ∼5 nt to ∼10 nt.SIGNIFICANCE STATEMENTThe “σ finger” of the bacterial initiation factor σ binds within the RNA polymerase active-center cleft and blocks the path of nascent RNA. It has been hypothesized that the σ finger must be displaced during initial transcription. By determining crystal structures defining successive steps in initial transcription, we demonstrate that the σ finger is displaced in stepwise fashion, driven by collision with the RNA 5’-end, as nascent RNA is extended from ∼5 nt to ∼10 nt during initial transcription, and we show that this is true for both the primary σ factor and alternate σ factors. Stepwise displacement of the σ finger can be conceptualized as stepwise compression of a “protein spring” that stores energy for subsequent breakage of protein-DNA and protein-protein interactions in promoter escape.

Promoter plays the critical role in regulating gene transcription, and dual-promoter has received the widespread attentions due to its high efficiency and continuity, here, we want to construct an efficient dual-promoter for protein production and metabolic pathway enhancement. Firstly, our results indicated that P43 promoter efficiently transcribed at logarithmic period, while the σB-type promoters (PylB, PgsiB, PykzA) were active at stationary phase. Then, several dual promoters were constructed by coupling these σB-type promoters with P43, and the attained dual-promoter PykzA-P43 showed the best performance, which led to 1.72-, 3.46- and 1.85-fold increases of green fluorescence intensity, red fluorescence intensity and α-amylase activity, compared with those of the recognized strong promoter P43, respectively. Furthermore, α-amylase activity was further increased to 389.65 U/mL by 32.20 % via optimizing sigma factor binding sites (-10 and -35 boxes) of PykzA-P43, attaining the optimized dual promoter Pdual3. Finally, Pdual3 was applied in metabolic pathway enhancement, and the yields of Poly γ-glutamic acid, acetoin and 2, 3-butanediol were respectively improved by 82.01 %, 17.09 % and 99.39 %. Our results indicated that dual-promoter significantly enhanced gene expression, and this study provided an energetic dual-promoter Pdual3 for efficient protein production and metabolic pathway enhancement in Bacillus licheniformis.

Background Transcriptional responses required to maintain cellular homeostasis or to adapt to environmental stress, is in part mediated by several nucleic-acid associated proteins. In this study, we sought to establish an affinity purification-mass spectrometry (AP-MS) approach that would enable the collective identification of nucleic acid-associated proteins in mycobacteria. We hypothesized that targeting the RNA polymerase complex through affinity purification would allow for the identification of RNA- and DNA-associated proteins that not only maintain the bacterial chromosome but also enable transcription and translation. Results AP-MS analysis of the RNA polymerase β-subunit cross-linked to nucleic acids identified 275 putative nucleic acid-associated proteins in the model organism Mycobacterium smegmatis under standard culturing conditions. The AP-MS approach successfully identified proteins that are known to make up the RNA polymerase complex, as well as several other known RNA polymerase complex-associated proteins such as a DNA polymerase, sigma factors, transcriptional regulators, and helicases. Gene ontology enrichment analysis of the identified proteins revealed that this approach selected for proteins with GO terms associated with nucleic acids and cellular metabolism. Importantly, we identified several proteins of unknown function not previously known to be associated with nucleic acids. Validation of several candidate nucleic acid-associated proteins demonstrated for the first time DNA association of ectopically expressed MSMEG_1060, MSMEG_2695 and MSMEG_4306 through affinity purification. Conclusions Effective identification of nucleic acid-associated proteins, which make up the RNA polymerase complex as well as other DNA- and RNA-associated proteins, was facilitated by affinity purification of the RNA polymerase β-subunit in M. smegmatis. The successful identification of several transcriptional regulators suggest that our approach could be sensitive enough to investigate the nucleic acid-associated proteins that maintain cellular functions and mediate transcriptional and translational change in response to environmental stress.

A major form of transcriptional regulation in bacteria occurs through the exchange of the primary σ factor of RNA polymerase (RNAP) with an alternative extracytoplasmic function (ECF) σ factor1. ECF σ factors are generally intrinsically active and are retained in an inactive state via the sequestration into σ factor-anti-σ factor complexes until their action is warranted2-20. Here, we report a previously uncharacterized mechanism of transcriptional regulation that relies on intrinsically inactive ECF σ factors, the activation of which and interaction with the β'-subunit of RNAP depends on σ factor phosphorylation. In Vibrio parahaemolyticus, the threonine kinase PknT phosphorylates the σ factor EcfP, which results in EcfP activation and expression of an essential polymyxin-resistant regulon. EcfP phosphorylation occurs at a highly conserved threonine residue, Thr63, positioned within a divergent region in the σ2.2 helix. Our data indicate that EcfP is intrinsically inactive and unable to bind the β'-subunit of RNAP due to the absence of a negatively charged DAED motif in this region. Furthermore, our results indicate that phosphorylation at residue Thr63 mimics this negative charge and licenses EcfP to interact with the β'-subunit in the formation of the RNAP holoenzyme, which in turn results in target gene expression. This regulatory mechanism is a previously unrecognized paradigm in bacterial signal transduction and transcriptional regulation, and our data suggest that it is widespread in bacteria.

