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NorR, as a single-target regulator, has been demonstrated to be involved in NO detoxification in bacteria under anaerobic conditions. Here, the norR gene was identified and deleted in the genome of Vibrio alginolyticus. The results showed that deletion of norR in Vibrio alginolyticus led to lower swarming motility and more biofilm formation on aerobic condition. Moreover, we proved that NorR from E. coli had a similar function in controlling motility. NorR overexpression led to increased resistance to oxidative stress and tetracycline. We also observed a reduced ability of the NorR-overexpressing strain to adapt to iron limitation condition. Transcriptome analysis showed that the genes responsible for bacterial motility and biofilm formation were affected by NorR. The expressions of several sigma factors (RpoS, RpoN, and RpoH) and response regulators (LuxR and MarR) were also controlled by NorR. Furthermore, Chip-qPCR showed that there is a direct binding between NorR and the promoter of rpoS. Based on these results, NorR appears to be a central regulator involved in biofilm formation and swarming motility in Vibrio alginolyticus., (© 2024. The Author(s).)

In cyanobacteria that perform oxygenic photosynthesis, alternative sigma factors can play critical roles in environmental acclimation at the transcriptional initiation step. Here, we found in Synechococcus elongatus PCC 7942 that transcription of the pilA1 gene, encoding the type IV pilin, is dependent on one of the group 3 sigma factors, SigF1. We analyzed the promoter sequence determinants and proposed herein that the -10 and -35 boxes upstream of the transcriptional start site are critical for transcription. Interestingly, while the pilA1 promoter is activated by illumination, RNA polymerase containing SigF1 is already located on the promoter region under dark conditions, prior to illumination. This strongly suggests that promoter activation by light follows the recruitment of RNA polymerase during transcriptional initiation.

Unlike Bacillus subtilis, Streptomyces coelicolor contains nine SigB homologues of the stress-response sigma factor SigB. By using a two-plasmid system, we previously identified promoters recognized by these sigma factors. Almost all promoters were recognized by several SigB homologues. However, no specific sequences of these promoters were found. One of these promoters, ssgBp, was selected to examine this cross-recognition in the native host. It controls the expression of the sporulation-specific gene ssgB. Using a luciferase reporter, the activity of this promoter in S. coelicolor and nine mutant strains lacking individual sigB homologous genes showed that sgBp is dependent on three sigma factors, SigH, SigN, and SigI. To determine which nucleotides in the-10 region are responsible for the selection of a specific SigB homologue, promoters mutated at the last three nucleotide positions were tested in the two-plasmid system. Some mutant promoters were specifically recognized by a distinct set of SigB homologues. Analysis of these mutant promoters in the native host showed the role of these nucleotides. A conserved nucleotide A at position 5 was essential for promoter activity, and two variable nucleotides at positions 4 and 6 were responsible for the partial selectivity of promoter recognition by SigB homologues., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

ECF (extracytoplasmic function) σ factors, members of the σ 70 -family, are the largest class of alternative σ factors which are stimulated in the presence of specific signals and direct RNA polymerase to transcribe a defined subset of genes. Thanks to them, bacterial pathogens can effectively reprogram their gene expression and, consequently, survive in the host and establish infection in a relatively short time. The number of ECF σ factors encoded within bacterial genomes is different depending on a given species and it reflects the likelihood that these bacteria will encounter harsh environmental conditions. The genome of L. interrogans, a zoonotic pathogen responsible for leptospirosis, is predicted to encode 11 ECF σ E -type factors, but none of them have been characterized biochemically to date and their functions are still unknown. Here, we focused on one of the leptospiral ECF σ factors, namely LIC_12757, which was previously found to be up-regulated at elevated temperatures and may be related to the expression of clpB encoding an important L. interrogans virulence factor. We report cloning of the coding sequence of the LIC_12757 gene, its expression with the pET system and biochemical characterization of LIC_12757. By performing EMSA and in vitro transcription assays, we provide strong evidence that LIC_12757 indeed functions as a transcriptional factor that enables RNA polymerase to bind to the specific σ E -type promoter and to initiate transcription. Interestingly, we demonstrate that LIC_12757 is autoregulated at the transcriptional level. Our study is a first step towards determining key aspects of LIC_12757 function in pathogenic Leptospira., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Promoters are key genetic elements in the initiation and regulation of gene expression. A limited number of natural promoters has been described for the control of gene expression in synthetic biology applications. Therefore, synthetic promoters have been developed to fine-tune the transcription for the desired amount of gene product. Mostly, synthetic promoters are characterized using promoter libraries that are constructed via mutagenesis of promoter sequences. The strength of promoters in the library is determined according to the expression of a reporter gene such as gfp encoding green fluorescent protein. Gene expression can be controlled using inducers. The majority of the studies on gram-negative bacteria are conducted using the expression system of the model organism Escherichia coli while that of the model organism Bacillus subtilis is mostly used in the studies on gram-positive bacteria. Additionally, synthetic promoters for the cyanobacteria, which are phototrophic microorganisms, are evaluated, especially using the model cyanobacterium Synechocystis sp. PCC 6803. Moreover, a variety of algorithms based on machine learning methods were developed to characterize the features of promoter elements. Some of these in silico models were verified using in vitro or in vivo experiments. Identification of novel synthetic promoters with improved features compared to natural ones contributes much to the synthetic biology approaches in terms of fine-tuning gene expression., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The specialized sigma factor RpoS mediates a general stress response in Escherichia coli and related bacteria, activating promoters that allow cells to survive stationary phase and many stresses. RpoS synthesis and stability are regulated at multiple levels. Translation of RpoS is positively regulated by multiple small RNAs in response to stress. Degradation of RpoS, dependent upon the adaptor protein RssB, is rapid during exponential growth and ceases upon starvation or other stresses, increasing accumulation of RpoS. E. coli carrying mutations that block the synthesis of polyamines were previously found to have low levels of RpoS, while levels increased rapidly when polyamines were added. We have used a series of reporters to examine the basis for the lack of RpoS in polyamine-deficient cells. The polyamine requirement was independent of small RNA-mediated positive regulation of RpoS translation. Mutations in rssB stabilize RpoS and significantly bypassed the polyamine deficit, suggesting that lack of polyamines might lead to rapid RpoS degradation. However, rates of degradation of mature RpoS were unaffected by polyamine availability. Codon optimization in rpoS partially relieved the polyamine dependence, suggesting a defect in RpoS translation in the absence of polyamines. Consistent with this, a hyperproofreading allele of ribosomal protein S12, encoded by rpsL, showed a decrease in RpoS levels, and this decrease was also suppressed by either codon optimization or blocking RpoS degradation. We suggest that rpoS codon usage leads it to be particularly sensitive to slowed translation, due to either lack of polyamines or hyperproofreading, leading to cotranslational degradation. We dedicate this study to Herb Tabor and his foundational work on polyamines, including the basis for this study., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Published by Elsevier Inc.)

