scholarly journals SirR, a Novel Iron-Dependent Repressor inStaphylococcus epidermidis

1998 ◽  
Vol 66 (9) ◽  
pp. 4123-4129 ◽  
Author(s):  
Philip J. Hill ◽  
Alan Cockayne ◽  
Patrick Landers ◽  
Julie A. Morrissey ◽  
Catriona M. Sims ◽  
...  
Keyword(s):  
Binding Site ◽  
Dna Sequences ◽  
Blot Analysis ◽  
Target Genes ◽  
Consensus Sequence ◽  
Dependent Manner ◽  
Chromosomal Dna ◽  
Iron Starvation ◽  
Mobility Shift ◽  
Western Blots

ABSTRACT In Staphylococcus epidermidis and Staphylococcus aureus, a number of cell wall- and cytoplasmic membrane-associated lipoproteins are induced in response to iron starvation. To gain insights into the molecular basis of iron-dependent gene regulation in the staphylococci, we sequenced the DNA upstream of the 3-kb S. epidermidis sitABC operon, which Northern blot analysis indicates is transcriptionally regulated by the growth medium iron content. We identified two DNA sequences which are homologous to elements of the Corynebacterium diphtheriae DtxR regulon, which controls, in response to iron stress, for example, production of diphtheria toxin, siderophore, and a heme oxygenase. Upstream of thesitABC operon and divergently transcribed lies a 645-bp open reading frame (ORF), which codes for a polypeptide of approximately 25 kDa with homology to the DtxR family of metal-dependent repressor proteins. This ORF has been designated SirR (staphylococcal iron regulator repressor). Within thesitABC promoter/operator region, we also located a region of dyad symmetry overlapping the transcriptional start ofsitABC which shows high homology to the DtxR operator consensus sequence, suggesting that this region, termed the Sir box, is the SirR-binding site. The SirR protein was overexpressed, purified, and used in DNA mobility shift assays; SirR retarded the migration of a synthetic oligonucleotide based on the Sir box in a metal (Fe2+ or Mn2+)-dependent manner, providing confirmatory evidence that this motif is the SirR-binding site. Furthermore, Southern blot analysis of staphylococcal chromosomal DNA with the synthetic Sir box as a probe confirmed that there are at least five Sir boxes in the S. epidermidis genome and at least three in the genome of S. aureus, suggesting that SirR controls the expression of multiple target genes. Using a monospecific polyclonal antibody raised against SirR to probe Western blots of whole-cell lysates of S. aureus, S. carnosus,S. epidermidis, S. hominis, S. cohnii, S. lugdunensis, and S. haemolyticus, we identified an approximately 25-kDa cross-reactive protein in each of the staphylococcal species examined. Taken together, these data suggest that SirR functions as a divalent metal cation-dependent transcriptional repressor which is widespread among the staphylococci.

scholarly journals MisR/MisS Two-Component Regulon in Neisseria meningitidis

2007 ◽  
Vol 76 (2) ◽  
pp. 704-716 ◽  
Author(s):  
Yih-Ling Tzeng ◽  
Charlene M. Kahler ◽  
Xinjian Zhang ◽  
David S. Stephens
Keyword(s):  
Neisseria Meningitidis ◽  
Target Genes ◽  
Inner Core ◽  
Consensus Sequence ◽  
Meningococcal Infection ◽  
Type I ◽  
Mobility Shift ◽  
Iron Assimilation ◽  
Wide Range ◽  
Two Component