Background: Plastid-encoded eubacterial-type RNA polymerase (PEP) plays a critical role in the transcription of photosynthesis genes in chloroplasts. Notably, some of the reaction center genes, including psaA, psaB, psbA, and psbD genes, are differentially transcribed by PEP in mature chloroplasts. However, the molecular mechanism of promoter selection in the reaction center gene transcription by PEP is not well understood. Objective: Sigma factor proteins direct promoter selection by a core PEP in chloroplasts as well as bacteria. AtSIG5 is a unique chloroplast sigma factor essential for psbD light-responsive promoter (psbD LRP) activity. To analyze the role of AtSIG5 in chloroplast transcription in more detail, we assessed the effect of AtSIG5 hyper-expression on the transcription of plastid-encoded genes in chloroplast transgenic plants. Results: The chloroplast transgenic tobacco (CpOX-AtSIG5) accumulates AtSIG5 protein at extremely high levels in chloroplasts. Due to the extremely high-level expression of recombinant AtSIG5, most PEP holoenzymes are most likely to include the recombinant AtSIG5 in the CpOXAtSIG5 chloroplasts. Thus, we can assess the promoter preference of AtSIG5 in vivo. The overexpression of AtSIG5 significantly increased the expression of psbD LRP transcripts encoding PSII reaction center D2 protein and psaA/B operon transcripts encoding PSI core proteins. Furthermore, run-on transcription analyses revealed that AtSIG5 preferentially recognizes the psaA/B promoter, as well as the psbD LRP. Moreover, we found that psbD LRP is constitutively active in CpOX-AtSIG5 plants irrespective of light and dark. Conclusion: AtSIG5 probably plays a significant role in differential transcription of reaction center genes in mature chloroplasts.

Diverse sigma factors associate with the RNA polymerase (RNAP) core enzyme to initiate transcription of specific target genes in bacteria. σ54-Mediated transcription uses AAA+ activators that utili...

The evolution of microorganisms often involves changes of unclear relevance, such as transient phenotypes and sequential development of multiple adaptive mutations in hotspot genes. Previously, we showed that ageing colonies of an E. coli mutant unable to produce cAMP when grown on maltose, accumulated mutations in the crp gene (encoding a global transcription factor) and in genes involved in pyrimidine metabolism such as cmk; combined mutations in both crp and cmk enabled fermentation of maltose (which usually requires cAMP-mediated Crp activation for catabolic pathway expression). Here, we study the sequential generation of hotspot mutations in those genes, and uncover a regulatory role of pyrimidine nucleosides in carbon catabolism. Cytidine binds to the cytidine regulator CytR, modifies the expression of sigma factor 32 (RpoH), and thereby impacts global gene expression. In addition, cytidine binds and activates a Crp mutant directly, thus modulating catabolic pathway expression, and could be the catabolite modulating factor whose existence was suggested by Jacques Monod and colleagues in 1976. Therefore, transcription factor Crp appears to work in concert with CytR and RpoH, serving a dual role in sensing both carbon availability and metabolic flux towards DNA and RNA. Our findings show how certain alterations in metabolite concentrations (associated with colony ageing and/or due to mutations in metabolic or regulatory genes) can drive the evolution in non-growing cells., Microbial evolution often involves transient phenotypes and sequential development of multiple mutations of unclear relevance. Here, the authors show that the evolution of non-growing E. coli cells can be driven by alterations in pyrimidine nucleoside levels associated with colony ageing and/or due to mutations in metabolic or regulatory genes.

