Publications
95 results found
Thompson CC, Griffiths C, Nicod SS, et al., 2015, The Rsb phosphoregulatory network controls availability of the primary sigma factor in Chlamydia trachomatis and influences the kinetics of growth and development, PLOS Pathogens, Vol: 11, Pages: 1-22, ISSN: 1553-7366
Chlamydia trachomatis is the leading cause of both bacterial sexually transmitted infection and infection-derived blindness world-wide. No vaccine has proven protective to date in humans. C. trachomatis only replicates from inside a host cell, and has evolved to acquire a variety of nutrients directly from its host. However, a typical human immune response will normally limit the availability of a variety of essential nutrients. Thus, it is thought that the success of C. trachomatis as a human pathogen may lie in its ability to survive these immunological stress situations by slowing growth and development until conditions in the cell have improved. This mode of growth is known as persistence and how C. trachomatis senses stress and responds in this manner is an important area of research. Our report characterizes a complete signaling module, the Rsb network, that is capable of controlling the growth rate or infectivity of Chlamydia. By manipulating the levels of different pathway components, we were able to accelerate and restrict the growth and development of this pathogen. Our results suggest a mechanism by which Chlamydia can tailor its growth rate to the conditions within the host cell. The disruption of this pathway could generate a strain incapable of surviving a typical human immune response and would represent an attractive candidate as an attenuated growth vaccine.
Thomas MS, Wigneshweraraj S, 2015, Regulation of virulence gene expression, VIRULENCE, Vol: 5, Pages: 832-834, ISSN: 2150-5594
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- Citations: 30
Nicod SS, Weinzierl RO, Burchell L, et al., 2014, Systematic mutational analysis of the LytTR DNA binding domain of Staphylococcus aureus virulence gene transcription factor AgrA, Nucleic Acids Research, Vol: 42, Pages: 12523-12536, ISSN: 1362-4962
Most DNA-binding bacterial transcription factors contact DNA through a recognition α-helix in their DNA-binding domains. An emerging class of DNA-binding transcription factors, predominantly found in pathogenic bacteria interact with the DNA via a relatively novel type of DNA-binding domain, called the LytTR domain, which mainly comprises β strands. Even though the crystal structure of the LytTR domain of the virulence gene transcription factor AgrA from Staphylococcus aureus bound to its cognate DNA sequence is available, the contribution of specific amino acid residues in the LytTR domain of AgrA to transcription activation remains elusive. Here, for the first time, we have systematically investigated the role of amino acid residues in transcription activation in a LytTR domain-containing transcription factor. Our analysis, which involves in vivo and in vitro analyses and molecular dynamics simulations of S. aureus AgrA identifies a highly conserved tyrosine residue, Y229, as a major amino acid determinant for maximal activation of transcription by AgrA and provides novel insights into structure-function relationships in S. aureus AgrA.
Painter KL, Krishna A, Wigneshweraraj S, et al., 2014, What role does the quorum-sensing accessory gene regulator system play during Staphylococcus aureus bacteremia?, Trends in Microbiology, Vol: In Press, ISSN: 0966-842X
Brown DR, Barton G, Pan Z, et al., 2014, Combinatorial stress responses: direct coupling of two major stress responses in Escherichia coli, Microbial Cell, Vol: 1, Pages: 315-317, ISSN: 2311-2638
Nitrogen is an essential element for all life, and this isno different for the bacterial cell. Numerous cellularmacromolecules contain nitrogen, including proteins,nucleic acids and cell wall components. In Escherichiacoli and related bacteria, the nitrogen stress (Ntr) responseallows cells to rapidly sense and adapt to nitrogenlimitation by scavenging for alternative nitrogensources through the transcriptional activation oftransport systems and catabolic and biosynthetic operonsby the global transcriptional regulator NtrC. Nitrogen-starvedbacterial cells also synthesize the(p)ppGpp effector molecules of a second global bacterialstress response - the stringent response. Recently,we showed that the transcription of relA, the genewhich encodes the major (p)ppGpp synthetase in E.coli, is activated by NtrC during nitrogen starvation.Our results revealed that in E. coli and related bacteria,NtrC functions in combinatorial stress and serves tocouple two major stress responses, the Ntr responseand stringent response.