The aim of this investigation was to discover the promoters that drive expression of the sig genes encoding sigma factors of RNA polymerase in Rhodococcus erythropolis CCM2595 and classify these promoters according to the sigma factors which control their activity. To analyze the regulation of major sigma factors, which control large regulons that also contain genes expressed under exponential growth and non-stressed conditions, we used the R. erythropolis CCM2595 culture, which grew rapidly in minimal medium. The transcriptional start sites (TSSs) of the genes sigA, sigB, sigD, sigE, sigG, sigH, sigJ, and sigK were detected by primary 5'-end-specific RNA sequencing. The promoters localized upstream of the detected TSSs were defined by their -35 and -10 elements, which were identical or closely similar to these sequences in the related species Corynebacterium glutamicum and Mycobacterium tuberculosis. Regulation of the promoter activities by different sigma factors was demonstrated by two independent techniques (in vivo and in vitro). All analyzed sig genes encoding the sigma factors with extracytoplasmic function (ECF) were found to be also driven from additional housekeeping promoters. Based on the classification of the sig gene promoters, a model of the basic sigma transcriptional regulatory network in R. erythropolis was designed., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

In contrast to Bacillus subtilis , Streptomyces coelicolor A3(2) contains nine homologues of stress response sigma factor SigB with a major role in differentiation and osmotic stress response. The aim of this study was to further characterize these SigB homologues. We previously established a two-plasmid system to identify promoters recognized by sigma factors and used it to identify promoters recognized by the three SigB homologues, SigF, SigG, and SigH from S. coelicolor A3(2). Here, we used this system to identify 14 promoters recognized by SigB. The promoters were verified in vivo in S. coelicolor A3(2) under osmotic stress conditions in sigB and sigH operon mutants, indicating some cross-recognition of these promoters by these two SigB homologues. This two-plasmid system was used to examine the recognition of all identified SigB-, SigF-, SigG-, and SigH-dependent promoters with all nine SigB homologues. The results confirmed this cross-recognition. Almost all 24 investigated promoters were recognized by two or more SigB homologues and data suggested some distinguishing groups of promoters recognized by these sigma factors. However, analysis of the promoters did not reveal any specific sequence characteristics for these recognition groups. All promoters showed high similarity in the -35 and -10 regions. Immunoblot analysis revealed the presence of SigB under osmotic stress conditions and SigH during morphological differentiation. Together with the phenotypic analysis of sigB and sigH operon mutants in S. coelicolor A3(2), the results suggest a dominant role for SigB in the osmotic stress response and a dual role for SigH in the osmotic stress response and morphological differentiation. These data suggest a complex regulation of the osmotic stress response in relation to morphological differentiation in S. coelicolor A3(2).

Sequence rewriting enables low-cost genome synthesis and the design of biological systems with orthogonal genetic codes. The error-free, robust rewriting of nucleotide sequences can be achieved with a complete annotation of gene regulatory elements. Here, we compare transcription in Caulobacter crescentus to transcription from plasmid-borne segments of the synthesized genome of C. ethensis 2.0. This rewritten derivative contains an extensive amount of supposedly neutral mutations, including 123'562 synonymous codon changes. The transcriptional landscape refines 60 promoter annotations, exposes 18 termination elements and links extensive transcription throughout the synthesized genome to the unintentional introduction of sigma factor binding motifs. We reveal translational regulation for 20 CDS and uncover an essential translational regulatory element for the expression of ribosomal protein RplS. The annotation of gene regulatory elements allowed us to formulate design principles that improve design schemes for synthesized DNA, en route to a bright future of iteration-free programming of biological systems.

Cyanobacterial genomes encode several isoforms of the D1 (PsbA) subunit of Photosystem II (PSII). The distinct regulation of each isoform ensures adaptation under changing environmental conditions. Uncovering the missing elements of signal transduction pathways and psbA gene expression could open new avenues in engineering programs of cyanobacterial strains., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Laboratory strains of Bacillus subtilis encode many alternative sigma factors, each dedicated to expressing a unique regulon such as those involved in stress resistance, sporulation, and motility. The ancestral strain of B. subtilis also encodes an additional sigma factor homolog, ZpdN, not found in lab strains due to being encoded on the large, low-copy-number plasmid pBS32, which was lost during domestication. DNA damage triggers pBS32 hyperreplication and cell death in a manner that depends on ZpdN, but how ZpdN mediates these effects is unknown. Here, we show that ZpdN is a bona fide sigma factor that can direct RNA polymerase to transcribe ZpdN-dependent genes, and we rename ZpdN SigN accordingly. Rend-seq (end-enriched transcriptome sequencing) analysis was used to determine the SigN regulon on pBS32, and the 5' ends of transcripts were used to predict the SigN consensus sequence. Finally, we characterize the regulation of SigN itself and show that it is transcribed by at least three promoters: P sigN1 , a strong SigA-dependent LexA-repressed promoter; P sigN2 , a weak SigA-dependent constitutive promoter; and P sigN3 , a SigN-dependent promoter. Thus, in response to DNA damage SigN is derepressed and then experiences positive feedback. How cells die in a pBS32-dependent manner remains unknown, but we predict that death is the product of expressing one or more genes in the SigN regulon. IMPORTANCE Sigma factors are utilized by bacteria to control and regulate gene expression. Some sigma factors are activated during times of stress to ensure the survival of the bacterium. Here, we report the presence of a sigma factor that is encoded on a plasmid that leads to cellular death after DNA damage., (Copyright © 2019 Burton et al.)

Mycobacterial σ B belongs to the group II family of sigma factors, which are widely considered to transcribe genes required for stationary-phase survival and the response to stress. Here we explored the mechanism underlying the observed hypersensitivity of Δ sigB deletion mutants of Mycobacterium smegmatis , M. abscessus , and M. tuberculosis during exponential growth of mycobacteria that complements the function of the primary sigma factor, σ B during exponential growth of mycobacteria that complements the function of the primary sigma factor, σ A Using chromatin immunoprecipitation sequencing (ChIP-Seq), we show that during exponential phase, σ B binds to over 200 promoter regions, including those driving expression of essential housekeeping genes, like the rRNA gene. ChIP-Seq of ectopically expressed σ A -FLAG demonstrated that at least 61 promoter sites are recognized by both σ A and σ B These results together suggest that RNA polymerase holoenzymes containing either σ A or σ B transcribe housekeeping genes in exponentially growing mycobacteria. The RIF sensitivity of the Δ sigB mutant possibly reflects a decrease in the effective housekeeping holoenzyme pool, which results in susceptibility of the mutant to lower doses of RIF. Consistent with this model, overexpression of σ A restores the RIF tolerance of the Δ sigB mutant to that of the wild type, concomitantly ruling out a specialized role of σ B in RIF tolerance. Although the properties of mycobacterial σ B parallel those of Escherichia coli σ 38 in its ability to transcribe a subset of housekeeping genes, σ B presents a clear departure from the E. coli paradigm, wherein the cellular levels of σ 38 are tightly controlled during exponential growth, such that the transcription of housekeeping genes is initiated exclusively by a holoenzyme containing σ 70 All mycobacteria encode a group II sigma factor, σ 70 , closely related to the group I principal housekeeping sigma factor, σ IMPORTANCE All mycobacteria encode a group II sigma factor, σ B , closely related to the group I principal housekeeping sigma factor, σ A Group II sigma factors are widely believed to play specialized roles in the general stress response and stationary-phase transition in the bacteria that encode them. Contrary to this widely accepted view, we show an additional housekeeping function of σ B that complements the function of σ A in logarithmically growing cells. These findings implicate a novel and dynamic partnership between σ A and σ B in maintaining the expression of housekeeping genes in mycobacteria and can perhaps be extended to other bacterial species that possess multiple group II sigma factors., (Copyright © 2019 Hurst-Hess et al.)