Maintenance of phospholipid (PL) and lipopoly- or lipooligo-saccharide (LPS or LOS) asymmetry in the outer membrane (OM) of Gram-negative bacteria is essential but poorly understood. The Yersinia pestis OM Ail protein was required to maintain lipid homeostasis and cell integrity at elevated temperature (37° C). Loss of this protein had pleiotropic effects. A Y. pestis Δail mutant and KIM6+ wild- type were systematically compared for (i) growth requirements at 37° C, (ii) cell structure, (iii) antibiotic and detergent sensitivity, (iv) proteins released into supernates, (v) induction of the heat shock response, and (vi) physiological and genetic suppressors that restored the wild- type phenotype. The Δail mutant grew normally at 28° C but lysed at 37° C when it entered stationary phase as shown by cell count, SDS-PAGE of cell supernatants, and electron microscopy. Immuno-fluorescent microscopy showed that the Δail mutant did not assemble Caf1 capsule. Expression of heat shock promoters rpoE or rpoH fused to a lux operon reporter were not induced when the Δail mutant was shifted from the 28° C to 37° C (p

Spx is a global transcriptional regulator in Gram-positive bacteria and has been inferred to efficiently activate transcription upon oxidative stress by engaging RNA polymerase (RNAP) and promoter DNA. However, the precise mechanism by which it interacts with RNAP and promoter DNA to initiate transcription remains obscure. Here, we report the cryo-EM structure of an intact Spx-dependent transcription activation complex (Spx–TAC) from Bacillus subtilis at 4.2 Å resolution. The structure traps Spx in an active conformation and defines key interactions accounting for Spx-dependent transcription activation. Strikingly, an oxidized Spx monomer engages RNAP by simultaneously interacting with the C-terminal domain of RNAP alpha subunit (αCTD) and σA. The interface between Spx and αCTD is distinct from those previously reported activators, indicating αCTD as a multiple target for the interaction between RNAP and various transcription activators. Notably, Spx specifically wraps the conserved –44 element of promoter DNA, thereby stabilizing Spx–TAC. Besides, Spx interacts extensively with σA through three different interfaces and promotes Spx-dependent transcription activation. Together, our structural and biochemical results provide a novel mechanistic framework for the regulation of bacterial transcription activation and shed new light on the physiological roles of the global Spx-family transcription factors.

Listeria monocytogenesis a facultative anaerobe which can cause a severe food-borne infection known as listeriosis. Rhamnose is a deoxyhexose sugar abundant in a range of environments, including the human intestine, and can be degraded byL. monocytogenesin aerobic and anaerobic conditions into lactate, acetate and 1,2-propanediol. Our previous study showed that addition of vitamin B12 stimulates anaerobic growth ofL. monocytogeneson rhamnose due to the activation of bacterial microcompartment (BMC)-dependent 1,2-propanediol utilization with concomitant production of propionate and propanol. Notably, anaerobic propanediol metabolism has been linked to virulence of enteric pathogens includingSalmonellaspp. andL. monocytogenes. In this study we investigate the impact of B12 on aerobic and anerobic growth ofL. monocytogeneson rhamnose, and observed growth stimulation andpduBMC activation only in anaerobically grown cells with B12 added to the medium. Comparative Caco-2 virulence assays, showed that thesepduBMC induced cells have significantly higher translocation efficiency compared to aerobically grown cells (without and with added B12) and non-induced anaerobically grown cells, while adhesion and invasion capacity is similar for all cells. Comparative proteomics analysis showed specific and overlapping responses linked to metabolic shifts, activation of stress defense proteins and virulence factors, with RNA polymerase sigma factor SigL; teichoic acids export ATP-binding protein, TagH; DNA repair and protection proteins RadA and DPS; and glutathione synthase GshAB previously linked to activation of virulence response inL. monocytogenes, uniquely upregulated in anaerobically rhamnose grownpduBMC induced cells. Our results shed new light into B12 impact onL. monocytogenescompetitive fitness and virulence.

Significance Diverse bacterial lifestyle transitions are controlled by the nucleotide second messenger c-di-GMP, including virulence, motility, and biofilm formation. To control such fundamentally distinct processes, the set of genes under c-di-GMP control must have gone through several shifts during bacterial evolution. Here we show that the same σ–(c-di-GMP)–anti-σ switch has been co-opted during evolution to regulate distinct biological functions in unicellular and filamentous bacteria, controlling type IV pilus production in the genus Rubrobacter and the differentiation of reproductive hyphae into spores in Streptomyces. Moreover, we show that the anti-σ likely originated as a homodimer and evolved to become a monomer through an intragenic duplication event. This study thus describes the structural and functional evolution of a c-di-GMP regulatory switch., Filamentous actinobacteria of the genus Streptomyces have a complex lifecycle involving the differentiation of reproductive aerial hyphae into spores. We recently showed c-di-GMP controls this transition by arming a unique anti-σ, RsiG, to bind the sporulation-specific σ, WhiG. The Streptomyces venezuelae RsiG–(c-di-GMP)2–WhiG structure revealed that a monomeric RsiG binds c-di-GMP via two E(X)3S(X)2R(X)3Q(X)3D repeat motifs, one on each helix of an antiparallel coiled-coil. Here we show that RsiG homologs are found scattered throughout the Actinobacteria. Strikingly, RsiGs from unicellular bacteria descending from the most basal branch of the Actinobacteria are small proteins containing only one c-di-GMP binding motif, yet still bind their WhiG partners. Our structure of a Rubrobacter radiotolerans (RsiG)2–(c-di-GMP)2–WhiG complex revealed that these single-motif RsiGs are able to form an antiparallel coiled-coil through homodimerization, thereby allowing them to bind c-di-GMP similar to the monomeric twin-motif RsiGs. Further data show that in the unicellular actinobacterium R. radiotolerans, the (RsiG)2–(c-di-GMP)2–WhiG regulatory switch controls type IV pilus expression. Phylogenetic analysis indicates the single-motif RsiGs likely represent the ancestral state and an internal gene-duplication event gave rise to the twin-motif RsiGs inherited elsewhere in the Actinobacteria. Thus, these studies show how the anti-σ RsiG has evolved through an intragenic duplication event from a small protein carrying a single c-di-GMP binding motif, which functions as a homodimer, to a larger protein carrying two c-di-GMP binding motifs, which functions as a monomer. Consistent with this, our structures reveal potential selective advantages of the monomeric twin-motif anti-σ factors.

