The Response Regulator BvgA and RNA Polymerase α Subunit C-Terminal Domain Bind Simultaneously to Different Faces of the Same Segment of Promoter DNA

Boucher, Philip E.; Maris, A.E.; Yang, Mei-Shin; Stibitz, Scott

doi:10.1016/s1097-2765(03)00007-8

Cited by 57 publications

(89 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Thus, Boucher et al (39) recently used our constructs to study the position of ␣CTD at a promoter that is activated by the Bordetella pertussis BvgA protein. One advantage of this approach is that it can give signals due to ␣CTD binding that makes little or no contribution to promoter activity, and thus cannot be detected by genetic means.…”

Section: Discussionmentioning

confidence: 99%

Exploitation of a Chemical Nuclease to Investigate the Location and Orientation of the Escherichia coli RNA Polymerase α Subunit C-terminal Domains at Simple Promoters That Are Activated by Cyclic AMP Receptor Protein

Lee

Busby

Lloyd

2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

The C-terminal domain of the ␣ subunit (␣CTD) of bacterial RNA polymerase plays an important role in promoter recognition. It is known that ␣CTD binds to the DNA minor groove at different locations at different promoters via a surface-exposed determinant, the 265 determinant. Here we describe experiments that permit us to determine the location and orientation of binding of ␣CTD at any promoter. In these experiments, a DNA cleavage reagent is attached to specific locations on opposite faces of the RNA polymerase ␣ subunit. After incorporation of the tagged ␣ subunits into holo-RNA polymerase, patterns of DNA cleavage due to the reagent are determined in open complexes. The locations of DNA cleavage due to the reagent attached at different positions allow the position and orientation of ␣CTD to be deduced. Here we present data from experiments with simple Escherichia coli promoters that are activated by the cyclic AMP receptor protein.RNA synthesis in bacteria is due to holo-RNA polymerase (RNAP), 1 which is a multisubunit complex with subunit composition ␣ 2 ␤␤Ј. It is well known that the major factor ensuring "correct" gene expression in bacteria is the efficiency with which RNAP recognizes the promoters of different genes. Although the principal determinants for promoter recognition reside in the RNAP subunit, at many promoters, the ␣ subunits also play a key role (1-3). Each RNAP ␣ subunit consists of two independently folding domains connected by a flexible linker. The N-terminal domain (␣NTD; residues 8 -235 in Escherichia coli ␣) carries determinants for the interaction of the ␣ subunits with the rest of RNAP, whereas the C-terminal domain (␣CTD; residues 249 -329 in E. coli ␣) carries determinants for interaction with promoter DNA elements and with certain transcription factors (4, 5). At some promoters, the interaction of ␣CTD with specific DNA sequence elements (known as UP elements) is essential for optimal transcription initiation. At other promoters, optimal expression depends on activator proteins that function by making a direct contact with ␣CTD, thereby recruiting RNAP to the promoter (3).High resolution structures for E. coli ␣CTD, either free or bound to the UP element DNA, have been obtained (6 -8). When bound to DNA, ␣CTD contacts the minor groove (9, 10). Genetic analyses, together with the structural studies, have identified residues in two helix-loop-helix motifs that are responsible for DNA binding. Thus, Arg 265 , Asn 268 , Gly 296 , Lys 298 , and Ser 299 appear to be the crucial ␣ subunit residues that make contact with the DNA minor groove (reviewed in Ref.3 and see Ref. 8). These residues are part of a small segment of the surface of ␣CTD known as the 265 determinant. The linker joining ␣NTD and ␣CTD contains at least 13 amino acids and appears to be very flexible and unstructured (11). A consequence of this is that ␣CTD can bind at different locations at different promoters. At promoters where RNAP initiates transcription without the need for an activator protein, one ␣CTD contacts t...

show abstract

Section: Discussionmentioning

confidence: 99%

Exploitation of a Chemical Nuclease to Investigate the Location and Orientation of the Escherichia coli RNA Polymerase α Subunit C-terminal Domains at Simple Promoters That Are Activated by Cyclic AMP Receptor Protein

