scholarly journals Irr regulates brucebactin and 2,3-dihydroxybenzoic acid biosynthesis, and is implicated in the oxidative stress resistance and intracellular survival of Brucella abortus

Microbiology ◽  
2006 ◽  
Vol 152 (9) ◽  
pp. 2591-2598 ◽  
Author(s):  
Marcela Martínez ◽  
Rodolfo A. Ugalde ◽  
Marta Almirón
Keyword(s):  
Brucella Abortus ◽  
Mammalian Cells ◽  
Wild Type Strain ◽  
Upstream Region ◽  
Dihydroxybenzoic Acid ◽  
Acid Biosynthesis ◽  
Mobility Shift ◽  
Iron Deprivation ◽  
Haem Biosynthesis ◽  
Mobility Shift Assay

Brucella abortus faces iron deprivation in both nature and the host. To overcome this limitation, Brucella secretes the siderophores 2,3-dihydroxybenzoic acid and brucebactin. A Fur-like protein named Irr has previously been characterized in B. abortus; this protein is present in the α-2 group of Proteobacteria only, where it negatively regulates haem biosynthesis when iron is scarce. Additional evidence that Irr also regulates the synthesis of both siderophores is presented here. Transcriptional lacZ fusion and chemical determinations revealed that Irr induced the transcription of the operon involved in the synthesis of the catecholic siderophores, which were consequently secreted under conditions of iron limitation. Irr was able to bind the upstream region of the operon, as shown by electrophoretic mobility shift assay. A B. abortus irr mutant showed higher intracellular haem content, catalase activity and resistance to hydrogen peroxide than the wild-type strain. The mutation also improved the replication and survival of iron-depleted bacteria within cultured mammalian cells. Although the pathogenesis of Brucella correlates with its ability to replicate intracellularly, pathogenicity was not attenuated when assayed in a murine model.

scholarly journals The AraC-Like Transcriptional Regulator DhbR Is Required for Maximum Expression of the 2,3-Dihydroxybenzoic Acid Biosynthesis Genes in Brucella abortus 2308 in Response to Iron Deprivation

2007 ◽  
Vol 190 (5) ◽  
pp. 1838-1842 ◽  
Author(s):  
Eric S. Anderson ◽  
James T. Paulley ◽  
R. Martin Roop
Keyword(s):  
Brucella Abortus ◽  
Transcriptional Regulator ◽  
Dihydroxybenzoic Acid ◽  
Acid Biosynthesis ◽  
Iron Deprivation ◽  
Isogenic Mutants ◽  
Maximum Expression

ABSTRACT Phenotypic evaluation of isogenic mutants derived from Brucella abortus 2308 indicates that the AlcR homolog DhbR (2,3-dihydroxybenzoic acid [2,3-DHBA] biosynthesis regulator) modulates the expression of the genes involved in 2,3-DHBA production, employing 2,3-DHBA or brucebactin as a coinducer.

scholarly journals DNA-binding specificity of GATA family transcription factors.

1993 ◽  
Vol 13 (7) ◽  
pp. 3999-4010 ◽  
Author(s):  
M Merika ◽  
S H Orkin
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Mammalian Cells ◽  
Chimeric Protein ◽  
Binding Specificity ◽  
Mobility Shift ◽  
Binding Domains ◽  
Dna Binding Specificity ◽  
Mobility Shift Assay ◽  
Gata Family

GATA-binding proteins constitute a family of transcription factors that recognize a target site conforming to the consensus WGATAR (W = A or T and R = A or G). Here we have used the method of polymerase chain reaction-mediated random site selection to assess in an unbiased manner the DNA-binding specificity of GATA proteins. Contrary to our expectations, we show that GATA proteins bind a variety of motifs that deviate from the previously assigned consensus. Many of the nonconsensus sequences bind protein with high affinity, equivalent to that of conventional GATA motifs. By using the selected sequences as probes in the electrophoretic mobility shift assay, we demonstrate overlapping, but distinct, sequence preferences for GATA family members, specified by their respective DNA-binding domains. Furthermore, we provide additional evidence for interaction of amino and carboxy fingers of GATA-1 in defining its binding site. By performing cotransfection experiments, we also show that transactivation parallels DNA binding. A chimeric protein containing the finger domain of areA and the activation domains of GATA-1 is capable of activating transcription in mammalian cells through GATA motifs. Our findings suggest a mechanism by which GATA proteins might selectively regulate gene expression in cells in which they are coexpressed.

