scholarly journals The Pseudomonas aeruginosa Ribbon-Helix-Helix DNA-Binding Protein AlgZ (AmrZ) Controls Twitching Motility and Biogenesis of Type IV Pili

2006 ◽  
Vol 188 (1) ◽  
pp. 132-140 ◽  
Author(s):  
Patricia J. Baynham ◽  
Deborah M. Ramsey ◽  
Borys V. Gvozdyev ◽  
Ellen M. Cordonnier ◽  
Daniel J. Wozniak
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Dna Binding ◽  
Dna Binding Protein ◽  
Binding Activity ◽  
Type Iv Pili ◽  
Twitching Motility ◽  
Whole Cell ◽  
Dna Binding Activity ◽  
Type Iv ◽  
Cell Extracts

ABSTRACT Pseudomonas aeruginosa is an opportunistic pathogen that is commonly found in water and soil. In order to colonize surfaces with low water content, P. aeruginosa utilizes a flagellum-independent form of locomotion called twitching motility, which is dependent upon the extension and retraction of type IV pili. This study demonstrates that AlgZ, previously identified as a DNA-binding protein absolutely required for transcription of the alginate biosynthetic operon, is required for twitching motility. AlgZ may be required for the biogenesis or function of type IV pili in twitching motility. Transmission electron microscopy analysis of an algZ deletion in nonmucoid PAO1 failed to detect surface pili. To examine expression and localization of PilA (the major pilin subunit), whole-cell extracts and cell surface pilin preparations were analyzed by Western blotting. While the PilA levels present in whole-cell extracts were similar for wild-type P. aeruginosa and P. aeruginosa with the algZ deletion, the amount of PilA on the surface of the cells was drastically reduced in the algZ mutant. Analysis of algZ and algD mutants indicates that the DNA-binding activity of AlgZ is essential for the regulation of twitching motility and that this is independent of the role of AlgZ in alginate expression. These data show that AlgZ DNA-binding activity is required for twitching motility independently of its role in alginate production and that this involves the surface localization of type IV pili. Given this new role in twitching motility, we propose that algZ (PA3385) be designated amrZ (alginate and motility regulator Z).

scholarly journals A simple method to measure CLOCK-BMAL1 DNA binding activity in tissue and cell extracts

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 1316 ◽  
Author(s):  
Maud Gillessen ◽  
Pieter Bas Kwak ◽  
Alfred Tamayo
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Casein Kinase I ◽  
Simple Method ◽  
Dna Binding Activity ◽  
Tissue Extracts ◽  
Cell Extracts ◽  
Specialized Equipment ◽  
Dna Binding Assay

The proteins CLOCK and BMAL1 form a heterodimeric transcription factor essential to circadian rhythms in mammals.  Daily rhythms of CLOCK-BMAL1 DNA binding activity are known to oscillate with target gene expression in vivo. Here we present a highly sensitive assay that recapitulates native CLOCK-BMAL1 DNA binding rhythms from crude tissue extracts, which we call the Clock Protein-DNA Binding Assay (CPDBA). This method can detect less than 2-fold differences in DNA binding activity, and can deliver results in two hours or less using 10 microliters or less of crude extract, while requiring neither specialized equipment nor expensive probes. To demonstrate the sensitivity and versatility of this assay, we show that enzymatic removal of phosphate groups from proteins in tissue extracts or pharmacological inhibition of casein kinase I in cell culture increased CLOCK-BMAL1 DNA binding activity by ~1.5 to ~2 fold, as measured by the CPDBA. In addition, we show that the CPDBA can measure CLOCK-BMAL1 binding to reconstituted chromatin. The CPDBA is a sensitive, fast, efficient and versatile probe of clock function.

scholarly journals Redox regulation of DNA binding activity of DREF (DNA replication-related element binding factor) in Drosophila

2004 ◽  
Vol 378 (3) ◽  
pp. 833-838 ◽  
Author(s):  
Tae-Yeong CHOI ◽  
S. Young PARK ◽  
Ho-Sung KANG ◽  
Jae-Hun CHEONG ◽  
Han-Do KIM ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Redox State ◽  
Redox Regulation ◽  
Binding Activity ◽  
Dna Binding Activity ◽  
Cell Extracts ◽  
Binding Factor ◽  
Related Element ◽  
Intracellular Redox