The gene transcription of bacteria starts with a promoter sequence being recognized by a transcription factor found in the RNAP enzyme, this process is assisted through the conservation of nucleotides as well as other factors governing these intergenic regions. Faced with this, the coding of genetic information into physical aspects of the DNA such as enthalpy, stability, and base-pair stacking could suggest promoter activity as well as protrude differentiation of promoter and non-promoter data. In this work, a total of 3131 promoter sequences associated to six different sigma factors in the bacterium E. coli were converted into numeric attributes, a strong set of control sequences referring to a shuffled version of the original sequences as well as coding regions is provided. Then, the parameterized genetic information was normalized, exhaustively analyzed through statistical tests. The results suggest that strong signals in the promoter sequences match the binding site of transcription factor proteins, indicating that promoter activity is well represented by its conversion into physical attributes. Moreover, the features tested in this report conveyed significant variances between promoter and control data, enabling these features to be employed in bacterial promoter classification. The results produced here may aid in bacterial promoter recognition by providing a robust set of biological inferences.

The alternative sigma factor σ54 has been shown to regulate the expression of a wide array of virulence-associated genes, as well as central metabolism, in bacterial pathogens. In Gram-positive organisms, the σ54 is commonly associated with carbon metabolism. In this study, we show that the Enterococcus faecalis alternative sigma factor σ54 (RpoN) and its cognate enhancer binding protein MptR are essential for mannose utilization and are primary contributors to glucose uptake through the Mpt phosphotransferase system. To gain further insight into how RpoN contributes to global transcriptional changes, we performed microarray transcriptional analysis of strain V583 and an isogenic rpoN mutant grown in a chemically defined medium with glucose as the sole carbon source. Transcripts of 340 genes were differentially affected in the rpoN mutant; the predicted functions of these genes mainly related to nutrient acquisition. These differentially expressed genes included those with predicted c atabolite- r esponsive e lement (cre) sites, consistent with loss of repression by the major carbon catabolite repressor CcpA. To determine if the inability to efficiently metabolize glucose/mannose affected infection outcome, we utilized two distinct infection models. We found that the rpoN mutant is significantly attenuated in both rabbit endocarditis and murine catheter-associated urinary tract infection (CAUTI). Here, we examined a ccpA mutant in the CAUTI model and showed that the absence of carbon catabolite control also significantly attenuates bacterial tissue burden in this model. Our data highlight the contribution of central carbon metabolism to growth of E. faecalis at various sites of infection.IMPORTANCE Hospital-acquired infections account for 2 billion dollars annually in increased health care expenses and cause more than 100,000 deaths in the United States alone. Enterococci are the second leading cause of hospital-acquired infections. They form biofilms at surgical sites and are often associated with infections of the urinary tract following catheterization. Nutrient uptake and growth are key factors that influence their ability to cause disease. Our research identified a large set of genes that illuminate nutrient uptake pathways in enterococci. Perturbation of the metabolic circuit reduces virulence in a rabbit endocarditis model, as well as in catheter-associated urinary tract infection in mice. Targeting metabolic pathways that are important in infection may lead to new treatments against multidrug-resistant enterococcal infections.

