scholarly journals Functional analysis of the integration host factor site of the σ54Pu promoter of Pseudomonas putida by in vivo UV imprinting

2011 ◽  
Vol 82 (3) ◽  
pp. 591-601 ◽  
Author(s):  
Marc Valls ◽  
Rafael Silva-Rocha ◽  
Ildefonso Cases ◽  
Amalia Muñoz ◽  
Víctor de Lorenzo
Keyword(s):  
Functional Analysis ◽  
Pseudomonas Putida ◽  
Integration Host Factor ◽  
Host Factor

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 Structure and function of the Pseudomonas putida integration host factor.

1996 ◽  
Vol 178 (21) ◽  
pp. 6319-6326 ◽  
Author(s):  
R Calb ◽  
A Davidovitch ◽  
S Koby ◽  
H Giladi ◽  
D Goldenberg ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
Integration Host Factor ◽  
Structure And Function ◽  
Host Factor ◽  
And Function

scholarly journals m-Xylene-Responsive Pu-PnifH Hybrid σ54 Promoters That Overcome Physiological Control in Pseudomonas putida KT2442

2005 ◽  
Vol 187 (1) ◽  
pp. 125-134 ◽  
Author(s):  
Manuel Carmona ◽  
Silvia Fernández ◽  
María J. Rodríguez ◽  
Víctor de Lorenzo
Keyword(s):  
Pseudomonas Putida ◽  
Promoter Sequence ◽  
Growth Conditions ◽  
Integration Host Factor ◽  
Vector System ◽  
Wild Type ◽  
Physiological Control ◽  
Accurate Comparison ◽  
Rnap Binding

ABSTRACT The sequences surrounding the −12/−24 motif of the m-xylene-responsive σ54 promoter Pu of the Pseudomonas putida TOL plasmid pWW0 were replaced by various DNA segments of the same size recruited from PnifH σ54 promoter variants known to have various degrees of efficacy and affinity for σ54-RNA polymerase (RNAP). In order to have an accurate comparison of the output in vivo of each of the hybrids, the resulting promoters were recombined at the same location of the chromosome of P. putida KT2442 with a tailored vector system. The promoters included the upstream activation sequence (UAS) for the cognate regulator of the TOL system (XylR) fused to the −12/−24 region of the wild-type PnifH and its higher σ54-RNAP affinity variants PnifH049 and PnifH319. As a control, the downstream region of the glnAp2 promoter (lacking integration host factor) was fused to the XylR UAS as well. When the induction patterns of the corresponding lacZ fusion strains were compared in vivo, we observed that promoters bearing the RNAP binding site of PnifH049 and PnifH319 were not silenced during exponential growth, as is distinctly the case for the wild-type Pu promoter or for the Pu-PnifH variant. Taken together, our results indicate that the promoter sequence(s) spanning the −12/−24 region of Pu dictates the coupling of promoter output to growth conditions.

scholarly journals Xis Protein Binding to the Left Arm Stimulates Excision of Conjugative Transposon Tn916

2002 ◽  
Vol 184 (8) ◽  
pp. 2088-2099 ◽  
Author(s):  
Kevin M. Connolly ◽  
Mizuho Iwahara ◽  
Robert T. Clubb
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Integration Host Factor ◽  
Host Factor ◽  
Human Pathogens ◽  
Bacteriophage Λ ◽  
Factor For Inversion Stimulation ◽  
Protein Nucleic Acid ◽  
Clinically Significant

ABSTRACT Tn916 and related conjugative transposons are clinically significant vectors for the transfer of antibiotic resistance among human pathogens, and they excise from their donor organisms using the transposon-encoded integrase (Tn916 Int) and excisionase (Tn916 Xis) proteins. In this study, we have investigated the role of the Tn916 Xis protein in stimulating excisive recombination. The functional relevance of Tn916 Xis binding sites on the arms of the transposon has been assessed in vivo using a transposon excision assay. Our results indicate that in Escherichia coli the stimulatory effect of the Tn916 Xis protein is mediated by sequence-specific binding to either of its two binding sites on the left arm of the transposon. These sites lie in between the core and arm sites recognized by Tn916 Int, suggesting that the Tn916 Xis protein enhances excision in a manner similar to the excisionase protein of bacteriophage λ, serving an architectural role in the stabilization of protein-nucleic acid structures required for strand synapsis. However, our finding that excision in E. coli is significantly enhanced by the host factor HU, but does not depend on the integration host factor or the factor for inversion stimulation, defines clear mechanistic differences between Tn916 and bacteriophage λ recombination.

