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Amos B. Oppenheim - One of the best experts on this subject based on the ideXlab platform.

  • Compaction of single DNA molecules induced by binding of Integration Host Factor (IHF).
    Proceedings of the National Academy of Sciences of the United States of America, 2001
    Co-Authors: B. M. Jaffar Ali, Amos B. Oppenheim, Roee Amit, Ido Braslavsky, Opher Gileadi, Joel Stavans
    Abstract:

    We studied the interaction between the Integration Host Factor (IHF), a major nucleoid-associated protein in bacteria, and single DNA molecules. Force–extension measurements of λ DNA and an analysis of the Brownian motion of small beads tethered to a surface by single short DNA molecules, in equilibrium with an IHF solution, indicate that: (i) the DNA–IHF complex retains a random, although more compact, coiled configuration for zero or small values of the tension, (ii) IHF induces DNA compaction by binding to multiple DNA sites with low specificity, and (iii) with increasing tension on the DNA, the elastic properties of bare DNA are recovered. This behavior is consistent with the predictions of a statistical mechanical model describing how proteins bending DNA are driven off by an applied tension on the DNA molecule. Estimates of the amount of bound IHF in DNA–IHF complexes obtained from the model agree very well with independent measurements of this quantity obtained from the analysis of DNA–IHF crosslinking. Our findings support the long-held view that IHF and other histone-like proteins play an important role in shaping the long-scale structure of the bacterial nucleoid.

  • Isolation and characterization of the Integration Host Factor genes of Pasteurella haemolytica.
    Fems Microbiology Letters, 1997
    Co-Authors: Sarah K Highlander, Orlando Garza, Billie Jo Brown, Simi Koby, Amos B. Oppenheim
    Abstract:

    Using a bacteriophage lambda complementation system in Escherichia coli, we cloned genes encoding subunits of the heterodimeric DNA binding/bending protein, Integration Host Factor, from the bovine pathogen, Pasteurella haemolytica. Complementation of ihfA and ihfB mutations in E. coli demonstrated that the P. haemolytica gene products form functional heterologous heterodimers. The ihfA and ihfB genes encode polypeptides predicted to be 99 and 93 amino acids long, respectively, and are very similar to Integration Host Factor subunits from other Gram-negative bacteria, although phylogenetic analysis indicated that the P. haemolytica sequences are distantly related to those from other bacteria. Most significant amino acid differences were restricted to the amino-terminal domains of the predicted peptides.

  • Genetic and biochemical analysis of the Integration Host Factor of Escherichia coli
    Journal of molecular biology, 1993
    Co-Authors: Galina Mengeritsky, Itai Mendelson, Hilla Giladi, Daniel Goldenberg, Amos B. Oppenheim
    Abstract:

    Abstract Integration Host Factor (IHF) is a small, heterodimeric DNA-binding protein of Escherichia coli composed of two subunits, α and β, encoded by the himA and hip genes, respectively. IHF binds to the minor groove at a consensus sequence and bends DNA. We mutagenized the hip gene and studied the activity of the mutant IHF proteins in vivo and in vitro. Substitutions at the C-terminal α-helix (α-helix 3) reduced IHF activity and relaxed the specificity to DNA without abolishing the ability of IHF to bend DNA. These results indicate that the C-terminal region of Hip participates in determining IHF specificity. Alanine substitutions in β-strands 2 and 3 generally had no effect on IHF activity in vivo suggesting that individually, many of these residues make only small contributions to the binding of IHF to DNA. Replacing the single amino acid of Hip that differs from HU in a highly conserved region of the arm did not affect IHF activity. This finding led us to conclude that this region of Hip does not contribute to specific DNA recognition by IHF. The binding of IHF to DNA is probably not restricted to one domain, but requires the co-operative participation of a number of regions of the protein.

  • Integration Host Factor binds to a unique class of complex repetitive extragenic DNA sequences in Escherichia coli.
    Molecular microbiology, 1993
    Co-Authors: Amos B. Oppenheim, Itai Mendelson, Kenneth E. Rudd, Dinah Teff
    Abstract:

    Interspersed repeated DNA sequences are characteristic features of both prokaryotic and eukaryotic genomes. REP sequences are defined as conserved repetitive extragenic palindromic sequences and are found in Escherichia coli, Salmonella typhimurium and other closely related enteric bacteria. These REP sequences may participate in the folding of the bacterial chromosome. In this work we describe a unique class of 28 conserved complex REP clusters, about 100bp long, in which two inverted REPs are separated by a singular Integration Host Factor (IHF) recognition sequence. We term these sequences RIP (for repetitive IHF-binding palindromic) elements and demonstrate that IHF binds to them specifically. It is estimated that there are about 70 RIP elements in E. coli. Our analysis shows that the RIP elements are evenly distributed around the bacterial chromosome. The possible function of the RIP element is discussed.