14 p.-6 fig.-1 tab., Promoter recognition by RNA polymerase is a key step in the regulation of gene expression. The bacterial RNA polymerase core enzyme is a complex of five subunits that interacts transitory with one of a set of sigma factors forming the RNA polymerase holoenzyme. The sigma factor confers promoter specificity to the RNA polymerase. In the Gram-positive pathogenic bacterium Streptococcus pneumoniae, most promoters are likely recognized by SigA, a poorly studied housekeeping sigma factor. Here we present a sequence conservation analysis and show that SigA has similar protein architecture to Escherichia coli and Bacillus subtilis homologs, namely the poorly conserved N-terminal 100 residues and well-conserved rest of the protein (domains 2, 3, and 4). Further, we have purified the native (untagged) SigA protein encoded by the pneumococcal R6 strain and reconstituted an RNA polymerase holoenzyme composed of the E. coli core enzyme and the sigma factor SigA (RNAP-SigA). By in vitro transcription, we have found that RNAP-SigA was able to recognize particular promoters, not only from the pneumococcal chromosome but also from the S. agalactiae promiscuous antibiotic-resistance plasmid pMV158. Specifically, SigA was able to direct the RNA polymerase to transcribe genes involved in replication and conjugative mobilization of plasmid pMV158. Our results point to the versatility of SigA in promoter recognition and its contribution to the promiscuity of plasmid pMV158., This study was financially supported by grants BIO2016-76412-C2-2-R (AEI/FEDER, UE) to AB from the Spanish Ministry of Economy and Competitiveness, and PID2019-104553RB-C21 to AB from the Spanish Ministry of Science and Innovation.

International audience; Clostridioides difficile infections are associated with gut microbiome dysbiosis and are the leading cause of hospital-acquired diarrhoea. The infectious process is strongly influenced by the microbiota and successful infection relies on the absence of specific microbiota-produced metabolites. Deoxycholate and short-chain fatty acids are microbiota-produced metabolites that limit the growth of C. difficile and protect the host against this infection. In a previous study, we showed that deoxycholate causes C. difficile to form strongly adherent biofilms after 48 h. Here, our objectives were to identify and characterize key molecules and events required for biofilm formation in the presence of deoxycholate. We applied time-course transcriptomics and genetics to identify sigma factors, metabolic processes and type IV pili that drive biofilm formation. These analyses revealed that extracellular pyruvate induces biofilm formation in the presence of deoxycholate. In the absence of deoxycholate, pyruvate supplementation was sufficient to induce biofilm formation in a process that was dependent on pyruvate uptake by the membrane protein CstA. In the context of the human gut, microbiota-generated pyruvate is a metabolite that limits pathogen colonization. Taken together our results suggest that pyruvate-induced biofilm formation might act as a key process driving C. difficile persistence in the gut.

Newly formed spores are essential for persistence of C. difficile in the host, transmission to a new susceptible host (Deakin et al., 2012b) and recurrence of CDI. BclA3 and BclA2 Spore surface proteins are expressed during sporulation under the control of mother-cell specific sigma factors of the RNA polymerase, SigE and SigK. Deletion of bclA3 leads to spores with an electron-dense exosporium layer that lacks bump-like structures in the electron-dense layer and hair-like projections, both structures typically found in the wild type spore. Therefore, in this work, we have addressed the role of the exosporium collagen-like BclA3 glycoprotein in the assembly of the exosporium layer. Immunogold labelling of BclA2CTD and BclA3CTD indicates that both proteins are located in the hairs, with BclA2 located outermost of BclA3. Absence of BclA3 leads to spores with no hair-like projections, and absence of bumps in thick exosporium spores, a phenotype also expressed in by the deletion of the collagen-like region of BclA3. Overall, these results provide insights into the role of BclA3 in the assembly of the exosporium layer of C. difficile spores.

A recent paper by Gallagher et al. (2020) demonstrates that c-di-GMP controls spore formation in Streptomyces venezuelae through sequestering the sporulation sigma factor σWhiG and presents the crystal structure of a ternary complex between c-di-GMP, σWhiG, and its anti-sigma factor, RsiG.

One of the promising strategies in the development of efficient antibacterial drugs is the use of antisense oligonucleotides specifically interacting with mRNAs of genes controlling the key functions of the bacterial cell such as survival and reproduction. The present paper reports the development of an algorithm for searching and testing the antibacterial activity of antisense oligonucleotides using mRNA of the rpoD gene encoding the sigma factor of the Staphylococcus aureus RNA polymerase. Based on the calculated thermodynamic parameters and the rpoD mRNA 5'-fragment (1–377 nt region) secondary structure model, 11 antisense oligonucleotides 18–20 nt in length complementary to the rpoD mRNA regions with unstable secondary structure have been selected. Hybridization of the selected oligonucleotides with the total S. aureus RNA followed by selective in vitro digestion of antisense oligonucleotide/mRNA rpoD heteroduplexes with RNase H and assessment of rpoD mRNA digestion rate using PCR revealed six candidate sequences which reduced the rpoD mRNA level by 2.2–4 times. Phosphorothioate oligonucleotides targeted to the regions 1–20, 30–47, and 82–101 of the rpoD mRNA synthesized on the basis of these sequences efficiently suppressed the growth of S. aureus bacteria in vitro. It has been demonstrated that the efficiency of penetration of these oligonucleotides into the bacterial cell may be significantly increased by introducing arginine-rich peptide into the oligonucleotide structure. As a result of this work, we have developed a simple and efficient algorithm for selection of antisense oligonucleotides against bacterial RNAs encompassing all stages starting from the binding stage and ending with the analysis of biological activity.

