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Charles J. Dorman - One of the best experts on this subject based on the ideXlab platform.
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Regulation of Transcription in Bacteria by DNA Supercoiling
Bacterial Physiology, 2008Co-Authors: Charles J. DormanAbstract:Understanding mechanisms of gene regulation is a major goal of modern molecular biology and much work has focused on the central roles of DNA binding proteins in controlling the key events in gene expression. This chapter takes a different approach by considering the contribution of the genetic material itself to gene regulatory processes. DNA is often regarded as a passive partner in the gene regulatory relationship, a mere substrate on which the proteins act. Here, we will examine evidence that the conformation of DNA has a significant influence on the gene expression process, at least at the level of transcription. The focus of this review is on transcription regulation in bacteria by DNA Supercoiling, with an emphasis on the Gram-negative organism, Escherichia coli, and its close relatives, not least because much of the relevant groundbreaking work has been conducted in these microbes.
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Roles for DNA Supercoiling and the Fis protein in modulating expression of virulence genes during intracellular growth of Salmonella enterica serovar Typhimurium
Molecular microbiology, 2006Co-Authors: Tadhg Ó Cróinín, Ronan K. Carroll, Arlene Kelly, Charles J. DormanAbstract:Adaptation of bacterial pathogens to an intracellular environment requires resetting of the expression levels of a wide range of both virulence and housekeeping genes. We investigated the possibility that changes in DNA Supercoiling could modulate the expression of genes known to be important in the intracellular growth of the pathogen Salmonella enterica serovar Typhimurium. Our data show that DNA becomes relaxed when Salmonella grows in murine macrophage but not in epithelial cells, indicating that DNA Supercoiling plays a role in discrimination between two types of intracellular environment. The ssrA regulatory gene within the SPI-2 pathogenicity island that is required for survival in macrophage was found to be upregulated by DNA relaxation. This enhancement of expression also required the Fis nucleoid-associated protein. Manipulating the level of the Fis protein modulated both the level of DNA Supercoiling and ssrA transcription. We discuss a model of bacterial intracellular adaptation in which Fis and DNA Supercoiling collaborate to fine-tune virulence gene expression.
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DNA Supercoiling and bacterial gene expression
Science Progress, 2006Co-Authors: Charles J. DormanAbstract:DNA in bacterial cells is maintained in a negatively supercoiled state. This contributes to the organization of the bacterial nucleoid and also influences the global gene expression pattern in the cell through modulatory effects on transcription. Supercoiling arises as a result of changes to the linking number of the relaxed double-stranded DNA molecule and is set and reset by the action of DNA topoisomerases. This process is subject to a multitude of influences that are usually summarized as environmental stress. Responsiveness of linking number change to stress offers the promise of a mechanism for the wholesale adjustment of the transcription programme of the cell as the bacterium experiences different environments. Recent data from DNA microarray experiments support this proposition. The emerging picture is one of DNA Supercoiling acting at or near the apex of a regulatory hierarchy where it collaborates with nucleoid-associated proteins and transcription factors to determine the gene expression profile of the cell.
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The site‐specific recombination system regulating expression of the Type 1 fimbrial subunit gene of Escherichia coli is sensitive to changes in DNA Supercoiling
Molecular microbiology, 1994Co-Authors: Simon L. Dove, Charles J. DormanAbstract:Summary We have studied the effect of altering the in vivo level of DNA Supercoiling on the phase-variable expression of the Escherichia coli fimA gene. Transcription from the fimA promoter was unaffected by changes in DNA Supercoiling whether caused by the introduction of a topA::7n10 mutation or by inhibition of DNA gyrase with the antibiotic novobiocin. However, inversion of the fimA promoter fragment was altered in response to perturbation of DNA Supercoiling. Specifically, inactivation of topA reduced the rate of promoter fragment inversion in both the ON-to-OFF and the OFF-to-ON directions. This effect correlated with the loss of functional topA and not with the global level of DNA Supercoiling. Inhibition of DNA gyrase introduced a bias in favour of the OFF-to-ON inversion; the ON-to-OFF inversion was affected only slightly. Changes in expression of fimB, the gene coding for the recombinase that catalyses fimA promoter fragment inversion in the strains used In this study, did not correlate with effects on fimA phase variation: we found that transcription of fimB was inhibited by loss of functional topA and was enhanced by inhibition of DNA gyrase in a manner that correlated well with the global level of in vivo DNA Supercoiling. A model is presented to account for the effects of lost topoisomerase function on fimA gene expression.