Coxiella burnetii , the etiological agent of Q fever, undergoes a unique biphasic developmental cycle where bacteria transition from a replicating (exponential-phase) large cell variant (LCV) form to a nonreplicating (stationary-phase) small cell variant (SCV) form. The alternative sigma factor RpoS is an essential regulator of stress responses and stationary-phase physiology in several bacterial species, including Legionella pneumophila , which has a developmental cycle superficially similar to that of C. burnetii Here, we used a C. burnetii Δ rpoS mutant to define the role of RpoS in intracellular growth and SCV development. Growth yields following infection of Vero epithelial cells or THP-1 macrophage-like cells with the rpoS mutant in the SCV form, but not the LCV form, were significantly lower than that of wild-type bacteria. RNA sequencing and whole-cell mass spectrometry of the C. burnetii Δ rpoS mutant revealed that a substantial portion of the C. burnetii genome is regulated by RpoS during SCV development. Regulated genes include those involved in stress responses, arginine transport, peptidoglycan remodeling, and synthesis of the SCV-specific protein ScvA. Genes comprising the dot/icm locus, responsible for production of the Dot/Icm type 4B secretion system, were also dysregulated in the rpoS mutant. These data were corroborated with independent assays demonstrating that the C. burnetii Δ rpoS strain has increased sensitivity to hydrogen peroxide and carbenicillin and a thinner cell wall/outer membrane complex. Collectively, these results demonstrate that RpoS is an important regulator of genes involved in C. burnetii SCV development and intracellular growth. IMPORTANCE The Q fever bacterium Coxiella burnetii has spore-like environmental stability, a characteristic that contributes to its designation as a potential bioweapon. Stability is likely conferred by a highly resistant, small cell variant (SCV) stationary-phase form that arises during a biphasic developmental cycle. Here, we define the role of the alternative sigma factor RpoS in regulating genes associated with SCV development. Genes involved in stress responses, amino acid transport, cell wall remodeling, and type 4B effector secretion were dysregulated in the rpoS mutant. Cellular impairments included defects in intracellular growth, cell wall structure, and resistance to oxidants. These results support RpoS as a central regulator of the Coxiella developmental cycle and identify developmentally regulated genes involved in morphological differentiation., (Copyright © 2019 American Society for Microbiology.)

Vibrio splendidus is an important aquatic pathogen that can cause typical symptoms of skin ulceration syndrome (SUS) in sea cucumber Apostichopus japonicus. A metalloprotease Vsm from V. splendidus, has been reported to be an important virulence factor for SUS outbreak. In the present study, the mRNA expression level of vsm was found to correlate to temperature with a peak expression at 28 °C. In contrast, the expression of a sigma factor rpoD, was significantly repressed at 28 °C. A predicted RpoD binding site in the promoter region of vsm revealed the potential regulation of RpoD on vsm expression. Electrophoretic mobility shift assay showed that the purified recombinant RpoD could specifically bind to the promoter region of vsm. Co-transfection of vsm promotor and pT3-rpoD into E. coli significantly inhibited the β-Galactosidase activities in a temperature-dependent manner, and the activities were 0.41-, 0.89- and 0.18-fold at 10 °C, 28 °C and 37 °C compared to the control DH5α/pT3. A rpoD overexpression strain Vs/JRTc rpoD was constructed to further examine the effect of RpoD on the expression of vsm in vivo. By real time RT-PCR analysis, vsm expression level was 0.47-fold in Vs/JRTc rpoD compared to that in Vs/JRTc. Consistently, the metalloprotease activities in Vs/JRTc rpoD was decreased by 18% compared to that in Vs/JRTc. All the results suggested that the sigma factor RpoD, showed a negative regulation on expression of vsm gene by directly interacting with the promoter region of vsm., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The σ I sigma factor from Bacillus subtilis is a σ factor associated with RNA polymerase (RNAP) that was previously implicated in adaptation of the cell to elevated temperature. Here, we provide a comprehensive characterization of this transcriptional regulator. By transcriptome sequencing (RNA-seq) of wild-type (wt) and σ I -null strains at 37°C and 52°C, we identified ∼130 genes affected by the absence of σ I Further analysis revealed that the majority of these genes were affected indirectly by σ I The σ I regulon, i.e., the genes directly regulated by σ I , consists of 16 genes, of which eight (the dhb and yku operons) are involved in iron metabolism. The involvement of σ I in iron metabolism was confirmed phenotypically. Next, we set up an in vitro transcription system and defined and experimentally validated the promoter sequence logo that, in addition to -35 and -10 regions, also contains extended -35 and -10 motifs. Thus, σ I -dependent promoters are relatively information rich in comparison with most other promoters. In summary, this study supplies information about the least-explored σ factor from the industrially important model organism B. subtilis IMPORTANCE In bacteria, σ factors are essential for transcription initiation. Knowledge about their regulons (i.e., genes transcribed from promoters dependent on these σ factors) is the key for understanding how bacteria cope with the changing environment and could be instrumental for biotechnologically motivated rewiring of gene expression. Here, we characterize the σ I regulon from the industrially important model Gram-positive bacterium Bacillus subtilis We reveal that σ I affects expression of ∼130 genes, of which 16 are directly regulated by σ I , including genes encoding proteins involved in iron homeostasis. Detailed analysis of promoter elements then identifies unique sequences important for σ I -dependent transcription. This study thus provides a comprehensive view on this underexplored component of the B. subtilis transcription machinery., (Copyright © 2018 American Society for Microbiology.)

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, σ G and σ E , during spore formation by Bacillus subtilis. CsfB assembles into a symmetrical homodimer, with each monomer bound to a single Zn 2+ ion via a treble-clef zinc finger fold. Directed mutagenesis indicates that dimer formation is critical for CsfB-mediated inhibition of both σ G and σ E , 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., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Activation of signaling cascades is critical for Mycobacterium tuberculosis (Mtb) to adapt the macrophage lifestyle. Parallel to several signal systems, sigma factor systems, especially the extra-cytoplasmic function sigma factors, are crucial for Mtb signaling. Most sigma factors lack a signal sensory domain and often are activated by various proteins that perceive the environmental cues and relay the signals through variegated post-translational modifications via the activity of antisigma factor, protein kinase and related transcriptional regulators. Antisigma factors are further controlled by multiple mechanisms. SigK senses the environmental redox state directly. Phosphorylation and lysine acetylation added another dimension to the regulatory hierarchy. This review will provide insights into Mtb pathogenesis, and lay the foundation for the discovery of novel approaches for therapeutic interventions.

