The sigma factor sigma 70 of E. coli RNA polymerase acts not only in initiation, but also at an early stage of elongation to induce a transcription pause, and simultaneously to allow the phage lambda gene Q transcription antiterminator to act. We identify the signal in DNA that induces early pausing to be a version of the sigma 70 -10 promoter consensus, and we show that sigma 70 is both necessary for pausing and present in the paused transcription complex. Regions 2 and 3 of sigma 70 suffice to induce pausing. Since pausing is induced by the nontemplate DNA strand of the open transcription bubble, we conclude that RNA polymerase containing sigma 70 carries out base-specific recognition of the nontemplate strand as single stranded DNA. We suggest that sigma 70 remains bound to core RNA polymerase when the -10 promoter contacts are broken, and then moves to the pause-inducing sequence.

σE is one of the 13 sigma factors encoded by the Mycobacterium tuberculosis chromosome, and its involvement in stress response and virulence has been extensively characterized. Several sigma factors are post-translationally regulated by proteins named anti-sigma factors, which prevent their binding to RNA polymerase. Rv1222 (RseA), whose gene lays immediately downstream sigE, has been proposed in the past as the σE-specific anti sigma factor. However, its role as anti-sigma factor was recently challenged and a new mechanism of action was hypothesized predicting RseA binding to RNA polymerase and DNA to slow down RNA transcription in a not specific way. In this manuscript, using specific M. tuberculosis mutants, we showed that by changing the levels of RseA expression, M. tuberculosis growth rate does not change (as hypothesized in case of non-specific decrease of RNA transcription) and has an impact only on the transcription level of genes whose transcriptional control is under σE, supporting a direct role of RseA as a specific anti-σE factor.

To engineer synthetic gene circuits, molecular building blocks are developed which can modulate gene expression without interference, mutually or with the host's cell machinery. As the complexity of gene circuits increases, automated design tools and tailored building blocks to ensure perfect tuning of all components in the network are required. Despite the efforts to develop prediction tools that allow forward engineering of promoter transcription initiation frequency (TIF), such a tool is still lacking. Here, we use promoter libraries of E. coli sigma factor 70 (σ70)- and B. subtilis σB-, σF- and σW-dependent promoters to construct prediction models, capable of both predicting promoter TIF and orthogonality of the σ-specific promoters. This is achieved by training a convolutional neural network with high-throughput DNA sequencing data from fluorescence-activated cell sorted promoter libraries. This model functions as the base of the online promoter design tool (ProD), providing tailored promoters for tailored genetic systems.

Bacteria are known to cope with environmental changes by using alternative sigma factors binding to RNA polymerase core enzyme. Sigma factor is one of the targets to modify transcription regulation in bacteria and to influence production capacities. In this study, the effect of overexpressing each annotated sigma factor gene in Corynebacterium glutamicum WT was assayed using an IPTG inducible plasmid system and different IPTG concentrations. It was revealed that growth was severely decreased when sigD or sigH were overexpressed with IPTG concentrations higher than 50 μM. Overexpression of sigH led to an obvious phenotypic change, a yellow-colored supernatant. High performance liquid chromatography analysis revealed that riboflavin was excreted to the medium when sigH was overexpressed and DNA microarray analysis confirmed increased expression of riboflavin biosynthesis genes. In addition, genes for enzymes related to the pentose phosphate pathway and for enzymes dependent on flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), or NADPH as cofactor were upregulated when sigH was overexpressed. To test if sigH overexpression can be exploited for production of riboflavin-derived FMN or FAD, the endogenous gene for bifunctional riboflavin kinase/FMN adenyltransferase was co-expressed with sigH from a plasmid. Balanced expression of sigH and ribF improved accumulation of riboflavin (19.8 ± 0.3 μM) and allowed for its conversion to FMN (33.1 ± 1.8 μM) in the supernatant. While a proof-of-concept was reached, conversion was not complete and titers were not high. This study revealed that inducible and gradable overexpression of sigma factor genes is an interesting approach to switch gene expression profiles and to discover untapped potential of bacteria for chemical production.

Sigma factors are an important class of bacterial transcription factors that lend specificity to RNA polymerases by binding to distinct promoter elements for genes in their regulons. Here we show that activation of the general stress sigma factor, σB, in Bacillus subtilis paradoxically leads to dramatic induction of translation for a subset of its regulon genes. These genes are translationally repressed when transcribed by the housekeeping sigma factor, σA, owing to extended RNA secondary structures as determined in vivo using DMS-MaPseq. Transcription from σB-dependent promoters ablates the secondary structures and activates translation, leading to dual induction. Translation efficiencies between σB- and σA-dependent RNA isoforms can vary by up to 100-fold, which in multiple cases exceeds the magnitude of transcriptional induction. These results highlight the role of long-range RNA folding in modulating translation and demonstrate that a transcription factor can regulate protein synthesis beyond its effects on transcript levels.