DREF [DRE (DNA replication-related element) binding factor] is an 80 kDa polypeptide homodimer which plays an important role in regulating cell proliferation-related genes. Both DNA binding and dimer formation activities are associated with residues 16–115 of the N-terminal region. However, the mechanisms by which DREF dimerization and DNA binding are regulated remain unknown. Here, we report that the DNA binding activity of DREF is regulated by a redox mechanism, and that the cysteine residues are involved in this regulation. Electrophoretic mobility shift analysis using Drosophila Kc cell extracts or recombinant DREF proteins indicated that the DNA binding domain is sufficient for redox regulation. Site-directed mutagenesis and transient transfection assays showed that Cys59 and/or Cys62 are critical both for DNA binding and for redox regulation, whereas Cys91 is dispensable. In addition, experiments using Kc cells indicated that the DNA binding activity and function of DREF are affected by the intracellular redox state. These findings give insight into the exact nature of DREF function in the regulation of target genes by the intracellular redox state.

scholarly journals Mucin inhibitsPseudomonas aeruginosabiofilm formation by significantly enhancing twitching motility

2014 ◽  
Vol 60 (3) ◽  
pp. 155-166 ◽  
Author(s):  
Cecily L. Haley ◽  
Cassandra Kruczek ◽  
Uzma Qaisar ◽  
Jane A. Colmer-Hamood ◽  
Abdul N. Hamood
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Iv Pili ◽  
Dependent Manner ◽  
Twitching Motility ◽  
Strain Pao1 ◽  
Concentration Dependent Manner ◽  
Dependent Effect ◽  
Type Iv ◽  
Biofilm Development

In Pseudomonas aeruginosa, type IV pili (TFP)-dependent twitching motility is required for development of surface-attached biofilm (SABF), yet excessive twitching motility is detrimental once SABF is established. In this study, we show that mucin significantly enhanced twitching motility and decreased SABF formation in strain PAO1 and other P. aeruginosa strains in a concentration-dependent manner. Mucin also disrupted partially established SABF. Our analyses revealed that mucin increased the amount of surface pilin and enhanced transcription of the pilin structural gene pilA. Mucin failed to enhance twitching motility in P. aeruginosa mutants defective in genes within the pilin biogenesis operons pilGHI/pilJK-chpA-E. Furthermore, mucin did not enhance twitching motility nor reduce biofilm development by chelating iron. We also examined the role of the virulence factor regulator Vfr in the effect of mucin. In the presence or absence of mucin, PAOΔvfr produced a significantly reduced SABF. However, mucin partially complemented the twitching motility defect of PAOΔvfr. These results suggest that mucin interferes with SABF formation at specific concentrations by enhancing TFP synthesis and twitching motility, that this effect, which is iron-independent, requires functional Vfr, and only part of the Vfr-dependent effect of mucin on SABF development occurs through twitching motility.

scholarly journals DNA Binding: a Novel Function of Pseudomonas aeruginosa Type IV Pili

2005 ◽  
Vol 187 (4) ◽  
pp. 1455-1464 ◽  
Author(s):  
Erin J. van Schaik ◽  
Carmen L. Giltner ◽  
Gerald F. Audette ◽  
David W. Keizer ◽  
Daisy L. Bautista ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Dna Binding ◽  
Biofilm Formation ◽  
Quaternary Structure ◽  
Natural Transformation ◽  
Ex Vivo ◽  
Specific Binding ◽  
Type Iv Pili ◽  
Type Iv ◽  
Bacteriophage Infection

ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa produces multifunctional, polar, filamentous appendages termed type IV pili. Type IV pili are involved in colonization during infection, twitching motility, biofilm formation, bacteriophage infection, and natural transformation. Electrostatic surface analysis of modeled pilus fibers generated from P. aeruginosa strain PAK, K122-4, and KB-7 pilin monomers suggested that a solvent-exposed band of positive charge may be a common feature of all type IV pili. Several functions of type IV pili, including natural transformation and biofilm formation, involve DNA. We investigated the ability of P. aeruginosa type IV pili to bind DNA. Purified PAK, K122-4, and KB-7 pili were observed to bind both bacterial plasmid and salmon sperm DNA in a concentration-dependent and saturable manner. PAK pili had the highest affinity for DNA, followed by K122-4 and KB-7 pili. DNA binding involved backbone interactions and preferential binding to pyrimidine residues even though there was no evidence of sequence-specific binding. Pilus-mediated DNA binding was a function of the intact pilus and thus required elements present in the quaternary structure. However, binding also involved the pilus tip as tip-specific, but not base-specific, antibodies inhibited DNA binding. The conservation of a Thr residue in all type IV pilin monomers examined to date, along with the electrostatic data, implies that DNA binding is a conserved function of type IV pili. Pilus-mediated DNA binding could be important for biofilm formation both in vivo during an infection and ex vivo on abiotic surfaces.

scholarly journals The retinoblastoma gene product RB stimulates Sp1-mediated transcription by liberating Sp1 from a negative regulator.

