Identification of a core sequence for the binding of BosR to the rpoS promoter region in Borrelia burgdorferi

The alternative sigma factor RpoS in Borrelia burgdorferi plays a central role in modulating host adaptive responses when spirochaetes cycle between ticks and mammals. The transcriptional activation of σ54-dependent rpoS requires a Fur homologue designated BosR. Previously, BosR was shown to directly activate rpoS transcription by binding to the rpoS promoter. However, many other DNA binding features of BosR have remained obscure. In particular, the precise DNA sequence targeted by BosR has not yet been completely elucidated. The prediction of a putative Per box within the rpoS promoter region has further confounded the identification of the BosR binding sequence. Herein, by using electrophoretic mobility shift assays, we demonstrate that the putative Per box predicted in the rpoS promoter region is not involved in the binding of BosR. Rather, a 13 bp palindromic sequence (ATTTAANTTAAAT) with dyad symmetry, which we denote as the ‘BosR box’, functions as the core sequence recognized by BosR in the rpoS promoter region of Borrelia burgdorferi. Similar to a Fur box and a Per box, the BosR box probably comprises a 6–1–6 inverted repeat composed of two hexamers (ATTTAA) in a head-to-tail orientation. Selected mutations in the BosR box prevented recombinant BosR from binding to rpoS. In addition, we found that sequences neighbouring the BosR box also are required for the formation of BosR–DNA complexes. Identification of the BosR box advances our understanding of how BosR recognizes its DNA target(s), and provides new insight into the mechanistic details behind the unique regulatory function of BosR.

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C/EBPα activates the transcription of triacylglycerol hydrolase in 3T3-L1 adipocytes

Biochemical Journal ◽

10.1042/bj20041442 ◽

2005 ◽

Vol 388 (3) ◽

pp. 959-966 ◽

Cited By ~ 13

Author(s):

Enhui WEI ◽

Richard LEHNER ◽

Dennis E. VANCE

Keyword(s):

Transcriptional Activation ◽

Promoter Region ◽

Ectopic Expression ◽

3T3 Cells ◽

Mobility Shift ◽

Protein Levels ◽

Triacylglycerol Hydrolase ◽

Enhancer Binding Protein ◽

Nih 3T3 ◽

Electrophoretic Mobility Shift Assays

TGH (triacylglycerol hydrolase) catalyses the lipolysis of intracellular stored triacylglycerol. To explore the mechanisms that regulate TGH expression in adipose tissue, we studied the expression of TGH during the differentiation of 3T3-L1 adipocytes. TGH mRNA and protein levels increased dramatically in 3T3-L1 adipocytes compared with pre-adipocytes. Electrophoretic mobility shift assays demonstrated enhanced binding of nuclear proteins of adipocytes to the distal murine TGH promoter region (−542/−371 bp), yielding one adipocyte-specific migrating complex. Competitive and supershift assays demonstrated that the distal TGH promoter fragment bound C/EBPα (CCAAT/enhancer-binding protein α). Transient transfections of different mutant TGH promoter–luciferase constructs into 3T3-L1 adipocytes and competitive electromobility shift assays showed that the C/EBP-binding elements at positions −470/−459 bp and −404/−390 bp are important for transcriptional activation. Co-transfection with C/EBPα cDNA and TGH promoter constructs in 3T3-L1 pre-adipocytes demonstrated that C/EBPα increased TGH promoter activity. Ectopic expression of C/EBPα in NIH 3T3 cells activated TGH mRNA expression without causing differentiation into adipocytes. These experiments directly link increased TGH expression in adipocytes to transcriptional regulation by C/EBPα. This is the first evidence that C/EBPα participates directly in the regulation of an enzyme associated with lipolysis.

