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 The Sequence-specific DNA Binding of NF-κB Is Reversibly Regulated by the Automodification Reaction of Poly (ADP-ribose) Polymerase 1

2001 ◽  
Vol 276 (50) ◽  
pp. 47664-47670 ◽  
Author(s):  
Woo-Jin Chang ◽  
Rafael Alvarez-Gonzalez
Keyword(s):  
Dna Binding ◽  
Hela Cells ◽  
Specific Binding ◽  
High Specificity ◽  
Immunoblot Analysis ◽  
Mobility Shift ◽  
Electrophoretic Mobility Shift Assays ◽  
Parp 1

Recent studies suggest that the synthesis of protein-bound ADP-ribose polymers catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1) regulates eucaryotic gene expression, including the NF-κB-dependent pathway. Here, we report the molecular mechanism by which PARP-1 activates the sequence-specific binding of NF-κB to its oligodeoxynucleotide. We co-incubated pure recombinant human PARP-1 and the p50 subunit of NF-κB (NF-κB-p50) in the presence or absence of βNAD+in vitro.Electrophoretic mobility shift assays showed that, when PARP-1 was present, NF-κB-p50 DNA binding was dependent on the presence of βNAD+. DNA binding by NF-κB-p50 was not efficient in the absence of βNAD+. In fact, the binding was not efficient in the presence of 3-aminobenzamide (3-AB) either. Thus, we conclude that NF-κB-p50 DNA binding is protein-poly(ADP-ribosyl)ation dependent. Co-immunoprecipitation and immunoblot analysis revealed that PARP-1 physically interacts with NF-κB-p50 with high specificity in the absence of βNAD+. Because NF-kB-p50 was not an efficient covalent target for poly(ADP-ribosyl)ation, our results are consistent with the conclusion that the auto-poly(ADP-ribosyl)ation reaction catalyzed by PARP-1 facilitates the binding of NF-κB-p50 to its DNA by inhibiting the specific protein·protein interactions between NF-κB-p50 and PARP-1. We also report the activation of NF-κB DNA binding by the automodification reaction of PARP-1 in cultured HeLa cells following exposure to H2O2. In these experiments, preincubation of HeLa cells with 3-AB, prior to oxidative damage, strongly inhibited NF-κB activationin vivoas well.

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 The ClgR Protein Regulates Transcription of the clpP Operon in Bifidobacterium breve UCC 2003

2005 ◽  
Vol 187 (24) ◽  
pp. 8411-8426 ◽  
Author(s):  
Marco Ventura ◽  
Ziding Zhang ◽  
Michelle Cronin ◽  
Carlos Canchaya ◽  
John G. Kenny ◽  
...  
Keyword(s):  
Promoter Region ◽  
Specific Binding ◽  
Three Dimensional ◽  
Binding Mode ◽  
Dna Interaction ◽  
Dimensional Structure ◽  
Mobility Shift ◽  
Significant Similarity ◽  
Bifidobacterium Breve

ABSTRACT Five clp genes (clpC, clpB, clpP1, clpP2, and clpX), representing chaperone- and protease-encoding genes, were previously identified in Bifidobacterium breve UCC 2003. In the present study, we characterize the B. breve UCC 2003 clpP locus, which consists of two paralogous genes, designated clpP1 and clpP2, whose deduced protein products display significant similarity to characterized ClpP peptidases. Transcriptional analyses showed that the clpP1 and clpP2 genes are transcribed in response to moderate heat shock as a bicistronic unit with a single promoter. The role of a clgR homologue, known to control the regulation of clpP gene expression in Streptomyces lividans and Corynebacterium glutamicum, was investigated by gel mobility shift assays and DNase I footprint experiments. We show that ClgR, which in its purified form appears to exist as a dimer, requires a proteinaceous cofactor to assist in specific binding to a 30-bp region of the clpP promoter region. In pull-down experiments, a 56-kDa protein copurified with ClgR, providing evidence that the two proteins also interact in vivo and that the copurified protein represents the cofactor required for ClgR activity. The prediction of the ClgR three-dimensional structure provides further insights into the binding mode of this protein to the clpP1 promoter region and highlights the key amino acid residues believed to be involved in the protein-DNA interaction.

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 The Bacteroides fragilis BtgA Mobilization Protein Binds to the oriT Region of pBFTM10

1998 ◽  
Vol 180 (18) ◽  
pp. 4922-4928 ◽  
Author(s):  
Leonid A. Sitailo ◽  
Alexander M. Zagariya ◽  
Patrick J. Arnold ◽  
Gayatri Vedantam ◽  
David W. Hecht
Keyword(s):  
Specific Binding ◽  
Bacteroides Fragilis ◽  
Binding Activity ◽  
Inverted Repeats ◽  
Mobility Shift ◽  
E Coli ◽  
Dna Binding Activity ◽  
Footprint Analysis ◽  
Nick Site ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT The Bacteroides fragilis conjugal plasmid pBFTM10 contains two genes, btgA and btgB, and a putative oriT region necessary for transfer inBacteroides fragilis and Escherichia coli. The BtgA protein was predicted to contain a helix-turn-helix motif, indicating possible DNA binding activity. DNA sequence analysis of the region immediately upstream of btgA revealed three sets of inverted repeats, potentially locating the oriTregion. A 304-bp DNA fragment comprising this putative oriT region was cloned and confirmed to be the functional pBFTM10 oriT by bacterial conjugation experiments using E. coli and B. fragilis. btgAwas cloned and overexpressed in E. coli, and the purified protein was used in electrophoretic mobility shift assays, demonstrating specific binding of BtgA protein to its cognateoriT. DNase I footprint analysis demonstrated that BtgA binds apparently in a single-stranded fashion to theoriT-containing fragment, overlapping inverted repeats I, II, and III and the putative nick site.