Sam Meyer - One of the best experts on this subject based on the ideXlab platform.
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Bacterial promoter opening underpins ubiquitous transcriptional regulation by DNA Supercoiling
2020Co-Authors: Raphael Forquet, William Nasser, Sylvie Reverchon, Maiwenn Pineau, Sam MeyerAbstract:DNA Supercoiling acts as a basal transcriptional regulator, which contributes to the quick and global transcriptional response of bacteria to many environmental changes. In spite of this importance, mechanistic models explaining the differential response of promoters to global topological variations of the chromosome remain essentially lacking. Here, we present the first quantitative transcriptional regulatory model by DNA Supercoiling, focusing on the specific step of promoter opening during transcription initiation. Based on the known physico-chemical properties of DNA denaturation, it involves only one global adjustable parameter and is yet able to predict the global Supercoiling response of promoters in a wide range of bacteria, based on the sequence content of their "discriminator" element. We first show that it quantitatively predicts both \emph{in vitro} and \emph{in vivo} data from transcription assays focusing on individual model promoters. We then assess the universality of the mechanism by analyzing transcriptomes of phylogenetically distant bacteria under conditions of Supercoiling variation: (1) by gyrase-inhibiting antibiotics, (2) by biologically relevant environmental stresses, (3) naturally acquired and inherited in the longest-running evolution experiment. The model robustly predicts a significant contribution of the entire transcriptomic response to transient or inherited Supercoiling variations in various species. This study strongly suggests that the proposed physical mechanism is used as an ubiquitous regulatory mechanism in the whole prokaryotic kingdom, based on the fundamental mechanical properties of the double-helix. Importance: DNA Supercoiling acts as a global yet underestimated transcriptional regulator in bacteria. We propose the first quantitative model of this regulation mode, based on the specific step of promoter opening during transcription initiation, explaining the differential response of promoters to global topological variations of the chromosome. In contrast to classical mechanisms requiring dedicated regulatory molecules to bind target promoters, we show that global deformations of the DNA template itself underpin a selective response of each particular promoter, according to its "discriminator" sequence, by modulating the ability of RNA Polymerase to initiate transcription. This study defines the first systematic rule underpinning the ubiquitous regulatory action of DNA Supercoiling on the core transcriptional machinery, in particular in response to quick environmental changes.
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bacterial genome architecture shapes global transcriptional regulation by DNA Supercoiling
Nucleic Acids Research, 2019Co-Authors: Bilal El Houdaigui, Raphael Forquet, Thomas Hindre, Dominique Schneider, William Nasser, Sylvie Reverchon, Sam MeyerAbstract:DNA Supercoiling acts as a global transcriptional regulator in bacteria, that plays an important role in adapting their expression programme to environmental changes, but for which no quantitative or even qualitative regulatory model is available. Here, we focus on spatial Supercoiling heterogeneities caused by the transcription process itself, which strongly contribute to this regulation mode. We propose a new mechanistic modeling of the transcription-Supercoiling dynamical coupling along a genome, which allows simulating and quantitatively reproducing in vitro and in vivo transcription assays, and highlights the role of genes' local orientation in their su-percoiling sensitivity. Consistently with predictions, we show that chromosomal relaxation artificially induced by gyrase inhibitors selectively activates con-vergent genes in several enterobacteria, while conversely , an increase in DNA Supercoiling naturally selected in a long-term evolution experiment with Escherichia coli favours divergent genes. Simulations show that these global expression responses to changes in DNA Supercoiling result from fundamental mechanical constraints imposed by transcription, independently from more specific regulation of each promoter. These constraints underpin a significant and predictable contribution to the complex rules by which bacteria use DNA Supercoiling as a global but fine-tuned transcriptional regulator.
