Rns, a virulence regulator within the AraC family, requires binding sites upstream and downstream of its own promoter to function as an activator

ABSTRACT The expression of CS1 pili by enterotoxigenic strains ofEscherichia coli is regulated at the transcriptional level and requires the virulence regulator Rns, a member of the AraC family of regulatory proteins. Rns binds at two separate sites upstream of Pcoo (the promoter of CS1 pilin genes), which were identified in vitro with an MBP::Rns fusion protein in gel mobility and DNase I footprinting assays. At each site, Rns recognizes asymmetric nucleotide sequences in two regions of the major groove and binds along one face of the DNA helix. Both binding sites are required for activation of Pcoo in vivo, because mutagenesis of either site significantly reduced the level of expression from this promoter. Thus, Rns regulates the expression of CS1 pilin genes directly, not via a regulatory cascade. Analysis of Rns-nucleotide interactions at each site suggests that binding sites for Rns and related virulence regulators are not easily identified because they do not bind palindromic or repeated sequences. A strategy to identify asymmetric binding sites is presented and applied to locate potential binding sites upstream of other genes that Rns can activate, including those encoding the CS2 and CFA/I pili of enterotoxigenic E. coli and the global regulator virB of Shigella flexneri.

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Rns, a virulence regulator within the AraC family, requires binding sites upstream and downstream of its own promoter to function as an activator

Molecular Microbiology ◽

10.1046/j.1365-2958.2000.01957.x ◽

2002 ◽

Vol 36 (6) ◽

pp. 1391-1402 ◽

Cited By ~ 54

Author(s):

George P. Munson ◽

June R. Scott

Keyword(s):

Binding Sites ◽

Virulence Regulator ◽

Arac Family

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CfaD-Dependent Expression of a Novel Extracytoplasmic Protein from Enterotoxigenic Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.00131-07 ◽

2007 ◽

Vol 189 (14) ◽

pp. 5060-5067 ◽

Cited By ~ 33

Author(s):

M. Carolina Pilonieta ◽

Maria D. Bodero ◽

George P. Munson

Keyword(s):

Escherichia Coli ◽

Binding Sites ◽

Secretory Pathway ◽

Dnase I ◽

Enterotoxigenic Escherichia Coli ◽

Watery Diarrhea ◽

Dnase I Footprinting ◽

Virulence Regulator

ABSTRACT H10407 is a strain of enterotoxigenic Escherichia coli (ETEC) that utilizes CFA/I pili to adhere to surfaces of the small intestine, where it elaborates toxins that cause profuse watery diarrhea in humans. Expression of the CFA/I pilus is positively regulated at the level of transcription by CfaD, a member of the AraC/XylS family. DNase I footprinting revealed that the activator has two binding sites upstream of the pilus promoter cfaAp. One site extends from positions −23 to −56, and the other extends from positions −73 to −103 (numbering relative to the transcription start site of cfaAp). Additional CfaD binding sites were predicted within the genome of H10407 by computational analysis. Two of these sites lie upstream of a previously uncharacterized gene, cexE. In vitro DNase I footprinting confirmed that both sites are genuine binding sites, and cexEp::lacZ reporters demonstrated that CfaD is required for the expression of cexE in vivo. The amino terminus of CexE contains a secretory signal peptide that is removed during translocation across the cytoplasmic membrane through the general secretory pathway. These studies suggest that CexE may be a novel ETEC virulence factor because its expression is controlled by the virulence regulator CfaD, and its distribution is restricted to ETEC.

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Salmonellaexpresses foreign genes during infection by degrading their silencer

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1912808117 ◽

2020 ◽

Vol 117 (14) ◽

pp. 8074-8082 ◽

Cited By ~ 3

Author(s):

Jeongjoon Choi ◽

Eduardo A. Groisman

Keyword(s):