Brown DR, Barton G, Pan Z, et al., 2014, Nitrogen stress response and stringent response are coupled in Escherichia coli, Nature Communications, Vol: 5, ISSN: 2041-1723
Assimilation of nitrogen is an essential process in bacteria. The nitrogen regulation stress response is an adaptive mechanism used by nitrogen-starved Escherichia coli to scavenge for alternative nitrogen sources and requires the global transcriptional regulator NtrC. In addition, nitrogen-starved E. coli cells synthesize a signal molecule, guanosine tetraphosphate (ppGpp), which serves as an effector molecule of many processes including transcription to initiate global physiological changes, collectively termed the stringent response. The regulatory mechanisms leading to elevated ppGpp levels during nutritional stresses remain elusive. Here, we show that transcription of relA, a key gene responsible for the synthesis of ppGpp, is activated by NtrC during nitrogen starvation. The results reveal that NtrC couples these two major bacterial stress responses to manage conditions of nitrogen limitation, and provide novel mechanistic insights into how a specific nutritional stress leads to elevating ppGpp levels in bacteria.
Liu B, Shadrin A, Sheppard C, et al., 2014, A bacteriophage transcription regulator inhibits bacterial transcription initiation by Sigma-factor displacement, Nucleic Acids Research, Vol: 42, Pages: 4294-4305, ISSN: 0305-1048
Bacteriophages (phages) appropriate essential processes of bacterial hosts to benefit their own development. The multisubunit bacterial RNA polymerase (RNAp) enzyme, which catalyses DNA transcription, is targeted by phage-encoded transcription regulators that selectively modulate its activity. Here, we describe the structural and mechanistic basis for the inhibition of bacterial RNAp by the transcription regulator P7 encoded by Xanthomonas oryzae phage Xp10. We reveal that P7 uses a two-step mechanism to simultaneously interact with the catalytic β and β’ subunits of the bacterial RNAp and inhibits transcription initiation by inducing the displacement of the σ70-factor on initial engagement of RNAp with promoter DNA. The new mode of interaction with and inhibition mechanism of bacterial RNAp by P7 underscore the remarkable variety of mechanisms evolved by phages to interfere with host transcription.
Sharma A, Leach RN, Gell C, et al., 2014, Domain movements of the enhancer-dependent sigma factor drive DNA delivery into the RNA polymerase active site: insights from single molecule studies, NUCLEIC ACIDS RESEARCH, Vol: 42, Pages: 5177-5190, ISSN: 0305-1048
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- Citations: 18
Liu B, Shadrin A, Sheppard C, et al., 2014, The sabotage of the bacterial transcription machinery by a small bacteriophage protein., Bacteriophage, Vol: 4, ISSN: 2159-7073
Many bacteriophages produce small proteins that specifically interfere with the bacterial host transcription machinery and thus contribute to the acquisition of the bacterial cell by the bacteriophage. We recently described how a small protein, called P7, produced by the Xp10 bacteriophage inhibits bacterial transcription initiation by causing the dissociation of the promoter specificity sigma factor subunit from the host RNA polymerase holoenzyme. In this addendum to the original publication, we present the highlights of that research.
