scholarly journals Fis negatively affects binding of Tn4652 transposase by out-competing IHF from the left end of Tn4652

Microbiology ◽  
2009 ◽  
Vol 155 (4) ◽  
pp. 1203-1214 ◽  
Author(s):  
Riho Teras ◽  
Julia Jakovleva ◽  
Maia Kivisaar
Keyword(s):  
Dnase I ◽  
Integration Host Factor ◽  
Mobile Dna ◽  
Mobility Shift ◽  
Transposase Gene ◽  
Transposition Activity ◽  
Global Regulators ◽  
Factor For Inversion Stimulation ◽  
Dna Elements ◽  
Gel Mobility Shift

Transposition activity in bacteria is generally maintained at a low level. The activity of mobile DNA elements can be controlled by bacterially encoded global regulators. Regulation of transposition of Tn4652 in Pseudomonas putida is one such example. Activation of transposition of Tn4652 in starving bacteria requires the stationary-phase sigma factor RpoS and integration host factor (IHF). IHF plays a dual role in Tn4652 translocation by activating transcription of the transposase gene tnpA of the transposon and facilitating TnpA binding to the inverted repeats of the transposon. Our previous results have indicated that besides IHF some other P. putida-encoded global regulator(s) might bind to the ends of Tn4652 and regulate transposition activity. In this study, employing a DNase I footprint assay we have identified a binding site of P. putida Fis (factor for inversion stimulation) centred 135 bp inside the left end of Tn4652. Our results of gel mobility shift and DNase I footprint studies revealed that Fis out-competes IHF from the left end of Tn4652, thereby abolishing the binding of TnpA. Thus, the results obtained in this study indicate that the transposition of Tn4652 is regulated by the cellular amount of P. putida global regulators Fis and IHF.

scholarly journals Transcription from Fusion Promoters Generated during Transposition of Transposon Tn4652 Is Positively Affected by Integration Host Factor in Pseudomonas putida

2000 ◽  
Vol 182 (3) ◽  
pp. 589-598 ◽  
Author(s):  
Riho Teras ◽  
Rita Hõrak ◽  
Maia Kivisaar
Keyword(s):  
Pseudomonas Putida ◽  
Specific Binding ◽  
Stationary Growth Phase ◽  
Integration Host Factor ◽  
Host Factor ◽  
Mobile Dna ◽  
Mobility Shift ◽  
Transposase Gene ◽  
Gel Mobility Shift

ABSTRACT We have previously shown that both ends of the Tn3family transposon Tn4652 contain integration host factor (IHF) binding sites and that IHF positively regulates expression of the Tn4652 transposase gene tnpA inPseudomonas putida (R. Hõrak, and M. Kivisaar, J. Bacteriol. 180:2822–2829, 1998). Tn4652 can activate silent genes by creating fusion promoters during the transposition. The promoters are created as fusions between the −35 hexamer provided by the terminal inverted repeats of Tn4652 and the −10 hexamers in the target DNA. Two fusion promoters, PRA1 and PLA1, that contain sequences of the right and left termini of Tn4652, respectively, were chosen for the study of mechanisms of transcription activation. Gel mobility shift analysis using crude extracts fromP. putida cells allowed us to detect specific binding ofP. putida IHF to the ends of the transposon Tn4652. We found that the rate of transcription from the fusion promoter PRA1 is enhanced by IHF. Notably, the positive effect of IHF on transcription from the promoter PRA1 appeared only when cells of P. putida reached the stationary growth phase. We speculate that the intracellular concentration of IHF might be critical for the in vivo effect of IHF on transcription from the fusion promoters in P. putida. In the case of PLA1, the mechanism of transcription modulation by IHF is different than that observed for PRA1. Our results demonstrate that transcription of neighboring genes from outwardly directed promoters at the ends of a mobile DNA element could be influenced by the same factors that control transposition of the element.

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 Integration Host Factor Positively Regulates Virulence Gene Expression in Vibrio cholerae

2008 ◽  
Vol 190 (13) ◽  
pp. 4736-4748 ◽  
Author(s):  
Emily Stonehouse ◽  
Gabriela Kovacikova ◽  
Ronald K. Taylor ◽  
Karen Skorupski
Keyword(s):  
Gene Expression ◽  
Vibrio Cholerae ◽  
Virulence Gene ◽  
Toxin Production ◽  
Dnase I ◽  
Integration Host Factor ◽  
Host Factor ◽  
Mobility Shift ◽  
Virulence Gene Expression ◽  
Dnase I Footprinting