scholarly journals Characterization of the Opposing Roles of H-NS and TraJ in Transcriptional Regulation of the F-Plasmid tra Operon

2006 ◽  
Vol 188 (2) ◽  
pp. 507-514 ◽  
Author(s):  
William R. Will ◽  
Laura S. Frost
Keyword(s):  
Escherichia Coli ◽  
Promoter Region ◽  
Regulatory Protein ◽  
Plasmid Transfer ◽  
Wild Type ◽  
F Plasmid ◽  
Transcriptional Fusion ◽  
Mobility Shift ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT The transfer (tra) operon of the conjugative F plasmid of Escherichia coli is a polycistronic 33-kb operon which encodes most of the proteins necessary for F-plasmid transfer. Here, we report that transcription from PY, the tra operon promoter, is repressed by the host nucleoid-associated protein, H-NS. Electrophoretic mobility shift assays indicate that H-NS binds preferentially to the tra promoter region, while Northern blot and transcriptional fusion analyses indicate that transcription of traY, the first gene in the tra operon, is derepressed in an hns mutant throughout growth. The plasmid-encoded regulatory protein TraJ is essential for transcription of the tra operon in wild-type Escherichia coli; however, TraJ is not necessary for plasmid transfer or traY operon transcription in an hns mutant. This indicates that H-NS represses transcription from PY directly and not indirectly via its effects on TraJ levels. These results suggest that TraJ functions to disrupt H-NS silencing at PY, allowing transcription of the tra operon.

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 Autoregulation of hpdR and its effect on CDA biosynthesis in Streptomyces coelicolor

Microbiology ◽  
2010 ◽  
Vol 156 (9) ◽  
pp. 2641-2648 ◽  
Author(s):  
Haihua Yang ◽  
Yang An ◽  
Linqi Wang ◽  
Shuli Zhang ◽  
Yue Zhang ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Promoter Region ◽  
Streptomyces Coelicolor ◽  
Binding Activity ◽  
Catabolic Gene ◽  
Mobility Shift ◽  
S1 Nuclease ◽  
Dna Binding Activity ◽  
Electrophoretic Mobility Shift Assays ◽  
Positive Effect

HpdR, an IclR-family regulator in Streptomyces coelicolor, is a substrate-dependent repressor for the tyrosine catabolic gene hppD. In this study, S1 nuclease protection assays revealed that hpdR is subject to a negative autoregulation. Purified HpdR showed specific DNA-binding activity for the promoter region of hpdR, indicating that the autoregulation of hpdR is performed directly. The disruption of hpdR led to reduced production of CDA by S. coelicolor J1501, suggesting a positive effect of hpdR on CDA biosynthesis. Electrophoretic mobility shift assays showed that HpdR specifically bound to the promoter region of hmaS (SCO3229 in the CDA gene cluster), encoding 4-hydroxymandelic acid synthase. Disruption of hmaS in J1501 abolished CDA production. It is possible that hpdR regulates CDA biosynthesis by controlling the transcription of hmaS.

scholarly journals An AbrB-Like Protein Regulates the Expression of the Bidirectional Hydrogenase in Synechocystis sp. Strain PCC 6803

2007 ◽  
Vol 190 (3) ◽  
pp. 1011-1019 ◽  
Author(s):  
Paulo Oliveira ◽  
Peter Lindblad
Keyword(s):  
Transcription Factor ◽  
Promoter Region ◽  
Hydrogen Metabolism ◽  
Mobility Shift ◽  
Unicellular Cyanobacterium ◽  
Show Evidence ◽  
State Regulator ◽  
Transition State Regulator ◽  
Electrophoretic Mobility Shift Assays ◽  
Pcc 6803

ABSTRACT In the unicellular cyanobacterium Synechocystis sp. strain PCC 6803, the pentameric bidirectional Ni-Fe hydrogenase (HoxEFUYH) is the sole enzyme involved in hydrogen metabolism. Recent investigations implicated the transcription factor LexA in the regulation of the hox genes in this cyanobacterium, suggesting the factor to work as an activator. In this work, we show evidence that LexA cannot account exclusively for the regulation of the hox genes in this cyanobacterium. Therefore, we investigated which additional transcription factors interact in and may regulate the expression of the hox genes in Synechocystis sp. strain PCC 6803. By using DNA affinity assays, a transcription factor with similarity to the transition state regulator AbrB from Bacillus subtilis was isolated. Electrophoretic mobility shift assays showed that the AbrB-like protein specifically interacts with the promoter region of the hox genes as well as with its own promoter region. In addition, results obtained with two genetically modified strains of Synechocystis sp. strain PCC 6803, one with a not fully segregated inactivation mutation of the abrB-like gene and the other overexpressing the same abrB-like gene, suggest that this transcription factor functions as a regulator of hox gene expression.

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.

Sign in / Sign up

Export Citation Format

Share Document