scholarly journals Transition State Regulator AbrB Inhibits Transcription of Bacillus amyloliquefaciens FZB45 Phytase through Binding at Two Distinct Sites Located within the Extended phyC Promoter Region

2008 ◽  
Vol 190 (19) ◽  
pp. 6467-6474 ◽  
Author(s):  
Oliwia Makarewicz ◽  
Svetlana Neubauer ◽  
Corinna Preusse ◽  
Rainer Borriss
Keyword(s):  
Bacillus Amyloliquefaciens ◽  
Promoter Region ◽  
Response Regulator ◽  
Dependent Manner ◽  
Coding Region ◽  
Mobility Shift ◽  
Dnase I Footprinting ◽  
Regulator Protein ◽  
Transition State Regulator ◽  
Gel Mobility Shift

ABSTRACT We have previously identified the phyC gene of Bacillus amyloliquefaciens FZB45, encoding extracellular phytase, as a member of the PhoP regulon, which is expressed only during phosphate starvation. Its σA-dependent promoter is positively and negatively regulated by the phosphorylated PhoP response regulator in a phosphate-dependent manner (O. Makarewicz, S. Dubrac, T. Msadek, and R. Borriss, J. Bacteriol. 188:6953-6965, 2006). Here, we provide experimental evidence that the transcription of phyC underlies a second control mechanism exerted by the global transient-phase regulator protein, AbrB, which hinders its expression during exponential growth. Gel mobility shift and DNase I footprinting experiments demonstrated that AbrB binds to two different regions in the phyC promoter region that are separated by about 200 bp. One binding site is near the divergently orientated yodU gene, and the second site is located downstream of the phyC promoter and extends into the coding region of the phyC gene. Cooperative binding to the two distant binding regions is necessary for the AbrB-directed repression of phyC transcription. AbrB does not affect the transcription of the neighboring yodU gene.

scholarly journals ScoC Regulates Bacilysin Production at the Transcription Level in Bacillus subtilis

2009 ◽  
Vol 191 (23) ◽  
pp. 7367-7371 ◽  
Author(s):  
Takashi Inaoka ◽  
Guojun Wang ◽  
Kozo Ochi
Keyword(s):  
Bacillus Subtilis ◽  
Genetic Engineering ◽  
Genome Sequencing ◽  
Promoter Region ◽  
Sequencing Analysis ◽  
Transcription Level ◽  
Mobility Shift ◽  
State Regulator ◽  
Transition State Regulator ◽  
Gel Mobility Shift

ABSTRACT Bacillus subtilis mutants with high expression of the bacilysin operon ywfBCDEFG were isolated. Comparative genome sequencing analysis revealed that all of these mutants have a mutation in the scoC gene. The disruption of scoC by genetic engineering also resulted in increased expression of ywfBCDEFG. Primer extension and gel mobility shift analyses showed that the ScoC protein binds directly to the promoter region of ywfBCDEFG. Our results indicate that the transition state regulator ScoC, together with CodY and AbrB, negatively regulates bacilysin production in B. subtilis.

scholarly journals The VirR Response Regulator from Clostridium perfringens Binds Independently to Two Imperfect Direct Repeats Located Upstream of the pfoA Promoter

2000 ◽  
Vol 182 (1) ◽  
pp. 57-66 ◽  
Author(s):  
Jackie K. Cheung ◽  
Julian I. Rood
Keyword(s):  
Binding Site ◽  
Clostridium Perfringens ◽  
Response Regulator ◽  
Toxin Production ◽  
Site Directed Mutagenesis ◽  
Direct Repeats ◽  
Mobility Shift ◽  
Perfringolysin O ◽  
Dnase I Footprinting ◽  
Gel Mobility Shift