Prokaryotic gene expression is largely regulated on transcriptional levels with the involvement of promoters, RNA polymerase and sigma factors. Developing new promoters to customize gene transcriptional regulation becomes increasingly demanded in synthetic biology and biotechnology. In this study, we designed synthetic promoters in the Gram-positive model bacterium Bacillus subtilis by interlocking the binding motifs of σA for house-keeping gene expression and that of two alternative sigma factors σH and σB which are involved in responding post-exponential growth and general stress, respectively. The developed promoters are recognized by multiple sigma factors and hence generate strong transcriptional strength when host cells grow under normal or stressed conditions. With green fluorescent protein as the reporter, a set of strong promoters were identified, in which the transcription activities of PHA-1, PHAB-4, PHAB-7 were 18.6, 4.1, 3.3 fold of that of the commonly used promoter P43, respectively. Moreover, some of the promoters such as PHA-1, PHAB-4, PHAB-7, PBA-2 displayed increased transcriptional activities in response to high salinity or low pH. The promoters developed in this study should enrich the biotechnological toolboxes of B. subtilis. Keywords: Synthetic biology, Transcription, Promoter engineering, Stress-responsive, Bacillus subtilis

Bacteria can rapidly and reversibly respond to changing environments via complex transcriptional and post-transcriptional regulatory mechanisms. Many of these adaptations are specific, with the regulatory output tailored to the inducing signal (for instance, repairing damage to cell components or improving acquisition and use of growth-limiting nutrients). However, the general stress response, activated in bacterial cells entering stationary phase or subjected to nutrient depletion or cellular damage, is unique in that its common, broad output is induced in response to many different signals. In many different bacteria, the key regulator for the general stress response is a specialized sigma factor, the promoter specificity subunit of RNA polymerase. The availability or activity of the sigma factor is regulated by complex regulatory circuits, the majority of which are post-transcriptional. In Escherichia coli, multiple small regulatory RNAs, each made in response to a different signal, positively regulate translation of the general stress response sigma factor RpoS. Stability of RpoS is regulated by multiple anti-adaptor proteins that are also synthesized in response to different signals. In this review, the modes of signaling to and levels of regulation of the E. coli general stress response are discussed. They are also used as a basis for comparison with the general stress response in other bacteria with the aim of extracting key principles that are common among different species and highlighting important unanswered questions.

Signal-specific activation of alternative sigma factors redirects RNA polymerase to induce transcription of distinct sets of genes conferring protection against the damage the signal and the related stresses incur. In Streptomyces coelicolor, σR (SigR), a member of ECF12 subfamily of Group IV sigma factors, responds to thiol-perturbing signals such as oxidants and electrophiles, as well as to translation-blocking antibiotics. Oxidants and electrophiles interact with and inactivate the zinc-containing anti-sigma factor, RsrA, via disulfide bond formation or alkylation of reactive cysteines, subsequently releasing σR for target gene induction. Translation-blocking antibiotics induce the synthesis of σR , via the WhiB-like transcription factor, WblC/WhiB7. Signal transduction via RsrA produces a dramatic transient response that involves positive feedback to produce more SigR as an unstable isoform σ R ' and negative feedbacks to degrade σ R ' , and reduce oxidized RsrA that subsequently sequester σR and σ R ' . Antibiotic stress brings about a prolonged response by increasing stable σR levels. The third negative feedback, which occurs via IF3, lowers the translation efficiency of the sigRp1 transcript that utilizes a non-canonical start codon. σR is a global regulator that directly activates > 100 transcription units in S. coelicolor, including genes for thiol homeostasis, protein quality control, sulfur metabolism, ribosome modulation and DNA repair. Close homologues in Actinobacteria, such as σH in Mycobacteria and Corynebacteria, show high conservation of the signal transduction pathways and target genes, thus reflecting the robustness of this type of regulation in response to redox and antibiotic stresses.