ABSTRACT Two-component regulatory systems are involved in processes important for bacterial pathogenesis. Inactivation of the misR/misS system in Neisseria meningitidis results in the loss of phosphorylation of the lipooligosaccharide inner core and causes attenuation in a mouse model of meningococcal infection. One hundred seventeen (78 up-regulated and 39 down-regulated) potential regulatory targets of the MisR/MisS (MisR/S) system were identified by transcriptional profiling of the NMBmisR mutant and the parental wild-type meningococcal strain NMB. The regulatory effect was further confirmed in a subset of target genes by quantitative real-time PCR and β-galactosidase transcriptional fusion reporter assays. The MisR regulon includes genes encoding proteins necessary for protein folding in the bacterial cytoplasm and periplasm, transcriptional regulation, metabolism, iron assimilation, and type I protein transport. Mutation in the MisR/S system caused increased sensitivity to oxidative stress and also resulted in decreased susceptibility to complement-mediated killing by normal human serum. To identify the direct targets of MisR regulation, electrophoretic mobility shift assays were carried out using purified MisR-His6 protein. Among 22 genes examined, misR directly interacted with 14 promoter regions. Six promoters were further investigated by DNase I protection assays, and a MisR-binding consensus sequence was proposed. Thus, the direct regulatory targets of MisR and the minimal regulon of the meningococcal MisR/S two-component signal transduction system were characterized. These data indicate that the MisR/S system influences a wide range of biological functions in N. meningitidis either directly or via intermediate regulators.

DNA-binding and transcriptional regulatory properties of hepatic leukemia factor (HLF) and the t(17;19) acute lymphoblastic leukemia chimera E2A-HLF

1994 ◽  
Vol 14 (9) ◽  
pp. 5986-5996
Author(s):  
S P Hunger ◽  
R Brown ◽  
M L Cleary
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Dna Sequences ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Lymphoblastic Leukemia ◽  
Consensus Sequence ◽  
Reporter Genes ◽  
Wild Type ◽  
Basic Leucine Zipper

The t(17;19) translocation in acute lymphoblastic leukemias results in creation of E2A-hepatic leukemia factor (HLF) chimeric proteins that contain the DNA-binding and protein dimerization domains of the basic leucine zipper (bZIP) protein HLF fused to a portion of E2A proteins with transcriptional activation properties. An in vitro binding site selection procedure was used to determine DNA sequences preferentially bound by wild-type HLF and chimeric E2A-HLF proteins isolated from various t(17;19)-bearing leukemias. All were found to selectively bind the consensus sequence 5'-GTTACGTAAT-3' with high affinity. Wild-type and chimeric HLF proteins also bound closely related sites identified previously for bZIP proteins of both the proline- and acidic amino acid-rich (PAR) and C/EBP subfamilies; however, E2A-HLF proteins were significantly less tolerant of certain deviations from the HLF consensus binding site. These differences were directly attributable to loss of an HLF ancillary DNA-binding domain in all E2A-HLF chimeras and were further exacerbated by a zipper mutation in one isolate. Both wild-type and chimeric HLF proteins displayed transcriptional activator properties in lymphoid and nonlymphoid cells on reporter genes containing HLF or C/EBP consensus binding sites. But on reporter genes with nonoptimal binding sites, their transcriptional properties diverged and E2A-HLF competitively inhibited activation by wild-type PAR proteins. These findings establish a spectrum of binding site-specific transcriptional properties for E2A-HLF which may preferentially activate expression of select subordinate genes as a homodimer and potentially antagonize expression of others through heteromeric interactions.

scholarly journals New Insights into the Pleiotropic Drug Resistance Network from Genome-Wide Characterization of the YRR1 Transcription Factor Regulation System

2002 ◽  
Vol 22 (8) ◽  
pp. 2642-2649 ◽  
Author(s):  
Stéphane Le Crom ◽  
Frédéric Devaux ◽  
Philippe Marc ◽  
Xiaoting Zhang ◽  
W. Scott Moye-Rowley ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Transcription Factor ◽  
Binding Site ◽  
Time Course ◽  
Target Genes ◽  
Regulation System ◽  
Dependent Manner ◽  
Pleiotropic Drug Resistance ◽  
Genome Wide

