scholarly journals Regulation of Type IV Secretion Apparatus Genes during Ehrlichia chaffeensis Intracellular Development by a Previously Unidentified Protein

2008 ◽  
Vol 190 (6) ◽  
pp. 2096-2105 ◽  
Author(s):  
Zhihui Cheng ◽  
Xueqi Wang ◽  
Yasuko Rikihisa
Keyword(s):  
Promoter Region ◽  
Hypothetical Protein ◽  
Type Iv Secretion ◽  
Ehrlichia Chaffeensis ◽  
Bacterial Lysate ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Specific Expression ◽  
Type Iv ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT The type IV secretion (T4S) system is critical for the virulence of several pathogens. In the rickettsial pathogen Ehrlichia chaffeensis, the virBD genes are split into two operons, the virB3-virB6 (preceded by sodB) and virB8-virD4 operons. Between these two operons, there are duplications of virB4, virB8, and virB9. In this study we found that transcription of all five loci was downregulated prior to the release of E. chaffeensis from host THP-1 cells and was upregulated at the initiation of exponential growth. Electrophoretic mobility shift assays revealed an E. chaffeensis-encoded protein that specifically bound to the promoter regions upstream of the virBD loci. The protein was purified from the bacterial lysate by affinity chromatography using a biotinylated promoter region upstream of sodB. Mass spectrometry identified the protein as an E. chaffeensis 12.3-kDa hypothetical protein, which was designated EcxR. Recombinant EcxR bound to the promoter regions upstream of five individual virBD loci. EcxR also activated transcription of all five virBD loci in lacZ reporter constructs. The expression of ecxR was positively autoregulated by EcxR. These results suggest that the five virBD loci are coordinately regulated by EcxR to allow developmental stage-specific expression of the T4S system in E. chaffeensis.

scholarly journals The Response Regulator CroR Modulates Expression of the Secreted Stress-Induced SalB Protein in Enterococcus faecalis

2006 ◽  
Vol 188 (7) ◽  
pp. 2636-2645 ◽  
Author(s):  
Cécile Muller ◽  
Yoann Le Breton ◽  
Thierry Morin ◽  
Abdellah Benachour ◽  
Yanick Auffray ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Promoter Region ◽  
Response Regulator ◽  
Signal Transduction System ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Dnase I Footprinting ◽  
Two Component ◽  
Two Component Signal Transduction ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT The Enterococcus faecalis two-component signal transduction system CroRS, also referred as the RR-HK05 pair, is required for intrinsic β-lactam resistance (Y. R. Comenge, R. Quintiliani, Jr., L. Li, L. Dubost, J. P. Brouard, J. E. Hugonnet, and M. Arthur, J. Bacteriol. 185:7184-7192, 2003) and is also suspected to be involved in the expression of salB (previously referred to as sagA), a gene important for resistance to environmental stress and cell morphology (Y. Le Breton, G. Boël, A. Benachour, H. Prévost, Y. Auffray, and A. Rincé, Environ. Microbiol. 5:329-337, 2003). In this report, we provide genetic and biochemical evidence that salB encodes a secreted protein that is expressed from a monocistronic stress-inducible operon. Consistent with CroR being a direct transcriptional activator of the salB expression, CroR was found to bind to the salB promoter region in electrophoretic mobility shift assays. Interestingly, we provide evidence that SalB does not play a role in the intrinsic β-lactam resistance associated with CroRS. We also show that the CroRS system is able to regulate its own expression. The sequence of the CroRS binding site in the salB and croR promoter regions was determined using DNase I footprinting assays.

scholarly journals The TraA relaxase autoregulates the putative type IV secretion-like system encoded by the broad-host-range Streptococcus agalactiae plasmid pIP501

