RESUMO
Vibrio cholerae biofilm formation/maintenance is controlled by myriad factors; chief among these are the regulator VpsR and cyclic di-guanosine monophosphate (c-di-GMP). VpsR has strong sequence similarity to enhancer binding proteins (EBPs) that activate RNA polymerase containing sigma factor σ54. However, we have previously shown that transcription from promoters within the biofilm biogenesis/maintenance pathways uses VpsR, c-di-GMP and RNA polymerase containing the primary sigma factor (σ70). Previous work suggested that phosphorylation of VpsR at a highly conserved aspartate, which is phosphorylated in other EBPs, might also contribute to activation. Using the biofilm biogenesis promoter PvpsL, we show that in the presence of c-di-GMP, either wild type or the phospho-mimic VpsR D59E activates PvpsL transcription, while the phospho-defective D59A variant does not. Furthermore, when c-di-GMP levels are low, acetyl phosphate (Acâ¼P) is required for significant VpsR activity in vivo and in vitro. Although these findings argue that VpsR phosphorylation is needed for activation, we show that VpsR is not phosphorylated or acetylated by Acâ¼P and either sodium phosphate or potassium phosphate, which are not phosphate donors, fully substitutes for Acâ¼P. We conclude that VpsR is an unusual regulator that senses phosphate directly, rather than through phosphorylation, to aid in the decision to form/maintain biofilm.
Assuntos
Vibrio cholerae , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Proteínas de Ligação a DNA/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Fosfatos/metabolismo , Fator sigma/genética , Fator sigma/metabolismo , Vibrio cholerae/metabolismoRESUMO
Nucleoid Associated Proteins (NAPs) organize the bacterial chromosome within the nucleoid. The interaction of the NAP H-NS with DNA also represses specific host and xenogeneic genes. Previously, we showed that the bacteriophage T4 early protein MotB binds to DNA, co-purifies with H-NS/DNA, and improves phage fitness. Here we demonstrate using atomic force microscopy that MotB compacts the DNA with multiple MotB proteins at the center of the complex. These complexes differ from those observed with H-NS and other NAPs, but resemble those formed by the NAP-like proteins CbpA/Dps and yeast condensin. Fluorescent microscopy indicates that expression of motB in vivo, at levels like that during T4 infection, yields a significantly compacted nucleoid containing MotB and H-NS. motB overexpression dysregulates hundreds of host genes; â¼70% are within the hns regulon. In infected cells overexpressing motB, 33 T4 late genes are expressed early, and the T4 early gene repEB, involved in replication initiation, is up â¼5-fold. We postulate that MotB represents a phage-encoded NAP that aids infection in a previously unrecognized way. We speculate that MotB-induced compaction may generate more room for T4 replication/assembly and/or leads to beneficial global changes in host gene expression, including derepression of much of the hns regulon.
Assuntos
Proteínas de Bactérias/metabolismo , Bacteriófago T4/genética , Proteínas de Ligação a DNA/metabolismo , Inativação Gênica , Proteínas de Bactérias/genética , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Viral/química , DNA Viral/genética , Proteínas de Ligação a DNA/genética , Escherichia coli , Interações Hospedeiro-Patógeno , RegulonRESUMO
Vibrio cholerae biofilm biogenesis, which is important for survival, dissemination, and persistence, requires multiple genes in the Vibrio polysaccharides (vps) operons I and II as well as the cluster of ribomatrix (rbm) genes. Transcriptional control of these genes is a complex process that requires several activators/repressors and the ubiquitous signaling molecule, cyclic di-GMP (c-di-GMP). Previously, we demonstrated that VpsR directly activates RNA polymerase containing σ70 (σ70-RNAP) at the vpsL promoter (P vpsL ), which precedes the vps-II operon, in a c-di-GMP-dependent manner by stimulating formation of the transcriptionally active, open complex. Using in vitro transcription, electrophoretic mobility shift assays, and DNase I footprinting, we show here that VpsR also directly activates σ70-RNAP transcription from other promoters within the biofilm formation cluster, including P vpsU , at the beginning of the vps-I operon, P rbmA , at the start of the rbm cluster, and P rbmF , which lies upstream of the divergent rbmF and rbmE genes. In this capacity, we find that VpsR is able to behave both as a class II activator, which functions immediately adjacent/overlapping the core promoter sequence (P vpsL and P vpsU ), and as a class I activator, which functions farther upstream (P rbmA and P rbmF ). Because these promoters vary in VpsR-DNA binding affinity in the absence and presence of c-di-GMP, we speculate that VpsR's mechanism of activation is dependent on both the concentration of VpsR and the level of c-di-GMP to increase transcription, resulting in finely tuned regulation.IMPORTANCEVibrio cholerae, the bacterial pathogen that is responsible for the disease cholera, uses biofilms to aid in survival, dissemination, and persistence. VpsR, which directly senses the second messenger c-di-GMP, is a major regulator of this process. Together with c-di-GMP, VpsR directly activates transcription by RNA polymerase containing σ70 from the vpsL biofilm biogenesis promoter. Using biochemical methods, we demonstrate for the first time that VpsR/c-di-GMP directly activates σ70-RNA polymerase at the first genes of the vps and ribomatrix operons. In this regard, it functions as either a class I or class II activator. Our results broaden the mechanism of c-di-GMP-dependent transcription activation and the specific role of VpsR in biofilm formation.
Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes , GMP Cíclico/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Fator sigma/metabolismo , Vibrio cholerae/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Óperon , Regiões Promotoras GenéticasRESUMO
The small molecule cyclic di-GMP (c-di-GMP) is known to affect bacterial gene expression in myriad ways. In Vibrio cholerae in vivo, the presence of c-di-GMP together with the response regulator VpsR results in transcription from PvpsL, a promoter of biofilm biosynthesis genes. VpsR shares homology with enhancer binding proteins that activate σ54-RNA polymerase (RNAP), but it lacks conserved residues needed to bind to σ54-RNAP and to hydrolyze adenosine triphosphate, and PvpsL transcription does not require σ54 in vivo. Consequently, the mechanism of this activation has not been clear. Using an in vitro transcription system, we demonstrate activation of PvspL in the presence of VpsR, c-di-GMP and σ70-RNAP. c-di-GMP does not significantly change the affinity of VpsR for PvpsL DNA or the DNase I footprint of VpsR on the DNA, and it is not required for VpsR to dimerize. However, DNase I and KMnO4 footprints reveal that the σ70-RNAP/VpsR/c-di-GMP complex on PvpsL adopts a different conformation from that formed by σ70-RNAP alone, with c-di-GMP or with VpsR. Our results suggest that c-di-GMP is required for VpsR to generate the specific protein-DNA architecture needed for activated transcription, a previously unrecognized role for c-di-GMP in gene expression.
Assuntos
Proteínas de Bactérias/fisiologia , Biofilmes/crescimento & desenvolvimento , GMP Cíclico/análogos & derivados , Proteínas de Ligação a DNA/fisiologia , Regulação Bacteriana da Expressão Gênica/genética , Regiões Promotoras Genéticas/genética , Iniciação da Transcrição Genética , Vibrio cholerae/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , GMP Cíclico/fisiologia , Pegada de DNA , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Ativação Enzimática , Ligação Proteica , Fator sigma/metabolismo , Relação Estrutura-Atividade , Vibrio cholerae/metabolismoRESUMO
During infection, bacteriophage T4 produces the MotA transcription factor that redirects the host RNA polymerase to the expression of T4 middle genes. The C-terminal 'double-wing' domain of MotA binds specifically to the MotA box motif of middle T4 promoters. We report the crystal structure of this complex, which reveals a new mode of protein-DNA interaction. The domain binds DNA mostly via interactions with the DNA backbone, but the binding is enhanced in the specific cognate structure by additional interactions with the MotA box motif in both the major and minor grooves. The linker connecting the two MotA domains plays a key role in stabilizing the complex via minor groove interactions. The structure is consistent with our previous model derived from chemical cleavage experiments using the entire transcription complex. α- and ß-d-glucosyl-5-hydroxymethyl-deoxycytosine replace cytosine in T4 DNA, and docking simulations indicate that a cavity in the cognate structure can accommodate the modified cytosine. Binding studies confirm that the modification significantly enhances the binding affinity of MotA for the DNA. Consequently, our work reveals how a DNA modification can extend the uniqueness of small DNA motifs to facilitate the specificity of protein-DNA interactions.