In bacteria, one primary and multiple alternative sigma (σ) factors associate with the RNA polymerase core enzyme (E) to form holoenzymes (Eσ) with different promoter recognition specificities. The alternative σ factor RpoS/σ S is produced in stationary phase and under stress conditions and reprograms global gene expression to promote bacterial survival. To date, the three-dimensional structure of a full-length free σ factor remains elusive. The current model suggests that extensive interdomain contacts in a free σ factor result in a compact conformation that masks the DNA-binding determinants of σ, explaining why a free σ factor does not bind double-stranded promoter DNA efficiently. Here, we explored the solution conformation of σ S using amide hydrogen/deuterium exchange coupled with mass spectrometry, NMR, analytical ultracentrifugation and molecular dynamics. Our data strongly argue against a compact conformation of free σ S Instead, we show that σ S adopts an open conformation in solution in which the folded σ 2 and σ 4 domains are interspersed by domains with a high degree of disorder. These findings suggest that E binding induces major changes in both the folding and domain arrangement of σ S and provide insights into the possible mechanisms of regulation of σ S activity by its chaperone Crl., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The model organism, Escherichia coli, contains a total of more than 4,500 genes, but the total number of RNA polymerase (RNAP) core enzyme or the transcriptase is only about 2,000 molecules per genome. The regulatory targets of RNAP are, however, modulated by changing its promoter selectivity through two-steps of protein-protein interplay with 7 species of the sigma factor in the first step, and then 300 species of the transcription factor (TF) in the second step. Scientists working in the field of prokaryotic transcription in Japan have made considerable contributions to the elucidation of genetic frameworks and regulatory modes of the genome transcription in E. coli K-12. This review summarizes the findings by this group, first focusing on three sigma factors, the stationary-phase sigma RpoS, the heat-shock sigma RpoH, and the flagellar-chemotaxis sigma RpoF, as examples. It also presents an overview of the current state of the systematic research being carried out to identify the regulatory functions of all TFs from a single and the same bacterium E. coli K-12, using the genomic SELEX and PS-TF screening systems. All these studies have been undertaken with the aim of understanding the genome regulation in E. coli K-12 as a whole.

The plant symbiotic α-proteobacterium Sinorhizobium meliloti has two RpoH-type sigma factors, RpoH1 and RpoH2. The former induces the synthesis of heat shock proteins and optimizes interactions with the host. Using a Western blot analysis, we examined time course changes in the intracellular contents of these factors upon a temperature upshift. The RpoH1 level was relatively high and constant, suggesting that its regulatory role in the heat shock response is attained through the activation of the pre-existing RpoH1 protein. In contrast, the RpoH2 level was initially undetectable, and gradually increased. These differential patterns reflect the functional diversification of these factors.

Sigma (σ) factors direct gene transcription by binding to and determining the promoter recognition specificity of RNA polymerase (RNAP) in bacteria. Genes transcribed under the control of alternative sigma factors allow cells to respond to stress and undergo developmental processes, such as sporulation in Bacillus subtilis , in which gene expression is controlled by a cascade of alternative sigma factors. Binding of sigma factors to RNA polymerase depends on the coiled-coil (or clamp helices) motif of the β' subunit. We have identified an amino acid substitution (L257P) in the coiled coil that markedly inhibits the function of σ H , the earliest-acting alternative sigma factor in the sporulation cascade. Cells with this mutant RNAP exhibited an early and severe block in sporulation but not in growth. The mutant was strongly impaired in σ H -directed gene expression but not in the activity of the stress-response sigma factor σ B Pulldown experiments showed that the mutant RNAP was defective in associating with σ H but could still associate with σ A and σ B The differential effects of the L257P substitution on sigma factor binding to RNAP are likely due to a conformational change in the β' coiled coil that is specifically detrimental for interaction with σ H This is the first example, to our knowledge, of an amino acid substitution in RNAP that exhibits a strong differential effect on a particular alternative sigma factor. IMPORTANCE In bacteria, all transcription is mediated by a single multisubunit RNA polymerase (RNAP) enzyme. However, promoter-specific transcription initiation necessitates that RNAP associates with a σ factor. Bacteria contain a primary σ factor that directs transcription of housekeeping genes and alternative σ factors that direct transcription in response to environmental or developmental cues. We identified an amino acid substitution (L257P) in the B. subtilis β' subunit whereby RNAP L257P associates with some σ factors (σ A and σ B ) and enables vegetative cell growth but is defective in utilization of σ H and is consequently blocked for sporulation. To our knowledge, this is the first identification of an amino acid substitution within the core enzyme that affects utilization of a specific sigma factor., (Copyright © 2017 American Society for Microbiology.)

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, which kills 1.8 million annually. Mtb RNA polymerase (RNAP) is the target of the first-line antituberculosis drug rifampin (Rif). We report crystal structures of Mtb RNAP, alone and in complex with Rif, at 3.8-4.4 Å resolution. The results identify an Mtb-specific structural module of Mtb RNAP and establish that Rif functions by a steric-occlusion mechanism that prevents extension of RNA. We also report non-Rif-related compounds-Nα-aroyl-N-aryl-phenylalaninamides (AAPs)-that potently and selectively inhibit Mtb RNAP and Mtb growth, and we report crystal structures of Mtb RNAP in complex with AAPs. AAPs bind to a different site on Mtb RNAP than Rif, exhibit no cross-resistance with Rif, function additively when co-administered with Rif, and suppress resistance emergence when co-administered with Rif., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Brucella abortus σ E1 is an EcfG family sigma factor that regulates the transcription of dozens of genes in response to diverse stress conditions and is required for maintenance of chronic infection in a mouse model. A putative ATP-binding cassette transporter operon, bab1_0223-bab1_0226 , is among the most highly activated gene sets in the σ E1 regulon. The proteins encoded by the operon resemble quaternary ammonium-compatible solute importers but are most similar in sequence to the broadly conserved YehZYXW system, which remains largely uncharacterized. Transcription of yehZYXW is activated by the general stress sigma factor σ S in Enterobacteriaceae , which suggests a functional role for this transport system in bacterial stress response across the classes Alphaproteobacteria and Gammaproteobacteria We present evidence that B. abortus YehZYXW does not function as an importer of known compatible solutes under physiological conditions and does not contribute to the virulence defect of a σ E1 -null strain. The sole in vitro phenotype associated with genetic disruption of this putative transport system is reduced growth in the presence of high Li + ion concentrations. A crystal structure of B. abortus YehZ revealed a class II periplasmic binding protein fold with significant structural homology to Archaeoglobus fulgidus ProX, which binds glycine betaine. However, the structure of the YehZ ligand-binding pocket is incompatible with high-affinity binding to glycine betaine. This is consistent with weak measured binding of YehZ to glycine betaine and related compatible solutes. We conclude that YehZYXW is a conserved, stress-regulated transport system that is phylogenetically and functionally distinct from quaternary ammonium-compatible solute importers. IMPORTANCE Brucella abortus σ E1 regulates transcription in response to stressors encountered in its mammalian host and is necessary for maintenance of chronic infection in a mouse model. The functions of the majority of genes regulated by σ E1 remain undefined. We present a functional/structural analysis of a conserved putative membrane transport system (YehZYXW) whose expression is strongly activated by σ E1 Though annotated as a quaternary ammonium osmolyte uptake system, experimental physiological studies and measured ligand-binding properties of the periplasmic binding protein (PBP), YehZ, are inconsistent with this function. A crystal structure of B. abortus YehZ provides molecular insight into differences between bona fide quaternary ammonium osmolyte importers and YehZ-related proteins, which form a distinct phylogenetic and functional group of PBPs., (Copyright © 2017 American Society for Microbiology.)