ABSTRACT Yrr1p is a recently described Zn2Cys6 transcription factor involved in the pleiotropic drug resistance (PDR) phenomenon. It is controlled in a Pdr1p-dependent manner and is autoregulated. We describe here a new genome-wide approach to characterization of the set of genes directly regulated by Yrr1p. We found that the time-course production of an artificial chimera protein containing the DNA-binding domain of Yrr1p activated the 15 genes that are also up-regulated by a gain-of-function mutant of Yrr1p. Gel mobility shift assays showed that the promoters of the genes AZR1, FLR1, SNG1, YLL056C, YLR346C, and YPL088W interacted with Yrr1p. The putative consensus Yrr1p binding site deduced from these experiments, (T/A)CCG(C/T)(G/T)(G/T)(A/T)(A/T), is strikingly similar to the PDR element binding site sequence recognized by Pdr1p and Pdr3p. The minor differences between these sequences are consistent with Yrr1p and Pdr1p and Pdr3p having different sets of target genes. According to these data, some target genes are directly regulated by Pdr1p and Pdr3p or by Yrr1p, whereas some genes are indirectly regulated by the activation of Yrr1p. Some genes, such as YOR1, SNQ2, and FLR1, are clearly directly controlled by both classes of transcription factor, suggesting an important role for the corresponding membrane proteins.

scholarly journals DNA-binding and transcriptional regulatory properties of hepatic leukemia factor (HLF) and the t(17;19) acute lymphoblastic leukemia chimera E2A-HLF.

1994 ◽  
Vol 14 (9) ◽  
pp. 5986-5996 ◽  
Author(s):  
S P Hunger ◽  
R Brown ◽  
M L Cleary
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Dna Sequences ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Lymphoblastic Leukemia ◽  
Consensus Sequence ◽  
Reporter Genes ◽  
Wild Type ◽  
Basic Leucine Zipper

The t(17;19) translocation in acute lymphoblastic leukemias results in creation of E2A-hepatic leukemia factor (HLF) chimeric proteins that contain the DNA-binding and protein dimerization domains of the basic leucine zipper (bZIP) protein HLF fused to a portion of E2A proteins with transcriptional activation properties. An in vitro binding site selection procedure was used to determine DNA sequences preferentially bound by wild-type HLF and chimeric E2A-HLF proteins isolated from various t(17;19)-bearing leukemias. All were found to selectively bind the consensus sequence 5'-GTTACGTAAT-3' with high affinity. Wild-type and chimeric HLF proteins also bound closely related sites identified previously for bZIP proteins of both the proline- and acidic amino acid-rich (PAR) and C/EBP subfamilies; however, E2A-HLF proteins were significantly less tolerant of certain deviations from the HLF consensus binding site. These differences were directly attributable to loss of an HLF ancillary DNA-binding domain in all E2A-HLF chimeras and were further exacerbated by a zipper mutation in one isolate. Both wild-type and chimeric HLF proteins displayed transcriptional activator properties in lymphoid and nonlymphoid cells on reporter genes containing HLF or C/EBP consensus binding sites. But on reporter genes with nonoptimal binding sites, their transcriptional properties diverged and E2A-HLF competitively inhibited activation by wild-type PAR proteins. These findings establish a spectrum of binding site-specific transcriptional properties for E2A-HLF which may preferentially activate expression of select subordinate genes as a homodimer and potentially antagonize expression of others through heteromeric interactions.

Enhancement of telomere-plasmid segregation by the X-telomere associated sequence in Saccharomyces cerevisiae involves SIR2, SIR3, SIR4 and ABF1.