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Citosina/análogos & derivados , Citosina/química , Citosina/metabolismo , DNA/química , Proteínas de Ligação a DNA/genética , Simulação de Acoplamento Molecular , Mutagênese , Conformação Proteica , Fatores de Transcrição/genética , Proteínas Virais/genéticaRESUMO
In Bordetella pertussis, two serologically distinct fimbriae, FIM2 and FIM3, undergo on/off phase variation independently of each other via variation in the lengths of C stretches in the promoters for their major subunit genes, fim2 and fim3 These two promoters are also part of the BvgAS virulence regulon and therefore, if in an on configuration, are activated by phosporylated BvgA (BvgA~P) under normal growth conditions (Bvg+ mode) but not in the Bvg- mode, inducible by growth in medium containing MgSO4 or other compounds, termed modulators. In the B. pertussis Tohama I strain (FIM2+ FIM3-), the fim3 promoter is in the off state. However, a high level of transcription of the fim3 gene is observed in the Bvg- mode. In this study, we provide an explanation for this anomalous behavior by defining a Bvg-repressed promoter (BRP), located approximately 400 bp upstream of the Pfim3 transcriptional start. Although transcription of the fim3 gene in the Bvg- mode resulted in Fim3 translation, as measured by LacZ translational fusions, no accumulation of Fim3 protein was detectable. We propose that Fim3 protein resulting from translation of mRNA driven by BRP in the Bvg- mode is unstable due to a lack of the fimbrial assembly apparatus encoded by the fimBC genes, located within the fha operon, and therefore is not expressed in the Bvg- mode.IMPORTANCE In Bordetella pertussis, the promoter Pfim3-15C for the major fimbrial subunit gene fim3 is activated by the two-component system BvgAS in the Bvg+ mode but not in the Bvg- mode. However, many transcriptional profiling studies have shown that fim3 is transcribed in the Bvg- mode even when Pfim3 is in a nonpermissive state (Pfim3-13C), suggesting the presence of a reciprocally regulated element upstream of Pfim3 Here, we provide evidence that BRP is the cause of this anomalous behavior of fim3 Although BRP effects vrg-like transcription of fim3 in the Bvg- mode, it does not lead to stable production of FIM3 fimbriae, because expression of the chaperone and usher proteins FimB and FimC occurs only in the Bvg+ mode.
Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Bordetella pertussis/genética , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/metabolismo , Regiões Promotoras Genéticas , Transativadores/genética , Fatores de Virulência de Bordetella/genética , Sequência de Aminoácidos , Antígenos de Bactérias/metabolismo , Sequência de Bases , Proteínas de Fímbrias/metabolismo , Regulação Bacteriana da Expressão Gênica , Óperon Lac , Sorogrupo , Fatores de Virulência de Bordetella/metabolismoRESUMO
Multisubunit RNA polymerases are complex protein machines that require a specificity factor for the recognition of a specific transcription start site. Although bacterial σ factors are thought to be quite different from the specificity factors employed in higher organisms, a comparison of the σ/RNA polymerase structures with recent structures of eukaryotic Pol II together with TFIIB highlights significant functional similarities. Other work reveals that both bacterial and eukaryotic promoters are composed of modular elements that are used in different combinations. Bacteria, archaea, and eukaryotes also utilize similar strategies to alter core promoter specificity, from specificity factor exchange to the employment of activators that bind close to or overlap core promoter sequences, directing the transcriptional machinery to a new start site. Here we examine the details of core promoter recognition in bacteria that reveal the transcriptional similarities throughout biology.