Structural Analysis and Identification of Cis-Elements of Rice osRACD Gene

2004 ◽  
Vol 36 (3) ◽  
pp. 191-198 ◽  
Author(s):  
Yun-Zhe Xu ◽  
Rui-Lin You ◽  
Nai-Hu Wu
Keyword(s):  
Oryza Sativa L ◽  
Small Gtpase ◽  
Inverse Pcr ◽  
Upstream Region ◽  
Male Sterile ◽  
Mobility Shift ◽  
Nuclear Extracts ◽  
Mobility Shift Assay ◽  
Photoperiod Sensitive ◽  
Gel Mobility Shift

Abstract The osRACD gene correlated with fertility transformation in the photoperiod sensitive genic male sterile rice (PGMR), Nongken 58S, encoded a rice (Oryza sativa L. ssp. japonica) small GTPase belonging to the Rac/Rho family. Inverse PCR was performed to amplify a fragment about 1.4 kb in 5′ upstream region of the osRACD promoter. Deletion mutation and gel mobility shift assay characterized two fragments (–799 to –686 nt, and –686 to –431 nt) in the osRACD promoter that could be involved in its transcriptional regulation. When these two deletion fragments were used as probe respectively, a retarded band appeared in the nuclear extracts of fertile 58S rice under short day (58S-SD). Whereas no retarded band was shown in the nuclear extracts of sterile 58S rice under long day (58S-LD). Competition assay indicated that the factors in the retarded bands binding to these two fragments were the same trans-acting factor (termed rice factor, RF). The binding affinity of RF was affected by phosphorylation and was higher in SD-growth rice than that of LD-growth rice.

An upstream element of the TamS1 gene is a site of DNA-protein interactions during differentiation to the merozoite in Theileria annulata

2000 ◽  
Vol 113 (12) ◽  
pp. 2243-2252 ◽  
Author(s):  
B. Shiels ◽  
M. Fox ◽  
S. McKellar ◽  
J. Kinnaird ◽  
D. Swan
Keyword(s):  
Gene Expression ◽  
Protein Interactions ◽  
Theileria Annulata ◽  
Upstream Region ◽  
Fundamental Aspect ◽  
Transcriptional Level ◽  
Mobility Shift ◽  
Nuclear Extracts ◽  
Mobility Shift Assay ◽  
High Level

Apicomplexan parasites are major pathogens of humans and domesticated animals. A fundamental aspect of apicomplexan biology, which may provide novel molecular targets for parasite control, is the regulation of stage differentiation. Studies carried out on Theileria annulata, a bovine apicomplexan parasite, have provided evidence that a stochastic process controls differentiation from the macroschizont to the merozoite stage. It was postulated that this process involves the presence of regulators of merozoite gene expression in the preceding stage of the life cycle, and that during differentiation a quantitative increase of these factors occurs. This study was carried out to test these postulations. Nuclear run-on analysis showed that TamS1 expression is controlled, at least in part, at the transcriptional level. The transcription start site showed homology with the consensus eukaryotic initiator motif, and study of the 5′ upstream region by the electrophoretic mobility-shift assay demonstrated that a 23 bp motif specifically bound factors from parasite-enriched nuclear extracts. Three complexes were shown to bind to a 9 bp core binding site (5′-TTTGTAGGG-3′). Two of these complexes were present in macroschizont extracts but were found at elevated levels during differentiation. Both complexes contain a polypeptide of the same molecular mass and may be related via the formation of homodimer or heterodimer complexes. The third complex appears to be distinct and was detected at time points associated with the transition to high level merozoite gene expression.

Regulation of NHE-1 promoter in mammalian myocardium

1996 ◽  
Vol 270 (1) ◽  
pp. H259-H266 ◽  
Author(s):  
W. Yang ◽  
J. R. Dyck ◽  
H. Wang ◽  
L. Fliegel
Keyword(s):  
Mammalian Cells ◽  
Ph Regulation ◽  
Integral Membrane Protein ◽  
Neonatal Rat ◽  
Mobility Shift ◽  
Rat Cardiomyocytes ◽  
Nuclear Extracts ◽  
Mobility Shift Assay ◽  
Gel Mobility Shift ◽  
Reduced Activity