Bacterial small RNAs (sRNAs) have been implicated in various aspects of post-transcriptional gene regulation. Here, we demonstrate that sRNAs also act at the level of transcription termination. We use the rpoS gene, which encodes a general stress sigma factor σ(S), as a model system, and show that sRNAs DsrA, ArcZ, and RprA bind the rpoS 5'UTR to suppress premature Rho-dependent transcription termination, both in vitro and in vivo. sRNA-mediated antitermination markedly stimulates transcription of rpoS during the transition to the stationary phase of growth, thereby facilitating a rapid adjustment of bacteria to global metabolic changes. Next generation RNA sequencing and bioinformatic analysis indicate that Rho functions as a global "attenuator" of transcription, acting at the 5'UTR of hundreds of bacterial genes, and that its suppression by sRNAs is a widespread mode of bacterial gene regulation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

To survive in the periodontal pocket, Porphyromonas gingivalis, the main causative agent of periodontal disease, must overcome oxidative and nitric oxide (NO) stress. Previously, we reported that, in the presence of NO comparable to stress conditions, the transcriptome of P. gingivalis was differentially expressed, and genes belonging to the PG1178-81 cluster were significantly upregulated. To further evaluate their role(s) in NO stress resistance, these genes were inactivated by allelic exchange mutagenesis. Isogenic mutants P. gingivalis FLL460 (ΔPG1181::ermF) and FLL461 (ΔPG1178-81::ermF) were black-pigmented, with gingipain and hemolytic activities comparable to that of the wild-type strain. Whereas the recovery of these isogenic mutants from NO stress was comparable to the wild-type, there was increased sensitivity to hydrogen peroxide-induced stress. RNA-Seq analysis under conditions of NO stress showed that approximately 5 and 8% of the genome was modulated in P. gingivalis FLL460 and FLL461, respectively. The PG1178-81 gene cluster was shown to be part of the same transcriptional unit and is inducible in response to NO stress. In the presence of NO, PG1181, a putative transcriptional regulator, was shown to bind to its own promoter region and that of several other NO responsive genes including PG0214 an extracytoplasmic function σ factor, PG0893 and PG1236. Taken together, the data suggest that PG1181 is a NO responsive transcriptional regulator that may play an important role in the NO stress resistance regulatory network in P. gingivalis., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Formation of a division septum near a randomly chosen pole during sporulation in Bacillus subtilis creates unequal sized daughter cells with dissimilar programs of gene expression. An unanswered question is how polar septation activates a transcription factor (σ(F)) selectively in the small cell. We present evidence that the upstream regulator of σ(F), the phosphatase SpoIIE, is compartmentalized in the small cell by transfer from the polar septum to the adjacent cell pole where SpoIIE is protected from proteolysis and activated. Polar recognition, protection from proteolysis, and stimulation of phosphatase activity are linked to oligomerization of SpoIIE. This mechanism for initiating cell-specific gene expression is independent of additional sporulation proteins; vegetative cells engineered to divide near a pole sequester SpoIIE and activate σ(F) in small cells. Thus, a simple model explains how SpoIIE responds to a stochastically-generated cue to activate σ(F) at the right time and in the right place.

Staphylococcus aureus is a major human pathogen. Hospital infections caused by methicillin-resistant strains (MRSA), which have acquired resistance to a broad spectrum of antibiotics through horizontal gene transfer (HGT), are of particular concern. In S. aureus, virulence and antibiotic resistance genes are often encoded on mobile genetic elements that are disseminated by HGT. Conjugation and phage transduction have long been known to mediate HGT in this species, but it is unclear whether natural genetic transformation contributes significantly to the process. Recently, it was reported that expression of the alternative sigma factor SigH induces the competent state in S. aureus. The transformation efficiency obtained, however, was extremely low, indicating that the optimal conditions for competence development had not been found. We therefore used transcriptome sequencing to determine whether the full set of genes known to be required for competence in other naturally transformable bacteria is part of the SigH regulon. Our results show that several essential competence genes are not controlled by SigH. This presumably explains the low transformation efficiency previously reported, and demonstrates that additional regulating mechanisms must be involved. We found that one such mechanism involves ComK1, a transcriptional activator that acts synergistically with SigH., (© 2014 John Wiley & Sons Ltd.)

The survival of bacteria to different environmental conditions depends on the activation of adaptive mechanisms, which are intricately driven through gene regulation. Because transcriptional initiation is considered to be the major step in the control of bacterial genes, we discuss the characteristics and roles of the sigma factors, addressing (1) their structural, functional and phylogenetic classification; (2) how their activity is regulated; and (3) the promoters recognized by these factors. Finally, we focus on a specific group of alternative sigma factors, the so-called σ(ECF) factors, in Bacillus subtilis and some of the main species that comprise the CMNR group, providing information on the roles they play in the microorganisms' physiology and indicating some of the genes whose transcription they regulate.

Primer extension with RNA from an RNase III null mutant of Streptomyces coelicolor M145 and a primer complementary to the polynucleotide phosphorylase gene revealed two major extension products. Two different extension products were observed using RNA from either wild type M145 or the null mutant with a primer complementary to rpsO. Mapping of the 5'-ends of these extension products to the rpsO-pnp intergenic region indicated that all four putative transcription start sites were preceded by possible promoter sequences. These putative promoters were synthesized by the PCR and cloned into pIPP2, a xylE-based streptomycete promoter probe vector. Transfer of the pIPP2 derivatives to S. coelicolor and catechol dioxygenase assays demonstrated that all four cloned fragments had promoter activity in vivo. The activities of the four promoters changed over the course of growth of S. coelicolor and studies in three sigma factor mutant strains demonstrated that three of the promoters were σ(B) dependent. Northern blotting studies showed that the levels of the rpsO-pnp transcripts remained relatively constant over the course of growth of S. coelicolor M145, but that on a molar basis, the levels of the readthrough and pnp transcripts were considerably lower than those of rpsO. PNPase is a cold shock protein in S. coelicolor and the activity of the rpsO-pnp promoters increased during cold shock at 10°, resulting in a two-fold increase in PNPase activity, compared with the activity at 30°., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Adjustment of gene expression during acclimation to stress conditions, such as bright light, in the cyanobacterium Synechocystis sp. PCC 6803 depends on four group 2 σ factors (SigB, SigC, SigD, SigE). A ΔsigCDE strain containing the stress-responsive SigB as the only functional group 2 σ factor appears twice as resistant to photoinhibition of photosystem II (PSII) as the control strain. Microarray analyses of the ΔsigCDE strain indicated that 77 genes in standard conditions and 79 genes in high light were differently expressed compared with the control strain. Analysis of possible photoprotective mechanisms revealed that high carotenoid content and up-regulation of the photoprotective flavodiiron operon flv4-sll0218-flv2 protected PSII in ΔsigCDE, while up-regulation of pgr5-like, hlipB or isiA genes in the mutant strain did not offer particular protection against photoinhibition. Photoinhibition resistance was lost if ΔsigCDE was grown in high CO2, where carotenoid and Flv4, Sll0218, and Flv2 contents were low. Additionally, photoinhibition resistance of the ΔrpoZ strain (lacking the omega subunit of RNA polymerase), with high carotenoid but low Flv4-Sll0218-Flv2 content, supported the importance of carotenoids in PSII protection. Carotenoids likely protect mainly by quenching of singlet oxygen, but efficient nonphotochemical quenching in ΔsigCDE might offer some additional protection. Comparison of photoinhibition kinetics in control, ΔsigCDE, and ΔrpoZ strains showed that protection by the flavodiiron operon was most efficient during the first minutes of high-light illumination.