SigR (sigma(R)) is a sigma factor responsible for inducing the thioredoxin system in response to oxidative stress in the antibiotic-producing, Gram-positive bacterium Streptomyces coelicolor A3(2). Here we identify a redox-sensitive, sigma(R)-specific anti-sigma factor, RsrA, which binds sigma(R) and inhibits sigma(R)-directed transcription in vitro only under reducing conditions. Exposure to H(2)O(2) or to the thiol-specific oxidant diamide caused the dissociation of the sigma(R)-RsrA complex, thereby allowing sigma(R)-dependent transcription. This correlated with intramolecular disulfide bond formation in RsrA. Thioredoxin was able to reduce oxidized RsrA, suggesting that sigma(R), RsrA and the thioredoxin system comprise a novel feedback homeostasis loop that senses and responds to changes in the intracellular thiol-disulfide redox balance.

Sigma factors are one of the components of RNA polymerase holoenzymes, and an essential factor of transcription initiation in bacteria. Corynebacterium glutamicum possesses seven genes coding for sigma factors, most of which have been studied to some detail; however, the role of SigD in transcriptional regulation in C. glutamicum has been mostly unknown.

Genetically identical individuals in bacterial populations can display significant phenotypic variability. This variability can be functional, for example by allowing a fraction of stress prepared cells to survive an otherwise lethal stress. The optimal fraction of stress prepared cells depends on environmental conditions. However, how bacterial populations modulate their level of phenotypic variability remains unclear. Here we show that the alternative sigma factor σV circuit in Bacillus subtilis generates functional phenotypic variability that can be tuned by stress level, environmental history and genetic perturbations. Using single-cell time-lapse microscopy and microfluidics, we find the fraction of cells that immediately activate σV under lysozyme stress depends on stress level and on a transcriptional memory of previous stress. Iteration between model and experiment reveals that this tunability can be explained by the autoregulatory feedback structure of the sigV operon. As predicted by the model, genetic perturbations to the operon also modulate the response variability. The conserved sigma-anti-sigma autoregulation motif is thus a simple mechanism for bacterial populations to modulate their heterogeneity based on their environment.

The focus of modern molecular biology turns from assigning functions to individual genes towards understanding the expression and regulation of complex sets of molecules. Here, we provide evidence that alternative sigma factor regulons in the pathogen Pseudomonas aeruginosa largely represent insulated functional modules which provide a critical level of biological organization involved in general adaptation and survival processes. Analysis of the operational state of the sigma factor network revealed that transcription factors functionally couple the sigma factor regulons and significantly modulate the transcription levels in the face of challenging environments. The threshold quality of newly evolved transcription factors was reached faster and more robustly in in silico testing when the structural organization of sigma factor networks was taken into account. These results indicate that the modular structures of alternative sigma factor regulons provide P. aeruginosa with a robust framework to function adequately in its environment and at the same time facilitate evolutionary change. Our data support the view that widespread modularity guarantees robustness of biological networks and is a key driver of evolvability.

Sigma factors bind and direct the RNA polymerase core to specific promoter sequences, and alternative sigma factors direct transcription of different regulons of genes. Here, we study the pBS32 plasmid-encoded sigma factor SigN of Bacillus subtilis to determine how it contributes to DNA damage-induced cell death. We find that SigN causes cell death when expressed at high levels and does so in the absence of its regulon suggesting it is intrinsically toxic. One way toxicity was relieved was by curing the pBS32 plasmid, which eliminated a positive feedback loop that led to SigN hyper-accumulation. Another way toxicity was relieved was through mutating the chromosomally encoded transcriptional repressor protein AbrB, thereby derepressing a potent antisense transcript that antagonized SigN expression. SigN efficiently competed with the vegetative sigma factor SigA in vitro, and SigN accumulation in the absence of positive feedback reduced SigA-dependent transcription suggesting that toxicity may be due to competitive inhibition of one or more essential transcripts. Why B. subtilis encodes a toxic sigma factor is unclear but SigN may function in host-inhibition during lytic conversion, as phage lysogen genes are also encoded on pBS32. IMPORTANCE Alternative sigma factors activate entire regulons of genes to improve viability in response to environmental stimuli. The pBS32 plasmid-encoded alternative sigma factor SigN of Bacillus subtilis however, is activated by the DNA damage response and leads to cellular demise. Here we find that SigN impairs viability by hyper-accumulating and outcompeting the vegetative sigma factor for the RNA polymerase core. Why B. subtilis retains a plasmid with a deleterious alternative sigma factor is unknown.