Assuntos
Archaea/genética , Bactérias/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Eucariotos/genética , Transcrição Gênica , Animais , Archaea/enzimologia , Bactérias/enzimologia , RNA Polimerases Dirigidas por DNA/genética , Eucariotos/enzimologia , Regulação da Expressão Gênica em Archaea , Regulação Bacteriana da Expressão Gênica , Humanos , Regiões Promotoras GenéticasRESUMO
The ability of RNA polymerase (RNAP) to select the right promoter sequence at the right time is fundamental to the control of gene expression in all organisms. However, there is only one crystallized structure of a complete activator/RNAP/DNA complex. In a process called σ appropriation, bacteriophage T4 activates a class of phage promoters using an activator (MotA) and a co-activator (AsiA), which function through interactions with the σ(70) subunit of RNAP. We have developed a holistic, structure-based model for σ appropriation using multiple experimentally determined 3D structures (Escherichia coli RNAP, the Thermus aquaticus RNAP/DNA complex, AsiA /σ(70) Region 4, the N-terminal domain of MotA [MotA(NTD)], and the C-terminal domain of MotA [MotA(CTD)]), molecular modeling, and extensive biochemical observations indicating the position of the proteins relative to each other and to the DNA. Our results visualize how AsiA/MotA redirects σ, and therefore RNAP activity, to T4 promoter DNA, and demonstrate at a molecular level how the tactful interaction of transcriptional factors with even small segments of RNAP can alter promoter specificity. Furthermore, our model provides a rational basis for understanding how a mutation within the ß subunit of RNAP (G1249D), which is far removed from AsiA or MotA, impairs σ appropriation.
Assuntos
Bacteriófago T4/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , DNA/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Transcrição Gênica , Sequência de Aminoácidos , Fenômenos Biofísicos , Reagentes de Ligações Cruzadas/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Luz , Modelos Moleculares , Peptídeos/química , Regiões Promotoras GenéticasRESUMO
Two-component systems [sensor kinase/response regulator (RR)] are major tools used by microorganisms to adapt to environmental conditions. RR phosphorylation is typically required for gene activation, but few studies have addressed how and if phosphorylation affects specific steps during transcription initiation. We characterized transcription complexes made with RNA polymerase and the Bordetella pertussis RR, BvgA, in its nonphosphorylated or phosphorylated (BvgAâ¼P) state at P(fim3), the promoter for the virulence gene fim3 (fimbrial subunit), using gel retardation, potassium permanganate and DNase I footprinting, cleavage reactions with protein conjugated with iron bromoacetamidobenzyl-EDTA, and in vitro transcription. Previous work has shown that the level of nonphosphorylated BvgA remains high in vivo under conditions in which BvgA is phosphorylated. Our results here indicate that surprisingly both BvgA and BvgAâ¼P form open and initiating complexes with RNA polymerase at P(fim3). However, phosphorylation of BvgA is needed to generate the correct conformation that can transition to competent elongation. Footprints obtained with the complexes made with nonphosphorylated BvgA are atypical; while the initiating complex with BvgA synthesizes short RNA, it does not generate full-length transcripts. Extended incubation of the BvgA/RNA polymerase initiated complex in the presence of heparin generates a stable, but defective species that depends on the initial transcribed sequence of fim3. We suggest that the presence of nonphosphorylated BvgA down-regulates P(fim3) activity when phosphorylated BvgA is present and may allow the bacterium to quickly adapt to the loss of inducing conditions by rapidly eliminating P(fim3) activation once the signal for BvgA phosphorylation is removed.
