scholarly journals Cooperative Function of TraJ and ArcA in Regulating the F PlasmidtraOperon

2018 ◽  
Vol 201 (1) ◽  
Author(s):  
Jun Lu ◽  
Yun Peng ◽  
Sereana Wan ◽  
Laura S. Frost ◽  
Tracy Raivio ◽  
...  
Keyword(s):  
Regulatory Region ◽  
Repeat Sequence ◽  
Bacterial Conjugation ◽  
Alternative Promoters ◽  
Mobility Shift ◽  
Bacterial Populations ◽  
Major Mechanism ◽  
Regulatory Circuit ◽  
Donor Ability ◽  
Electrophoretic Mobility Shift Assays

ABSTRACTThe F plasmidtraoperon encodes most of the proteins required for bacterial conjugation. TraJ and ArcA are known activators of thetraoperon promoter PY, which is subject to H-NS-mediated silencing. Donor ability and promoter activity assays indicated that PYis inactivated by silencers and requires both TraJ and ArcA for activation to support efficient F conjugation. The observed low-level, ArcA-independent F conjugation is caused bytraexpression from upstream alternative promoters. Electrophoretic mobility shift assays showed that TraJ alone weakly binds to PYregulatory DNA; however, TraJ binding is significantly enhanced by ArcA binding to the same DNA, indicating cooperativity of the two proteins. Analysis of binding affinities between ArcA and various DNA fragments in the PYregulatory region defined a 22-bp tandem repeat sequence (from −76 to −55 of PY) sufficient for optimal ArcA binding, which is immediately upstream of the predicted TraJ-binding site (from −54 to −34). Deletion analysis of the PYpromoter in strains deficient in TraJ, ArcA, and/or H-NS determined that sequences upstream of −103 are required by silencers including H-NS for PYsilencing, whereas sequences downstream of −77 are targeted by TraJ and ArcA for activation. TraJ and ArcA appear not only to counteract PYsilencers but also to directly activate PYin a cooperative manner. Our data reveal the cooperativity of TraJ and ArcA during PYactivation and provide insights into the regulatory circuit controlling F-family plasmid-mediated bacterial conjugation.IMPORTANCEConjugation is a major mechanism for dissemination of antibiotic resistance and virulence among bacterial populations. Thetraoperon in the F family of conjugative plasmids encodes most of the proteins involved in bacterial conjugation. This work reveals that activation oftraoperon transcription requires two proteins, TraJ and ArcA, to bind cooperatively to adjacent sites immediately upstream of the majortrapromoter PY. The interaction of TraJ and ArcA with thetraoperon not only relieves PYfrom silencers but also directly activates it. These findings provide insights into the regulatory circuit of the F-family plasmid-mediated bacterial conjugation.

SoxS regulates the expression of the Salmonella enterica serovar Typhimurium ompW gene

Microbiology ◽  
2009 ◽  
Vol 155 (8) ◽  
pp. 2490-2497 ◽  
Author(s):  
F. Gil ◽  
I. Hernández-Lucas ◽  
R. Polanco ◽  
N. Pacheco ◽  
B. Collao ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Regulatory Region ◽  
Transcription Start ◽  
Mobility Shift ◽  
Serovar Typhimurium ◽  
A Minor ◽  
Electrophoretic Mobility Shift Assays ◽  
Putative Binding Site ◽  
Transcriptional Fusions

OmpW of Salmonella enterica serovar Typhimurium has been described as a minor porin involved in osmoregulation, and is also affected by environmental conditions. Biochemical and genetic evidence from our laboratory indicates that OmpW is involved in efflux of and resistance towards paraquat (PQ), and its expression has been shown to be activated in response to oxidative stress. In this study we have explored ompW expression in response to PQ. Primer extension and transcriptional fusions showed that its expression was induced in the presence of PQ. In silico analyses suggested a putative binding site for the SoxS transcriptional factor at the ompW regulatory region. Electrophoretic mobility shift assays (EMSAs) and footprinting experiments showed that SoxS binds at a region that starts close to −54 and ends at about −197 upstream of the transcription start site. Transcriptional fusions support the relevance of this region in ompW activation. The SoxS site is in the forward orientation and its location suggests that the ompW gene has a class I SoxS-dependent promoter.