The bacterial flagellum is a complex molecular machine that is assembled by more than 30 proteins and is rotated to propel cells either through liquids or over solid surfaces. Flagellar gene expression is extensively regulated to co-ordinate flagellar assembly in both space and time. In Bacillus subtilis, the proteins of unknown function, SwrA and SwrB, and the alternative sigma factor σ(D) are required to activate expression of the flagellar filament protein, flagellin. Here we determine that in the absence of SwrA and SwrB, the phosphorylated form of the response regulator DegU inhibits σ(D) -dependent gene expression indirectly by binding to the P(flgM) promoter region and activating expression of the anti-sigma factor FlgM. We further demonstrate that DegU-P-dependent activation of FlgM is essential to inhibit flagellin expression when flagellar basal body assembly is disrupted. Regulation of FlgM is poorly understood outside of Salmonella, and differential control of FlgM expression may be a common means of coupling flagellin expression to flagellar assembly., (© 2011 Blackwell Publishing Ltd.)

Streptomyces are bacteria of industrial interest whose genome contains more than 73% of bases GC. In order to define, in these GC-rich bacteria, specific sequence features of strong promoters, a library of synthetic promoters of various sequence composition was constructed in Streptomyces. To do so, the sequences located upstream, between and downstream of the -35 and -10 consensus promoter sequences were completely randomized and some variability was introduced in the -35 (position 6) and -10 (positions 3, 4 and 5) hexamers recognized by the major vegetative sigma factor HrdB. The synthetic promoters were cloned into the promoter-probe plasmid pIJ487 just upstream of the promoter-less aphII gene that confers resistance to neomycin. This synthetic promoter library was transformed into Streptomyces lividans, and the resulting transformants were screened for their ability to grow in the presence of different concentrations of neomycin (20, 50, and 100 μgml(-1)). Promoter strengths varied up to 12-fold, in small increments of activity increase, as determined by reverse transcriptase-PCR. This collection of promoters of various strengths can be useful for the fine-tuning of gene expression in genetic engineering projects. Thirty-eight promoters were sequenced, and the sequences of the 14 weakest and 14 strongest promoters were compared using the WebLogo software with small sample correction. This comparison revealed that the -10 box, the -10 extended motif as well as the spacer of the strong Streptomyces promoters are more G rich than those of the weak promoters., (© Springer-Verlag 2011)

The Mycobacterium marinum G13 promoter is a sigma 70-like promoter that is more active by green fluorescent protein (gfp) differential fluorescence induction (DFI) assays when M. marium resides in an intracellular compartment as compared with growth in media. In assays using DFI, we found that the mycobacterial G13 promoter was also more active in a background of lower nutrient availability during logarithmic growth. This promoter, contained in an insert cloned upstream of a gfp reporter gene, is also active in Escherichia coli. When gfp expression assays of different plasmid constructs were performed in parallel with E. coli and M. marinum, expression in E. coli was maintained after deletion of both upstream and/or downstream regions proximal to the core promoter sequence. In M. marinum, however, although upstream deletions had no appreciable effect on gfp expression, promoter constructs with deleted downstream regions expressed 20- to 40-fold less gfp over all growth phases. The high-level expression of gfp was restored, however, in a clone containing approximately 100 bp downstream of the transcriptional start point. We have therefore utilized this gfp reporter assay of promoter activity to distinguish possible differences in requirements for gfp expression between different genera that utilize sigma 70-like promoter elements. We found that high levels of expression of gfp from the G13 promoter in M. marinum require downstream regions not necessary for gfp expression in E. coli.

The DNA binding protein from starved cells (Dps) is a general stress protein that provides Escherichia coli protection from osmotic, oxidative, and acid stresses. While Dps production and accumulation is primarily associated with stationary phase, during log phase, this protein protects against oxidative stress in an OxyR-dependent manner. In this study, evidence is provided that expands the role of Dps in acid tolerance to both log- and stationary-phase E. coli O157:H7. The transcription of dps occurred in log-phase cells without OxyR or stress and was upregulated during entry into stationary phase. The expression in log and stationary phase involved sigma70 and sigmas, respectively, with both sigma factors recognizing the same promoter region. Site-directed mutagenesis identified an extended -10 region that was essential to both sigma70 and sigmas transcription of dps. cAMP receptor protein (CRP) was found to repress dps expression as a crp mutant had a significant increase in the dps mRNA level. However, a CRP binding site was not found in the dps promoter and upregulation of dps in the crp mutant was absent in a crp rpoS double mutant. The findings from this study demonstrated that dps was expressed at a basal level during growth, both sigma70- and sigmas-driven transcription required an extended -10, and CRP repression is mediated through the alternative sigma factor sigmas (rpoS).

Lactobacilli and bifidobacteria are important members of the gastrointestinal microflora of humans and animals and are thought to have positive effects on human health. Therefore, there is an increasing interest in using these microorganisms as probiotics to be incorporated into either fermented dairy products or tablets. However, convincing scientific data that support claims of their health benefits are scarce. The effect of cell-free extracts of milk fermented by 10 probiotic bacteria (five Bifidobacterium strains and five Lactobacillus strains) on the expression of the flaA gene of Campylobacter jejuni was assessed using a fusion between the flaA sigma28 promoter and a promoterless luxCDABE cassette carried on the plasmid pRYluxCDABE, which resulted in strains with quantifiable luminescence linked to flaA sigma28 promoter activity. Cell-free extracts of milk fermented by all of the tested probiotic strains inhibited the growth of the C. jejuni and down-regulatedflaA sigma28 promoter activity. Two nonprobiotic lactic acid bacterial strains, Lactococcus lactis and Streptococcus thermophilus, were less inhibitory.

Two chlamydial homologues of the Yersinia lcrH chaperone for type III secretion system structural components are present within separate gene clusters. Quantitative transcriptional analyses demonstrated that each cluster is differentially regulated and expressed as an operon using major sigma factor elements, suggesting the presence of more elaborate developmental regulation mechanisms in chlamydiae.

Translational repression results from a complex choreography of macromolecular interactions interfering with the formation of translational initiation complexes. The relationship between the rate and extent of formation of these interactions to form repressed mRNA complexes determines the extent of repression. A novel analysis of repression mechanisms is presented here and it indicates that the reversibility of repressed complex formation influences the steady state balance of the distribution of translationally active and inactive complexes and therefore has an impact on the efficiency of repression. Reviewed here is evidence for three distinct translational repression mechanisms, regulating expression of the transcription factor sigma32, threonine tRNA synthetase and ribosomal proteins on the alpha operon in Escherichia coli. Efficient regulation of expression in these systems makes use of specific mRNA structures in quite different ways.