The Na+/H exchanger (NHE-1) is an integral membrane protein responsible for intracellular pH regulation in the myocardium and other tissues. The NHE-1 isoform is universally distributed in mammalian cells. We examined regulation of a 1.1-kb fragment of the NHE-1 promoter in neonatal rat cardiomyocytes. Deletion of most of the promoter up to an AP-2 site reduced activity 75%. Further deletion of the promoter or mutation of the AP-2 site reduced or eliminated activity almost completely. Gel mobility shift assay showed that purified AP-2 protein or AP-2-like protein from nuclear extracts of isolated myocytes can bind to DNA of the NHE-1 protein. External acidosis did not cause increased transcription from the promoter. Removal of serum from the medium reduced activity of the NHE-1 promoter. The elements responsible for activation of the promoter by serum were contained within both the 1.1-kb and AP-2-containing region. The results show that the cis-acting putative AP-2 site and the presence of serum are important in NHE-1 expression, whereas external acidosis had no direct effect on the promoter.

scholarly journals Synergistic Activation of the Pathogenicity-Related Proline Iminopeptidase Gene in Xanthomonas campestris pv. campestris by HrpX and a LuxR Homolog

2012 ◽  
Vol 78 (19) ◽  
pp. 7069-7074 ◽  
Author(s):  
Jingxi Zhang ◽  
Jinhong Kan ◽  
Jieqiong Zhang ◽  
Ping Guo ◽  
Xiaoying Chen ◽  
...  
Keyword(s):  
Xanthomonas Campestris ◽  
Fusion Gene ◽  
Inducible Promoter ◽  
Upstream Region ◽  
Mobility Shift ◽  
Content Type ◽  
Pulldown Assay ◽  
Synergistic Activation ◽  
Mobility Shift Assay ◽  
Proline Iminopeptidase

ABSTRACTXanthomonas campestrispv. campestris strain 8004 contains an orphan quorum-sensing (QS) locus,xccR-pipXcc, in which the proline iminopeptidase (pipXcc) gene (where “Xcc” indicates that thepipgene is fromX. campestrispv. campestris) is positively regulated by the LuxR homologue XccR by binding to theluxXcbox of thepipXccpromoter. The disruption ofpipXccsignificantly attenuated the virulence ofX. campestrispv. campestris. An imperfect plant-inducible promoter (PIP) box is located in the upstream region of thepipXccpromoter, which is the putative binding site of the transcriptional activator HrpX. To explore whether the expression of thepipXccgene is regulated by HrpX, the expression level of apipXccpromoter-gusAfusion gene was assayed in anhrpXdisruption mutant. The results showed that the lack of HrpX dramatically decreased the β-glucuronidase (GUS) activity. Further analyses using an electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP)-PCR indicated that the imperfect PIP box inX. campestrispv. campestris is specifically bound to HrpX. These data demonstrated that thepipXccgene belongs to the hrp regulon and that the imperfect PIP box of thepipXccpromoter could be aciselement for the HrpX protein. We further showed in a pulldown assay that XccR can bind HrpX, suggesting that these two regulatory proteins coactivate the virulence factor by binding to the differentciselements of thepipXccgene and adapt to the host environment duringX. campestrispv. campestris infection.

scholarly journals Dimeric Brucella abortus Irr protein controls its own expression and binds haem

Microbiology ◽  
2005 ◽  
Vol 151 (10) ◽  
pp. 3427-3433 ◽  
Author(s):  
Marcela Martínez ◽  
Rodolfo A. Ugalde ◽  
Marta Almirón
Keyword(s):  
Brucella Abortus ◽  
Biosynthetic Pathway ◽  
Response Regulator ◽  
Protoporphyrin Ix ◽  
Close Relative ◽  
Iron Availability ◽  
Iron Deprivation ◽  
Haem Biosynthesis ◽  
Insight Into

Brucella abortus needs to synthesize haem in order to replicate intracellularly and to produce virulence in mice. Thus, to gain insight into the pathogenesis of the bacterium, regulatory proteins of the haem biosynthetic pathway were sought. An iron response regulator (Irr) from Bradyrhizobium japonicum, which is a close relative of Brucella, was previously described as being involved in the coordination of haem biosynthesis and iron availability. The Bru. abortus genome was searched for an irr orthologue gene, and the Bru. abortus irr gene was cloned, sequenced and disrupted. A null mutant was constructed that accumulated protoporphyrin IX under conditions of iron deprivation. This phenotype was overcome by a complementing plasmid carrying the wild-type irr. Purified recombinant Bru. abortus Irr behaved as a stable dimer and bound haem. Interestingly, in vivo, Irr was only detected in cells obtained from iron-limited cultures and the protein downregulated its own transcription. Through lacZ fusion, it was demonstrated that iron did not regulate irr transcription. The data reported show that Bru. abortus Irr is a homodimeric protein that is accumulated in iron-limited cells, controls its own transcription and downregulates the biosynthesis of haem precursors.