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DNA Supercoiling: an Ancestral Regulator of Gene Expression in Pathogenic Bacteria?
Computational and Structural Biotechnology Journal, 2019Co-Authors: Shiny Martis B., Raphael Forquet, William Nasser, Sylvie Reverchon, Sam MeyerAbstract:DNA Supercoiling acts as a global and ancestral regulator of bacterial gene expression. In this review, we advocate that it plays a pivotal role in host-pathogen interactions by transducing environmental signals to the bacterial chromosome and coordinating its transcriptional response. We present available evidence that DNA Supercoiling is modulated by environmental stress conditions relevant to the infection process according to ancestral mechanisms , in zoopathogens as well as phytopathogens. We review the results of transcriptomics studies obtained in widely distant bacterial species, showing that such structural transitions of the chromosome are associated to a complex transcriptional response affecting a large fraction of the genome. Mechanisms and computational models of the transcriptional regulation by DNA Supercoiling are then discussed, involving both basal interactions of RNA Polymerase with promoter DNA, and more specific interactions with regulatory proteins. A final part is specifically focused on the regulation of virulence genes within pathogenicity islands of several pathogenic bacterial species.
Fenfei Leng - One of the best experts on this subject based on the ideXlab platform.
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Inhibition of the gyrA promoter by transcription-coupled DNA Supercoiling in Escherichia coli
Scientific reports, 2018Co-Authors: Samantha Dages, Xiaoduo Zhi, Kelley Dages, Fenfei LengAbstract:The E. coli gyrA promoter (PgyrA) is a DNA Supercoiling sensitive promoter, stimulated by relaxation of DNA templates, and inhibited by (−) DNA Supercoiling in bacteria. However, whether PgyrA can be inhibited by transient and localized transcription-coupled DNA Supercoiling (TCDS) has not been fully examined. In this paper, using different DNA templates including the E. coli chromosome, we show that transient and localized TCDS strongly inhibits PgyrA in E. coli. This result can be explained by a twin-supercoiled domain model of transcription in which (+) and (−) supercoiled domains are generated around the transcribing RNA polymerase. We also find that fluoroquinolones, such as ciprofloxacin, can substantially increase the expression of the firefly luciferase under the control of the PgyrA coupled to a divergent IPTG-inducible promoter in the presence of IPTG. This stimulation of PgyrA by fluoroquinolones can be also explained by the twin-supercoiled domain model of transcription. This unique property of TCDS may be configured into a high throughput-screening (HTS) assay to identify antimicrobial compounds targeting bacterial DNA gyrase.
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DNA Supercoiling Measurement in Bacteria
Methods in molecular biology (Clifton N.J.), 2017Co-Authors: Yingting Liu, Zhi-chun Hua, Fenfei LengAbstract:DNA Supercoiling plays critical roles in several essential DNA metabolic pathways, such as replication, transcription and recombination. Typically plasmid DNA molecules are used to measure DNA Supercoiling status inside bacterial cells. In this chapter, we describe how to isolate plasmid DNA molecules from E. coli cells and determine DNA Supercoiling density by 1% agarose gel electrophoresis containing chloroquine using plasmid pACYC184 as an example.
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Transient and dynamic DNA Supercoiling potently stimulates the leu-500 promoter in Escherichia coli.