The temporal and spatial expression of the yqfS gene of Bacillus subtilis, which encodes a type IV apurinic/apyrimidinic endonuclease, was studied. A reporter gene fusion to the yqfS opening reading frame revealed that this gene is not transcribed during vegetative growth but is transcribed during the last steps of the sporulation process and is localized to the developing forespore compartment. In agreement with these results, yqfS mRNAs were mainly detected by both Northern blotting and reverse transcription-PCR, during the last steps of sporulation. The expression pattern of the yqfS-lacZ fusion suggested that yqfS may be an additional member of the Esigma(G) regulon. A primer extension product mapped the transcriptional start site of yqfS, 54 to 55 bp upstream of translation start codon of yqfS. Such an extension product was obtained from RNA samples of sporulating cells but not from those of vegetatively growing cells. Inspection of the nucleotide sequence lying upstream of the in vivo-mapped transcriptional yqfS start site revealed the presence of a sequence with good homology to promoters preceding genes of the sigma(G) regulon. Although yqfS expression was temporally regulated, neither oxidative damage (after either treatment with paraquat or hydrogen peroxide) nor mitomycin C treatment induced the transcription of this gene.

We have modified an assay using two compatible vectors that coexist in Escherichia coli cells and applied it in the investigation of the transcriptional activity of Spo0A, a key regulator of sporulation in Bacillus subtilis. We have chosen the promoters of the Spo0A dependent genes, spoIIE and spoIIA, involved in sporulation, in order to study the transcription activity solely of the DNA binding domain of Spo0A. We have prepared the two-vector system so that one vector contained the cloned C-Spo0A under the control of an inducible promoter, and the second vector (the promoter probe vector), was composed of the Spo0A dependent spoIIE and spoIIA promoters. Using this two-vector system in E. coli, we proved that C-Spo0A is able to interact with the E. coli transcription apparatus, recognizes both promoters and activates transcription from these promoters., (Copyright 2002 Elsevier Science B.V.)

In the course of a Bacillus subtilis functional genomics project which involved screening for sporulation genes, we identified an open reading frame, yaaT, whose disruptant exhibits a sporulation defect. Twenty-four hours after the initiation of sporulation, most cells of the yaaT mutant exhibited stage 0 of sporulation, indicating that the yaaT mutation blocks sporulation at an early stage. Furthermore, the mutation in yaaT led to a significant decrease in transcription from a promoter controlled by Spo0A, a key response regulator required for the initiation of sporulation. However, neither the level of transcription of spo0A, the activity of sigma(H), which transcribes spo0A, nor the amount of Spo0A protein was severely affected by the mutation in yaaT. Bypassing the phosphorelay by introducing an spo0A mutation (sof-1) into the yaaT mutant suppressed the sporulation defect, suggesting that the yaaT mutation interferes with the phosphorelay process comprising Spo0F, Spo0B, and histidine kinases. We also observed that mutation of spo0E, which encodes the phosphatase that dephosphorylates Spo0A-P, suppressed the sporulation defect in the yaaT mutant. These results strongly suggest that yaaT plays a significant role in the transduction of signals to the phosphorelay for initiation of sporulation. Micrographs indicated that YaaT-green fluorescent protein localizes to the peripheral membrane, as well as to the septum, during sporulation.

Escherichia coli uses overlapping envelope stress responses to adapt to insults to the bacterial envelope that cause protein misfolding. The sigmaE and Cpx envelope stress responses are activated by both common and distinct envelope stresses and respond by increasing the expression of the periplasmic protease DegP as well as target genes unique to each response. The sigmaE pathway is involved in outer membrane protein (OMP) folding quality control whereas the Cpx pathway plays an important role in the assembly of at least one pilus. Previously, we identified the spy gene as a new Cpx regulon member of unknown function. Interestingly, induction of spy expression by severe envelope stresses such as spheroplasting is only partially dependent on an intact Cpx signalling pathway, unlike other Cpx-regulated genes. Here we show that the BaeS sensor kinase and BaeR response regulator also control expression of spy in response to envelope stress. BaeS and BaeR do not affect expression of other known Cpx-regulated genes, however, baeR cpxR double mutants show increased sensitivity to envelope stresses relative to either single mutant alone. We propose that the Bae signal transduction pathway controls a third envelope stress response in E. coli that induces expression of a distinct set of adaptive genes.

Silencing of the Escherichia coli bgl operon is mediated by histone-like protein H-NS and affected by other pleiotropic regulators, including sigma factor RpoS. Silencing is relieved and the bgl operon is activated in hns mutants and by mutations that map in the vicinity of the bgl promoter. However, the expression level of activated bgl operon derivatives varies with the strain background. Here it is shown that the repression of the bgl operon by RpoS requires Crl. Crl is a protein that is necessary for the RpoS-dependent expression of the csgBA operon and that enhances the expression of other RpoS-dependent genes. In a Crl-negative strain RpoS had no effect on the bgl operon. The crl gene maps close to the proBA locus in the lac operon region and is deleted in many commonly used E. coli strains. Crl may therefore account for some of the observed strain-dependent variations of bgl operon expression levels and effects of pleiotropic regulators on bgl operon regulation.

The HpmA haemolysin toxin of Proteus mirabilis is encoded by the hpmBA locus and its production is upregulated co-ordinately with the synthesis and assembly of flagella during differentiation into hyperflagellated swarm cells. Primer extension identified a sigma(70) promoter upstream of hpmB that was upregulated during swarming. Northern blotting indicated that this promoter region was also required for concomitant transcription of the immediately distal hpmA gene, and that the unstable hpmBA transcript generated a stable hpmA mRNA and an unstable hpmB mRNA. Transcriptional luxAB fusions to the DNA regions 5' of the hpmB and hpmA genes confirmed that hpmB sigma(70) promoter activity increased in swarm cells, and that there was no independent hpmA promoter. Increased transcription of the hpmBA operon in swarm cells was dependent upon a 125 bp sequence 5' of the sigma(70) promoter -35 hexamer. This sequence spans multiple putative binding sites for the leucine-responsive regulatory protein (Lrp), and band-shift assays with purified Lrp confirmed the presence of at least two such sites. The influence on hpmBA expression of the key swarming positive regulators FlhD(2)C(2) (encoded by the flagellar master operon), Lrp, and the membrane-located upregulator of the master operon, UmoB, was examined. Overexpression of each of these regulators moderately increased hpmBA transcription in wild-type P. mirabilis, and the hpmBA operon was not expressed in any of the flhDC, lrp or umoB mutants. Expression in the mutants was not recovered by cross-complementation, i.e. by overexpression of FlhD(2)C(2), Lrp or UmoB. Expression of the zapA protease virulence gene, which like hpmBA is also upregulated in swarm cells, did not require Lrp, but like flhDC it was upregulated by UmoB. The results indicate intersecting pathways of control linking virulence gene expression and swarm cell differentiation.

The response regulator Spo0A is the master control element in the initiation of sporulation in Bacillus subtilis. Like many other multi-domain response regulators, the latent activity of the effector, C-terminal domain is stimulated by phosphorylation on a conserved aspartic acid residue in the regulatory, N-terminal domain. If a threshold concentration of phosphorylated Spo0A is achieved, the transcription of genes required for sporulation is activated, whereas the genes encoding stationary phase sentinels are repressed, and sporulation proceeds. Despite detailed genetic, biochemical and structural characterisation, it is not understood how the phosphorylation signal in the receiver domain is transduced into DNA binding and transcription activation in the distal effector domain. An obstacle to our understanding of Spo0A function is the uncertainty concerning changes in quaternary structure that accompany phosphorylation. Here we have revisited this question and shown unequivocally that Spo0A forms dimers upon phosphorylation and that the subunit interactions in the dimer are mediated principally by the receiver domain. Purified dimers of two mutants of Spo0A, in which the phosphorylatable aspartic acid residue has been substituted, activate transcription from the spoIIG promoter in vitro, whereas monomers do not. This suggests that dimers represent the activated form of Spo0A., (Copyright 2002 Elsevier Science Ltd.)