scholarly journals DNA-binding specificity of GATA family transcription factors

1993 ◽  
Vol 13 (7) ◽  
pp. 3999-4010 ◽  
Author(s):  
M Merika ◽  
S H Orkin
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Mammalian Cells ◽  
Chimeric Protein ◽  
Binding Specificity ◽  
Mobility Shift ◽  
Binding Domains ◽  
Dna Binding Specificity ◽  
Mobility Shift Assay ◽  
Gata Family

GATA-binding proteins constitute a family of transcription factors that recognize a target site conforming to the consensus WGATAR (W = A or T and R = A or G). Here we have used the method of polymerase chain reaction-mediated random site selection to assess in an unbiased manner the DNA-binding specificity of GATA proteins. Contrary to our expectations, we show that GATA proteins bind a variety of motifs that deviate from the previously assigned consensus. Many of the nonconsensus sequences bind protein with high affinity, equivalent to that of conventional GATA motifs. By using the selected sequences as probes in the electrophoretic mobility shift assay, we demonstrate overlapping, but distinct, sequence preferences for GATA family members, specified by their respective DNA-binding domains. Furthermore, we provide additional evidence for interaction of amino and carboxy fingers of GATA-1 in defining its binding site. By performing cotransfection experiments, we also show that transactivation parallels DNA binding. A chimeric protein containing the finger domain of areA and the activation domains of GATA-1 is capable of activating transcription in mammalian cells through GATA motifs. Our findings suggest a mechanism by which GATA proteins might selectively regulate gene expression in cells in which they are coexpressed.

scholarly journals The Bacillus subtilis Response Regulator GenedegUIs Positively Regulated by CcpA and by Catabolite-Repressed Synthesis of ClpC

2012 ◽  
Vol 195 (2) ◽  
pp. 193-201 ◽  
Author(s):  
Hiroshi Ishii ◽  
Teruo Tanaka ◽  
Mitsuo Ogura
Keyword(s):  
Bacillus Subtilis ◽  
Response Regulator ◽  
Consensus Sequence ◽  
Protein A ◽  
Upstream Region ◽  
Sporulation Medium ◽  
Mobility Shift ◽  
Content Type ◽  
Cellular Processes ◽  
Mobility Shift Assay

ABSTRACTInBacillus subtilis, the response regulator DegU and its cognate kinase, DegS, constitute a two-component system that regulates many cellular processes, including exoprotease production and genetic competence. Phosphorylated DegU (DegU-P) activates its own promoter and is degraded by the ClpCP protease. We observed induction ofdegUby glucose in sporulation medium. This was abolished in two mutants: theccpA(catabolite control protein A) andclpCdisruptants. Transcription of the promoter of the operon containingclpC(PclpC) decreased in the presence of glucose, and the disruption ofccpAresulted in derepression of PclpC. However, this was not directly mediated by CcpA, because we failed to detect binding of CcpA to PclpC. Glucose decreased the expression ofclpC, leading to low cellular concentrations of the ClpCP protease. Thus,degUis induced through activation of autoregulation by a decrease in ClpCP-dependent proteolysis of DegU-P. An electrophoretic mobility shift assay showed that CcpA bound directly to thedegUupstream region, indicating that CcpA activatesdegUthrough binding. The bound region was narrowed down to 27 bases, which contained acre(catabolite-responsiveelement) sequence with a low match to thecreconsensus sequence. In a footprint analysis, CcpA specifically protected a region containing thecresequence from DNase I digestion. The induction ofdegUby glucose showed complex regulation of thedegUgene.

scholarly journals Repression of Antibiotic Downregulator WblA by AdpA in Streptomyces coelicolor

2013 ◽  
Vol 79 (13) ◽  
pp. 4159-4163 ◽  
Author(s):  
Han-Na Lee ◽  
Jin-Su Kim ◽  
Pil Kim ◽  
Heung-Shick Lee ◽  
Eung-Soo Kim
Keyword(s):  
Electrophoretic Mobility Shift Assay ◽  
Electrophoretic Mobility ◽  
Streptomyces Coelicolor ◽  
Upstream Region ◽  
Mobility Shift ◽  
Binding Motifs ◽  
Content Type ◽  
Disruption Mutant ◽  
Mobility Shift Assay

ABSTRACTThe upstream region of antibiotic downregulatorywblAinStreptomyces coelicolorwas found to contain AdpA binding motifs. A key morphological regulator, AdpA was shown to specifically bind these motifs by electrophoretic mobility shift assay. AnadpAdisruption mutant exhibited increasedwblAtranscription, suggesting that AdpA negatively regulateswblAtranscription inS. coelicolor.

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