The Journal of biological chemistry, 2017Co-Authors: Xiaoduo Zhi, Yingting Liu, Samantha Dages, Kelley Dages, Zi-chun Hua, John C. Makemson, Fenfei LengAbstract:The inactive prokaryotic leu-500 promoter (Pleu-500) contains a single A-to-G point mutation in the -10 region of the leucine operon promoter, which causes leucine auxotrophy. This promoter can be activated by (-) DNA Supercoiling in Escherichia coli topA strains. However, whether this activation arises from global, permanent, or transient, dynamic Supercoiling is still not fully understood. In this article, using a newly established in vivo system carrying a pair of divergently coupled promoters, i.e. an IPTG-inducible promoter and Pleu-500 that control the expression of lacZ and luc (the firefly luciferase gene), respectively, we demonstrate that transient, dynamic (-) DNA Supercoiling provided by divergent transcription in both wild-type and topA strains can potently activate Pleu-500 We found that this activation depended on the promoter strength and the length of RNA transcripts, which are functional characteristics of transcription-coupled DNA Supercoiling (TCDS) precisely predicted by the twin-supercoiled domain model of transcription in which a (+) supercoiled domain is produced ahead of the RNA polymerase and a (-) supercoiled domain behind it. We also demonstrate that TCDS can be generated on topologically open DNA molecules, i.e. linear DNA molecules, in Escherichia coli, suggesting that topological boundaries or barriers are not required for the production of TCDS in vivo This work demonstrates that transient, dynamic TCDS by RNA polymerases is a major chromosome remodeling force in E. coli and greatly influences the nearby, coupled promoters/transcription.
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Determining DNA Supercoiling enthalpy by isothermal titration calorimetry.
Biochimie, 2012Co-Authors: Xiaoduo Zhi, Fenfei LengAbstract:DNA Supercoiling plays a critical role in certain essential DNA transactions, such as DNA replication, recombination, and transcription. For this reason, exploring energetics of DNA Supercoiling is fundamentally important for understanding its biological functions. In this paper, using a unique property of DNA intercalators, such as ethidium bromide and daunorubicin, which bind to supercoiled, nicked, and relaxed DNA templates with different DNA-binding enthalpies, we determined DNA Supercoiling enthalpy of plasmid pXXZ6, a 4.5 kb plasmid to be about 11.5 kcal/mol per linking number change. This determination allowed us to partition the DNA Supercoiling free energy into enthalpic and entropic contributions where the unfavorable DNA Supercoiling free energy exclusively originated from the large positive Supercoiling enthalpy and was compensated by a large, favorable entropy term (TΔS).
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Coupling DNA Supercoiling to Transcription in Defined Protein Systems
The Journal of biological chemistry, 2004Co-Authors: Fenfei Leng, Luciana Amado, Roger McmackenAbstract:Transcription of closed circular DNA templates in the presence of DNA gyrase is known to stimulate negative DNA Supercoiling both in vivo and in vitro. It has proven elusive, however, to establish a general system in vitro that supports transcription-coupled DNA Supercoiling (TCDS) by the "twin-domain" mechanism (Liu, L. F. and Wang, J. C. (1987) Proc. Natl. Acad. Sci. USA 84, 7024-7027) that operates in bacteria. In this report, we examine the properties of TCDS in defined protein systems that minimally contained T7 RNA polymerase and DNA gyrase. Specifically designed plasmid DNA templates permitted us to control the location and length of RNA transcripts. We demonstrate that TCDS takes place by two separate, and apparently independent, mechanistic pathways in vitro. The first Supercoiling pathway, which is not likely to be significant in vivo, was found to be dependent on R-loop formation and could be suppressed by the presence of RNase H or bacterial HU protein. The second pathway for TCDS was much more potent, but became predominant in vitro only when sequence-specific DNA-bending proteins were present during transcription, and RNA transcript lengths exceeded 3 kb. This major Supercoiling route was shown to be resistant to RNase H and had functional properties consistent with those predicted for the twin-domain mechanism. For example, DNA Supercoiling activity was proportional to RNA transcript length and was greatly stimulated by macromolecular crowding agents. Under optimal conditions, the twin domain pathway of TCDS rapidly and efficiently generated superhelicity levels more than twice that typically found in vivo.
Tadhg Ó Cróinín - One of the best experts on this subject based on the ideXlab platform.
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Relaxation of DNA Supercoiling leads to increased invasion of epithelial cells and protein secretion by Campylobacter jejuni.