ABSTRACT Virulence gene expression in Vibrio cholerae is dependent upon a complex transcriptional cascade that is influenced by both specific and global regulators in response to environmental stimuli. Here, we report that the global regulator integration host factor (IHF) positively affects virulence gene expression in V. cholerae. Inactivation of ihfA and ihfB, the genes encoding the IHF subunits, decreased the expression levels of the two main virulence factors tcpA and ctx and prevented toxin-coregulated pilus and cholera toxin production. IHF was found to directly bind to and bend the tcpA promoter region at an IHF consensus site centered at position −162 by using gel mobility shift assays and DNase I footprinting experiments. Deletion or mutation of the tcpA IHF consensus site resulted in the loss of IHF binding and additionally disrupted the binding of the repressor H-NS. DNase I footprinting revealed that H-NS protection overlaps with both the IHF and the ToxT binding sites at the tcpA promoter. In addition, disruption of ihfA in an hns or toxT mutant background had no effect on tcpA expression. These results suggest that IHF may function at the tcpA promoter to alleviate H-NS repression.

scholarly journals Regulation of Expression of theyiaKLMNOPQRS Operon for Carbohydrate Utilization inEscherichia coli: Involvement of the Main Transcriptional Factors

2000 ◽  
Vol 182 (16) ◽  
pp. 4617-4624 ◽  
Author(s):  
Ester Ibañez ◽  
Evangelina Campos ◽  
Laura Baldoma ◽  
Juan Aguilar ◽  
Josefa Badia
Keyword(s):  
Mutant Strain ◽  
Integration Host Factor ◽  
Anaerobic Growth ◽  
Receptor Protein ◽  
Mobility Shift ◽  
Regulation Of Expression ◽  
Carbohydrate Utilization ◽  
In The Wild ◽  
Gel Mobility Shift ◽  
Transcriptional Fusions

ABSTRACT The yiaKLMNOPQRS (yiaK-S) gene cluster ofEscherichia coli is believed to be involved in the utilization of a hitherto unknown carbohydrate which generates the intermediate l-xylulose. Transcription ofyiaK-S as a single message from the unique promoter found upstream of yiaK is proven in this study. The 5′ end has been located at 60 bp upstream from the ATG. Expression of theyiaK-S operon is controlled in the wild-type strain by a repressor encoded by yiaJ. No inducer molecule of theyiaK-S operon has been identified among over 80 carbohydrate or derivative compounds tested, the system being expressed only in a mutant strain lacking the YiaJ repressor. ThelacZ transcriptional fusions in the genetic background of the mutant strain revealed that yiaK-S is modulated by the integration host factor and by the cyclic AMP (cAMP)-cAMP receptor protein (Crp) activator complex. A twofold increase in the induction was observed during anaerobic growth, which was independent of ArcA or Fnr. Gel mobility shift assays showed that the YiaJ repressor binds to a promoter fragment extending from −50 to +121. These studies also showed that the cAMP-Crp complex can bind to two different sites. ThelacZ transcriptional fusions of different fragments of the promoter demonstrated that binding of cAMP-Crp to the Crp site 1, centered at −106, is essential for yiaK-S expression. The 5′ end of the yiaJ gene was determined, and its promoter region was found to overlap with the divergent yiaK-Spromoter. Expression of yiaJ is autogenously regulated and reduced by the binding of Crp-cAMP to the Crp site 1 of theyiaK-S promoter.

scholarly journals The LysR-Type Virulence Activator AphB Regulates the Expression of Genes in Vibrio cholerae in Response to Low pH and Anaerobiosis

2010 ◽  
Vol 192 (16) ◽  
pp. 4181-4191 ◽  
Author(s):  
Gabriela Kovacikova ◽  
Wei Lin ◽  
Karen Skorupski
Keyword(s):  
Binding Site ◽  
Vibrio Cholerae ◽  
Chloride Channels ◽  
Low Ph ◽  
Mobility Shift ◽  
Global Regulators ◽  
Expression Of Genes ◽  
Number Of Genes ◽  
Chromosomal Genes ◽  
Gel Mobility Shift

ABSTRACT AphB is a LysR-type activator that initiates the expression of the virulence cascade in Vibrio cholerae by cooperating with the quorum-sensing-regulated activator AphA at the tcpPH promoter on the Vibrio pathogenicity island (VPI). To identify the ancestral chromosomal genes in V. cholerae regulated by AphB, we carried out a microarray analysis and show here that AphB influences the expression of a number of genes that are not associated with the VPI. One gene strongly activated by AphB is cadC, which encodes the ToxR-like transcriptional activator responsible for activating the expression of lysine decarboxylase, which plays an important role in survival at low pH. Other genes activated by AphB encode a Na+/H+ antiporter, a carbonic anhydrase, a member of the ClC family of chloride channels, and a member of the Gpr1/Fun34/YaaH family. AphB influences each of these genes directly by recognizing a conserved binding site within their promoters, as determined by gel mobility shift assays. Transcriptional lacZ fusions indicate that AphB activates the expression of these genes under aerobic conditions in response to low pH and also under anaerobic conditions at neutral pH. Further experiments show that the regulation of cadC by AphB in response to low pH and anaerobiosis is mirrored in the heterologous organism Escherichia coli, is independent of the global regulators Fnr and ArcAB, and depends upon the region of the promoter that contains the AphB binding site. These results raise the possibility that the activity of AphB is influenced by the pH and oxygen tension of the environment.

