Simon L Dove

Harvard Medical School, Boston, Massachusetts, United States

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Publications (48)348.39 Total impact

  • Amy E Rohlfing, Simon L Dove
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    ABSTRACT: In Francisella tularensis the putative DNA-binding protein PigR works in concert with the SspA protein family members MglA and SspA to control the expression of genes that are essential for the intramacrophage growth and survival of the organism. MglA and SspA form a complex that interacts with RNA polymerase (RNAP) and this interaction between the MglA-SspA complex and RNAP is thought to be critical to its regulatory function. How PigR works in concert with the MglA-SspA complex is not known; previously published findings differ over whether PigR interacts with the MglA-SspA complex leading to disparate models for how PigR and the MglA-SspA complex exert their regulatory effects. Here, using a combination of genetic assays, we identify mutants of MglA and SspA that are specifically defective for interaction with PigR. Analysis of the MglA and SspA mutants in F. tularensis reveals that interaction between PigR and the MglA-SspA complex is essential in order for PigR to work coordinately with MglA and SspA to positively regulate the expression of virulence genes. Our findings uncover a surface of the MglA-SspA complex that is important for interaction with PigR and support the idea that PigR exerts its regulatory effects through an interaction with the RNAP-associated MglA-SspA complex.
    Journal of bacteriology. 07/2014;
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    ABSTRACT: Pseudomonas aeruginosa contains two distinct members of H-NS family of nucleoid-structuring proteins: MvaT and MvaU. Together, these proteins bind to the same regions of the chromosome and function coordinately in the regulation of hundreds of genes. Due to their structural similarity, they can associate to form heteromeric complexes. These findings left us wondering whether they bear similar DNA binding properties that underlie their gene-silencing functions. Using single-molecule stretching and imaging experiments, we found striking similarities in the DNA organization modes of MvaU compared to the previously studied MvaT. MvaU can form protective nucleoprotein filaments that are insensitive to environmental factors, consistent with its role as a repressor of gene expression. Similar to MvaT, MvaU filament can mediate DNA bridging while excessive MvaU can cause DNA aggregation. The almost identical DNA organization modes of MvaU and MvaT explain their functional redundancy, and raise an interesting question regarding the evolutionary benefits of having multiple H-NS paralogues in the Pseudomonas genus.
    PLoS ONE 01/2014; 9(11):e112246. · 3.53 Impact Factor
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    ABSTRACT: Pathogenicity of Pseudomonas aeruginosa, a major cause of many acute and chronic human infections, is determined by tightly regulated expression of multiple virulence factors. Quorum sensing (QS) controls expression of many of these pathogenic determinants. Previous microarray studies have shown that the AmpC β-lactamase regulator AmpR, a member of the LysR family of transcription factors, also controls non-β-lactam resistance and multiple virulence mechanisms. Using RNA-Seq and complementary assays, this study further expands the AmpR regulon to include diverse processes such as oxidative stress, heat shock and iron uptake. Importantly, AmpR affects many of these phenotypes, in part, by regulating expression of non-coding RNAs such as rgP32, asRgsA, asPrrF1 and rgRsmZ. AmpR positively regulates expression of the major QS regulators LasR, RhlR and MvfR, and genes of the Pseudomonas quinolone system. Chromatin immunoprecipitation (ChIP)-Seq and ChIP-quantitative real-time polymerase chain reaction studies show that AmpR binds to the ampC promoter both in the absence and presence of β-lactams. In addition, AmpR directly binds the lasR promoter, encoding the QS master regulator. Comparison of the AmpR-binding sequences from the transcriptome and ChIP-Seq analyses identified an AT-rich consensus-binding motif. This study further attests to the role of AmpR in regulating virulence and physiological processes in P. aeruginosa.