Recent studies have shown that, early during sporulation in Bacillus subtilis, the temporary exclusion of 70% of the chromosome from the forespore compartment is critical to the regulated activation of two major transcription factors, sigma(F) and sigma(E).

A variety of stress situations may affect the activity and survival of plant-beneficial pseudomonads added to soil to control root diseases. This study focused on the roles of the sigma factor AlgU (synonyms, AlgT, RpoE, and sigma(22)) and the anti-sigma factor MucA in stress adaptation of the biocontrol agent Pseudomonas fluorescens CHA0. The algU-mucA-mucB gene cluster of strain CHA0 was similar to that of the pathogens Pseudomonas aeruginosa and Pseudomonas syringae. Strain CHA0 is naturally nonmucoid, whereas a mucA deletion mutant or algU-overexpressing strains were highly mucoid due to exopolysaccharide overproduction. Mucoidy strictly depended on the global regulator GacA. An algU deletion mutant was significantly more sensitive to osmotic stress than the wild-type CHA0 strain and the mucA mutant were. Expression of an algU'-'lacZ reporter fusion was induced severalfold in the wild type and in the mucA mutant upon exposure to osmotic stress, whereas a lower, noninducible level of expression was observed in the algU mutant. Overexpression of algU did not enhance tolerance towards osmotic stress. AlgU was found to be essential for tolerance of P. fluorescens towards desiccation stress in a sterile vermiculite-sand mixture and in a natural sandy loam soil. The size of the population of the algU mutant declined much more rapidly than the size of the wild-type population at soil water contents below 5%. In contrast to its role in pathogenic pseudomonads, AlgU did not contribute to tolerance of P. fluorescens towards oxidative and heat stress. In conclusion, AlgU is a crucial determinant in the adaptation of P. fluorescens to dry conditions and hyperosmolarity, two major stress factors that limit bacterial survival in the environment.

In Pseudomonas syringae strains, the hrp-hrc pathogenicity island consists of an HrpL-dependent regulon that encodes a type III protein translocation complex and translocated effector proteins required for pathogenesis. HrpR and HrpS function as positive regulatory factors for the hrpL promoter, but their mechanism of action has not been established. Both HrpR and HrpS are structurally related to enhancer-binding proteins, but they lack receiver domains and do not appear to require a cognate protein kinase for activity. hrpR and hrpS were shown to be expressed as an operon: a promoter was identified 5' to hrpR, and reverse transcriptase PCR detected the presence of an hrpRS transcript. The hrpR promoter and coding sequence were conserved among P. syringae strains. The coding sequences for hrpR and hrpS were cloned into compatible expression vectors, and their activities were monitored in Escherichia coli transformants carrying an hrpL'-lacZ fusion. HrpS could function as a weak activator of the hrpL promoter, but the activity was only 2.5% of the activity detected when both HrpR and HrpS were expressed in the reporter strain. This finding is consistent with a requirement for both HrpR and HrpS in the activation of the hrpL promoter. By using a yeast two-hybrid assay, an interaction between HrpR and HrpS was detected, suggestive of the formation of a heteromeric complex. Physical interaction of HrpR and HrpS was confirmed by column-binding experiments. The results show that HrpR and HrpS physically interact to regulate the sigma(54)-dependent hrpL promoter in P. syringae strains.

RpoH (Escherichia coli sigma(32) and its homologs) is the central regulator of the heat shock response in gram-negative proteobacteria. Here we studied salient regulatory features of RpoH in Agrobacterium tumefaciens by examining its synthesis, stability, and activity while increasing the temperature from 25 to 37 degrees C. Heat induction of RpoH synthesis occurred at the level of transcription from an RpoH-dependent promoter, coordinately with that of DnaK, and followed by an increase in the RpoH level. Essentially normal induction of heat shock proteins was observed even with a strain that was unable to increase the RpoH level upon heat shock. Moreover, heat-induced accumulation of dnaK mRNA occurred without protein synthesis, showing that preexisting RpoH was sufficient for induction of the heat shock response. These results suggested that controlling the activity, rather than the amount, of RpoH plays a major role in regulation of the heat shock response. In addition, increasing or decreasing the DnaK-DnaJ chaperones specifically reduced or enhanced the RpoH activity, respectively. On the other hand, the RpoH protein was normally stable and remained stable during the induction phase but was destabilized transiently during the adaptation phase. We propose that the DnaK-mediated control of RpoH activity plays a primary role in the induction of heat shock response in A. tumefaciens, in contrast to what has been found in E. coli.

The rpoH gene of Pseudomonas putida KT2440 encoding the heat-shock sigma factor sigma(32) was cloned and sequenced, and the translated gene product was predicted to be a protein of 32.5 kDa. The unambiguous role of the gene as a sigma factor was confirmed because the cloned P. putida gene complemented the growth defect, at 37 and 42 degrees C, of an Escherichia coli rpoH mutant strain. Primer extension analysis showed that in P. putida the rpoH gene is expressed from three promoters in cells growing at 30 degrees C. Two of them, P1 and P3, share homology with the sigma(70)-dependent promoters, while the third one, P2, shows a typical sigma(24)-consensus sequence. The pattern of transcription initiation of the rpoH gene did not change in response to different stresses, i.e. a sudden heat shock or the addition of aromatic compounds. However, the predicted secondary structure of the 5' region of the mRNA derived from the three different promoters suggests regulation at the level of translation efficiency and/or mRNA half-life. An inverted repeat sequence located 20 bp downstream of the rpoH stop codon was shown to function as a terminator in vivo in P. putida growing at temperatures from 18 to 42 degrees C.

Mycobacterium tuberculosis has demonstrated remarkable ability to survive in diverse conditions encountered during the infection process. These involve surviving the bactericidal stresses within the macrophage, the anaerobic and nutritionally altered environment of the granuloma, and the metabolically inactive latent state. Understanding the molecular basis of this adaptive behavior lies in the identification of genes (or virulence determinants) specifically expressed under these varied conditions. Transcriptional control plays a key role in regulating gene expression in response to environmental signals. However, even after decades of investigation our knowledge about the function of these regulatory mechanisms in mycobacteria remains meagre. But the elucidation of the genome sequence and implementation of sophisticated molecular genetic approaches to this organism have made a revolutionary impact on the study of mycobacterial pathogenesis. Deletion and complementation of individual genes can be done at will facilitating the comparative analysis of mutants and wild-type strains. Novel and powerful technologies such as DNA microarrays, fluorescent beacons and proteomics have made possible the analysis of the expression levels of multiple genes in in vitro systems. More technically challenging uses of these techniques is being undertaken to explore pathogen gene expression within the host. This will lead to the identification of virulence factors and give definitive insight into their regulatory signals.