scholarly journals Footprint Analysis of the RAG Protein Recombination Signal Sequence Complex for V(D)J Type Recombination

1998 ◽  
Vol 18 (1) ◽  
pp. 655-663 ◽  
Author(s):  
Fumikiyo Nagawa ◽  
Kei-ichiro Ishiguro ◽  
Akio Tsuboi ◽  
Tomoyuki Yoshida ◽  
Akiko Ishikawa ◽  
...  
Keyword(s):  
Signal Sequence ◽  
Dimethyl Sulfate ◽  
Dnase I ◽  
Mobility Shift ◽  
Partial Methylation ◽  
Recombination Signal ◽  
Recombination Activating Genes ◽  
Bottom Strand ◽  
Dnase I Footprinting ◽  
Gel Mobility Shift

ABSTRACT We have studied the interaction between recombination signal sequences (RSSs) and protein products of the truncated forms of recombination-activating genes (RAG) by gel mobility shift, DNase I footprinting, and methylation interference assays. Methylation interference with dimethyl sulfate demonstrated that binding was blocked by methylation in the nonamer at the second-position G residue in the bottom strand and at the sixth- and seventh-position A residues in the top strand. DNase I footprinting experiments demonstrated that RAG1 alone, or even a RAG1 homeodomain peptide, gave footprint patterns very similar to those obtained with the RAG1-RAG2 complex. In the heptamer, partial methylation interference was observed at the sixth-position A residue in the bottom strand. In DNase I footprinting, the heptamer region was weakly protected in the bottom strand by RAG1. The effects of RSS mutations on RAG binding were evaluated by DNA footprinting. Comparison of the RAG-RSS footprint data with the published Hin model confirmed the notion that sequence-specific RSS-RAG interaction takes place primarily between the Hin domain of the RAG1 protein and adjacent major and minor grooves of the nonamer DNA.

scholarly journals Affinities of Terminal Inverted Repeats to DNA Binding Domain of Transposase Affect the Transposition Activity of Bamboo Ppmar2 Mariner-Like Element

2019 ◽  
Vol 20 (15) ◽  
pp. 3692 ◽  
Author(s):  
Muthusamy Ramakrishnan ◽  
Mingbing Zhou ◽  
Chunfang Pan ◽  
Heikki Hänninen ◽  
Kim Yrjälä ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Domain ◽  
Inverted Repeats ◽  
Dna Binding Domain ◽  
Mobility Shift ◽  
Transposition Frequency ◽  
Terminal Inverted Repeats ◽  
Transposition Activity ◽  
Highly Active ◽  
Gel Mobility Shift

Mariner-like elements (MLE) are a super-family of DNA transposons widespread in animal and plant genomes. Based on their transposition characteristics, such as random insertions and high-frequency heterogeneous transpositions, several MLEs have been developed to be used as tools in gene tagging and gene therapy. Two active MLEs, Ppmar1 and Ppmar2, have previously been identified in moso bamboo (Phyllostachys edulis). Both of these have a preferential insertion affinity to AT-rich region and their insertion sites are close to random in the host genome. In Ppmar2 element, we studied the affinities of terminal inverted repeats (TIRs) to DNA binding domain (DBD) and their influence on the transposition activity. We could identify two putative boxes in the TIRs which play a significant role in defining the TIR’s affinities to the DBD. Seven mutated TIRs were constructed, differing in affinities based on similarities with those of other plant MLEs. Gel mobility shift assays showed that the TIR mutants with mutation sites G669A-C671A had significantly higher affinities than the mutants with mutation sites C657T-A660T. The high-affinity TIRs indicated that their transposition frequency was 1.5–2.0 times higher than that of the wild type TIRs in yeast transposition assays. The MLE mutants with low-affinity TIRs had relatively lower transposition frequency from that of wild types. We conclude that TIR affinity to DBD significantly affects the transposition activity of Ppmar2. The mutant MLEs highly active TIRs constructed in this study can be used as a tool for bamboo genetic studies.

scholarly journals Regulation of Expression of the Divergent ulaG and ulaABCDEF Operons Involved in l-Ascorbate Dissimilation in Escherichia coli