    Nucleic Acids Research 10/2013; · 8.81 Impact Factor
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    ABSTRACT: Pseudomonas aeruginosa PlcH degrades phosphatidylcholine (PC), an abundant lipid in cell membranes and lung surfactant. A ΔplcHR mutant, known to be defective in virulence in animal models, was less able to colonize epithelial cell monolayers and defective in biofilm formation on plastic when grown in lung surfactant. Microarray analyses found that strains defective in PlcH production had lower levels of Anr-regulated transcripts than the wild type. PC degradation stimulated the Anr regulon in an Anr-dependent manner under conditions where Anr activity was submaximal due to the presence of oxygen. Two PC catabolites, choline and glycine betaine, were sufficient to stimulate Anr activity, and their catabolism was required for Anr activation. The addition of choline or GB to glucose-containing medium did not alter Anr protein levels, growth rates, or respiratory activity, and Anr activation could not be attributed to the osmoprotectant functions of GB. The Δanr mutant was defective in virulence in a mouse pneumonia model. Several lines of evidence indicate that Anr is important for the colonization of biotic and abiotic surfaces in both P. aeruginosa PAO1 and PA14, and that increases in Anr activity resulted in enhanced biofilm formation. Our data suggest that PlcH activity promotes Anr activity in oxic environments, and that Anr activity contributes to virulence, even in acute infections where low oxygen tensions are not expected. This finding highlights the relationship between in vivo bacterial metabolism, the activity of the oxygen-sensitive regulator Anr, and virulence.
    Journal of bacteriology 05/2013; · 3.94 Impact Factor
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    Sandra Castang, Simon L Dove
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    ABSTRACT: Members of the histone-like nucleoid-structuring (H-NS) family of proteins have been shown to play important roles in silencing gene expression and in nucleoid compaction. In Pseudomonas aeruginosa, the two H-NS family members MvaT and MvaU are thought to bind the same AT-rich regions of the chromosome and function coordinately to control a common set of genes. Here we present evidence that the loss of both MvaT and MvaU cannot be tolerated because it results in the production of Pf4 phage that superinfect and kill cells or inhibit their growth. Using a ClpXP-based protein depletion system in combination with transposon mutagenesis, we identify mutants of P. aeruginosa that can tolerate the depletion of MvaT in an ΔmvaU mutant background. Many of these mutants contain insertions in genes encoding components, assembly factors, or regulators of type IV pili or contain insertions in genes of the prophage Pf4. We demonstrate that cells that no longer produce type IV pili or that no longer produce the replicative form of the Pf4 genome can tolerate the loss of both MvaT and MvaU. Furthermore, we show that the loss of both MvaT and MvaU results in an increase in expression of Pf4 genes and that cells that cannot produce type IV pili are resistant to infection by Pf4 phage. Our findings suggest that type IV pili are the receptors for Pf4 phage and that the essential activities of MvaT and MvaU are to repress the expression of Pf4 genes.
    Journal of bacteriology 07/2012; 194(18):5101-9. · 3.94 Impact Factor
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    ABSTRACT: MvaT from Pseudomonas aeruginosa is a member of the histone-like nucleoid structuring protein (H-NS) family of nucleoid-associated proteins widely spread among Gram-negative bacteria that functions to repress the expression of many genes. Recently, it was reported that H-NS from Escherichia coli can form rigid nucleoproteins filaments on DNA, which are important for their gene-silencing function. This raises a question whether the gene-silencing function of MvaT, which has only ∼18% sequence similarity to H-NS, is also based on the formation of nucleoprotein filaments. Here, using magnetic tweezers and atomic force microscopy imaging, we demonstrate that MvaT binds to DNA through cooperative polymerization to form a nucleoprotein filament that can further organize DNA into hairpins or higher-order compact structures. Furthermore, we studied DNA binding by MvaT mutants that fail to repress gene expression in P. aeruginosa because they are specifically defective for higher-order oligomer formation. We found that, although the mutants can organize DNA into compact structures, they fail to form rigid nucleoprotein filaments. Our findings suggest that higher-order oligomerization of MvaT is required for the formation of rigid nucleoprotein filaments that silence at least some target genes in P. aeruginosa. Further, our findings suggest that formation of nucleoprotein filaments provide a general structural basis for the gene-silencing H-NS family members.