Genetics ◽  
1994 ◽  
Vol 136 (3) ◽  
pp. 757-767 ◽  
Author(s):  
S Enomoto ◽  
M S Longtine ◽  
J Berman
Keyword(s):  
Binding Site ◽  
Consensus Sequence ◽  
Active Role ◽  
Repeat Sequence ◽  
Dependent Manner ◽  
Enhancer Element ◽  
Telomere Repeat ◽  
Plasmid Segregation ◽  
Telomere Function ◽  
Associated Sequences

Abstract We have previously shown that circular replicating plasmids that carry yeast telomere repeat sequence (TG1-3) tracts segregate efficiently relative to analogous plasmids lacking the TG1-3 tract and this efficient segregation is dependent upon RAP1. While a long TG1-3 tract is sufficient to improve plasmid segregation, the segregation efficiency of telomere plasmids (TEL-plasmids) is enhanced when the X-Telomere Associated Sequence (X-TAS) is also included on the plasmids. We now demonstrate that the enhancement of TEL-plasmid segregation by the X-TAS depends on SIR2, SIR3, SIR4 and ABF1 in trans and requires the Abf1p-binding site within the X-TAS. Mutation of the Abf1p-binding site within the X-TAS results in TEL-plasmids that are no longer affected by mutations in SIR2, SIR3 or SIR4, despite the fact that other Abf1p-binding sites are present on the plasmid. Mutation of the ARS consensus sequence within the X-TAS converts the X-TAS from an enhancer element to a negative element that interferes with TEL-plasmid segregation in a SIR-dependent manner. Thus, telomere associated sequences interact with TG1-3 tracts on the plasmid, suggesting that the TASs have an active role in modulating telomere function.

scholarly journals Incompatibility Protein IncC and Global Regulator KorB Interact in Active Partition of Promiscuous Plasmid RK2

2000 ◽  
Vol 182 (21) ◽  
pp. 6014-6026 ◽  
Author(s):  
Thomas M. Rosche ◽  
Azeem Siddique ◽  
Michelle H. Larsen ◽  
David H. Figurski
Keyword(s):  
Binding Site ◽  
Broad Host Range ◽  
Dependent Manner ◽  
Chromosomal Dna ◽  
Obvious Effect ◽  
Partition System ◽  
Stable Maintenance ◽  
Plasmid Rk2

ABSTRACT Replication of the broad-host-range, IncPα plasmid RK2 requires two plasmid loci: trfA, the replication initiator gene, andoriV, the origin of replication. While these determinants are sufficient for replication in a wide variety of bacteria, they do not confer the stable maintenance of parental RK2 observed in its hosts. The product of the incC gene has been proposed to function in the stable maintenance of RK2 because of its relatedness to the ParA family of ATPases, some of which are known to be involved in the active partition of plasmid and chromosomal DNA. Here we show that IncC has the properties expected of a component of an active partition system. The smaller polypeptide product of incC (IncC2) exhibits a strong, replicon-independent incompatibility phenotype with RK2. This incompatibility phenotype requires the global transcriptional repressor, KorB, and the target for incC-mediated incompatibility is a KorB-binding site (OB). We found that KorB and IncC interact in vivo by using the yeast two-hybrid system and in vitro by using partially purified proteins. Elevated expression of the incC and korB genes individually has no obvious effect on Escherichia coli cell growth, but their simultaneous overexpression is toxic, indicating a possible interaction of IncC-KorB complexes with a vital host target. A region of RK2 bearing incC, korB, and multiple KorB-binding sites is able to stabilize an unstable, heterologous plasmid in anincC-dependent manner. Finally, elevated levels of IncC2 cause RK2 to aggregate, indicating a possible role for IncC in plasmid pairing. These findings demonstrate that IncC, KorB, and at least one KorB-binding site are components of an active partition system for the promiscuous plasmid RK2.

scholarly journals Role of the IE62 Consensus Binding Site in Transactivation by the Varicella-Zoster Virus IE62 Protein

2010 ◽  
Vol 84 (8) ◽  
pp. 3767-3779 ◽  
Author(s):  
Kris White ◽  
Hua Peng ◽  
John Hay ◽  
William T. Ruyechan
Keyword(s):  
Dna Binding ◽  
Varicella Zoster Virus ◽  
Binding Site ◽  
Consensus Sequence ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Consensus Binding Site ◽  
Mobility Shift ◽  
Varicella Zoster ◽  
Mobility Shift Assay