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RbsR Activates Capsule but Represses therbsUDKOperon in Staphylococcus aureus

Journal of Bacteriology ◽

10.1128/jb.00640-15 ◽

2015 ◽

Vol 197 (23) ◽

pp. 3666-3675 ◽

Cited By ~ 13

Author(s):

Mei G. Lei ◽

Chia Y. Lee

Keyword(s):

Staphylococcus Aureus ◽

Virulence Factor ◽

Transcriptional Activation ◽

Promoter Region ◽

Mobility Shift ◽

Content Type ◽

Virulence Regulation ◽

Important Virulence Factor ◽

Dna Fragment ◽

Mobility Shift Assay

ABSTRACTStaphylococcus aureuscapsule is an important virulence factor that is regulated by a large number of regulators. Capsule genes are expressed from a major promoter upstream of thecapoperon. A 10-bp inverted repeat (IR) located 13 bp upstream of the −35 region of the promoter was previously shown to affect capsule gene transcription. However, little is known about transcriptional activation of thecappromoter. To search for potential proteins which directly interact with thecappromoter region (Pcap), we directly analyzed the proteins interacting with the PcapDNA fragment from shifted gel bands identified by electrophoretic mobility shift assay. One of these regulators, RbsR, was further characterized and found to positively regulatecapgene expression by specifically binding to thecappromoter region. Footprinting analyses showed that RbsR protected a DNA region encompassing the 10-bp IR. Our results further showed thatrbsRwas directly controlled by SigB and that RbsR was a repressor of therbsUDKoperon, involved in ribose uptake and phosphorylation. The repression ofrbsUDKby RbsR could be derepressed byd-ribose. However,d-ribose did not affect RbsR activation of capsule.IMPORTANCEStaphylococcus aureusis an important human pathogen which produces a large number of virulence factors. We have been using capsule as a model virulence factor to study virulence regulation. Although many capsule regulators have been identified, the mechanism of regulation of most of these regulators is unknown. We show here that RbsR activates capsule by direct promoter binding and that SigB is required for the expression ofrbsR. These results define a new pathway wherein SigB activates capsule through RbsR. Our results further demonstrate that RbsR inhibits therbsoperon involved in ribose utilization, thereby providing an example of coregulation of metabolism and virulence inS. aureus. Thus, this study further advances our understanding of staphylococcal virulence regulation.

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ATF4-Dependent Oxidative Induction of the DNA Repair Enzyme Ape1 Counteracts Arsenite Cytotoxicity and Suppresses Arsenite-Mediated Mutagenesis

Molecular and Cellular Biology ◽

10.1128/mcb.00974-07 ◽

2007 ◽

Vol 27 (24) ◽

pp. 8834-8847 ◽

Cited By ~ 33

Author(s):

Hua Fung ◽

Pingfang Liu ◽

Bruce Demple

Keyword(s):

Dna Repair ◽

Transcriptional Activation ◽

Activity Levels ◽

Cellular Mechanisms ◽

Mobility Shift ◽

Repair Enzyme ◽

Base Excision ◽

Transcription Suppression ◽

Tk6 Cells ◽

Electrophoretic Mobility Shift Assays

ABSTRACT Arsenite is a human carcinogen causing skin, bladder, and lung tumors, but the cellular mechanisms underlying these effects remain unclear. We investigated expression of the essential base excision DNA repair enzyme apurinic endonuclease 1 (Ape1) in response to sodium arsenite. In mouse 10T½ fibroblasts, Ape1 induction in response to arsenite occurred about equally at the mRNA, protein, and enzyme activity levels. Analysis of the APE1 promoter region revealed an AP-1/CREB binding site essential for arsenite-induced transcriptional activation in both mouse and human cells. Electrophoretic mobility shift assays indicated that an ATF4/c-Jun heterodimer was the responsible transcription factor. RNA interference targeting c-Jun or ATF4 eliminated arsenite-induced APE1 transcription. Suppression of Ape1 or ATF4 sensitized both mouse fibroblasts (10T½) and human lymphoblastoid cells (TK6) to arsenite cytotoxicity. Expression of Ape1 from a transgene did not efficiently restore arsenite resistance in ATF4-depleted cells but did offset initial accumulation of abasic DNA damage following arsenite treatment. Mutagenesis by arsenite (at the TK and HPRT loci in TK6 cells) was observed only for ATF4-depleted cells, which was strongly offset by Ape1 expression from a transgene. Therefore, the ATF4-mediated up-regulation of Ape1 and other genes plays a key role against arsenite-mediated toxicity and mutagenesis.