scholarly journals Regulation ofperRExpression by Iron and PerR in Campylobacter jejuni

2011 ◽  
Vol 193 (22) ◽  
pp. 6171-6178 ◽  
Author(s):  
Minkyeong Kim ◽  
Sunyoung Hwang ◽  
Sangryeol Ryu ◽  
Byeonghwa Jeon
Keyword(s):  
Oxidative Stress ◽  
Campylobacter Jejuni ◽  
Regulatory Region ◽  
Transcriptional Level ◽  
Mobility Shift ◽  
Content Type ◽  
Dnase I Footprinting ◽  
Transcriptional Changes ◽  
Electrophoretic Mobility Shift Assays ◽  
Binding Sequence

Campylobacter jejuniis a leading food-borne pathogen causing gastroenteritis in humans. Although OxyR is a widespread oxidative stress regulator in many Gram-negative bacteria,C. jejunilacks OxyR and instead possesses the metalloregulator PerR. Despite the important role played by PerR in oxidative stress defense, little is known about the factors influencingperRexpression inC. jejuni. In this study, aperRpromoter-lacZfusion assay demonstrated that iron significantly reduced the level ofperRtranscription, whereas other metal ions, such as copper, cobalt, manganese, and zinc, did not affectperRtranscription. Notably, aperRmutation substantially increased the level ofperRtranscription and intranscomplementation restored the transcriptional changes, suggestingperRis transcriptionally autoregulated inC. jejuni. In theperRmutant, iron did not repressperRtranscription, indicating the iron dependence ofperRexpression results fromperRautoregulation. Electrophoretic mobility shift assays showed that PerR binds to theperRpromoter, and DNase I footprinting assays identified a PerR binding site overlapping the −35 region of the twoperRpromoters, further supportingperRautoregulation at the transcriptional level. Alignment of the PerR binding sequence in theperRpromoter with the regulatory region of other PerR regulon genes ofC. jejunirevealed a 16-bp consensus PerR binding sequence, which shares high similarities to theBacillus subtilisPerR box. The results of this study demonstrated that PerR directly interacts with theperRpromoter and regulatesperRtranscription and thatperRautoregulation is responsible for the repression ofperRtranscription by iron inC. jejuni.

scholarly journals SpdR, a Response Regulator Required for Stationary-Phase Induction of Caulobacter crescentus cspD

2010 ◽  
Vol 192 (22) ◽  
pp. 5991-6000 ◽  
Author(s):  
Carolina A. P. T. da Silva ◽  
Heloise Balhesteros ◽  
Ricardo R. Mazzon ◽  
Marilis V. Marques
Keyword(s):  
Stationary Phase ◽  
Cold Shock ◽  
Caulobacter Crescentus ◽  
Response Regulator ◽  
Phosphorylation Site ◽  
Regulatory Region ◽  
Repeat Sequence ◽  
Medium Composition ◽  
Cold Shock Protein ◽  
Mobility Shift

ABSTRACT The cold shock protein (CSP) family includes small polypeptides that are induced upon temperature downshift and stationary phase. The genome of the alphaproteobacterium Caulobacter crescentus encodes four CSPs, with two being induced by cold shock and two at the onset of stationary phase. In order to identify the environmental signals and cell factors that are involved in cspD expression at stationary phase, we have analyzed cspD transcription during growth under several nutrient conditions. The results showed that expression of cspD was affected by the medium composition and was inversely proportional to the growth rate. The maximum levels of expression were decreased in a spoT mutant, indicating that ppGpp may be involved in the signalization for carbon starvation induction of cspD. A Tn5 mutant library was screened for mutants with reduced cspD expression, and 10 clones that showed at least a 50% reduction in expression were identified. Among these, a strain with a transposon insertion into a response regulator of a two-component system showed no induction of cspD at stationary phase. This protein (SpdR) was able to acquire a phosphate group from its cognate histidine kinase, and gel mobility shift assay and DNase I footprinting experiments showed that it binds to an inverted repeat sequence of the cspD regulatory region. A mutated SpdR with a substitution of the conserved aspartyl residue that is the probable phosphorylation site is unable to bind to the cspD regulatory region and to complement the spdR mutant phenotype.