Sigma factor, as a unit of RNA polymerase holoenzyme, is a critical factor in the process of gene transcriptional regulation. It recognizes the specific DNA sites and brings the core enzyme of RNA polymerase to the upstream regions of target genes. Therefore, the prediction of the promoters for a particular sigma factor is essential for interpreting functional genomic data and observation. This paper develops a new method to predict sigma-54 promoters in bacterial genomes. The new method organically integrates motif finding and machine learning strategies to capture the intrinsic features of sigma-54 promoters. The experiments on E. coli benchmark test set show that our method has good capability to distinguish sigma-54 promoters from surrounding or randomly selected DNA sequences. The applications of the other three bacterial genomes indicate the potential robustness and applicative power of our method on a large number of bacterial genomes. The source code of our method can be freely downloaded at https://github.com/maqin2001/PromotePredictor.

Natural genetic transformation via horizontal gene transfer enables rapid adaptation to dynamic environments and contributes to both antibiotic resistance and vaccine evasion among bacterial populations. In Streptococcus pneumoniae (pneumococcus), transformation occurs when cells enter competence, a transient state in which cells express the competence master regulator, SigX (σX), an alternative σ factor (σ), and a competence co-regulator, ComW. Together, ComW and σX facilitate expression of the genes required for DNA uptake and genetic recombination. SigX activity depends on ComW, as ΔcomW cells transcribe late genes and transform at levels 10- and 10,000-fold below that of WT cells, respectively. Previous findings suggest that ComW functions during assembly of the RNA polymerase-σX holoenzyme to help promote transcription from σX-targeted promoters. However, it remains unknown how ComW facilitates holoenzyme assembly. As ComW seems to be unique to Gram-positive cocci and has no sequence similarity with known transcriptional activators, here we used Rosetta to generate an ab initio model of pneumococcal ComW's 3D-structure. Using this model as a basis for further biochemical, biophysical, and genetic investigations into the molecular features important for its function, we report that ComW is a predicted globular protein and that it interacts with DNA, independently of DNA sequence. We also identified conserved motifs in ComW and show that key residues in these motifs contribute to DNA binding. Lastly, we provide evidence that ComW's DNA-binding activity is important for transformation in pneumococcus. Our findings begin to fill the gaps in understanding how ComW regulates σX activity during bacterial natural transformation.

The σ subunit of the bacterial RNA polymerase determines promoter specificity. The extracytoplasmic function σ factors (ECFs) represent the most abundant and diverse group of alternative σ factors and are present in the vast majority of bacterial genomes. Typically, ECFs are regulated by anti-σ factors that sequester their cognate ECFs, thereby preventing their interaction with the RNA polymerase. Beyond these ECF paradigms, a number of distinct modes of regulation have been proposed and experimentally investigated. Regulatory extensions represent one such alternative mechanism of ECF regulation that can be found in 18 phylogenetically distinct ECF groups. Here, the σ factors contain additional domains that are fused to the ECF core domains and are involved in stimulus perception and modulation of σ factor activity. We will summarize the current state of knowledge on regulating ECF activity by C-terminal extensions. We will also discuss newly identified ECF groups containing either N- or C-terminal extensions and propose possible mechanisms by which these extensions have been generated and affect ECF σ factor activity. Based on their modular architecture and the resulting physical connection between stimulus perception and transcriptional output, these ECFs are analogous to one-component systems, the primary mechanism of bacterial signal transduction.

Transcription is the fundamental process that enables the expression of genetic information. DNA-directed RNA polymerase (RNAP) uses one strand of the DNA duplex as template to produce complementary RNA molecules that serve in translation (rRNA, tRNA), protein synthesis (mRNA), and regulation (sRNA). Although the RNAP core is catalytically competent for RNA synthesis, the selectivity of transcription initiation requires a sigma (σ) factor for promoter recognition and opening. Expression of alternative σ factors provides a powerful mechanism to control the expression of discrete sets of genes (a σ regulon) in response to specific nutritional, developmental, or stress-related signals. Here, I review the key insights that led to the original discovery of σ factor 50 years ago and the subsequent discovery of alternative σ factors as a ubiquitous mechanism of bacterial gene regulation. These studies form a prelude to the more recent, genomics-enabled insights into the vast diversity of σ factors in Bacteria.

RNA polymerase can cleave a phosphodiester bond at the 3′ end of a nascent RNA in the presence of pyrophosphate producing NTP. Pyrophosphorolysis has been characterized during elongation steps of transcription where its rate is significantly slower than the forward rate of NMP addition. In contrast, we report here that pyrophosphorolysis can occur in a millisecond time scale during the transition of Escherichia coli RNA polymerase from initiation to elongation at the psbA2 promoter. This rapid pyrophosphorolysis occurs during productive RNA synthesis as opposed to abortive RNA synthesis. Dissociation of σ(70) or RNA extension beyond nine nucleotides dramatically reduces the rate of pyrophosphorolysis. We argue that the rapid pyrophosphorolysis allows iterative cycles of cleavage and re-synthesis of the 3′ phosphodiester bond by the productive complexes in the early stage of transcription. This iterative process may provide an opportunity for the σ(70) to dissociate from the RNA exit channel of the enzyme, enabling RNA to extend through the channel.