Assuntos
Adaptação Fisiológica/fisiologia , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Bordetella pertussis/genética , Proteínas de Fímbrias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Complexos Multiproteicos/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/fisiologia , Fatores de Virulência de Bordetella/metabolismo , Adaptação Fisiológica/genética , Antígenos de Bactérias/genética , Bordetella pertussis/patogenicidade , Pegada de DNA , Primers do DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Combinação de Medicamentos , Ensaio de Desvio de Mobilidade Eletroforética , Proteínas de Fímbrias/genética , Regulação Bacteriana da Expressão Gênica/genética , Complexos Multiproteicos/genética , Óleos , Fenóis , Fosforilação , Transcrição Gênica/genética , Virulência , Fatores de Virulência de Bordetella/genéticaRESUMO
The Bvg-regulated promoters for the fimbrial subunit genes fim2 and fim3 of Bordetella pertussis behave differently from each other both in vivo and in vitro. In vivoâ Pfim2 is significantly stronger than Pfim3 , even though predictions based on the DNA sequences of BvgA-binding motifs and core promoter elements would indicate the opposite. In vitroâ Pfim3 demonstrated robust BvgAâ¼P-dependent transcriptional activation, while none was seen with Pfim2 . This apparent contradiction was investigated further. By swapping sequence elements we created a number of hybrid promoters and assayed their strength in vivo. We found that, while Pfim3 promoter elements upstream of the +1 transcriptional start site do indeed direct Bvg-activated transcription more efficiently than those of Pfim2 , the overall promoter strength of Pfim3 â in vivo is reduced due to sequences downstream of +1 that inhibit transcription more than 250-fold. This element, the DRE (downstream repressive element), was mapped to the 15 bp immediately downstream of the Pfim3 +1. Placing the DRE in different promoter contexts indicated that its activity was not specific to fim promoters, or even to Bvg-regulated promoters. However it does appear to be specific to Bordetella species in that it did not function in Escherichia coli.
Assuntos
Antígenos de Bactérias/biossíntese , Bordetella pertussis/genética , Bordetella pertussis/metabolismo , Proteínas de Fímbrias/biossíntese , Regulação Bacteriana da Expressão Gênica , Elementos Reguladores de Transcrição , Transcrição Gênica , Fatores de Virulência de Bordetella/biossíntese , Antígenos de Bactérias/genética , Escherichia coli/genética , Proteínas de Fímbrias/genética , Engenharia Metabólica , Regiões Promotoras Genéticas , Recombinação Genética , Sítio de Iniciação de Transcrição , Fatores de Virulência de Bordetella/genéticaRESUMO
Gene expression can be regulated through factors that direct RNA polymerase to the correct promoter sequence at the correct time. Bacteriophage T4 controls its development in this way using phage proteins that interact with host RNA polymerase. Using a process called σ appropriation, the T4 co-activator AsiA structurally remodels the σ(70) subunit of host RNA polymerase, while a T4 activator, MotA, engages the C terminus of σ(70) and binds to a DNA promoter element, the MotA box. Structures for the N-terminal (NTD) and C-terminal (CTD) domains of MotA are available, but no structure exists for MotA with or without DNA. We report the first molecular map of the MotA/DNA interaction within the σ-appropriated complex, which we obtained by using the cleaving reagent, iron bromoacetamidobenzyl-EDTA (FeBABE). We conjugated surface-exposed, single cysteines in MotA with FeBABE and performed cleavage reactions in the context of stable transcription complexes. The DNA cleavage sites were analyzed using ICM Molsoft software and three-dimensional physical models of MotA(NTD), MotA(CTD), and the DNA to investigate shape complementarity between the protein and the DNA and to position MotA on the DNA. We found that the unusual "double wing" motif present within MotA(CTD) resides in the major groove of the MotA box. In addition, we have used surface plasmon resonance to show that MotA alone is in a very dynamic equilibrium with the MotA element. Our results demonstrate the utility of fine resolution FeBABE mapping to determine the architecture of protein-DNA complexes that have been recalcitrant to traditional structure analyses.
Assuntos
Bacteriófago T4/química , DNA Viral/química , Proteínas de Ligação a DNA/química , RNA Polimerases Dirigidas por DNA/química , Proteínas de Escherichia coli/química , Modelos Moleculares , Regiões Promotoras Genéticas , Fator sigma/química , Fatores de Transcrição/química , Proteínas Virais/química , Motivos de Aminoácidos , Bacteriófago T4/genética , Bacteriófago T4/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/virologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Estrutura Terciária de Proteína , Fator sigma/genética , Fator sigma/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
We have used protein electrophoresis through polyacrylamide gels derivatized with the proprietary ligand Phos-tag™ to separate the response regulator BvgA from its phosphorylated counterpart BvgAâ¼P. This approach has allowed us to readily ascertain the degree of phosphorylation of BvgA in in vitro reactions, or in crude lysates of Bordetella pertussis grown under varying laboratory conditions. We have used this technique to examine the kinetics of BvgA phosphorylation after shift of B. pertussis cultures from non-permissive to permissive conditions, or of its dephosphorylation following a shift from permissive to non-permissive conditions. Our results provide the first direct evidence that levels of BvgAâ¼Pâ in vivo correspond temporally to the expression of early and late BvgA-regulated virulence genes. We have also examined a number of other aspects of BvgA function predicted from previous studies and by analogy with other two-component response regulators. These include the site of BvgA phosphorylation, the exclusive role of the cognate BvgS sensor kinase in its phosphorylation in Bordetella pertussis, and the effect of the T194M mutation on phosphorylation. We also detected the phosphorylation of a small but consistent fraction of BvgA purified after expression in Escherichia coli.