scholarly journals Pancreatic-Duodenal Homeobox 1 Regulates Expression of Liver Receptor Homolog 1 during Pancreas Development

2003 ◽  
Vol 23 (19) ◽  
pp. 6713-6724 ◽  
Author(s):  
Jean-Sébastien Annicotte ◽  
Elisabeth Fayard ◽  
Galvin H. Swift ◽  
Lars Selander ◽  
Helena Edlund ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Regulatory Region ◽  
Mobility Shift ◽  
Pancreatic Development ◽  
Downstream Target ◽  
Regulatory Cascade ◽  
And Function ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT Liver receptor homolog 1 (LRH-1) and pancreatic-duodenal homeobox 1 (PDX-1) are coexpressed in the pancreas during mouse embryonic development. Analysis of the regulatory region of the human LRH-1 gene demonstrated the presence of three functional binding sites for PDX-1. Electrophoretic mobility shift assays and chromatin immunoprecipitation analysis showed that PDX-1 bound to the LRH-1 promoter, both in cultured cells in vitro and during pancreatic development in vivo. Retroviral expression of PDX-1 in pancreatic cells induced the transcription of LRH-1, whereas reduced PDX-1 levels by RNA interference attenuated its expression. Consistent with direct regulation of LRH-1 expression by PDX-1, PDX-1−/− mice expressed smaller amounts of LRH-1 mRNA in the embryonic pancreas. Taken together, our data indicate that PDX-1 controls LRH-1 expression and identify LRH-1 as a novel downstream target in the PDX-1 regulatory cascade governing pancreatic development, differentiation, and function.

Transcriptional regulation of the hamster Muc1gene: identification of a putative negative regulatory element

2003 ◽  
Vol 284 (1) ◽  
pp. L160-L168 ◽  
Author(s):  
Insong James Lee ◽  
Sang Won Hyun ◽  
Asit Nandi ◽  
K. Chul Kim
Keyword(s):  
Transcriptional Regulation ◽  
Regulatory Element ◽  
Regulatory Region ◽  
Chinese Hamster ◽  
Nuclear Extract ◽  
Mobility Shift ◽  
Transcriptional Initiation ◽  
Negative Regulatory Element ◽  
Muc1 Gene ◽  
Electrophoretic Mobility Shift Assays

The mucin gene Muc1 is expressed in glandular epithelial cells and is involved in lubricative and protective functions. It is also overexpressed in many carcinomas including breast and lung cancer cells. To study the transcriptional regulation of Muc1, we cloned a 2.4-kb fragment containing the promoter region of the hamster Muc1 gene and analyzed it for its ability to mediate transcription. Transcriptional initiation was localized to 22 base pairs downstream of the TATA box. We performed functional analysis of the Muc1 promoter in hamster (HP-1 and Chinese hamster ovary) and human cells (MCF-7, A549, and BEAS-2B) using deletion/reporter constructs. A positive regulatory region between bases −555 and −252 and a putative negative regulatory element (P-NRE) between nucleotides −1,652 and −1,614 were found to be active in transfected cells. The P-NRE contains a yin yang 1 (YY1) transcription factor binding site, and electrophoretic mobility shift assays with HP-1 cell nuclear extract revealed the binding of YY1 to this site. Our data suggest that YY1 may play an inhibitory role in the transcription of the Muc1 gene.

scholarly journals Characterization of EvgAS-YdeO-GadE Branched Regulatory Circuit Governing Glutamate-Dependent Acid Resistance in Escherichia coli