Lee

Busby

Lloyd

2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Two hundred forty-five genes were identified as positively regulated by BvgA~P (Table S1B). Category 1 (Tables 2 and S2) contains well-known vags, such as virulence determinants and accessory factors, that have been identified by previous genetic and microarray studies (9,10,31,(33)(34)(35)(36); these include filamentous hemagglutinin/adhesin (fhaB and fhaC), adenylate cyclase toxins (cyaABCDE), fimbriae (fimABCD), pertactin (prn), pertussis toxins (ptxABDEC), and type 4 secretion system proteins (ptlABCDIEFGH). RT-qPCR confirmed the significant increase in RNA levels of several of these vags (Fig.…”

Section: Analysis Of Thementioning

confidence: 99%

“…To investigate whether some of the Bvg(ϩ) genes, whose expression significantly increased in the presence of BvgA~P, are directly regulated by BvgA~P, we performed in vitro transcriptions using B. pertussis RNA polymerase (RNAP) alone or in the presence of BvgA or BvgA~P. The BvgA~P-dependent promoter for fhaB (36,43) was used as a positive control. Among the newly identified BvgA-regulated genes, we selected BP3441 (phasin-like protein, 7-FD), BP2142 (GntR-like regulator, 6-FD), and BP1005 (hypothetical protein, 5-FD).…”

Section: Analysis Of Thementioning

confidence: 99%

“…To determine whether BvgA~P directly binds to sites within P brpL , we performed iron bromoacetamidobenzyl-EDTA (FeBABE) footprinting using BvgA T194C -FeBABE and BvgA V148C -FeBABE (36). As BvgA residue 194 is located near the BvgA~P dimer interface, while residue 148 is located on the outside face of the dimer (36), DNA cleavage by FeBABE conjugated to these residues maps the outside and inside edges of each BvgA relative to the DNA.…”

Section: Analysis Of Thementioning

confidence: 99%

See 1 more Smart Citation

The BvgAS Regulon of Bordetella pertussis

Moon

Bonocora

Kim

et al. 2017

mBio

Self Cite

120

View full text Add to dashboard Cite

Nearly all virulence factors in Bordetella pertussis are activated by a master two-component system, BvgAS, composed of the sensor kinase BvgS and the response regulator BvgA. When BvgS is active, BvgA is phosphorylated (BvgA~P), and virulence-activated genes (vags) are expressed [Bvg(ϩ) mode]. When BvgS is inactive and BvgA is not phosphorylated, virulence-repressed genes (vrgs) are induced [Bvg(Ϫ) mode]. Here, we have used transcriptome sequencing (RNA-seq) and reverse transcription-quantitative PCR (RT-qPCR) to define the BvgAS-dependent regulon of B. pertussis Tohama I. Our analyses reveal more than 550 BvgA-regulated genes, of which 353 are newly identified. BvgA-activated genes include those encoding two-component systems (such as kdpED), multiple other transcriptional regulators, and the extracytoplasmic function (ECF) sigma factor brpL, which is needed for type 3 secretion system (T3SS) expression, further establishing the importance of BvgA~P as an apex regulator of transcriptional networks promoting virulence. Using in vitro transcription, we demonstrate that the promoter for brpL is directly activated by BvgA~P. BvgA-FeBABE cleavage reactions identify BvgA~P binding sites centered at positions Ϫ41.5 and Ϫ63.5 in bprL. Most importantly, we show for the first time that genes for multiple and varied metabolic pathways are significantly upregulated in the B. pertussis Bvg(Ϫ) mode. These include genes for fatty acid and lipid metabolism, sugar and amino acid transporters, pyruvate dehydrogenase, phenylacetic acid degradation, and the glycolate/glyoxylate utilization pathway. Our results suggest that metabolic changes in the Bvg(Ϫ) mode may be participating in bacterial survival, transmission, and/or persistence and identify over 200 new vrgs that can be tested for function.IMPORTANCE Within the past 20 years, outbreaks of whooping cough, caused by Bordetella pertussis, have led to respiratory disease and infant mortalities, despite good vaccination coverage. This is due, at least in part, to the introduction of a less effective acellular vaccine in the 1990s. It is crucial, then, to understand the molecular basis of B. pertussis growth and infection. The two-component system BvgA (response regulator)/BvgS (histidine kinase) is the master regulator of B. pertussis virulence genes. We report here the first RNA-seq analysis of the BvgAS regulon in B. pertussis, revealing that more than 550 genes are regulated by BvgAS. We show that genes for multiple and varied metabolic pathways are highly regulated in the Bvg(Ϫ) mode (absence of BvgA phosphorylation). Our results suggest that metabolic changes in the Bvg(Ϫ) mode may be participating in bacterial survival, transmission, and/or persistence.