The Gram-negative envelope is a complex structure that consists of the inner membrane, the periplasm, peptidoglycan and the outer membrane, and protects the bacterial cell from the environment. Changing environmental conditions can cause damage, which triggers the envelope stress responses to maintain cellular homeostasis. In this Review, we explore the causes, both environmental and intrinsic, of envelope stress, as well as the cellular stress response pathways that counter these stresses. Furthermore, we discuss the damage to the cell that occurs when these pathways are aberrantly activated either in the absence of stress or to an excessive degree. Finally, we review the mechanisms whereby the σE response constantly acts to prevent cell death caused by highly toxic unfolded outer membrane proteins. Together, the recent work that we discuss has provided insights that emphasize the necessity for proper levels of stress response activation and the detrimental consequences that can occur in the absence of proper regulation.

Vibriosis is one of the major bacterial diseases observed in cultured marine fish worldwide. An epidemic trait of the Vibrio derived from cage-cultured marine fish is still lacking. To identify the Vibrio and discriminate ten Vibrio species from cage-cultured marine fish, we designed specific primers targeting the RNA polymerase sigma factor (rpoD), recombination protein F (recF), transcriptional regulator (toxR) genes (known as housekeeping genes), and two multiplex PCR sets (PCR set 1 for V. anguillarum, V. gigantis, V. atlanticus, V. harveyi, and V. scophthalmi; and PCR set 2 for V. lentus, V. splendidus, Photoacterium piscicola, V. alginolyticus and P. damselae). Moreover, each multiplex PCR assay comprised internal control primers that enabled the detection of the Vibrio. Designed primers yielded specific diagnostic results to target the Vibrio and ten Vibrio species in the multiplex PCR assays. The minimal detection limits of the multiplex PCR were each 5 ng DNA μL−1, which showed ten-fold sensitivity compared to a single PCR reaction. The novel multiplex PCR assays could contribute to detecting the Vibrio and discriminate the major Vibrio species in cage-cultured marine fish, and could be used as a reliable and informative diagnostic tool in the aquaculture industry.

The σ70 family alternative σI factors and their cognate anti-σI factors are widespread in Clostridia and Bacilli and play a role in heat stress response, virulence, and polysaccharide sensing. Multiple σI/anti-σI factors exist in some lignocellulolytic clostridial species, specifically for regulation of components of a multienzyme complex, termed the cellulosome. The σI and anti-σI factors are unique, because the C-terminal domain of σI (SigIC) and the N-terminal inhibitory domain of anti-σI (RsgIN) lack homology to known proteins. Here, we report structure and interaction studies of a pair of σI and anti-σI factors, SigI1 and RsgI1, from the cellulosome-producing bacterium, Clostridium thermocellum. In contrast to other known anti-σ factors that have N-terminal helical structures, RsgIN has a β-barrel structure. Unlike other anti-σ factors that bind both σ2 and σ4 domains of the σ factors, RsgIN binds SigIC specifically. Structural analysis showed that SigIC contains a positively charged surface region that recognizes the promoter –35 region, and the synergistic interactions among multiple interfacial residues result in the specificity displayed by different σI/anti-σI pairs. We suggest that the σI/anti-σI factors represent a distinctive mode of σ/anti-σ complex formation, which provides the structural basis for understanding the molecular mechanism of the intricate σI/anti-σI system.

The stationary phase promoter specificity subunit σS (RpoS) is delivered to the ClpXP machinery for degradation dependent on the adaptor RssB. This adaptor-specific degradation of σS provides a major point for regulation and transcriptional reprogramming during the general stress response. RssB is an atypical response regulator and the only known ClpXP adaptor that is inhibited by multiple but dissimilar antiadaptors (IraD, IraP, and IraM). These are induced by distinct stress signals and bind to RssB in poorly understood manners to achieve stress-specific inhibition of σS turnover. Here we present the first crystal structure of RssB bound to an antiadaptor, the DNA damage-inducible IraD. The structure reveals that RssB adopts a compact closed architecture with extensive interactions between its N-terminal and C-terminal domains. The structural data, together with mechanistic studies, suggest that RssB plasticity, conferred by an interdomain glutamate-rich flexible linker, is critical for regulation of σS degradation. Structural modulation of interdomain linkers may thus constitute a general strategy for tuning response regulators.