Integration Host Factor IHF facilitates homologous recombination and mutagenic processes in Pseudomonas putida

DNA Repair ◽  
2020 ◽  
Vol 85 ◽  
pp. 102745 ◽  
Author(s):  
Katren Mikkel ◽  
Mari Tagel ◽  
Kärt Ukkivi ◽  
Heili Ilves ◽  
Maia Kivisaar
Keyword(s):  
Homologous Recombination ◽  
Pseudomonas Putida ◽  
Integration Host Factor ◽  
Host Factor

scholarly journals Contributions of [4Fe-4S]-FNR and Integration Host Factor to fnr Transcriptional Regulation

2007 ◽  
Vol 189 (8) ◽  
pp. 3036-3043 ◽  
Author(s):  
Erin L. Mettert ◽  
Patricia J. Kiley
Keyword(s):  
Binding Site ◽  
In Vitro Transcription ◽  
Integration Host Factor ◽  
Host Factor ◽  
Upstream Region ◽  
Anaerobic Conditions ◽  
Protein Levels ◽  
Dnase I Footprinting

ABSTRACT Maintaining appropriate levels of the global regulator FNR is critical to its function as an O2 sensor. In this study, we examined the mechanisms that control transcription of fnr to increase our understanding of how FNR protein levels are regulated. Under anaerobic conditions, one mechanism that controls fnr expression is negative autoregulation by the active [4Fe-4S] form of FNR. Through DNase I footprinting and in vitro transcription experiments, we observed that direct binding of [4Fe-4S]-FNR to the predicted downstream FNR binding site is sufficient for repression of the fnr promoter in vitro. In addition, the downstream FNR binding site was required for repression of transcription from fnr′-lacZ fusions in vivo. No repression of fnr was observed in vivo or in vitro with the apoprotein form of FNR, indicating that repression requires the dimeric, Fe-S cluster-containing protein. Furthermore, our in vitro and in vivo data suggest that [4Fe-4S]-FNR does not bind to the predicted upstream FNR binding site within the fnr promoter. Rather, we provide evidence that integration host factor binds to this upstream region and increases in vivo expression of Pfnr under both aerobic and anaerobic conditions.

scholarly journals In Vivo and In Vitro Effects of Integration Host Factor at the DmpR-Regulated ς54-Dependent Po Promoter

2001 ◽  
Vol 183 (9) ◽  
pp. 2842-2851 ◽  
Author(s):  
Chun Chau Sze ◽  
Andrew D. Laurie ◽  
Victoria Shingler
Keyword(s):  
Rna Polymerase ◽  
Binding Site ◽  
Binding Sites ◽  
Integration Host Factor ◽  
Host Factor ◽  
Physical Interaction ◽  
Rich Region ◽  
Polymerase Binding

ABSTRACT Transcription from the Pseudomonas CF600-derived ς54-dependent promoter Po is controlled by the aromatic-responsive activator DmpR. Here we examine the mechanism(s) by which integration host factor (IHF) stimulates DmpR-activated transcriptional output of the Po promoter both in vivo and in vitro. In vivo, the Po promoter exhibits characteristics that typify many ς54-dependent promoters, namely, a phasing-dependent tolerance with respect to the distance from the regulator binding sites to the distally located RNA polymerase binding site, and a strong dependence on IHF for optimal promoter output. IHF is shown to affect transcription via structural repercussions mediated through binding to a single DNA signature located between the regulator and RNA polymerase binding sites. In vitro, using DNA templates that lack the regulator binding sites and thus bypass a role of IHF in facilitating physical interaction between the regulator and the transcriptional apparatus, IHF still mediates a DNA binding-dependent stimulation of Po transcription. This stimulatory effect is shown to be independent of previously described mechanisms for the effects of IHF at ς54 promoters such as aiding binding of the regulator or recruitment of ς54-RNA polymerase via UP element-like DNA. The effect of IHF could be traced to promotion and/or stabilization of open complexes within the nucleoprotein complex that may involve an A+T-rich region of the IHF binding site and promoter-upstream DNA. Mechanistic implications are discussed in the context of a model in which IHF binding results in transduction of DNA instability from an A+T-rich region to the melt region of the promoter.

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