ABSTRACT Regulation of toxin production in the gram-positive anaerobeClostridium perfringens occurs at the level of transcription and involves a two-component signal transduction system. The sensor histidine kinase is encoded by the virS gene, while its cognate response regulator is encoded by the virRgene. We have constructed a VirR expression plasmid inEscherichia coli and purified the resultant His-tagged VirR protein. Gel mobility shift assays demonstrated that VirR binds to the region upstream of the pfoA gene, which encodes perfringolysin O, but not to regions located upstream of the VirR-regulated plc, colA, and pfoRgenes, which encode alpha-toxin, collagenase, and a putativepfoA regulator, respectively. The VirR binding site was shown by DNase I footprinting to be a 52-bp core sequence situated immediately upstream of the pfoA promoter. When this region was deleted, VirR was no longer able to bind to the pfoApromoter. The binding site was further localized to two imperfect direct repeats (CCCAGTTNTNCAC) by site-directed mutagenesis. Binding and protection analysis of these mutants indicated that VirR had the ability to bind independently to the two repeated sequences. Based on these observations it is postulated that the VirR positively regulates the synthesis of perfringolysin O by binding directly to a region located immediately upstream of the pfoA promoter and activating transcription.

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 Expression of nodD1 and nodD2 in Sinorhizobium fredii, a Nitrogen-Fixing Symbiont of Soybean and Other Legumes

1998 ◽  
Vol 11 (5) ◽  
pp. 375-382 ◽  
Author(s):  
Daphne Machado ◽  
Steven G. Pueppke ◽  
José Maria Vinardel ◽  
José E. Ruiz-Sainz ◽  
Hari B. Krishnan
Keyword(s):  
Protein Binding ◽  
Dna Sequences ◽  
Root Nodules ◽  
Nitrogen Fixing ◽  
Coding Region ◽  
Mobility Shift ◽  
Sinorhizobium Fredii ◽  
Dnase I Footprinting ◽  
Gel Mobility Shift ◽  
Dna Complex

The ability of Sinorhizobium fredii strains USDA191 and USDA257 to form nitrogen-fixing root nodules on legume plants is regulated by nodD1 and nodD2, which sense flavonoid signals from host roots and then activate the expression of inducible nodulation genes. We assessed the interactions between these two loci with nodD1-negative and nodD2-negative mutants and with strains containing extra copies of these genes. Although both nodD1 and nodD2 are expressed constitutively, levels of nodD1 are much higher, as measured by RNA dot blots. Extra plasmid-borne copies of nodD2 reduced transcription of nodD1 to below the level of detection. We employed gel mobility shift assays to demonstrate that cellular proteins from flavonoid-treated cultures of strain USDA191 bind to DNA sequences that lie upstream from nodD1, but not to the corresponding region upstream from nodD2. Extra plasmid-borne copies of nodD2 enhanced protein binding to the nodD1-associated region and rendered the process flavonoid independent. DNase I footprinting analysis and gel retardation experiments with an oligonucleotide DNA probe localized the protein-binding site to a nod box-like sequence that lies just upstream from the nodD1 coding region. Antibodies raised against a NodD2 fusion protein and capable of reacting both with NodD1 and NodD2 blocked formation of the retarded protein-DNA complex.

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.

Multiple silencer elements are involved in regulating the chicken vimentin gene

1994 ◽  
Vol 14 (2) ◽  
pp. 934-943
Author(s):  
R J Garzon ◽  
Z E Zehner
Keyword(s):  
Intermediate Filament Protein ◽  
Mobility Shift ◽  
Specific Expression ◽  
Cis Acting ◽  
Dnase I Footprinting ◽  
Silencer Elements ◽  
Gel Mobility Shift ◽  
Filament Protein ◽  
Silencer Element ◽  
Vimentin Gene

Vimentin, a member of the intermediate filament protein family, exhibits tissue- as well as development-specific expression. Transcription factors that are involved in expression of the chicken vimentin gene have been described and include a cis-acting silencer element (SE3) that is involved in the down-regulation of this gene (F. X. Farrell, C. M. Sax, and Z. E. Zehner, Mol. Cell. Biol. 10:2349-2358, 1990). In this study, we report the identification of two additional silencer elements (SE1 and SE2). We show by transfection analysis that all three silencer elements are functionally active and that optimal silencing occurs when multiple (at least two) silencer elements are present. In addition, the previously identified SE3 can be divided into three subregions, each of which is moderately active alone. By gel mobility shift assays, all three silencer elements plus SE3 subregions bind a protein which by Southwestern (DNA-protein) blot analysis is identical in molecular mass (approximately 95 kDa). DNase I footprinting experiments indicate that this protein binds to purine-rich sites. Therefore, multiple elements appear to be involved in the negative regulation of the chicken vimentin gene, which may be important in the regulation of other genes as well.