ABSTRACT The varicella-zoster virus (VZV) IE62 protein is the major transcriptional activator. IE62 is capable of associating with DNA both nonspecifically and in a sequence-specific manner via a consensus binding site (5′-ATCGT-3′). However, the function of the consensus site is poorly understood, since IE62 efficiently transactivates promoter elements lacking this sequence. In the work presented here, sequence analysis of the VZV genome revealed the presence of 245 IE62 consensus sites throughout the genome. Some 54 sites were found to be present within putative VZV promoters. Electrophoretic mobility shift assay (EMSA) experiments using an IE62 fragment containing the IE62 DNA-binding domain and duplex oligonucleotides that did or did not contain the IE62 consensus binding sequence yielded KD (equilibrium dissociation constant) values in the nanomolar range. Further, the IE62 DNA binding domain was shown to have a 5-fold-increased affinity for its consensus site compared to nonconsensus sequences. The effect of consensus site presence and position on IE62-mediated activation of native VZV and model promoters was examined using site-specific mutagenesis and transfection and superinfection reporter assays. In all promoters examined, the consensus sequence functioned as a distance-dependent repressive element. Protein recruitment assays utilizing the VZV gI promoter indicated that the presence of the consensus site increased the recruitment of IE62 but not Sp1. These data suggest a model where the IE62 consensus site functions to down-modulate IE62 activation, and interaction of IE62 with this sequence may result in loss or decrease of the ability of IE62 to recruit cellular factors needed for full promoter activation.

scholarly journals DNA Sequence and Functional Analysis of Homologous ARS Elements of Saccharomyces cerevisiae and S. carlsbergensis

Genetics ◽  
1999 ◽  
Vol 152 (3) ◽  
pp. 943-952
Author(s):  
James F Theis ◽  
Chen Yang ◽  
Christopher B Schaefer ◽  
Carol S Newlon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Consensus Sequence ◽  
Chromosomal Dna ◽  
Functional Elements ◽  
Sequence Comparisons ◽  
Cis Acting ◽  
Homologous Sequences ◽  
Ars Elements

Abstract ARS elements of Saccharomyces cerevisiae are the cis-acting sequences required for the initiation of chromosomal DNA replication. Comparisons of the DNA sequences of unrelated ARS elements from different regions of the genome have revealed no significant DNA sequence conservation. We have compared the sequences of seven pairs of homologous ARS elements from two Saccharomyces species, S. cerevisiae and S. carlsbergensis. In all but one case, the ARS308-ARS308carl pair, significant blocks of homology were detected. In the cases of ARS305, ARS307, and ARS309, previously identified functional elements were found to be conserved in their S. carlsbergensis homologs. Mutation of the conserved sequences in the S. carlsbergensis ARS elements revealed that the homologous sequences are required for function. These observations suggested that the sequences important for ARS function would be conserved in other ARS elements. Sequence comparisons aided in the identification of the essential matches to the ARS consensus sequence (ACS) of ARS304, ARS306, and ARS310carl, though not of ARS310.

Orphan nuclear receptor SHP interacts with and represses hepatocyte nuclear factor-6 (HNF-6) transactivation

2008 ◽  
Vol 413 (3) ◽  
pp. 559-569 ◽  
Author(s):  
Yong-Soo Lee ◽  
Don-Kyu Kim ◽  
Yong Deuk Kim ◽  
Ki Cheol Park ◽  
Minho Shong ◽  
...  
Keyword(s):  
Nuclear Receptor ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Target Genes ◽  
Consensus Sequence ◽  
Nuclear Factor ◽  
Orphan Nuclear Receptor ◽  
Hepatocyte Nuclear Factor ◽  
Mobility Shift ◽  
Endocrine Differentiation