1994 ◽  
Vol 14 (7) ◽  
pp. 4380-4389 ◽  
Author(s):  
L I Chen ◽  
T Nishinaka ◽  
K Kwan ◽  
I Kitabayashi ◽  
K Yokoyama ◽  
...  
Keyword(s):  
Dna Binding ◽  
Gene Product ◽  
Binding Activity ◽  
Negative Regulator ◽  
Control Element ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Dna Binding Activity ◽  
Cell Extracts ◽  
Mobility Shift Assay

Studies have demonstrated that the retinoblastoma susceptibility gene product, RB, can either positively or negatively regulate expression of several genes through cis-acting elements in a cell-type-dependent manner. The nucleotide sequence of the retinoblastoma control element (RCE) motif, GCCACC or CCACCC, and the Sp1 consensus binding sequence, CCGCCC, can confer equal responsiveness to RB. Here, we report that RB activates transcription of the c-jun gene through the Sp1-binding site within the c-jun promoter. Preincubation of crude nuclear extracts with monoclonal antibodies to RB results in reduction of Sp1 complexes in a mobility shift assay, while addition of recombinant RB in mobility shift assay mixtures with CCL64 cell extracts leads to an enhancement of DNA-binding activity of SP1. These results suggest that RB is directly or indirectly involved in Sp1-DNA binding activity. A mechanism by which RB regulates transactivation is indicated by our detection of a heat-labile and protease-sensitive Sp1 negative regulator(s) (Sp1-I) that specifically inhibits Sp1 binding to a c-jun Sp1 site. This inhibition is reversed by addition of recombinant RB proteins, suggesting that RB stimulates Sp1-mediated transactivation by liberating Sp1 from Sp1-I. Additional evidence for Sp1-I involvement in Sp1-mediated transactivation was demonstrated by cotransfection of RB, GAL4-Sp1, and a GAL4-responsive template into CV-1 cells. Finally, we have identified Sp1-I, a approximately 20-kDa protein(s) that inhibits the Sp1 complexes from binding to DNA and that is also an RB-associated protein. These findings provide evidence for a functional link between two distinct classes of oncoproteins, RB and c-Jun, that are involved in the control of cell growth, and also define a novel mechanism for the regulation of c-jun expression.

scholarly journals Silencing of the inhibitor of DNA binding protein 4 (ID4) contributes to the pathogenesis of mouse and human CLL

Blood ◽  
2011 ◽  
Vol 117 (3) ◽  
pp. 862-871 ◽  
Author(s):  
Shih-Shih Chen ◽  
Rainer Claus ◽  
David M. Lucas ◽  
Lianbo Yu ◽  
Jiang Qian ◽  
...  
Keyword(s):  
Dna Binding ◽  
Promoter Methylation ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Binding Activity ◽  
Lymphocytic Leukemia ◽  
Dominant Negative ◽  
Helix Loop Helix ◽  
Dna Binding Activity ◽  
Inhibitor Of Dna Binding

Abstract Inhibitor of DNA binding protein 4 (ID4) is a member of the dominant-negative basic helix-loop-helix transcription factor family that lacks DNA binding activity and has tumor suppressor function. ID4 promoter methylation has been reported in acute myeloid leukemia and chronic lymphocytic leukemia (CLL), although the expression, function, and clinical relevance of this gene have not been characterized in either disease. We demonstrate that the promoter of ID4 is consistently methylated to various degrees in CLL cells, and increased promoter methylation in a univariable analysis correlates with shortened patient survival. However, ID4 mRNA and protein expression is uniformly silenced in CLL cells irrespective of the degree of promoter methylation. The crossing of ID4+/− mice with Eμ-TCL1 mice triggers a more aggressive murine CLL as measured by lymphocyte count and inferior survival. Hemizygous loss of ID4 in nontransformed TCL1-positive B cells enhances cell proliferation triggered by CpG oligonucleotides and decreases sensitivity to dexamethasone-mediated apoptosis. Collectively, this study confirms the importance of the silencing of ID4 in murine and human CLL pathogenesis.

scholarly journals cpc-1, the general regulatory gene for genes of amino acid biosynthesis in Neurospora crassa, is differentially expressed during the asexual life cycle

1991 ◽  
Vol 11 (2) ◽  
pp. 928-934 ◽  
Author(s):  
D J Ebbole ◽  
J L Paluh ◽  
M Plamann ◽  
M S Sachs ◽  
C Yanofsky
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Neurospora Crassa ◽  
Regulatory Gene ◽  
Regulatory Protein ◽  
Binding Activity ◽  
Recognition Sequence ◽  
Dna Binding Activity ◽  
Cell Extracts