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Roles of RpoS and PsrA in cyst formation and alkylresorcinol synthesis in Azotobacter vinelandii

Microbiology ◽

10.1099/mic.0.046268-0 ◽

2011 ◽

Vol 157 (6) ◽

pp. 1685-1693 ◽

Cited By ~ 17

Author(s):

Miguel Cocotl-Yañez ◽

Arístides Sampieri ◽

Soledad Moreno ◽

Cinthia Núñez ◽

Miguel Castañeda ◽

...

Keyword(s):

Promoter Region ◽

Azotobacter Vinelandii ◽

Specific Binding ◽

Cyst Formation ◽

Sigma Factors ◽

Mobility Shift ◽

Alternative Sigma Factors ◽

Main Regulator ◽

Sigma E ◽

Electrophoretic Mobility Shift Assays

Azotobacter vinelandii is a soil bacterium that undergoes differentiation to form cysts that are resistant to desiccation. Upon induction of cyst formation, the bacterium synthesizes alkylresorcinols that are present in cysts but not in vegetative cells. Alternative sigma factors play important roles in differentiation. In A. vinelandii, AlgU (sigma E) is involved in controlling the loss of flagella upon induction of encystment. We investigated the involvement of the sigma factor RpoS in cyst formation in A. vinelandii. We analysed the transcriptional regulation of the rpoS gene by PsrA, the main regulator of rpoS in Pseudomonas species, which are closely related to A. vinelandii. Inactivation of rpoS resulted in the inability to form cysts resistant to desiccation and to produce cyst-specific alkylresorcinols, whereas inactivation of psrA reduced by 50 % both production of alkylresorcinols and formation of cysts resistant to desiccation. Electrophoretic mobility shift assays revealed specific binding of PsrA to the rpoS promoter region and that inactivation of psrA reduced rpoS transcription by 60 %. These results indicate that RpoS and PsrA are involved in regulation of encystment and alkylresorcinol synthesis in A. vinelandii.

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Transcriptional Activation of Multiple Operons Involved inpara-Nitrophenol Degradation by Pseudomonas sp. Strain WBC-3

Applied and Environmental Microbiology ◽

10.1128/aem.02720-14 ◽

2014 ◽

Vol 81 (1) ◽

pp. 220-230 ◽

Cited By ~ 22

Author(s):

Wen-Mao Zhang ◽

Jun-Jie Zhang ◽

Xuan Jiang ◽

Hongjun Chao ◽

Ning-Yi Zhou

Keyword(s):

Transcriptional Activation ◽

Transcriptional Regulator ◽

Pseudomonas Sp ◽

Mobility Shift ◽

Content Type ◽

High Affinity ◽

Dnase I Footprinting ◽

Nitrophenol Degradation ◽

Transcriptional Start Sites ◽

Electrophoretic Mobility Shift Assays

ABSTRACTPseudomonassp. strain WBC-3 utilizespara-nitrophenol (PNP) as a sole carbon and energy source. The genes involved in PNP degradation are organized in the following three operons:pnpA,pnpB, andpnpCDEFG. How the expression of the genes is regulated is unknown. In this study, an LysR-type transcriptional regulator (LTTR) is identified to activate the expression of the genes in response to the specific inducer PNP. While the LTTR coding genepnpRwas found to be not physically linked to any of the three catabolic operons, it was shown to be essential for the growth of strain WBC-3 on PNP. Furthermore, PnpR positively regulated its own expression, which is different from the function of classical LTTRs. A regulatory binding site (RBS) with a 17-bp imperfect palindromic sequence (GTT-N11-AAC) was identified in allpnpA,pnpB,pnpC, andpnpRpromoters. Through electrophoretic mobility shift assays and mutagenic analyses, this motif was proven to be necessary for PnpR binding. This consensus motif is centered at positions approximately −55 bp relative to the four transcriptional start sites (TSSs). RBS integrity was required for both high-affinity PnpR binding and transcriptional activation ofpnpA,pnpB, andpnpR. However, this integrity was essential only for high-affinity PnpR binding to the promoter ofpnpCDEFGand not for its activation. Intriguingly, unlike other LTTRs studied, no changes in lengths of the PnpR binding regions of thepnpAandpnpBpromoters were observed after the addition of the inducer PNP in DNase I footprinting.