Binding Sites ◽

Bacterial Species ◽

Foreign Gene ◽

Regulatory Sequences ◽

Intracellular Pathogen ◽

Heat Stable ◽

Serovar Typhimurium ◽

Foreign Dna ◽

Foreign Genes ◽

Virulence Regulator

The heat-stable nucleoid structuring (H-NS, also referred to as histone-like nucleoid structuring) protein silences transcription of foreign genes in a variety of Gram-negative bacterial species. To take advantage of the products encoded in foreign genes, bacteria must overcome the silencing effects of H-NS. Because H-NS amounts are believed to remain constant, overcoming gene silencing has largely been ascribed to proteins that outcompete H-NS for binding to AT-rich foreign DNA. However, we report here that the facultative intracellular pathogenSalmonella entericaserovar Typhimurium decreases H-NS amounts 16-fold when inside macrophages. This decrease requires both the protease Lon and the DNA-binding virulence regulator PhoP. The decrease in H-NS abundance reduces H-NS binding to foreign DNA, allowing transcription of foreign genes, including those required for intramacrophage survival. The purified Lon protease degraded free H-NS but not DNA-bound H-NS. By displacing H-NS from DNA, the PhoP protein promoted H-NS proteolysis, thereby de-repressing foreign genes—even those whose regulatory sequences are not bound by PhoP. The uncovered mechanism enables a pathogen to express foreign virulence genes during infection without the need to evolve binding sites for antisilencing proteins at each foreign gene.

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Novel Group of Virulence Activators within the AraC Family That Are Not Restricted to Upstream Binding Sites

Infection and Immunity ◽

10.1128/iai.69.1.186-193.2001 ◽

2001 ◽

Vol 69 (1) ◽

pp. 186-193 ◽

Cited By ~ 43

Author(s):

George P. Munson ◽

Lisa G. Holcomb ◽

June R. Scott

Keyword(s):

Binding Site ◽

Virulence Factors ◽

Binding Sites ◽

Pathogenic Bacteria ◽

Small Region ◽

Start Site ◽

Transcription Start ◽

Family Control ◽

Expression Of Genes ◽

Arac Family

ABSTRACT Several regulators within the AraC family control the expression of genes known or thought to be required for virulence of bacterial pathogens. One of these, Rns, activates transcription from an unprecedented variety of binding-site locations. Although nearly all prokaryotic activators bind within a small region upstream and adjacent to the promoter that they regulate, Rns does not bind within this region to activate its own promoter, Prns. Instead, to activate Prns, Rns requires one binding site 224.5 bp upstream and one downstream of the transcription start site. We show in this study that several other AraC family activators recognize the same binding sites as Rns and share with it the ability to utilize a downstream binding site. Like Rns, other members of this group of activators positively regulate the expression of virulence factors in pathogenic bacteria. These regulators are also able to activate transcription from promoter-proximal upstream binding sites since they are able to substitute for Rns at Pcoo, a promoter with only upstream binding sites. Thus, Rns is the prototype for a group of regulators, which include CfaR, VirF, AggR, and CsvR and which activate transcription from locations that are more diverse than those of any other known activator.

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A new class of inhibitors of the AraC family virulence regulator Vibrio cholerae ToxT

Scientific Reports ◽

10.1038/srep45011 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 9

Author(s):

Anne K. Woodbrey ◽

Evans O. Onyango ◽

Maria Pellegrini ◽

Gabriela Kovacikova ◽

Ronald K. Taylor ◽

...

Keyword(s):

Vibrio Cholerae ◽

New Class ◽

Virulence Regulator ◽

Arac Family

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Conformation of Ribosomes from Escherichia Coli

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100051062 ◽

1974 ◽

Vol 32 ◽

pp. 210-211

Author(s):

M. Boublik ◽

W. Hellmann ◽

F. Jenkins

Keyword(s):

Binding Sites ◽

Ribosomal Proteins ◽

Fluorescent Dyes ◽

Protein Biosynthesis ◽

Three Dimensional ◽

Spatial Arrangement ◽

Dimensional Structure ◽

Complete Understanding ◽

And Function

The present knowledge of the three-dimensional structure of ribosomes is far too limited to enable a complete understanding of the various roles which ribosomes play in protein biosynthesis. The spatial arrangement of proteins and ribonuclec acids in ribosomes can be analysed in many ways. Determination of binding sites for individual proteins on ribonuclec acid and locations of the mutual positions of proteins on the ribosome using labeling with fluorescent dyes, cross-linking reagents, neutron-diffraction or antibodies against ribosomal proteins seem to be most successful approaches. Structure and function of ribosomes can be correlated be depleting the complete ribosomes of some proteins to the functionally inactive core and by subsequent partial reconstitution in order to regain active ribosomal particles.