Molecular microbiology, 2017Co-Authors: Eoin Scanlan, Laura Ardill, Matthew V. X. Whelan, Claire Shortt, Jarlath E. Nally, Billy Bourke, Tadhg Ó CróinínAbstract:Summary Invasion of intestinal epithelial cells by Campylobacter jejuni is a critical step during infection of the intestine by this important human pathogen. In this study we investigated the role played by DNA Supercoiling in the regulation of invasion of epithelial cells and the mechanism by which this could be mediated. A significant correlation between more relaxed DNA Supercoiling and an increased ability of C. jejuni strains to penetrate human epithelial cells was demonstrated. Directly inducing relaxation of DNA Supercoiling in C. jejuni was shown to significantly increase invasion of epithelial cells. Mutants in the fibronectin binding proteins CadF and FlpA still displayed an increased invasion after treatment with novobiocin suggesting these proteins were not essential for the observed phenotype. However, a large increase in protein secretion from multiple C. jejuni strains upon relaxation of DNA Supercoiling was demonstrated. This increase in protein secretion was not mediated by outer membrane vesicles and appeared to be dependent on an intact flagellar structure. This study identifies relaxation of DNA Supercoiling as playing a key role in enhancing C. jejuni pathogenesis during infection of the human intestine and identifies proteins present in a specific invasion associated secretome induced by relaxation of DNA Supercoiling.
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DNA Supercoiling Regulates the Motility of Campylobacter jejuni and Is Altered by Growth in the Presence of Chicken Mucus
mBio, 2016Co-Authors: Claire Shortt, Eoin Scanlan, Billy Bourke, Amber Hilliard, Chiara E. Cotroneo, Tadhg Ó CróinínAbstract:ABSTRACT Campylobacter jejuni is the leading cause of bacterial gastroenteritis in humans, but relatively little is known about the global regulation of virulence factors during infection of chickens or humans. This study identified DNA Supercoiling as playing a key role in regulating motility and flagellar protein production and found that this Supercoiling-controlled regulon is induced by growth in chicken mucus. A direct correlation was observed between motility and resting DNA Supercoiling levels in different strains of C. jejuni, and relaxation of DNA Supercoiling resulted in decreased motility. Transcriptional analysis and Western immunoblotting revealed that a reduction in motility and DNA Supercoiling affected the two-component regulatory system FlgRS and was associated with reduced FlgR expression, increased FlgS expression, and aberrant expression of flagellin subunits. Electron microscopy revealed that the flagellar structure remained intact. Growth in the presence of porcine mucin resulted in increased negative Supercoiling, increased motility, increased FlgR expression, and reduced FlgS expression. Finally, this Supercoiling-dependent regulon was shown to be induced by growth in chicken mucus, and the level of activation was dependent on the source of the mucus from within the chicken intestinal tract. In conclusion, this study reports for the first time the key role played by DNA Supercoiling in regulating motility in C. jejuni and indicates that the induction of this Supercoiling-induced regulon in response to mucus from different sources could play a critical role in regulating motility in vivo . IMPORTANCE Although Campylobacter jejuni is the leading cause of bacterial gastroenteritis, very little is understood about how this pathogen controls the expression of genes involved in causing disease. This study for the first time identifies DNA Supercoiling as a key regulator of motility in C. jejuni, which is essential for both pathogenesis and colonization. Altering the level of DNA Supercoiling results in changes in motility levels, as well as changes in the expression of genes involved in flagellar gene regulation. Furthermore, spontaneous clones of the organism with different motility profiles have altered DNA Supercoiling levels. Finally, mucus was identified as a key stimulator of changes in DNA Supercoiling, and it was shown that mucus from different sites in the chicken intestine induced different levels of DNA Supercoiling. In conclusion, this study implicates DNA Supercoiling as a key regulator of motility in C. jejuni in vivo during colonization of the mucus layer.