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Regulation of the Bacillus subtilis mannitol utilization genes: promoter structure and transcriptional activation by the wild-type regulator (MtlR) and its mutants

Microbiology ◽

10.1099/mic.0.071233-0 ◽

2014 ◽

Vol 160 (1) ◽

pp. 91-101 ◽

Cited By ~ 10

Author(s):

Kambiz Morabbi Heravi ◽

Josef Altenbuchner

Keyword(s):

Bacillus Subtilis ◽

Transcriptional Activation ◽

Transcription Initiation ◽

Phosphotransferase System ◽

Mobility Shift ◽

Dnase I Footprinting ◽

Proposed Model ◽

Rna Polymerase Holoenzyme ◽

Electrophoretic Mobility Shift Assays

Expression of mannitol utilization genes in Bacillus subtilis is directed by P mtlA , the promoter of the mtlAFD operon, and P mtlR , the promoter of the MtlR activator. MtlR contains phosphoenolpyruvate-dependent phosphotransferase system (PTS) regulation domains, called PRDs. The activity of PRD-containing MtlR is mainly regulated by the phosphorylation/dephosphorylation of its PRDII and EIIBGat-like domains. Replacing histidine 342 and cysteine 419 residues, which are the targets of phosphorylation in these two domains, by aspartate and alanine provided MtlR-H342D C419A, which permanently activates P mtlA in vivo. In the mtlR-H342D C419A mutant, P mtlA was active, even when the mtlAFD operon was deleted from the genome. The mtlR-H342D C419A allele was expressed in an Escherichia coli strain lacking enzyme I of the PTS. Electrophoretic mobility shift assays using purified MtlR-H342D C419A showed an interaction between the MtlR double-mutant and the Cy5-labelled P mtlA and P mtlR DNA fragments. These investigations indicate that the activated MtlR functions regardless of the presence of the mannitol-specific transporter (MtlA). This is in contrast to the proposed model in which the sequestration of MtlR by the MtlA transporter is necessary for the activity of MtlR. Additionally, DNase I footprinting, construction of P mtlA -P licB hybrid promoters, as well as increasing the distance between the MtlR operator and the −35 box of P mtlA revealed that the activated MtlR molecules and RNA polymerase holoenzyme likely form a class II type activation complex at P mtlA and P mtlR during transcription initiation.

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Identification of a core sequence for the binding of BosR to the rpoS promoter region in Borrelia burgdorferi

Microbiology ◽

10.1099/mic.0.000060 ◽

2015 ◽

Vol 161 (4) ◽

pp. 931-931 ◽

Cited By ~ 2

Author(s):

Zhiming Ouyang ◽

Ranjit K. Deka ◽

Michael V. Norgard ◽

Jianli Zhou ◽

Chad A. Brautigam

Keyword(s):

Borrelia Burgdorferi ◽

Promoter Region ◽

Core Sequence

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Regulation ofperRExpression by Iron and PerR in Campylobacter jejuni

Journal of Bacteriology ◽

10.1128/jb.05493-11 ◽

2011 ◽

Vol 193 (22) ◽

pp. 6171-6178 ◽

Cited By ~ 17

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.