Microbiology ◽  
2006 ◽  
Vol 152 (3) ◽  
pp. 637-645 ◽  
Author(s):  
Brigitta Kurenbach ◽  
Jolanta Kopeć ◽  
Marion Mägdefrau ◽  
Kristin Andreas ◽  
Walter Keller ◽  
...  
Keyword(s):  
Type Iv Secretion ◽  
Transcriptional Level ◽  
Broad Host Range ◽  
Rt Pcr ◽  
Mobility Shift ◽  
Transcriptional Terminator ◽  
Type Iv ◽  
Dnase I Footprinting ◽  
In Trans ◽  
Electrophoretic Mobility Shift Assays

The conjugative multiple antibiotic resistance plasmid pIP501 can be transferred and stably maintained in a variety of Gram-positive genera, including multicellular Streptomyces lividans, as well as in Gram-negative Escherichia coli. The 15 putative pIP501 transfer (tra) genes are organized in an operon-like structure terminating in a strong transcriptional terminator. This paper reports co-transcription of the pIP501 tra genes in exponentially growing Enterococcus faecalis JH2-2 cells, as shown by RT-PCR. The tra genes are expressed throughout the life cycle of Ent. faecalis, and the expression level is independent of the growth phase. Electrophoretic mobility shift assays indicated that the TraA relaxase, the first gene of the tra operon, binds to the tra promoter P tra , which partially overlaps with the origin of transfer (oriT). DNase I footprinting experiments further delimited the TraA binding region and defined the nucleotides bound by TraA. β-Galactosidase assays with P tra–lacZ fusions proved P tra promoter activity, which was strongly repressed when TraA was supplied in trans. Thus, it is concluded that the pIP501 tra operon is negatively autoregulated at the transcriptional level by the conjugative DNA relaxase TraA.

Positive and negative transcriptional elements of the human type IV collagenase gene

1990 ◽  
Vol 10 (12) ◽  
pp. 6524-6532
Author(s):  
S M Frisch ◽  
J H Morisaki
Keyword(s):  
Core Promoter ◽  
Type Iv Collagenase ◽  
Mobility Shift ◽  
Specific Expression ◽  
Enhancer Element ◽  
Type Iv ◽  
Dnase I Footprinting ◽  
Gel Mobility Shift ◽  
Transcriptional Elements ◽  
Mcf 7

Proteolysis by type IV collagenase (T4) has been implicated in the process of tumor metastasis. The T4 gene is expressed in fibroblasts, but not in normal epithelial cells, and its expression is specifically repressed by the E1A oncogene of adenovirus. We present an investigation of the transcriptional elements responsible for basal, E1A-repressible, and tissue-specific expression. 5'-Deletion analysis, DNase I footprinting, and gel mobility shift assays revealed a strong, E1A-repressible enhancer element, r2, located about 1,650 bp upstream of the start site. This enhancer bound a protein with binding specificity very similar to that of the transcription factor AP-2. A potent silencer sequence was found 2 to 5 bp downstream of this enhancer. The silencer repressed transcription from either r2 or AP-1 enhancer elements and in the context of either type IV collagenase or thymidine kinase (tk) gene core promoters; enhancerless transcription from the latter core promoter was also repressed. Comprising the silencer were two contiguous, autonomously functioning silencer elements. Negative regulation of T4 transcription by at least two factors was demonstrated. mcf-7 proteins specifically binding both elements were detected by gel mobility shift assays; a protein of approximately 185 kDa that bound to one of these elements was detected by DNA-protein cross-linking. The silencer repressed transcription, in an r2 enhancer-tk promoter context, much more efficiently in T4-nonproducing cells (mcf-7 or HeLa) than in T4-producing cells (HT1080), suggesting that cell type-specific silencing may contribute to the regulation of this gene.

scholarly journals ApoFnr Binds as a Monomer to Promoters Regulating the Expression of Enterotoxin Genes of Bacillus cereus