Identification of a cell-type-specific transcriptional repressor in the promoter region of the mouse hepatocyte growth factor gene

1994 ◽  
Vol 14 (11) ◽  
pp. 7046-7058
Author(s):  
Y Liu ◽  
A B Beedle ◽  
L Lin ◽  
A W Bell ◽  
R Zarnegar
Keyword(s):  
Epithelial Cells ◽  
Promoter Region ◽  
Nuclear Protein ◽  
Transcriptional Repressor ◽  
Cell Type ◽  
Mobility Shift ◽  
A Cell ◽  
Cell Type Specific ◽  
Gel Mobility Shift ◽  
Hepatocyte Growth

Hepatocyte growth factor (HGF), a cytokine with multiple functions, exhibits cell-type-specific as well as cytokine- and steroid hormone-regulated expression. The HGF gene is known to be expressed predominately in mesenchymal but not in epithelial cells. In this study, we report the identification of a cell-type-specific transcriptional repressor in the promoter region of the mouse HGF gene, which is evidently responsible for the suppression of HGF expression in epithelial cells. Gel mobility shift assays and DNase I footprinting studies revealed that a 27-bp element (-16 to +11) around the transcription initiation site is responsible for the binding of a nuclear protein which is present in epithelial but not in mesenchymally derived cells. Further analysis of the binding activity of the DNA region with nuclear protein revealed that an approximately 19-bp sequence containing a unique palindromic structure (5'-AACCGACCGGTT-3') overlapped by a CAP box is essential for binding. Substitution of a single base (the contact site) within this region by site-directed mutagenesis resulted in total abrogation of the binding of the nuclear protein and a concomitant increase in the transcriptional activity of various lengths of HGF-chloramphenicol acetyltransferase fused genes when transfected into the epithelial cell line RL95-2 but not the mesenchymal cell line NIH 3T3. Southwestern (DNA-protein) analyses revealed that the nuclear protein which binds to this repressor element is a single polypeptide of approximately 70 kDa. Analysis of the nuclear extract prepared from regenerating mouse liver at various times after two-thirds partial hepatectomy by gel mobility shift assay revealed a substantial reduction (more than 75% within 3 h) in the binding of the repressor to its cognate binding site. Our results suggest that a cis-acting transcriptional repressor in the promoter region of the mouse HGF gene is involved in cell-type-specific regulation through binding to its cognate trans-acting protein which exists in epithelial cells but is absent in fibroblast cells.

The suppression of testis-brain RNA binding protein and kinesin heavy chain disrupts mRNA sorting in dendrites

1999 ◽  
Vol 112 (21) ◽  
pp. 3691-3702 ◽  
Author(s):  
W.L. Severt ◽  
T.U. Biber ◽  
X. Wu ◽  
N.B. Hecht ◽  
R.J. DeLorenzo ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Antisense Oligonucleotides ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Neuronal Remodeling ◽  
Kinesin Heavy Chain ◽  
Gel Mobility Shift

Ribonucleoprotein particles (RNPs) are thought to be key players in somato-dendritic sorting of mRNAs in CNS neurons and are implicated in activity-directed neuronal remodeling. Here, we use reporter constructs and gel mobility shift assays to show that the testis brain RNA-binding protein (TB-RBP) associates with mRNPs in a sequence (Y element) dependent manner. Using antisense oligonucleotides (anti-ODN), we demonstrate that blocking the TB-RBP Y element binding site disrupts and mis-localizes mRNPs containing (alpha)-calmodulin dependent kinase II (alpha)-CAMKII) and ligatin mRNAs. In addition, we show that suppression of kinesin heavy chain motor protein alters only the localization of (alpha)-CAMKII mRNA. Thus, differential sorting of mRNAs involves multiple mRNPs and selective motor proteins permitting localized mRNAs to utilize common mechanisms for shared steps.