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Electron Probe Analysis of Muscle

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100054583 ◽

1982 ◽

Vol 40 ◽

pp. 398-401

Author(s):

A. V. Somlyo ◽

H. Shuman ◽

A. P. Somlyo

Keyword(s):

Skeletal Muscle ◽

Smooth Muscle ◽

Sarcoplasmic Reticulum ◽

Resting State ◽

Binding Sites ◽

Electron Probe ◽

Frog Skeletal Muscle ◽

Electron Probe Analysis ◽

Probe Analysis

Electron probe analysis of frozen dried cryosections of frog skeletal muscle, rabbit vascular smooth muscle and of isolated, hyperpermeab1 e rabbit cardiac myocytes has been used to determine the composition of the cytoplasm and organelles in the resting state as well as during contraction. The concentration of elements within the organelles reflects the permeabilities of the organelle membranes to the cytoplasmic ions as well as binding sites. The measurements of [Ca] in the sarcoplasmic reticulum (SR) and mitochondria at rest and during contraction, have direct bearing on their role as release and/or storage sites for Ca in situ.

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Sites of Insulin Action Using Ferritin Labeling

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100066759 ◽

1971 ◽

Vol 29 ◽

pp. 540-541

Author(s):

Burton B. Silver ◽

Ronald S. Nelson

Keyword(s):

Electron Microscopy ◽

Binding Sites ◽

Anterior Pituitary ◽

Insulin Action ◽

Diabetic Rats ◽

Insulin Antibody ◽

Fat Pad ◽

Target Organs ◽

Uranium Salts ◽

Sugar Levels

Some investigators feel that insulin does not enter cells but exerts its influence in some manner on the cell surface. Ferritin labeling of insulin and insulin antibody was used to determine if binding sites of insulin to specific target organs could be seen with electron microscopy.Alloxanized rats were considered diabetic if blood sugar levels were in excess of 300 mg %. Test reagents included ferritin, ferritin labeled insulin, and ferritin labeled insulin antibody. Target organs examined were were diaphragm, kidney, gastrocnemius, fat pad, liver and anterior pituitary. Reagents were administered through the left common carotid. Survival time was at least one hour in test animals. Tissue incubation studies were also done in normal as well as diabetic rats. Specimens were fixed in gluteraldehyde and osmium followed by staining with lead and uranium salts. Some tissues were not stained.

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Fluorescence ratio imaging of dynamic intracellular ionic signals

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100102298 ◽

1988 ◽

Vol 46 ◽

pp. 42-43

Author(s):

R. Y. Tsien ◽

A. Minta ◽

M. Poenie ◽

J.P.Y. Kao ◽

A. Harootunian

Keyword(s):

Binding Sites ◽

Living Cells ◽

Molecular Engineering ◽

Ph Indicators ◽

Highly Sensitive ◽

Fluorescence Ratio Imaging ◽

Ratio Imaging ◽

Technical Advances ◽

Indicator Dyes ◽

Fluorescein Dyes

Recent technical advances now enable the continuous imaging of important ionic signals inside individual living cells with micron spatial resolution and subsecond time resolution. This methodology relies on the molecular engineering of indicator dyes whose fluorescence is strong and highly sensitive to ions such as Ca2+, H+, or Na+, or Mg2+. The Ca2+ indicators, exemplified by fura-2 and indo-1, derive their high affinity (Kd near 200 nM) and selectivity for Ca2+ to a versatile tetracarboxylate binding site3 modeled on and isosteric with the well known chelator EGTA. The most commonly used pH indicators are fluorescein dyes (such as BCECF) modified to adjust their pKa's and improve their retention inside cells. Na+ indicators are crown ethers with cavity sizes chosen to select Na+ over K+: Mg2+ indicators use tricarboxylate binding sites truncated from those of the Ca2+ chelators, resulting in a more compact arrangement of carboxylates to suit the smaller ion.

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