Alternative sigma factors trigger various adaptive responses. Lactobacillus sakei, a non-sporulating meat-borne bacterium, carries an alternative sigma factor seemingly orthologous to σ(H) of Bacillus subtilis, best known for its contribution to the initiation of a large starvation response ultimately leading to sporulation. As the role of σ(H)-like factors has been little studied in non-sporulating bacteria, we investigated the function of σ(H) in L. sakei.

Global changes in bacterial gene expression can be orchestrated by the coordinated activation/deactivation of alternative sigma (σ) factor subunits of RNA polymerase. Sigma factors themselves are regulated in myriad ways, including via anti-sigma factors. Here, we have determined the solution structure of anti-sigma factor CsfB, responsible for inhibition of two alternative sigma factors, σG and σE, during spore formation by Bacillus subtilis. CsfB assembles into a symmetrical homodimer, with each monomer bound to a single Zn2+ ion via a treble-clef zinc finger fold. Directed mutagenesis indicates that dimer formation is critical for CsfB-mediated inhibition of both σG and σE, and we have characterized these interactions in vitro. This work represents an advance in our understanding of how CsfB mediates inhibition of two alternative sigma factors to drive developmental gene expression in a bacterium.

Synthetic genetic sensors and circuits enable programmable control over timing and conditions of gene expression and, as a result, are increasingly incorporated into the control of complex and multi-gene pathways. Size and complexity of genetic circuits are growing, but stay limited by a shortage of regulatory parts that can be used without interference. Therefore, orthogonal expression and regulation systems are needed to minimize undesired crosstalk and allow for dynamic control of separate modules. This work presents a set of orthogonal expression systems for use in Escherichia coli based on heterologous sigma factors from Bacillus subtilis that recognize specific promoter sequences. Up to four of the analyzed sigma factors can be combined to function orthogonally between each other and toward the host. Additionally, the toolbox is expanded by creating promoter libraries for three sigma factors without loss of their orthogonal nature. As this set covers a wide range of transcription initiation frequencies, it enables tuning of multiple outputs of the circuit in response to different sensory signals in an orthogonal manner. This sigma factor toolbox constitutes an interesting expansion of the synthetic biology toolbox and may contribute to the assembly of more complex synthetic genetic systems in the future.

Sigma factors are dissociable subunits of bacterial RNA polymerase that ensure efficient transcription initiation from gene promoters. Owing to their prokaryotic origin, chloroplasts possess a typical bacterial RNA polymerase together with its sigma factor subunit. The higher plant Arabidopsis thaliana contain as many as six sigma factors for the hundred or so of its chloroplast genes. The role of this relatively large number of transcription initiation factors for the miniature chloroplast genome, however, is not fully understood. Using two Arabidopsis T-DNA insertion mutants, we show that sigma factor 1 (SIG1) initiates transcription of a specific subset of chloroplast genes. We further show that the photosynthetic control of PSI reaction center gene transcription requires complementary regulation of the nuclear SIG1 gene at the transcriptional level. This SIG1 gene regulation is dependent on both a plastid redox signal and a light signal transduced by the phytochrome photoreceptor.

Cell-free transcription-translation provides a simplified prototyping environment to rapidly design and study synthetic networks. Despite the presence of a well characterized toolbox of genetic elements, examples of genetic networks that exhibit complex temporal behavior are scarce. Here, we present a genetic oscillator implemented in an E. coli-based cell-free system under steady-state conditions using microfluidic flow reactors. The oscillator has an activator-repressor motif that utilizes the native transcriptional machinery of E. coli: the RNAP and its associated sigma factors. We optimized a kinetic model with experimental data using an evolutionary algorithm to quantify the key regulatory model parameters. The functional modulation of the RNAP was investigated by coupling two oscillators driven by competing sigma factors, allowing the modification of network properties by means of passive transcriptional regulation.