show abstract

“…BvgS, the sensor in the cytoplasmic membrane, is thought to directly sense changes in the environment and, through a phosphorylation-transfer mechanism, activates BvgA, the response regulator [24][25][26]. Once BvgA is activated (Bvg + phase), it binds to high and low affinity motifs in the genome, resulting in increased expression of the genes encoding toxins and adhesins, while expression of Bvg 2 phase genes involved in motility and uptake of certain nutrients are repressed; the opposite occurs in the Bvg 2 phase [27][28][29][30][31][32]. An intermediate phase has been described in which a subset of virulence factors are expressed, along with a unique set of factors [33][34][35]; however the Bvg + phase has been shown to be necessary and sufficient for host colonization [36].…”

Section: Introductionmentioning

confidence: 99%

Enzymatic Carbon Dioxide Capture

Bandyopadhyay¹

2014

Carbon Capture and Storage

View full text Add to dashboard Cite

Sensing the environment allows pathogenic bacteria to coordinately regulate gene expression to maximize survival within or outside of a host. Here we show that Bordetella species regulate virulence factor expression in response to carbon dioxide levels that mimic in vivo conditions within the respiratory tract. We found strains of Bordetella bronchiseptica that did not produce adenylate cyclase toxin (ACT) when grown in liquid or solid media with ambient air aeration, but produced ACT and additional antigens when grown in air supplemented to 5% CO 2 . Transcriptome analysis and quantitative real time-PCR analysis revealed that strain 761, as well as strain RB50, increased transcription of genes encoding ACT, filamentous hemagglutinin (FHA), pertactin, fimbriae and the type III secretion system in 5% CO 2 conditions, relative to ambient air. Furthermore, transcription of cyaA and fhaB in response to 5% CO 2 was increased even in the absence of BvgS. In vitro analysis also revealed increases in cytotoxicity and adherence when strains were grown in 5% CO 2 . The human pathogens B. pertussis and B. parapertussis also increased transcription of several virulence factors when grown in 5% CO 2 , indicating that this response is conserved among the classical bordetellae. Together, our data indicate that Bordetella species can sense and respond to physiologically relevant changes in CO 2 concentrations by regulating virulence factors important for colonization, persistence and evasion of the host immune response.

show abstract

The Response Regulator BvgA and RNA Polymerase α Subunit C-Terminal Domain Bind Simultaneously to Different Faces of the Same Segment of Promoter DNA

Cited by 57 publications

References 28 publications

Exploitation of a Chemical Nuclease to Investigate the Location and Orientation of the Escherichia coli RNA Polymerase α Subunit C-terminal Domains at Simple Promoters That Are Activated by Cyclic AMP Receptor Protein

Exploitation of a Chemical Nuclease to Investigate the Location and Orientation of the Escherichia coli RNA Polymerase α Subunit C-terminal Domains at Simple Promoters That Are Activated by Cyclic AMP Receptor Protein

The BvgAS Regulon of Bordetella pertussis

Enzymatic Carbon Dioxide Capture

Contact Info

Product

Resources

About