Assuntos
Proteínas de Bactérias/metabolismo , Bordetella pertussis/metabolismo , Bordetella pertussis/patogenicidade , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Ácido Aspártico/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Bordetella pertussis/efeitos dos fármacos , Bordetella pertussis/genética , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Cinética , Sulfato de Magnésio/farmacologia , Dados de Sequência Molecular , Mutação/genética , Fosforilação/efeitos dos fármacos , Fosfotreonina/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Virulência/efeitos dos fármacos , Virulência/genéticaRESUMO
Efficient DNA replication involves coordinated interactions among DNA polymerase, multiple factors, and the DNA. From bacteriophage T4 to eukaryotes, these factors include a helicase to unwind the DNA ahead of the replication fork, a single-stranded binding protein (SSB) to bind to the ssDNA on the lagging strand, and a helicase loader that associates with the fork, helicase, and SSB. The previously reported structure of the helicase loader in the T4 system, gene product (gp)59, has revealed an N-terminal domain, which shares structural homology with the high mobility group (HMG) proteins from eukaryotic organisms. Modeling of this structure with fork DNA has suggested that the HMG-like domain could bind to the duplex DNA ahead of the fork, whereas the C-terminal portion of gp59 would provide the docking sites for helicase (T4 gp41), SSB (T4 gp32), and the ssDNA fork arms. To test this model, we have used random and targeted mutagenesis to generate mutations throughout gp59. We have assayed the ability of the mutant proteins to bind to fork, primed fork, and ssDNAs, to interact with SSB, to stimulate helicase activity, and to function in leading and lagging strand DNA synthesis. Our results provide strong biochemical support for the role of the N-terminal gp59 HMG motif in fork binding and the interaction of the C-terminal portion of gp59 with helicase and SSB. Our results also suggest that processive replication may involve the switching of gp59 between its interactions with helicase and SSB.
Assuntos
Bacteriófago T4/genética , DNA Helicases/genética , DNA de Cadeia Simples/genética , DNA Viral/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Ligação a DNA/química , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Proteínas Virais/químicaRESUMO
LF82, an adherent invasive Escherichia coli pathobiont, is associated with ileal Crohn's disease, an inflammatory bowel disease of unknown etiology. Although LF82 contains no virulence genes, it carries several genetic differences, including single nucleotide polymorphisms (SNPs), that distinguish it from nonpathogenic E. coli. We have identified and investigated an extremely rare SNP that is within the highly conserved rpoD gene, encoding σ70, the primary sigma factor for RNA polymerase. We demonstrate that this single residue change (D445V) results in specific transcriptome and phenotypic changes that are consistent with multiple phenotypes observed in LF82, including increased antibiotic resistance and biofilm formation, modulation of motility, and increased capacity for methionine biosynthesis. Our work demonstrates that a single residue change within the bacterial primary sigma factor can lead to multiple alterations in gene expression and phenotypic changes, suggesting an underrecognized mechanism by which pathobionts and other strain variants with new phenotypes can emerge.