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VraR Binding to the Promoter Region of agr Inhibits Its Function in Vancomycin-Intermediate Staphylococcus aureus (VISA) and Heterogeneous VISA

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02740-16 ◽

2017 ◽

Vol 61 (5) ◽

Cited By ~ 8

Author(s):

Yuanyuan Dai ◽

Wenjiao Chang ◽

Changcheng Zhao ◽

Jing Peng ◽

Liangfei Xu ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Promoter Region ◽

Reference Strain ◽

Vancomycin Resistance ◽

Mobility Shift ◽

Content Type ◽

Promoter Sequences ◽

Dnase I Footprinting ◽

Resistant Strains ◽

Electrophoretic Mobility Shift Assays

ABSTRACT Acquisition of vancomycin resistance in Staphylococcus aureus is often accompanied by a reduction in virulence, but the mechanisms underlying this change remain unclear. The present study was undertaken to investigate this process in a clinical heterogeneous vancomycin-intermediate S. aureus (hVISA) strain, 10827; an hVISA reference strain, Mu3; and a VISA reference strain, Mu50, along with their respective series of vancomycin-induced resistant strains. In these strains, increasing MICs of vancomycin were associated with increased expression of the vancomycin resistance-associated regulator gene (vraR) and decreased expression of virulence genes (hla, hlb, and coa) and virulence-regulated genes (RNAIII, agrA, and saeR). These results suggested that VraR might have a direct or indirect effect on virulence in S. aureus. In electrophoretic mobility shift assays, VraR did not bind to promoter sequences of hla, hlb, and coa genes, but it did bind to the agr promoter region. In DNase I footprinting assays, VraR protected a 15-nucleotide (nt) sequence in the intergenic region between the agr P2 and P3 promoters. These results indicated that when S. aureus is subject to induction by vancomycin, expression of vraR is upregulated, and VraR binding inhibits the function of the Agr quorum-sensing system, causing reductions in the virulence of VISA/hVISA strains. Our results suggested that VraR in S. aureus is involved not only in the regulation of vancomycin resistance but also in the regulation of virulence.

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Genomic organization, chromosomal mapping and promoter analysis of the mouse selenocysteine tRNA gene transcription-activating factor (mStaf) gene

Biochemical Journal ◽

10.1042/bj3460045 ◽

2000 ◽

Vol 346 (1) ◽

pp. 45-51 ◽

Cited By ~ 2

Author(s):

Kazushige ADACHI ◽

Masato KATSUYAMA ◽

Shigeaki SONG ◽

Takami OKA

Keyword(s):

Transcriptional Activation ◽

Trna Gene ◽

Zinc Finger Protein ◽

Regulatory Elements ◽

Chromosomal Mapping ◽

Proximal Promoter ◽

Mobility Shift ◽

Responsive Element ◽

Specificity Protein ◽

Electrophoretic Mobility Shift Assays

mStaf is a zinc-finger protein that activates the transcription of the mouse selenocysteine tRNA gene. The mStaf gene is approx. 35 kb long and split into 16 exons. All exon-intron junction sequences conform to the GT/AG rule. The transcription start site is located 83 bp upstream of the initiation codon. Chromosomal mapping localized the gene to mouse chromosome 7, region E3-F1. Sequence analysis of the proximal promoter region revealed several potential regulatory elements; these include the recognition elements of Sp1, Nkx, CP2, E2A, SIF (SIS-inducible factor), TFII-I and cAMP-responsive element (CRE), but no TATA sequences. Transfection experiments demonstrated that the 5ʹ-flanking region (-1894 to +37) of the mStaf gene drives transcription in mouse NMuMG cells and that a construct containing a fragment from -387 to +37 showed the highest transcriptional activity. Deletion and mutation experiments suggested that four Sp1 sites played an important role for the basal promoter activity. Furthermore, electrophoretic mobility-shift assays demonstrated that Sp3 but not other Sp (specificity protein) family members binds to three of the Sp1 sites. Our present study suggests that Sp3 is involved in the basal transcriptional activation of the mStaf gene.

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