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Roles for DNA Supercoiling and the Fis protein in modulating expression of virulence genes during intracellular growth of Salmonella enterica serovar Typhimurium
Molecular microbiology, 2006Co-Authors: Tadhg Ó Cróinín, Ronan K. Carroll, Arlene Kelly, Charles J. DormanAbstract:Adaptation of bacterial pathogens to an intracellular environment requires resetting of the expression levels of a wide range of both virulence and housekeeping genes. We investigated the possibility that changes in DNA Supercoiling could modulate the expression of genes known to be important in the intracellular growth of the pathogen Salmonella enterica serovar Typhimurium. Our data show that DNA becomes relaxed when Salmonella grows in murine macrophage but not in epithelial cells, indicating that DNA Supercoiling plays a role in discrimination between two types of intracellular environment. The ssrA regulatory gene within the SPI-2 pathogenicity island that is required for survival in macrophage was found to be upregulated by DNA relaxation. This enhancement of expression also required the Fis nucleoid-associated protein. Manipulating the level of the Fis protein modulated both the level of DNA Supercoiling and ssrA transcription. We discuss a model of bacterial intracellular adaptation in which Fis and DNA Supercoiling collaborate to fine-tune virulence gene expression.
Ming Tan - One of the best experts on this subject based on the ideXlab platform.
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Differential Effects of DNA Supercoiling on Chlamydia Early Promoters Correlate with Expression Patterns in Midcycle
Journal of bacteriology, 2012Co-Authors: Eric Cheng, Ming TanAbstract:Changes in DNA Supercoiling levels during the chlamydial developmental cycle have been proposed as a global mechanism to upregulate midcycle genes, but the effects on early genes are not known. We examined the promoters for 10 Chlamydia trachomatis early genes and found that they could be separated into two subsets based on their responses to DNA Supercoiling in vitro. Furthermore, the type of Supercoiling response correlated with the in vivo expression pattern for each early gene. One subset of seven early genes had promoters that were transcribed in a Supercoiling-insensitive manner over the physiologic range of Supercoiling levels that have been measured in Chlamydia. In vivo transcripts for these genes were detected at similar levels during early-stage and midstage times. In contrast, a second subset, represented in our study by three early genes, had Supercoiling-dependent promoters that were transcribed at higher levels from more-supercoiled templates, which is the response observed for midcycle genes. Genes in this subset were expressed at higher levels at midstage times than at early times in vivo. We propose that this second subset represents a novel class of chlamydial developmental genes with features of both early and midcycle genes. We hypothesize that expression of these Supercoiling-dependent early genes is upregulated by increased chlamydial Supercoiling levels in midcycle via their Supercoiling-responsive promoters in a manner similar to that for midcycle genes. Thus, we propose that DNA Supercoiling is utilized in Chlamydia as a general mechanism to regulate genes in the midstage of the developmental cycle and not just midcycle genes.
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DNA Supercoiling-Dependent Gene Regulation in Chlamydia
Journal of bacteriology, 2008Co-Authors: Eike Niehus, Eric Cheng, Ming TanAbstract:The intracellular pathogen Chlamydia has an unusual developmental cycle marked by temporal expression patterns whose mechanisms of regulation are largely unknown. To examine if DNA topology can regulate chlamydial gene expression, we tested the in vitro activity of five chlamydial promoters at different superhelical densities. We demonstrated for the first time that individual chlamydial promoters show a differential response to changes in DNA Supercoiling that correlates with the temporal expression pattern. The promoters for two midcycle genes, ompA and pgk, were responsive to alterations in Supercoiling, and promoter activity could be regulated more than eightfold. In contrast, the promoters for three late transcripts, omcAB, hctA, and ltuB, were relatively insensitive to Supercoiling, with promoter activity varying by no more than 2.2-fold over a range of superhelicities. To obtain a measure of how DNA Supercoiling levels vary during the chlamydial developmental cycle, we recovered the cryptic chlamydial plasmid at different times after infection and assayed its superhelical density. The chlamydial plasmid was most negatively supercoiled at midcycle, with an approximate superhelical density of −0.07. At early and late times, the plasmid was more relaxed, with an approximate superhelicity of −0.03. Thus, we found a correlation between the responsiveness to Supercoiling shown by the two midcycle promoters and the increased level of negative Supercoiling during mid time points in the developmental cycle. Our results support a model in which the response of individual promoters to alterations in DNA Supercoiling can provide a mechanism for global patterns of temporal gene expression in Chlamydia.