Pader V, James EH, Painter KL, et al., 2014, The Agr quorum-sensing system regulates fibronectin-binding but not hemolysis in the absence of a functional electron transport chain., Infection and Immunity
Bae B, Davis E, Brown D, et al., 2013, Phage T7 Gp2 inhibition of Escherichia coli RNA polymerase involves misappropriation of σ70 domain 1.1., Proceedings of the National Academy of Sciences, Vol: 110, Pages: 19772-19777
James EH, Edwards AM, Wigneshweraraj S, 2013, Transcriptional downregulation of agr expression in Staphylococcus aureus during growth in human serum can be overcome by constitutively active mutant forms of the sensor kinase AgrC, FEMS Microbiology Letters, Vol: 349, Pages: 153-162, ISSN: 0378-1097
The temporal and cell density-dependent regulation of expression of virtually all the Staphylococcus aureus virulon is under the control of the agr (accessory gene regulatory) operon. The expression of the agr operon is subject to transcriptional regulation by the AgrA/C two-component response regulator/sensor kinase pair. During bacteraemia, a frequent syndrome caused by methicillin-resistant S. aureus (MRSA), the transcriptional downregulation of agr expression has been attributed to the sequestration of the quorum-signalling molecule auto-inducing peptide (AIP) by the human serum component apolipoprotein B as part of an innate immune response to infection. However, it is not known whether transcriptional downregulation of agr expression during growth in human serum is additionally subjected to regulation by transcription regulatory proteins that either directly or indirectly affect transcription from the agr operon promoters. Here, using chromosomal fluorescence reporters of agr expression in S. aureus, we show that the transcriptional downregulation of agr expression in human serum can be overcome using constitutive active mutant forms of AgrC. Therefore, it seems that the sequestration of the AIP is likely to be the only mechanism by which the host innate immune response limits agr expression at the transcriptional level to maintain the host–pathogen balance towards a noninvasive outcome.
Schumacher J, Behrends V, Pan Z, et al., 2013, Nitrogen and Carbon Status Are Integrated at the Transcriptional Level by the Nitrogen Regulator NtrC <i>In Vivo</i>, MBIO, Vol: 4, ISSN: 2150-7511
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- Citations: 45
Sheppard C, James E, Barton G, et al., 2013, A non-bacterial transcription factor inhibits bacterial transcription by a multipronged mechanism, RNA BIOLOGY, Vol: 10, Pages: 495-501, ISSN: 1547-6286
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- Citations: 9
Shadrin A, Sheppard C, Savalia D, et al., 2013, Overexpression of <i>Escherichia coli udk</i> mimics the absence of T7 Gp2 function and thereby abrogates successful infection by T7 phage, MICROBIOLOGY-SGM, Vol: 159, Pages: 269-274, ISSN: 1350-0872
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- Citations: 7
Drennan A, Kraemer M, Capp M, et al., 2012, Key Roles of the Downstream Mobile Jaw of <i>Escherichia coli</i> RNA Polymerase in Transcription Initiation, BIOCHEMISTRY, Vol: 51, Pages: 9447-9459, ISSN: 0006-2960
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- Citations: 18
Shadrin A, Sheppard C, Severinov K, et al., 2012, Substitutions in the <i>Escherichia coli</i> RNA polymerase inhibitor T7 Gp2 that allow inhibition of transcription when the primary interaction interface between Gp2 and RNA polymerase becomes compromised, MICROBIOLOGY-SGM, Vol: 158, Pages: 2753-2764, ISSN: 1350-0872
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- Citations: 11
James E, Liu M, Sheppard C, et al., 2012, Structural and Mechanistic Basis for the Inhibition of <i>Escherichia coli</i> RNA Polymerase by T7 Gp2, MOLECULAR CELL, Vol: 47, Pages: 755-766, ISSN: 1097-2765
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- Citations: 34
Chakraborty A, Wang D, Ebright YW, et al., 2012, Opening and Closing of the Bacterial RNA Polymerase Clamp, SCIENCE, Vol: 337, Pages: 591-595, ISSN: 0036-8075
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- Citations: 158
Drennan A, Saecker R, Heitkamp S, et al., 2012, E. Coli RNA Polymerase: A Molecular DNA Opening Machine, BIOPHYSICAL JOURNAL, Vol: 102, Pages: 286A-286A, ISSN: 0006-3495