RESUMO
Sigma factors, the specificity subunits of RNA polymerase, are involved in interactions with promoter DNA, the core subunits of RNA polymerase, and transcription factors. The bacteriophage T4-encoded activator, MotA, is one such factor, which engages the C terminus of the Escherichia coli housekeeping sigma factor, σ(70). MotA functions in concert with a phage-encoded co-activator, AsiA, as a molecular switch. This process, termed sigma appropriation, inhibits host transcription while activating transcription from a class of phage promoters. Previous work has demonstrated that MotA contacts the C terminus of σ(70), H5, a region that is normally bound within RNA polymerase by its interaction with the ß-flap tip. To identify the specific σ(70) residues responsible for interacting with MotA and the ß-flap tip, we generated single substitutions throughout the C terminus of σ(70). We find that MotA targets H5 residues that are normally engaged by the ß-flap. In two-hybrid assays, the interaction of σ(70) with either the ß-flap tip or MotA is impaired by alanine substitutions at residues Leu-607, Arg-608, Phe-610, Leu-611, and Asp-613. Transcription assays identify Phe-610 and Leu-611 as the key residues for MotA/AsiA-dependent transcription. Phe-610 is a crucial residue in the H5/ß-flap tip interaction using promoter clearance assays with RNA polymerase alone. Our results show how the actions of small transcriptional factors on a defined local region of RNA polymerase can fundamentally change the specificity of polymerase.
Assuntos
Bacteriófago T4/metabolismo , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Fator sigma/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Virais/metabolismo , Bacteriófago T4/genética , Proteínas de Ligação a DNA/genética , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/genética , Escherichia coli/virologia , Proteínas de Escherichia coli/genética , Regiões Promotoras Genéticas/fisiologia , Estrutura Terciária de Proteína , Fator sigma/genética , Especificidade por Substrato , Fatores de Transcrição/genética , Transcrição Gênica/fisiologia , Proteínas Virais/genéticaRESUMO
Bordetella pertussis causes whooping cough, an infectious disease that is reemerging despite widespread vaccination. A more complete understanding of B. pertussis pathogenic mechanisms will involve unravelling the regulation of its impressive arsenal of virulence factors. Here we review the action of the B. pertussis response regulator BvgA in the context of what is known about bacterial RNA polymerase and various modes of transcription activation. At most virulence gene promoters, multiple dimers of phosphorylated BvgA (BvgA~P) bind upstream of the core promoter sequence, using a combination of high- and low-affinity sites that fill through cooperativity. Activation by BvgA~P is typically mediated by a novel form of class I/II mechanisms, but two virulence genes, fim2 and fim3, which encode serologically distinct fimbrial subunits, are regulated using a previously unrecognized RNA polymerase/activator architecture. In addition, the fim genes undergo phase variation because of an extended cytosine (C) tract within the promoter sequences that is subject to slipped-strand mispairing during replication. These sophisticated systems of regulation demonstrate one aspect whereby B. pertussis, which is highly clonal and lacks the extensive genetic diversity observed in many other bacterial pathogens, has been highly successful as an obligate human pathogen.
Assuntos
Proteínas de Bactérias/metabolismo , Bordetella pertussis/genética , Regulação Bacteriana da Expressão Gênica , Fatores de Transcrição/metabolismo , Humanos , Modelos Biológicos , Regiões Promotoras Genéticas , Ligação Proteica , Multimerização ProteicaRESUMO
Transcription initiation is a dynamic process in which RNA polymerase (RNAP) and promoter DNA act as partners, changing in response to one another, to produce a polymerase/promoter open complex (RPo) competent for transcription. In Escherichia coli RNAP, region 1.1, the N-terminal 100 residues of sigma(70), is thought to occupy the channel that will hold the DNA downstream of the transcription start site; thus, region 1.1 must move from this channel as RPo is formed. Previous work has also shown that region 1.1 can modulate RPo formation depending on the promoter. For some promoters region 1.1 stimulates the formation of open complexes; at the P(minor) promoter, region 1.1 inhibits this formation. We demonstrate here that the AT-rich P(minor) spacer sequence, rather than promoter recognition elements or downstream DNA, determines the effect of region 1.1 on promoter activity. Using a P(minor) derivative that contains good sigma(70)-dependent DNA elements, we find that the presence of a more GC-rich spacer or a spacer with the complement of the P(minor) sequence results in a promoter that is no longer inhibited by region 1.1. Furthermore, the presence of the P(minor) spacer, the GC-rich spacer, or the complement spacer results in different mobilities of promoter DNA during gel electrophoresis, suggesting that the spacer regions impart differing conformations or curvatures to the DNA. We speculate that the spacer can influence the trajectory or flexibility of DNA as it enters the RNAP channel and that region 1.1 acts as a "gatekeeper" to monitor channel entry.