    Nucleic Acids Research 07/2012; 40(18):8942-8952. · 8.81 Impact Factor
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    ABSTRACT: Prokaryotic and eukaryotic RNA polymerases can use 2- to ∼4-nt RNAs, "nanoRNAs," to prime transcription initiation in vitro. It has been proposed that nanoRNA-mediated priming of transcription can likewise occur under physiological conditions in vivo and influence transcription start site selection and gene expression. However, no direct evidence of such regulation has been presented. Here we demonstrate in Escherichia coli that nanoRNAs prime transcription in a growth phase-dependent manner, resulting in alterations in transcription start site selection and changes in gene expression. We further define a sequence element that determines, in part, whether a promoter will be targeted by nanoRNA-mediated priming. By establishing that a significant fraction of transcription initiation is primed in living cells, our findings contradict the conventional model that all cellular transcription is initiated using nucleoside triphosphates (NTPs) only. In addition, our findings identify nanoRNAs as a previously undocumented class of regulatory small RNAs that function by being directly incorporated into a target transcript.
    Genes & development 07/2012; 26(13):1498-507. · 12.08 Impact Factor
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    ABSTRACT: The type VI secretion system (T6SS) has emerged as an important mediator of interbacterial interactions. A T6SS from Pseudomonas aeruginosa targets at least three effector proteins, type VI secretion exported 1-3 (Tse1-3), to recipient Gram-negative cells. The Tse2 protein is a cytoplasmic effector that acts as a potent inhibitor of target cell proliferation, thus providing a pronounced fitness advantage for P. aeruginosa donor cells. P. aeruginosa utilizes a dedicated immunity protein, type VI secretion immunity 2 (Tsi2), to protect against endogenous and intercellularly-transferred Tse2. Here we show that Tse2 delivered by the T6SS efficiently induces quiescence, not death, within recipient cells. We demonstrate that despite direct interaction of Tsi2 and Tse2 in the cytoplasm, Tsi2 is dispensable for targeting the toxin to the secretory apparatus. To gain insights into the molecular basis of Tse2 immunity, we solved the 1.00 Å X-ray crystal structure of Tsi2. The structure shows that Tsi2 assembles as a dimer that does not resemble previously characterized immunity or antitoxin proteins. A genetic screen for Tsi2 mutants deficient in Tse2 interaction revealed an acidic patch distal to the Tsi2 homodimer interface that mediates toxin interaction and immunity. Consistent with this finding, we observed that destabilization of the Tsi2 dimer does not impact Tse2 interaction. The molecular insights into Tsi2 structure and function garnered from this study shed light on the mechanisms of T6 effector secretion, and indicate that the Tse2-Tsi2 effector-immunity pair has features distinguishing it from previously characterized toxin-immunity and toxin-antitoxin systems.
    PLoS Pathogens 04/2012; 8(4):e1002613. · 8.14 Impact Factor
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    ABSTRACT: Expression of the pneumococcal type 1 pilus is bistable and positively regulated by the transcription factor RlrA. RlrA is also known to positively control its own expression. Here we present evidence that bistable expression of the type 1 pilus is mediated by the positive-feedback loop controlling rlrA expression.
    Journal of bacteriology 12/2011; 194(5):1088-91. · 3.94 Impact Factor
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    Heather R McManus, Simon L Dove
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    ABSTRACT: The CgrA and CgrC proteins of Pseudomonas aeruginosa are coregulators that are required for the phase-variable expression of the cupA fimbrial genes. Neither CgrA nor CgrC resembles a classical transcription regulator, and precisely how these proteins exert their regulatory effects on cupA gene expression is poorly understood. Here, we show that CgrA and CgrC interact with one another directly. We identify a mutant of CgrC that is specifically defective for interaction with CgrA and demonstrate that this mutant cannot restore the phase-variable expression of the cupA fimbrial genes to cells of a cgrC mutant strain. Using this mutant, we also show that CgrC associates with the cupA promoter regardless of whether or not it interacts with CgrA. Our findings establish that interaction between CgrA and CgrC is required for the phase-variable expression of the cupA fimbrial genes and suggest that CgrC exerts its regulatory effects directly at the cupA promoter, possibly by recruiting CgrA. Because the regions of CgrA and CgrC that we have identified as interacting with one another are highly conserved among orthologs, our findings raise the possibility that CgrA- and CgrC-related regulators present in other bacteria function coordinately through a direct protein-protein interaction.