Mekler V, Minakhin L, Sheppard C, et al., 2011, Molecular Mechanism of Transcription Inhibition by Phage T7 gp2 Protein, JOURNAL OF MOLECULAR BIOLOGY, Vol: 413, Pages: 1016-1027, ISSN: 0022-2836
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- Citations: 26
Sheppard C, Camara B, Shadrin A, et al., 2011, Reprint of: Inhibition of Escherichia coli RNAp by T7 Gp2 protein: Role of Negatively Charged Strip of Amino Acid Residues in Gp2, JOURNAL OF MOLECULAR BIOLOGY, Vol: 412, Pages: 832-841, ISSN: 0022-2836
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- Citations: 3
Reynolds J, Wigneshweraraj S, 2011, Molecular Insights into the Control of Transcription Initiation at the <i>Staphylococcus aureus</i> agr operon, JOURNAL OF MOLECULAR BIOLOGY, Vol: 412, Pages: 862-881, ISSN: 0022-2836
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- Citations: 21
Sheppard C, Camara B, Shadrin A, et al., 2011, Inhibition of <i>Escherichia coli</i> RNAp by T7 Gp2 Protein: Role of Negatively Charged Strip of Amino Acid Residues in Gp2, JOURNAL OF MOLECULAR BIOLOGY, Vol: 407, Pages: 623-632, ISSN: 0022-2836
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- Citations: 9
Jovanovic M, Burrows PC, Bose D, et al., 2011, Activity map of the Escherichia coli RNA polymerase bridge helix, J Biol Chem, Vol: 286, Pages: 14469-14479, ISSN: 1083-351X
Transcription, the synthesis of RNA from a DNA template, is performed by multisubunit RNA polymerases (RNAPs) in all cellular organisms. The bridge helix (BH) is a distinct feature of all multisubunit RNAPs and makes direct interactions with several active site-associated mobile features implicated in the nucleotide addition cycle and RNA and DNA binding. Because the BH has been captured in both kinked and straight conformations in different crystals structures of RNAP, recently supported by molecular dynamics studies, it has been proposed that cycling between these conformations is an integral part of the nucleotide addition cycle. To further evaluate the role of the BH, we conducted systematic alanine scanning mutagenesis of the Escherichia coli RNAP BH to determine its contributions to activities required for transcription. Combining our data with an atomic model of E. coli RNAP, we suggest that alterations in the interactions between the BH and (i) the trigger loop, (ii) fork loop 2, and (iii) switch 2 can help explain the observed changes in RNAP functionality associated with some of the BH variants. Additionally, we show that extensive defects in E. coli RNAP functionality depend upon a single previously not studied lysine residue (Lys-781) that is strictly conserved in all bacteria. It appears that direct interactions made by the BH with other conserved features of RNAP are lost in some of the E. coli alanine substitution variants, which we infer results in conformational changes in RNAP that modify RNAP functionality.
Cordes T, Santoso Y, Tomescu AI, et al., 2010, Sensing DNA Opening in Transcription Using Quenchable Forster Resonance Energy Transfer, BIOCHEMISTRY, Vol: 49, Pages: 9171-9180, ISSN: 0006-2960
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- Citations: 34
Camara B, Liu M, Reynolds J, et al., 2010, T7 phage protein Gp2 inhibits the <i>Escherichia coli</i> RNA polymerase by antagonizing stable DNA strand separation near the transcription start site, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 107, Pages: 2247-2252, ISSN: 0027-8424
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- Citations: 53
Zhang N, Joly N, Burrows PC, et al., 2009, The role of the conserved phenylalanine in the σ<SUP>54</SUP>-interacting GAFTGA motif of bacterial enhancer binding proteins, NUCLEIC ACIDS RESEARCH, Vol: 37, Pages: 5981-5992, ISSN: 0305-1048
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- Citations: 28
Xiao Y, Wigneshweraraj SR, Weinzierl R, et al., 2009, Construction and functional analyses of a comprehensive Σ<SUP>54</SUP> site-directed mutant library using alanine-cysteine mutagenesis, NUCLEIC ACIDS RESEARCH, Vol: 37, Pages: 4482-4497, ISSN: 0305-1048
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- Citations: 7
Burrows PC, Joly N, Cannon WV, et al., 2009, Coupling σ Factor Conformation to RNA Polymerase Reorganisation for DNA Melting, JOURNAL OF MOLECULAR BIOLOGY, Vol: 387, Pages: 306-319, ISSN: 0022-2836
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- Citations: 12
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