2004 ◽  
Vol 186 (6) ◽  
pp. 1720-1728 ◽  
Author(s):  
Evangelina Campos ◽  
Laura Baldoma ◽  
Juan Aguilar ◽  
Josefa Badia
Keyword(s):  
Escherichia Coli ◽  
Integration Host Factor ◽  
The Other ◽  
Receptor Protein ◽  
Loop Formation ◽  
Mobility Shift ◽  
Regulation Of Expression ◽  
Global Regulators ◽  
Conserved Sequence ◽  
Inducer Exclusion

ABSTRACT The ula regulon, responsible for the utilization of l-ascorbate in Escherichia coli, is formed by two divergently transcribed operons, ulaG and ulaABCDEF. The regulon is negatively regulated by a repressor of the DeoR family which is encoded by the constitutive gene ulaR located downstream of ulaG. Full repression of the ula regulon requires simultaneous interaction of the repressor with both divergent promoters and seems to be dependent on repressor-mediated DNA loop formation, which is helped by the action of integration host factor. Two operator sites have been identified in each promoter. Lack of either of the two sets of operators partially relieved the repression of the other operon; thus, each promoter is dependent on the UlaR operator sites of the other promoter to enhance repression. Electrophoretic mobility shift assays with purified UlaR protein and promoter deletion analyses revealed a conserved sequence, present in each of the four operators, acting as a UlaR binding site. Glucose represses the ula regulon via at least two mechanisms, one dependent on cyclic AMP (cAMP)-cAMP receptor protein (CRP) and the other (possibly inducer exclusion) independent of it. Glucose effects mediated by other global regulators cannot be ruled out with the present information. Changes in cAMP-CRP levels affected only the expression of the ulaABCDEF operon.

scholarly journals Binding of Escherichia coli integration host factor (IHF) to the origin segment of p15A plasmid.

1995 ◽  
Vol 42 (1) ◽  
pp. 103-108
Author(s):  
E Hiszczyńska-Sawicka ◽  
J Kur
Keyword(s):  
Escherichia Coli ◽  
Consensus Sequence ◽  
Integration Host Factor ◽  
Host Factor ◽  
Mobility Shift ◽  
Consensus Sequences ◽  
Report Data ◽  
Functional Dna ◽  
Gel Mobility Shift ◽  
Dna Binding And Bending

The integration host factor (IHF) is a sequence-specific, histone-like, multi-functional DNA-binding and -bending protein of Escherichia coli. Characterization and functional analysis of this protein has been carried out mainly in bacteriophage lambda and other mobile genetic elements. In this paper we report data concerning the binding of IHF protein to the plasmid orip15A region. IHF binds to the single site of the DNA fragment containing the orip15A, as shown by the gel mobility shift assays and footprinting experiment. On the basis of the ihf consensus sequences published, we have been able to identify one sequence of putative ihf site into the orip15A sequence with two mismatches in relation to the consensus sequence of Kur et al., 1989, Gene 81, 1-15. One ihf binding site was also found in the oriColE1 region sequence with three mismatches in relation to this consensus sequence.

scholarly journals The DNA Binding Domains of P1 ParB and the Architecture of the P1 Plasmid Partition Complex

2001 ◽  
Vol 276 (15) ◽  
pp. 12385-12394 ◽  
Author(s):  
Jennifer A. Surtees ◽  
Barbara E. Funnell
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Integration Host Factor ◽  
Mobility Shift ◽  
Dna Binding Domains ◽  
High Affinity ◽  
Binding Domains ◽  
Dna Binding Activity ◽  
C Terminus ◽  
Gel Mobility Shift

Stable maintenance of P1 plasmids inEscherichia coliis mediated by a high affinity nucleoprotein complex called the partition complex, which consists of ParB and theE. coliintegration host factor (IHF) bound specifically to the P1parSsite. IHF strongly stimulates ParB binding toparS, and the minimal partition complex contains a single dimer of ParB. To examine the architecture of the partition complex, we have investigated the DNA binding activity of various ParB fragments. Gel mobility shift and DNase I protection assays showed that the first 141 residues of ParB are dispensable for the formation of the minimal, high affinity partition complex. A fragment missing only the last 16 amino acids of ParB bound specifically toparS, but binding was weak and was no longer stimulated by IHF. The ability of IHF to stimulate ParB binding toparScorrelated with the ability of ParB to dimerize via its C terminus. Using full and partialparSsites, we show that two regions of ParB, one in the center and the other near the C terminus of the protein, interact with distinct sequences withinparS. Based on these data, we have proposed a model of how the ParB dimer bindsparSto form the minimal partition complex.

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