2004 ◽  
Vol 186 (21) ◽  
pp. 7378-7389 ◽  
Author(s):  
Zhuo Ma ◽  
Nobuhisa Masuda ◽  
John W. Foster
Keyword(s):  
Escherichia Coli ◽  
Response Regulator ◽  
Resistance Mechanism ◽  
Acid Resistance ◽  
Mobility Shift ◽  
E Coli ◽  
Regulatory Circuit ◽  
Regulatory Circuits ◽  
Normal Expression ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT Escherichia coli prefers growth in neutral pH environments but can withstand extremely acidic conditions (pH 2) for long periods. Of the four E. coli systems that contribute to acid resistance, one, the glutamate-dependent system, is remarkable in its efficacy and regulatory complexity. The resistance mechanism involves the intracellular consumption of protons by the glutamate decarboxylase isozymes GadA and GadB. The antiporter GadC then exports the product, γ-aminobutyric acid, in exchange for fresh glutamate. A microarray study using overexpressed regulators uncovered evgAS and ydeO as potential regulators of gadE, now known to encode the essential activator of the gadA and gadBC genes. Examination of evgA and ydeO under normal expression conditions revealed that their products do activate gadE expression but only under specific conditions. They were important during exponential growth in acidified minimal medium containing glucose but were unnecessary for gadE expression in stationary-phase cells grown in complex medium. The response regulator EvgA activates gadE directly and indirectly via induction of the AraC-like regulator ydeO. Evidence obtained using gadE-lacZ operon fusions also revealed that GadE was autoinduced. Electrophoretic mobility shift assays indicated that EvgA, YdeO, and GadE bind to different regions upstream of gadE, indicating they all act directly at the gadE promoter. Since GadE controls the expression of numerous genes besides gadA and gadBC, the relevance of these regulatory circuits extends beyond acid resistance.

scholarly journals QseA Directly Activates Transcription of LEE1 in Enterohemorrhagic Escherichia coli

2007 ◽  
Vol 75 (5) ◽  
pp. 2432-2440 ◽  
Author(s):  
Faith C. Sharp ◽  
Vanessa Sperandio
Keyword(s):  
Escherichia Coli ◽  
Regulatory Region ◽  
Enterohemorrhagic Escherichia Coli ◽  
Proximal Promoter ◽  
Transcription Activator ◽  
Mobility Shift ◽  
Activation Of Transcription ◽  
Enterocyte Effacement ◽  
K 12 ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT Quorum sensing (QS) in enterohemorrhagic Escherichia coli (EHEC) regulates the expression of the locus of enterocyte effacement (LEE). The LEE contains five major operons named LEE1 through LEE5. QseA was previously shown to be activated through QS and to activate the transcription of LEE1. The LEE1 operon encodes Ler, the transcription activator of all other LEE genes, and has two promoters: a distal promoter (P1) and a proximal promoter (P2). We have previously reported that QseA acts on P1 and not P2. To identify the minimal region of LEE1 that is necessary for QseA-mediated activation, a series of nested-deletion constructs of the LEE1 promoter fused to a lacZ reporter were constructed in both the EHEC and E. coli K-12 backgrounds. In an EHEC background, QseA-dependent activation of LEE1 can be observed for the entire regulatory region (beginning at nucleotide −393 and ending at nucleotide −123). In contrast to what occurred in EHEC, in K-12 there was no QseA-dependent activation of LEE1 transcription between base pairs −393 and −343. These data indicate that a QseA-dependent EHEC-specific regulator is required for the activation of transcription in this region. We also observed QseA-dependent LEE1 activation from nucleotides −218 to −123 in K-12, similar to results of the nested-deletion analysis performed with EHEC. Electrophoretic mobility shift assays established that QseA directly binds to the region of LEE1 from bp −173 to −42 and not to the region from bp −393 to −343. These studies suggest that QseA activates the transcription of LEE1 by directly binding upstream of its P1 promoter region.

scholarly journals Temporal Regulation of Enhancer Function in Intestinal Epithelium

2006 ◽  
Vol 281 (43) ◽  
pp. 32263-32271 ◽  
Author(s):  
Elizabeth A. Maier ◽  
Mary R. Dusing ◽  
Dan A. Wiginton
Keyword(s):  
Expression Patterns ◽  
Regulatory Region ◽  
Specific Gene ◽  
Temporal Region ◽  
Mobility Shift ◽  
Temporal Regulation ◽  
Gata Factors ◽  
Dnase I Footprinting ◽  
Duodenal Epithelium ◽  
Electrophoretic Mobility Shift Assays