CPCI, the principal regulatory protein required for cross-pathway control of amino acid biosynthetic genes in Neurospora crassa, contains a domain similar to the DNA-binding domain of GCN4, the corresponding general regulator in Saccharomyces cerevisiae. We examined binding by CPC1 synthesized in vitro and by CPC1 present in N. crassa whole-cell extracts. CPCI from both sources was shown to bind to the DNA sequence 5'-ATGACTCAT-3', which is also the preferred recognition sequence of GCN4, CPC1 was confirmed as the source of DNA-binding activity in extracts by immunoblotting. Slightly mobility differences between DNA complexes containing CPCI synthesized in vitro and CPC1 in mycelial extracts were observed. Analyses of N. crassa extracts from different stages of asexual development revealed that CPC1 was abundant immediately following spore germination and through early mycelial growth but was scarce subsequently. CPC1 levels could be increased at any time by imposing amino acid starvation. Copies of the CPC1 response element are located upstream of several genes regulated by cross-pathway control, including cpc-1 itself.

The CUP2 gene product, regulator of yeast metallothionein expression, is a copper-activated DNA-binding protein

1989 ◽  
Vol 9 (9) ◽  
pp. 4091-4095 ◽  
Author(s):  
C Buchman ◽  
P Skroch ◽  
J Welch ◽  
S Fogel ◽  
M Karin
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Regulatory Gene ◽  
Point Mutations ◽  
Dna Binding Protein ◽  
Binding Activity ◽  
Dna Binding Domain ◽  
Yeast Saccharomyces Cerevisiae ◽  
Dna Binding Activity ◽  
Yeast Strains

CUP2 is a regulatory gene controlling expression of CUP1, which encodes the Cu-binding yeast metallothionein. CUP2, which is identical to the ACE1 gene, encodes a Cu-regulated DNA-binding protein. The CUP2 protein contains a cysteine-rich DNA-binding domain dependent on Cu+ and Ag+ ions which bind the cysteine residues and direct the refolding of the metal-free apoprotein. CUP2 mutant alleles from Cu-sensitive yeast strains have point mutations affecting the DNA-binding activity. These results establish CUP2 as the primary sensor of intracellular Cu+ in the yeast Saccharomyces cerevisiae, functioning as a Cu+-regulated transcriptional activator.

Characterization of the mouse metal-regulatory-element-binding proteins, metal element protein-1 and metal regulatory transcription factor-1

2001 ◽  
Vol 353 (3) ◽  
pp. 591-601 ◽  
Author(s):  
Olivier LAROCHELLE ◽  
Gale STEWART ◽  
Pierre MOFFATT ◽  
Véronique TREMBLAY ◽  
Carl SÉGUIN
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Binding Activity ◽  
Dependent Manner ◽  
Rnase Protection ◽  
Dna Binding Activity ◽  
Cell Extracts ◽  
Metal Element ◽  
Transcription Factor 1

Metal activation of metallothionein gene transcription depends mainly on the presence of regulatory DNA sequences termed metal-regulatory elements (MREs) and involves MRE-binding transcription factor-1 (MTF-1) interacting with the MREs in a Zn2+-dependent manner. We previously identified and characterized a nuclear protein, termed metal element protein-1 (MEP-1), specifically binding with high affinity to MRE elements. The precise relationship between MTF-1 and MEP-1 was unclear, and to determine whether MEP-1 and MTF-1 were distinct protein species, we performed DNA binding analyses to characterize the binding properties of both proteins. Electrophoretic mobility-shift assays showed that MTF-1, produced in COS cells, produces a slower-migrating band compared with that obtained with purified MEP-1. Using an anti-MTF-1 antibody, we showed that both the MTF-1–MRE and the MEP-1–MRE complexes are supershifted by an anti-MTF-1 antibody, thus demonstrating that MEP-1 is antigenically related to MTF-1. RNase protection analyses carried out with RNA prepared from different tissues and cell lines failed to reveal the presence of MTF-1 splicing variants. This indicates that MEP-1 may be a proteolytic fragment of MTF-1. MTF-1 DNA-binding activity was rapidly activated in vivo by Zn2+ ions but not by Cd2+, UV irradiation or PMA, and occurred on ice as well as at 21°C. In control and Zn2+-treated cell extracts, DNA-binding activity was not enhanced in vitro following the addition of exogenous Zn2+ or a preincubation at 37°C. However, recombinant MTF-1 produced in vitro required Zn2+ activation for DNA binding. Interestingly, treatment of nuclear extracts with calf intestine phosphatase completely abrogated MTF-1 DNA-binding activity, thus suggesting that phosphorylation is involved in the regulation of MTF-1 activity.

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