Rivastigmine (Riv) is a potent and selective cholinesterase (acetylcholinesterase, AChE and butyrylcholinesterase, BuChE) inhibitor developed for the treatment of Alzheimer's disease (AD). To elucidate whether Riv causes neuronal differentiation, we examined its effect on nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. At concentrations of 0-100 μM, Riv was non-toxic in PC12 cells. Riv caused dose-dependent (10-100 μM) enhancement of NGF-induced neurite outgrowth, which was completely inhibited by the TrkA antagonist GW-441756. By contrast, Riv-mediated enhancement of neurite outgrowth was not blocked by the acetylcholine receptor antagonists, scopolamine and hexamethonium. However, the sigma-1 receptor (Sig-1R) antagonist NE-100 and sigma-2 receptor (Sig-2R) antagonist SM-21 each blocked about half of the Riv-mediated enhancement of NGF-induced neurite outgrowth. Interestingly, the simultaneous application of NE-100 and SM-21 completely blocked the enhancement of NGF-induced neurite outgrowth by Riv. These findings suggest that both Sig-1R and Sig-2R play important roles in NGF-induced neurite outgrowth through TrkA and that Riv may contribute to neuronal repair via Sig-1R and Sig-2R in AD therapy.

Salmonella enterica is the second most common foodborne pathogen. The use of biocides is crucial to prevent spread of foodborne pathogens, and it would be devastating for food safety if Salmonella would become resistant to the disinfectants used. Another concern is that exposure to disinfectants might lead to decreased susceptibility to antibiotics. The current study aimed to identify genetic changes causing high level triclosan resistance in S. enterica serovar Typhimurium and evaluate how these affected antibiotic resistance and efflux pump activity.

Next to the two-component and quorum sensing systems, cell-surface signaling (CSS) has been recently identified as an important regulatory system in Pseudomonas aeruginosa. CSS systems sense signals from outside the cell and transmit them into the cytoplasm. They generally consist of a TonB-dependent outer membrane receptor, a sigma factor regulator (or anti-sigma factor) in the cytoplasmic membrane, and an extracytoplasmic function (ECF) sigma factor. Upon perception of the extracellular signal by the receptor the ECF sigma factor is activated and promotes the transcription of a specific set of gene(s). Although most P. aeruginosa CSS systems are involved in the regulation of iron uptake, we have identified a novel system involved in the regulation of virulence. This CSS system, which has been designated PUMA3, has a number of unusual characteristics. The most obvious difference is the receptor component which is considerably smaller than that of other CSS outer membrane receptors and lacks a beta-barrel domain. Homology modeling of PA0674 shows that this receptor is predicted to be a bilobal protein, with an N-terminal domain that resembles the N-terminal periplasmic signaling domain of CSS receptors, and a C-terminal domain that resembles the periplasmic C-terminal domains of the TolA/TonB proteins. Furthermore, the sigma factor regulator both inhibits the function of the ECF sigma factor and is required for its activity. By microarray analysis we show that PUMA3 regulates the expression of a number of genes encoding potential virulence factors, including a two-partner secretion (TPS) system. Using zebrafish (Danio rerio) embryos as a host we have demonstrated that the P. aeruginosa PUMA3-induced strain is more virulent than the wild-type. PUMA3 represents the first CSS system dedicated to the transcriptional activation of virulence functions in a human pathogen.

Chloroplasts are a common feature of plant cells and aspects of their metabolism, including photosynthesis, are influenced by low-temperature conditions. Chloroplasts contain a small circular genome that encodes essential components of the photosynthetic apparatus and chloroplast transcription/translation machinery. Here, we show that in Arabidopsis, a nuclear-encoded sigma factor that controls chloroplast transcription (SIGMA FACTOR5) contributes to adaptation to low-temperature conditions. This process involves the regulation of SIGMA FACTOR5 expression in response to cold by the bZIP transcription factors ELONGATED HYPOCOTYL5 and ELONGATED HYPOCOTYL5 HOMOLOG. The response of this pathway to cold is gated by the circadian clock, and it enhances photosynthetic efficiency during long-term cold and freezing exposure. We identify a process that integrates low-temperature and circadian signals, and modulates the response of chloroplasts to low-temperature conditions.

SigE represents one of the best characterized alternative sigma factors of Mycobacterium tuberculosis, playing a major role in the response to several environmental stresses and essential for growth in macrophages and virulence. In previous work we demonstrated that a mutant of M. tuberculosis in which the sigE gene was disrupted by a cassette conferring hygromycin resistance is a promising vaccine candidate conferring better protection than Mycobacterium bovis BCG in a mouse model of infection. In this work we describe the construction of a new unmarked mutant in which the entire sigE gene was disrupted in order to fulfill the requirements of the Geneva consensus to enter clinical trials. After showing that the phenotype of this mutant is superimposable to that of the previous one, we further characterized the role of SigE in the M tuberculosis intracellular behavior showing that it is dispensable for replication in human pneumocytes, while it is essential for the arrest of phagosome maturation in THP-1-derived macrophages.