    Journal of bacteriology 09/2011; 193(22):6152-61. · 3.94 Impact Factor
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    ABSTRACT: It is often presumed that, in vivo, the initiation of RNA synthesis by DNA-dependent RNA polymerases occurs using NTPs alone. Here, using the model Gram-negative bacterium Pseudomonas aeruginosa, we demonstrate that depletion of the small-RNA-specific exonuclease, Oligoribonuclease, causes the accumulation of oligoribonucleotides 2 to ∼4 nt in length, "nanoRNAs," which serve as primers for transcription initiation at a significant fraction of promoters. Widespread use of nanoRNAs to prime transcription initiation is coupled with global alterations in gene expression. Our results, obtained under conditions in which the concentration of nanoRNAs is artificially elevated, establish that small RNAs can be used to initiate transcription in vivo, challenging the idea that all cellular transcription occurs using only NTPs. Our findings further suggest that nanoRNAs could represent a distinct class of functional small RNAs that can affect gene expression through direct incorporation into a target RNA transcript rather than through a traditional antisense-based mechanism.
    Molecular cell 06/2011; 42(6):817-25. · 14.61 Impact Factor
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    Bryce E Nickels, Simon L Dove
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    ABSTRACT: It has been widely assumed that all transcription in cells occur using NTPs only (i.e., de novo). However, it has been known for several decades that both prokaryotic and eukaryotic RNA polymerases can utilize small (2 to ∼5 nt) RNAs to prime transcription initiation in vitro, raising the possibility that small RNAs might also prime transcription initiation in vivo. A new study by Goldman et al. has now provided the first evidence that priming with so-called "nanoRNAs" (i.e., 2 to ∼5 nt RNAs) can, in fact, occur in vivo. Furthermore, this study provides evidence that altering the extent of nanoRNA-mediated priming of transcription initiation can profoundly influence global gene expression. In this perspective, we summarize the findings of Goldman et al. and discuss the prospect that nanoRNA-mediated priming of transcription initiation represents an underappreciated aspect of gene expression in vivo.
    Journal of Molecular Biology 06/2011; 412(5):772-81. · 3.91 Impact Factor
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    ABSTRACT: The pneumococcal type 1 pilus, which is present in 25 to 30% of clinical isolates, has been associated with increased adherence and inflammatory responses and is being evaluated as a potential vaccine candidate. Here we show that expression of the pilus is bistable as a result of the molecular interaction between the transcription activator RrlA and a structural component of the pilus called RrgA. Sampling various clinical pneumococcal isolates that harbor the type 1 pilus-encoding islet, we show that distinct populations of cells can be identified with either undetectable or prominent pilus expression. When these two populations are separated and regrown in liquid medium, they are phenotypically different: the nonexpressing population reverts to the previous bimodal distribution, whereas the expressing population retains the same high level of pilus expression. Controlled exogenous expression of the regulatory pilus gene rlrA in a strain from which the endogenous version has been deleted increases pilus expression steadily, suggesting that the bistable expression of the pilus observed in wild-type cells is dependent on the native rlrA promoter. Finally, we demonstrate that RrgA is a negative regulator of pilus expression and that this repression is likely mediated through direct interaction with RlrA. We conclude that type 1 pilus expression in pneumococcus exhibits a bistable phenotype, which is dependent upon the molecular interplay between the RlrA and RrgA proteins. We suggest that this flexibility in expression may assist adaptation to a range of immune conditions, such as evasion of antipilus antibodies, within potential hosts.