2008 ◽  
Vol 190 (12) ◽  
pp. 4242-4251 ◽  
Author(s):  
Julia Esbelin ◽  
Yves Jouanneau ◽  
Jean Armengaud ◽  
Catherine Duport
Keyword(s):  
Fusion Protein ◽  
Bacillus Cereus ◽  
Regulatory Protein ◽  
Disulfide Bridge ◽  
Oligomeric State ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Enterotoxin Genes ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT Bacillus cereus Fnr is a member of the Crp/Fnr (cyclic AMP-binding protein/fumarate nitrate reduction regulatory protein) family of helix-turn-helix transcriptional regulators. It is essential for the expression of hbl and nhe enterotoxin genes independently of the oxygen tension in the environment. We studied aerobic Fnr binding to target sites in promoters regulating the expression of enterotoxin genes. B. cereus Fnr was overexpressed and purified as either a C-terminal His-tagged (FnrHis) fusion protein or an N-terminal fusion protein tagged with the Strep-tag (IBA BioTAGnology) (StrepFnr). Both recombinant Fnr proteins were produced as apoforms (clusterless) and occurred as mixtures of monomers and oligomers in solution. However, apoFnrHis was mainly monomeric, while apoStrepFnr was mainly oligomeric, suggesting that the His-tagged C-terminal extremity may interfere with oligomerization. The oligomeric state of apoStrepFnr was dithiothreitol sensitive, underlining the importance of a disulfide bridge for apoFnr oligomerization. Electrophoretic mobility shift assays showed that monomeric apoFnr, but not oligomeric apoFnr, bound to specific sequences located in the promoter regions of the enterotoxin regulators fnr, resDE, and plcR and the structural genes hbl and nhe. The question of whether apoFnr binding is regulated in vivo by redox-dependent oligomerization is discussed.

scholarly journals Gata4 Regulates Testis Expression of Dmrt1

2004 ◽  
Vol 24 (1) ◽  
pp. 377-388 ◽  
Author(s):  
Ning Lei ◽  
Leslie L. Heckert
Keyword(s):  
Transient Transfection ◽  
Mobility Shift ◽  
Specific Expression ◽  
Dnase I Footprinting ◽  
Unknown Factor ◽  
Related Transcription Factor ◽  
Important Regulator ◽  
Functional Relevance ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT The doublesex and mab-3 related transcription factor 1 (Dmrt1) is a putative transcriptional regulator that is expressed exclusively in the gonads and is required for postnatal testis differentiation. Here we describe the transcriptional mechanisms regulating testis-specific expression of the Dmrt1 gene. Transient-transfection analysis identified a region of the promoter between kb −3.2 and −2.8 that is important for Sertoli cell-specific expression. DNase I footprinting revealed four sites of DNA-protein interaction within this region, three of which were prominent in primary Sertoli cells. Analysis of these sites, using electrophoretic mobility shift assays, revealed that Gata4 and another unknown factor bound within these regions. Further transient-transfection assays of various mutant promoters established the functional relevance of the Gata4-response and unknown factor-response elements, while studies of Dmrt1 expression in 13.5 days postcoitum Fog2 null gonads supported the in vivo importance of Gata4's regulation. As a whole, these studies identify Gata4 as an important regulator in the Dmrt1 transcriptional machinery that is responsible for robust expression of Dmrt1 in the testis.

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

Microbiology ◽  
2011 ◽  
Vol 157 (6) ◽  
pp. 1685-1693 ◽  
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.

scholarly journals Four VirB6 Paralogs and VirB9 Are Expressed and Interact in Ehrlichia chaffeensis-Containing Vacuoles

2008 ◽  
Vol 191 (1) ◽  
pp. 278-286 ◽  
Author(s):  
Weichao Bao ◽  
Yumi Kumagai ◽  
Hua Niu ◽  
Mamoru Yamaguchi ◽  
Koshiro Miura ◽  
...  
Keyword(s):  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Human Leukemia ◽  
Ehrlichia Chaffeensis ◽  
Tick Cell ◽  
Type Iv ◽  
Important Virulence Factor ◽  
Human Monocytic Ehrlichiosis ◽  
First Time