An intestine-specific gene regulatory region was previously identified near the second exon of the human adenosine deaminase (ADA) gene. In mammalian intestine, ADA is expressed at high levels only along the villi of the duodenal epithelium, principally if not exclusively in enterocytes. Within the ADA intestinal regulatory region, a potent duodenum-specific enhancer was identified that controls this pattern of expression. This enhancer has been shown to rely on PDX-1, GATA factors, and Cdx factors for its function. Upstream of the enhancer, a separate temporal regulatory region was identified that has no independent enhancer capability but controls the timing of enhancer activation. DNase I footprinting and electrophoretic mobility shift assays were used to begin to characterize temporal region function at the molecular level. In this manner, binding sites for the Onecut (OC) family of factors, YY1, and NFI family members were identified. Identification of the OC site was especially interesting, because almost nothing is known about the function of OC factors in intestine. In transgenic mice, mutation of the OC site to ablate binding resulted in a delay of 2–3 weeks in enhancer activation in the developing postnatal intestine, a result very similar to that observed previously when the entire temporal region was deleted. In mammals, the OC family is comprised of OC-1/HNF-6, OC-2, and OC-3. An examination of intestinal expression patterns showed that all three OC factors are expressed at detectable levels in adult mouse duodenum, with OC-2 predominant. In postnatal day 2 mice only OC-2 and OC-3 were detected in intestine, with OC-2 again predominant.

A mammalian factor that binds telomeric TTAGGG repeats in vitro

1992 ◽  
Vol 12 (11) ◽  
pp. 4834-4843 ◽  
Author(s):  
Z Zhong ◽  
L Shiue ◽  
S Kaplan ◽  
T de Lange
Keyword(s):  
Sodium Dodecyl ◽  
Binding Activity ◽  
Repeat Sequence ◽  
Mobility Shift ◽  
Dna Binding Activity ◽  
Nuclear Extracts ◽  
Circular Dnas ◽  
Electrophoretic Mobility Shift Assays ◽  
Ttaggg Repeat

We have identified a DNA-binding activity with specificity for the TTAGGG repeat arrays found at mammalian telomeres. This factor, called TTAGGG repeat factor (TRF), is present in nuclear extracts of human, mouse, and monkey cells. TRF from HeLa cells was characterized in detail by electrophoretic mobility shift assays. It binds double-stranded TTAGGG repeats in linear and circular DNAs. Single-stranded repeats are not recognized. The optimal site for TRF appears to contain more than six contiguous TTAGGG repeats. Tandem arrays of TAGGG, TTTAGGG, TTTTAGGG, TTGGGG, and TTAGGC repeats do not bind TRF well, indicating that TRF preferentially recognizes the telomeric repeat sequence present at mammalian chromosome ends. The apparent molecular mass of this factor, based on recovery of TRF from sodium dodecyl sulfate-polyacrylamide gels, is approximately 50 kDa. We suggest that TRF binds along the length of mammalian telomeres.

scholarly journals A mammalian factor that binds telomeric TTAGGG repeats in vitro.

1992 ◽  
Vol 12 (11) ◽  
pp. 4834-4843 ◽  
Author(s):  
Z Zhong ◽  
L Shiue ◽  
S Kaplan ◽  
T de Lange
Keyword(s):  
Sodium Dodecyl ◽  
Binding Activity ◽  
Repeat Sequence ◽  
Mobility Shift ◽  
Dna Binding Activity ◽  
Nuclear Extracts ◽  
Circular Dnas ◽  
Electrophoretic Mobility Shift Assays ◽  
Ttaggg Repeat

We have identified a DNA-binding activity with specificity for the TTAGGG repeat arrays found at mammalian telomeres. This factor, called TTAGGG repeat factor (TRF), is present in nuclear extracts of human, mouse, and monkey cells. TRF from HeLa cells was characterized in detail by electrophoretic mobility shift assays. It binds double-stranded TTAGGG repeats in linear and circular DNAs. Single-stranded repeats are not recognized. The optimal site for TRF appears to contain more than six contiguous TTAGGG repeats. Tandem arrays of TAGGG, TTTAGGG, TTTTAGGG, TTGGGG, and TTAGGC repeats do not bind TRF well, indicating that TRF preferentially recognizes the telomeric repeat sequence present at mammalian chromosome ends. The apparent molecular mass of this factor, based on recovery of TRF from sodium dodecyl sulfate-polyacrylamide gels, is approximately 50 kDa. We suggest that TRF binds along the length of mammalian telomeres.

Sign in / Sign up

Export Citation Format

Share Document