SHP (small heterodimer partner; NR0B2) is an atypical orphan NR (nuclear receptor) that functions as a transcriptional co-repressor by interacting with a diverse set of NRs and transcriptional factors. HNF-6 (hepatocyte nuclear factor-6) is a key regulatory factor in pancreatic development, endocrine differentiation and the formation of the biliary tract, as well as glucose metabolism. In this study, we have investigated the function of SHP as a putative repressor of HNF-6. Using transient transfection assays, we have shown that SHP represses the transcriptional activity of HNF-6. Confocal microscopy revealed that both SHP and HNF-6 co-localize in the nuclei of cells. SHP physically interacted with HNF-6 in protein–protein association assays in vitro. EMSAs (electrophoretic mobility-shift assays) and ChIP (chromatin immunoprecipitation) assays demonstrated that SHP inhibits the DNA-binding activity of HNF-6 to an HNF-6-response element consensus sequence, and the HNF-6 target region of the endogenous G6Pase (glucose 6-phosphatase) promoter respectively. Northern blot analysis of HNF-6 target genes in cells infected with adenoviral vectors for SHP and SHP siRNAs (small inhibitory RNAs) indicated that SHP represses the expression of endogenous G6Pase and PEPCK (phosphoenolpyruvate carboxykinase). Our results suggest that HNF-6 is a novel target of SHP in the regulation of gluconeogenesis.

scholarly journals RifZ (AMED_0655) Is a Pathway-Specific Regulator for Rifamycin Biosynthesis in Amycolatopsis mediterranei

2017 ◽  
Vol 83 (8) ◽  
Author(s):  
Chen Li ◽  
Xinqiang Liu ◽  
Chao Lei ◽  
Han Yan ◽  
Zhihui Shao ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Dna Sequences ◽  
Consensus Sequence ◽  
Biosynthetic Gene Cluster ◽  
High Yield ◽  
Biosynthetic Gene ◽  
Amycolatopsis Mediterranei ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Content Type

ABSTRACT Rifamycin and its derivatives are particularly effective against the pathogenic mycobacteria Mycobacterium tuberculosis and Mycobacterium leprae. Although the biosynthetic pathway of rifamycin has been extensively studied in Amycolatopsis mediterranei, little is known about the regulation in rifamycin biosynthesis. Here, an in vivo transposon system was employed to identify genes involved in the regulation of rifamycin production in A. mediterranei U32. In total, nine rifamycin-deficient mutants were isolated, among which three mutants had the transposon inserted in AMED_0655 (rifZ, encoding a LuxR family regulator). The rifZ gene was further knocked out via homologous recombination, and the transcription of genes in the rifamycin biosynthetic gene cluster (rif cluster) was remarkably reduced in the rifZ null mutant. Based on the cotranscription assay results, genes within the rif cluster were grouped into 10 operons, sharing six promoter regions. By use of electrophoretic mobility shift assay and DNase I footprinting assay, RifZ was proved to specially bind to all six promoter regions, which was consistent with the fact that RifZ regulated the transcription of the whole rif cluster. The binding consensus sequence was further characterized through alignment using the RifZ-protected DNA sequences. By use of bionformatic analysis, another five promoters containing the RifZ box (CTACC-N8-GGATG) were identified, among which the binding of RifZ to the promoter regions of both rifK and orf18 (AMED_0645) was further verified. As RifZ directly regulates the transcription of all operons within the rif cluster, we propose that RifZ is a pathway-specific regulator for the rif cluster. IMPORTANCE To this day, rifamycin and its derivatives are still the first-line antituberculosis drugs. The biosynthesis of rifamycin has been extensively studied, and most biosynthetic processes have been characterized. However, little is known about the regulation of the transcription of the rifamycin biosynthetic gene cluster (rif cluster), and no regulator has been characterized. Through the employment of transposon screening, we here characterized a LuxR family regulator, RifZ, as a direct transcriptional activator for the rif cluster. As RifZ directly regulates the transcription of the entire rif cluster, it is considered a pathway-specific regulator for rifamycin biosynthesis. Therefore, as the first regulator characterized for direct regulation of rif cluster transcription, RifZ may provide a new clue for further engineering of high-yield industrial strains.

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