    Infection and immunity 05/2011; 79(8):2974-83. · 4.21 Impact Factor
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    ABSTRACT: The tubulin-like FtsZ protein initiates assembly of the bacterial cytokinetic machinery by polymerizing into a ring structure, the Z ring, at the prospective site of division. To block Z-ring formation over the nucleoid and help coordinate cell division with chromosome segregation, Escherichia coli employs the nucleoid-associated division inhibitor, SlmA. Here, we investigate the mechanism by which SlmA regulates FtsZ assembly. We show that SlmA disassembles FtsZ polymers in vitro. In addition, using chromatin immunoprecipitation (ChIP), we identified 24 SlmA-binding sequences (SBSs) on the chromosome. Remarkably, SlmA binding to SBSs dramatically enhanced its ability to interfere with FtsZ polymerization, and ChIP studies indicate that SlmA regulates FtsZ assembly at these sites in vivo. Because of the dynamic and highly organized nature of the chromosome, coupling SlmA activation to specific DNA binding provides a mechanism for the precise spatiotemporal control of its anti-FtsZ activity within the cell.
    Proceedings of the National Academy of Sciences 02/2011; 108(9):3773-8. · 9.81 Impact Factor
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    ABSTRACT: Comprehensive clone sets representing the entire genome now exist for a large number of organisms. The Gateway entry clone sets are a particularly useful means to study gene function, given the ease of introduction into any Gateway-suitable destination vector. We have adapted a bacterial two-hybrid system for use with Gateway entry clone sets, such that potential interactions between proteins encoded within these clone sets can be determined by new destination vectors. We show that utilizing the Gateway clone sets for Francisella tularensis and Vibrio cholerae, known interactions between F. tularensis IglA and IglB and V. cholerae VipA and VipB could be confirmed with these destination vectors. Moreover, the introduction of unique tags into each vector allowed for visualization of the expressed hybrid proteins via Western immunoblot. This Gateway-suitable bacterial two-hybrid system provides a new tool for rapid screening of protein-protein interactions.
    BioTechniques 11/2010; 49(5):831-3. · 2.40 Impact Factor
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    Sandra Castang, Simon L Dove
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    ABSTRACT: H-NS is an abundant DNA-binding protein that has been implicated in the silencing of foreign DNA in several different bacteria. The ability of H-NS dimers to form higher-order oligomers is thought to aid the polymerization of the protein across AT-rich stretches of DNA and facilitate gene silencing. Although the oligomerization of H-NS from enteric bacteria has been the subject of intense investigation, little is known regarding the oligomerization of H-NS family members from bacteria outside of the enterobacteriaceae, many of which share little sequence similarity with their enteric counterparts. Here we show that MvaT, a member of the H-NS family of proteins from Pseudomonas aeruginosa, can form both dimers and higher-order oligomers, and we identify a region within MvaT that mediates higher-order oligomer formation. Using genetic assays we identify mutants of MvaT that are defective for higher-order oligomer formation. We present evidence that these mutants are functionally impaired and exhibit DNA-binding defects because of their inability to form higher-order oligomers. Our findings support a model in which the ability of MvaT to bind efficiently to the DNA depends upon protein-protein interactions between MvaT dimers and suggest that the ability to form higher-order oligomers is a conserved and essential feature of H-NS family members.
    Molecular Microbiology 11/2010; 78(4):916-31. · 5.03 Impact Factor
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    ABSTRACT: Phenotypic variation within an isogenic bacterial population is thought to ensure the survival of a subset of cells in adverse conditions. The opportunistic pathogen Pseudomonas aeruginosa variably expresses several phenotypes, including antibiotic resistance, biofilm formation, and the production of CupA fimbriae. Here we describe a previously unidentified bistable switch in P. aeruginosa. This switch controls the expression of a diverse set of genes, including aprA, which encodes the secreted virulence factor alkaline protease. We present evidence that bistable expression of PA2432, herein named bexR (bistable expression regulator), which encodes a LysR-type transcription regulator, controls this switch. In particular, using DNA microarrays, quantitative RT-PCR analysis, chromatin immunoprecipitation, and reporter gene fusions, we identify genes directly under the control of BexR and show that these genes are bistably expressed. Furthermore, we show that bexR is itself bistably expressed and positively autoregulated. Finally, using single-cell analyses of a GFP reporter fusion, we present evidence that positive autoregulation of bexR is necessary for bistable expression of the BexR regulon. Our findings suggest that a positive feedback loop involving a LysR-type transcription regulator serves as the basis for an epigenetic switch that controls virulence gene expression in P. aeruginosa.