scholarly journals HpdR Is a Transcriptional Activator of Sinorhizobium meliloti hpdA, Which Encodes a Herbicide-Targeted 4-Hydroxyphenylpyruvate Dioxygenase

2007 ◽  
Vol 189 (9) ◽  
pp. 3660-3664 ◽  
Author(s):  
Suvit Loprasert ◽  
Wirongrong Whangsuk ◽  
James M. Dubbs ◽  
Ratiboot Sallabhan ◽  
Kumpanart Somsongkul ◽  
...  
Keyword(s):  
Sinorhizobium Meliloti ◽  
Transcriptional Activator ◽  
Direct Repeat ◽  
Dnase I ◽  
Mobility Shift ◽  
Intergenic Space ◽  
Repeat Sequences ◽  
Dnase I Footprinting ◽  
Gel Mobility Shift

ABSTRACT Sinorhizobium meliloti hpdA, which encodes the herbicide target 4-hydroxyphenylpyruvate dioxygenase, is positively regulated by HpdR. Gel mobility shift and DNase I footprinting analyses revealed that HpdR binds to a region that spans two conserved direct-repeat sequences within the hpdR-hpdA intergenic space. HpdR-dependent hpdA transcription occurs in the presence of 4-hydroxyphenylpyruvate, tyrosine, and phenylalanine, as well as during starvation.

scholarly journals Identification of a cell-type-specific transcriptional repressor in the promoter region of the mouse hepatocyte growth factor gene.

1994 ◽  
Vol 14 (11) ◽  
pp. 7046-7058 ◽  
Author(s):  
Y Liu ◽  
A B Beedle ◽  
L Lin ◽  
A W Bell ◽  
R Zarnegar
Keyword(s):  
Epithelial Cells ◽  
Promoter Region ◽  
Nuclear Protein ◽  
Transcriptional Repressor ◽  
Cell Type ◽  
Mobility Shift ◽  
A Cell ◽  
Cell Type Specific ◽  
Gel Mobility Shift ◽  
Hepatocyte Growth

Hepatocyte growth factor (HGF), a cytokine with multiple functions, exhibits cell-type-specific as well as cytokine- and steroid hormone-regulated expression. The HGF gene is known to be expressed predominately in mesenchymal but not in epithelial cells. In this study, we report the identification of a cell-type-specific transcriptional repressor in the promoter region of the mouse HGF gene, which is evidently responsible for the suppression of HGF expression in epithelial cells. Gel mobility shift assays and DNase I footprinting studies revealed that a 27-bp element (-16 to +11) around the transcription initiation site is responsible for the binding of a nuclear protein which is present in epithelial but not in mesenchymally derived cells. Further analysis of the binding activity of the DNA region with nuclear protein revealed that an approximately 19-bp sequence containing a unique palindromic structure (5'-AACCGACCGGTT-3') overlapped by a CAP box is essential for binding. Substitution of a single base (the contact site) within this region by site-directed mutagenesis resulted in total abrogation of the binding of the nuclear protein and a concomitant increase in the transcriptional activity of various lengths of HGF-chloramphenicol acetyltransferase fused genes when transfected into the epithelial cell line RL95-2 but not the mesenchymal cell line NIH 3T3. Southwestern (DNA-protein) analyses revealed that the nuclear protein which binds to this repressor element is a single polypeptide of approximately 70 kDa. Analysis of the nuclear extract prepared from regenerating mouse liver at various times after two-thirds partial hepatectomy by gel mobility shift assay revealed a substantial reduction (more than 75% within 3 h) in the binding of the repressor to its cognate binding site. Our results suggest that a cis-acting transcriptional repressor in the promoter region of the mouse HGF gene is involved in cell-type-specific regulation through binding to its cognate trans-acting protein which exists in epithelial cells but is absent in fibroblast cells.

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