ABSTRACT The type IV secretion system is an important virulence factor in several host cell-associated pathogens, as it delivers various bacterial macromolecules to target eukaryotic cells. Genes homologous to several virB genes and virD4 of Agrobacterium tumefaciens are found in an intravacuolar pathogen Ehrlichia chaffeensis, the tick-borne causative agent of human monocytic ehrlichiosis. In particular, despite its small genome size, E. chaffeensis has four tandem virB6 paralogs (virB6-1, -2, -3, and -4) that are 3- to 10-fold larger than A. tumefaciens virB6. The present study for the first time illustrates the relevance of the larger quadruple VirB6 paralogs by demonstrating the protein expression and interaction in E. chaffeensis. All four virB6 paralogs were cotranscribed in THP-1 human leukemia and ISE6 tick cell cultures. The four VirB6 proteins and VirB9 were expressed by E. chaffeensis in THP-1 cells, and amounts of these five proteins were similar in isolated E. chaffeensis-containing vacuoles and vacuole-free E. chaffeensis. In addition, an 80-kDa fragment of VirB6-2 was detected, which was strikingly more prevalent in E. chaffeensis-containing vacuoles than in vacuole-free E. chaffeensis. Coimmunoprecipitation analysis revealed VirB9 interaction with VirB6-1 and VirB6-2; VirB6-4 interaction with VirB6-1, VirB6-2, and VirB6-3; and VirB6-2 80-kDa fragment interaction with VirB6-3 and VirB6-4. The interaction of VirB9 and VirB6-2 was confirmed by far-Western blotting. The results suggest that E. chaffeensis VirB9, the quadruple VirB6 proteins, and the VirB6-2 80-kDa fragment form a unique molecular subassembly to cooperate in type IV secretion.

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

2005 ◽  
Vol 388 (3) ◽  
pp. 959-966 ◽  
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.

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

2017 ◽  
Vol 61 (5) ◽  
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.

scholarly journals Positive and negative transcriptional elements of the human type IV collagenase gene.

1990 ◽  
Vol 10 (12) ◽  
pp. 6524-6532 ◽  
Author(s):  
S M Frisch ◽  
J H Morisaki
Keyword(s):  
Core Promoter ◽  
Type Iv Collagenase ◽  
Mobility Shift ◽  
Specific Expression ◽  
Enhancer Element ◽  
Type Iv ◽  
Dnase I Footprinting ◽  
Gel Mobility Shift ◽  
Transcriptional Elements ◽  
Mcf 7

Proteolysis by type IV collagenase (T4) has been implicated in the process of tumor metastasis. The T4 gene is expressed in fibroblasts, but not in normal epithelial cells, and its expression is specifically repressed by the E1A oncogene of adenovirus. We present an investigation of the transcriptional elements responsible for basal, E1A-repressible, and tissue-specific expression. 5'-Deletion analysis, DNase I footprinting, and gel mobility shift assays revealed a strong, E1A-repressible enhancer element, r2, located about 1,650 bp upstream of the start site. This enhancer bound a protein with binding specificity very similar to that of the transcription factor AP-2. A potent silencer sequence was found 2 to 5 bp downstream of this enhancer. The silencer repressed transcription from either r2 or AP-1 enhancer elements and in the context of either type IV collagenase or thymidine kinase (tk) gene core promoters; enhancerless transcription from the latter core promoter was also repressed. Comprising the silencer were two contiguous, autonomously functioning silencer elements. Negative regulation of T4 transcription by at least two factors was demonstrated. mcf-7 proteins specifically binding both elements were detected by gel mobility shift assays; a protein of approximately 185 kDa that bound to one of these elements was detected by DNA-protein cross-linking. The silencer repressed transcription, in an r2 enhancer-tk promoter context, much more efficiently in T4-nonproducing cells (mcf-7 or HeLa) than in T4-producing cells (HT1080), suggesting that cell type-specific silencing may contribute to the regulation of this gene.

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