Assuntos
RNA Polimerases Dirigidas por DNA/fisiologia , Proteínas de Escherichia coli/fisiologia , Escherichia coli/genética , Regiões Promotoras Genéticas , Fator sigma/fisiologia , Transcrição Gênica , DNA Bacteriano/química , RNA Polimerases Dirigidas por DNA/química , Conformação de Ácido Nucleico , Fator sigma/químicaRESUMO
Background: : Although many bacteriophage T4 early genes are nonessential with unknown functions, they are believed to aid in the takeover of the Escherichia coli host. Understanding the functions of these genes could be helpful to develop novel antibacterial strategies. MotB, encoded by a previously uncharacterized T4 early gene, is a DNA-binding protein that compacts the host nucleoid and alters host gene expression. Methods: : MotB structure was predicted by AlphaFold 2. RNA-seq and mass spectrometry (MS) analyses were performed to determine RNA and protein changes when motB was overexpressed in E. coli BL21(DE3) ±5 min T4 infection. Results: : MotB structure is predicted to be a two-domain protein with N-terminal Kyprides-Onzonis-Woese and C-terminal oligonucleotide/oligosaccharide-fold domains. In E. coli B, motB overexpression during infection does not affect T4 RNAs, but affects the expression of host genes, including the downregulation of 21 of the 84 chargeable host tRNAs. Many of these tRNAs are used less frequently by T4 or have a counterpart encoded within the T4 genome. The MS analyses indicate that the levels of multiple T4 proteins are changed by motB overexpression. Conclusion: : Our results suggest that in this E. coli B host, motB is involved in establishing a more favorable tRNA pool for the phage during infection.
RESUMO
The BvgAS two-component system regulates virulence gene expression in Bordetella pertussis. Although precise three-dimensional structural information is not available for the response regulator BvgA, its sequence conservation with E. coli NarL and previous studies have indicated that it is composed of 3 domains: an N-terminal domain (NTD) containing the phosphorylation site, a linker, and a DNA-binding C-terminal domain (CTD). Previous work has determined how BvgACTD dimers interact with the promoter (P fhaB ) of fhaB, the gene encoding the virulence adhesin filamentous hemagglutinin. Here we use molecular modeling, FeBABE footprinting, and crosslinking to show that within the transcription complex of phosphorylated BvgA (BvgA â¼ P), B. pertussis RNAP, and P fhaB , the NTDs displace from the CTDs and are positioned at specific locations relative to the three BvgA â¼ P binding sites. Our work identifies a patch of the NTD that faces the DNA and suggests that BvgA â¼ P undergoes a conformational rearrangement that relocates the NTD to allow productive interaction of the CTD with the DNA.
RESUMO
The bacteriophage T4 early gene product MotB binds tightly but nonspecifically to DNA, copurifies with the host Nucleoid Associated Protein (NAP) H-NS in the presence of DNA and improves T4 fitness. However, the T4 transcriptome is not significantly affected by a motB knockdown. Here we have investigated the phylogeny of MotB and its predicted domains, how MotB and H-NS together interact with DNA, and how heterologous overexpression of motB impacts host gene expression. We find that motB is highly conserved among Tevenvirinae. Although the MotB sequence has no homology to proteins of known function, predicted structure homology searches suggest that MotB is composed of an N-terminal Kyprides-Onzonis-Woese (KOW) motif and a C-terminal DNA-binding domain of oligonucleotide/oligosaccharide (OB)-fold; either of which could provide MotB's ability to bind DNA. DNase I footprinting demonstrates that MotB dramatically alters the interaction of H-NS with DNA in vitro. RNA-seq analyses indicate that expression of plasmid-borne motB up-regulates 75 host genes; no host genes are down-regulated. Approximately 1/3 of the up-regulated genes have previously been shown to be part of the H-NS regulon. Our results indicate that MotB provides a conserved function for Tevenvirinae and suggest a model in which MotB functions to alter the host transcriptome, possibly by changing the association of H-NS with the host DNA, which then leads to conditions that are more favorable for infection.