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 Characterization of Bacteriophage Lambda Excisionase Mutants Defective in DNA Binding

2000 ◽  
Vol 182 (20) ◽  
pp. 5807-5812 ◽  
Author(s):  
Eun Hee Cho ◽  
Renato Alcaraz ◽  
Richard I. Gumport ◽  
Jeffrey F. Gardner
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Glutamic Acid ◽  
Cooperative Interaction ◽  
Mutant Proteins ◽  
Amino Terminal ◽  
Factor For Inversion Stimulation ◽  
Α Helix

ABSTRACT The bacteriophage λ excisionase (Xis) is a sequence-specific DNA binding protein required for excisive recombination. Xis binds cooperatively to two DNA sites arranged as direct repeats on the phage DNA. Efficient excision is achieved through a cooperative interaction between Xis and the host-encoded factor for inversion stimulation as well as a cooperative interaction between Xis and integrase. The secondary structure of the Xis protein was predicted to contain a typical amphipathic helix that spans residues 18 to 28. Several mutants, defective in promoting excision in vivo, were isolated with mutations at positions encoding polar amino acids in the putative helix (T. E. Numrych, R. I. Gumport, and J. F. Gardner, EMBO J. 11:3797–3806, 1992). We substituted alanines for the polar amino acids in this region. Mutant proteins with substitutions for polar amino acids in the amino-terminal region of the putative helix exhibited decreased excision in vivo and were defective in DNA binding. In addition, an alanine substitution at glutamic acid 40 also resulted in altered DNA binding. This indicates that the hydrophilic face of the α-helix and the region containing glutamic acid 40 may form the DNA binding surfaces of the Xis protein.

scholarly journals Inhibition of IS2transposition by factor for inversion stimulation

2007 ◽  
Vol 275 (1) ◽  
pp. 98-105 ◽  
Author(s):  
Guang-Sheng Lei ◽  
Chii-Jaan Chen ◽  
Hanna S. Yuan ◽  
Shao-Hung Wang ◽  
Shiau-Ting Hu
Keyword(s):  
Factor For Inversion Stimulation

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.

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