    PLoS Genetics 12/2009; 5(12):e1000779. · 8.52 Impact Factor
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    ABSTRACT: In Francisella tularensis, the SspA protein family members MglA and SspA form a complex that associates with RNA polymerase (RNAP) to positively control the expression of virulence genes critical for the intramacrophage growth and survival of the organism. Although the association of the MglA-SspA complex with RNAP is evidently central to its role in controlling gene expression, the molecular details of how MglA and SspA exert their effects are not known. Here we show that in the live vaccine strain of F. tularensis (LVS), the MglA-SspA complex works in concert with a putative DNA-binding protein we have called PigR, together with the alarmone guanosine tetraphosphate (ppGpp), to regulate the expression of target genes. In particular, we present evidence that MglA, SspA, PigR and ppGpp regulate expression of the same set of genes, and show that mglA, sspA, pigR and ppGpp null mutants exhibit similar intramacrophage growth defects and are strongly attenuated for virulence in mice. We show further that PigR interacts directly with the MglA-SspA complex, suggesting that the central role of the MglA and SspA proteins in the control of virulence gene expression is to serve as a target for a transcription activator. Finally, we present evidence that ppGpp exerts its effects by promoting the interaction between PigR and the RNAP-associated MglA-SspA complex. Through its responsiveness to ppGpp, the contact between PigR and the MglA-SspA complex allows the integration of nutritional cues into the regulatory network governing virulence gene expression.
    PLoS Pathogens 10/2009; 5(10):e1000641. · 8.14 Impact Factor
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    ABSTRACT: We report here the results of an analysis of the regulatory range of the GacS/GacA two-component system in Pseudomonas aeruginosa. Using microarrays, we identified a large number of genes that are regulated by the system, and detected a near complete overlap of these genes with those regulated by two small RNAs (sRNAs), RsmY and RsmZ, suggesting that the expression of all GacA-regulated genes is RsmY/Z-dependent. Using genome-wide DNA-protein interaction analyses, we identified only two genomic regions that associated specifically with GacA, located upstream of the rsmY and rsmZ genes. These results demonstrate that in P. aeruginosa, the GacS/GacA system transduces the regulatory signals to downstream genes exclusively by directly controlling the expression of only two genes rsmY and rsmZ. These two sRNAs serve as intermediates between the input signals and the output at the level of mRNA stability, although additional regulatory inputs can influence the levels of these two riboregulators. We show that the A+T-rich DNA segment upstream of rsmZ is bound and silenced by MvaT and MvaU, the global gene regulators of the H-NS family. This work highlights the importance of post-transcriptional mechanisms involving sRNAs in controlling gene expression during bacterial adaptation to different environments.
    Molecular Microbiology 08/2009; 73(3):434-45. · 5.03 Impact Factor
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    ABSTRACT: An Escherichia coli system was used to produce the human membrane proteins presenilin 1 and amyloid precursor protein and to analyse their interaction. Our data indicate that the main binding site for amyloid precursor protein is located in the N-terminal three-transmembrane segments of presenilin and not in the proposed active site containing the two conserved aspartate residues. The data also suggest the presence of an additional segment of sufficient hydrophobicity at the C-terminus of PS1 to act potentially as a transmembrane segment. The implications of these findings for the function of gamma-secretase are discussed.
    Molecular Membrane Biology 07/2009; 21(6):373-83. · 3.13 Impact Factor

Publication Stats

2k Citations
348.39 Total Impact Points

Institutions

  • 1997–2012
    • Harvard Medical School
      • Department of Microbiology and Immunobiology
      Boston, Massachusetts, United States
  • 2009
    • Center for Molecular Genetics
      Gif, Île-de-France, France
  • 2008
    • Boston Children's Hospital
      • Division of Infectious Diseases
      Boston, MA, United States
  • 2002
    • The Rockefeller University
      • Laboratory of Molecular Biophysics
      New York City, NY, United States