  • Stimulation of the phage λ pL promoter by Integration Host Factor requires the carboxy terminus of the α-subunit of RNA polymerase
    Journal of molecular biology, 1992
    Co-Authors: Hilla Giladi, Kazuhiko Igarashi, Akira Ishihama, Amos B. Oppenheim
    Abstract:

    Abstract Escherichia coli Integration Host Factor (IHF) binds with high affinity to two tandem IHF consensus sequences located upstream from the pL promoter of bacteriophage lambda. IHF was shown to stimulate transcription initiation from the pL promoter by increasing close complex formation (KB). We show here, by the use of reconstituted mutant RNA polymerases, that the C-terminal portion of the α subunit of RNA polymerase plays an essential role in the stimulation of transcription by IHF. Our results are in agreement with the hypothesis that IHF, like the cAMP-CRP activator, increases the affinity of RNA polymerase to the promoter by protein-protein interaction.

Jeffrey F. Gardner - One of the best experts on this subject based on the ideXlab platform.

  • Integration Host Factor: Putting a Twist on Protein–DNA Recognition
    Journal of molecular biology, 2003
    Co-Authors: Thomas W Lynch, Erik K. Read, Aras N. Mattis, Jeffrey F. Gardner, Phoebe A. Rice
    Abstract:

    Abstract Integration Host Factor (IHF) is a DNA–bending protein that recognizes its cognate sites through indirect readout. Previous studies have shown that binding of wild-type (WT)-IHF is disrupted by a T to A mutation at the center position of a conserved TTR motif in its binding site, and that substitution of βGlu44 with Ala prevented IHF from discriminating between A and T at this position. We have determined the crystal structures and relative binding affinities for all combinations of WT-IHF and IHF-βGlu44Ala bound to the WT and mutant DNAs. Comparison of these structures reveals that DNA twist plays a major role in DNA recognition by IHF, and that this geometric parameter is dependent on the dinucleotide step and not on the bound IHF variant.

  • Integration Host Factor putting a twist on protein dna recognition
    Journal of Molecular Biology, 2003
    Co-Authors: Thomas W Lynch, Erik K. Read, Aras N. Mattis, Jeffrey F. Gardner, Phoebe A. Rice
    Abstract:

    Abstract Integration Host Factor (IHF) is a DNA–bending protein that recognizes its cognate sites through indirect readout. Previous studies have shown that binding of wild-type (WT)-IHF is disrupted by a T to A mutation at the center position of a conserved TTR motif in its binding site, and that substitution of βGlu44 with Ala prevented IHF from discriminating between A and T at this position. We have determined the crystal structures and relative binding affinities for all combinations of WT-IHF and IHF-βGlu44Ala bound to the WT and mutant DNAs. Comparison of these structures reveals that DNA twist plays a major role in DNA recognition by IHF, and that this geometric parameter is dependent on the dinucleotide step and not on the bound IHF variant.

  • Site-Specific Recombination of Bacteriophage P22 Does Not Require Integration Host Factor
    Journal of bacteriology, 1999
    Co-Authors: Eun Hee Cho, Chan Eun Nam, Renato Alcaraz, Jeffrey F. Gardner
    Abstract:

    Site-specific recombination by phages λ and P22 is carried out by multiprotein-DNA complexes. Integration Host Factor (IHF) facilitates λ site-specific recombination by inducing DNA bends necessary to form an active recombinogenic complex. Mutants lacking IHF are over 1,000-fold less proficient in supporting λ site-specific recombination than wild-type cells. Although the attP region of P22 contains strong IHF binding sites, in vivo measurements of Integration and excision frequencies showed that infecting P22 phages can perform site-specific recombination to its maximum efficiency in the absence of IHF. In addition, a plasmid Integration assay showed that integrative recombination occurs equally well in wild-type and ihfA mutant cells. P22 integrative recombination is also efficient in Escherichia coli in the absence of functional IHF. These results suggest that nucleoprotein structures proficient for recombination can form in the absence of IHF or that another Factor(s) can substitute for IHF in the formation of complexes.

  • Mutants of Escherichia coli Integration Host Factor: DNA-binding and recombination properties
    Biochimie, 1994
    Co-Authors: L. M. Hales, Richard I. Gumport, Jeffrey F. Gardner
    Abstract:

    Abstract Integration Host Factor (IHF) is a protein encoded by Escherichia coli which was first discovered as a requirement for bacteriophage λ site-specific recombination. In this study, we characterized mutants of IHF for their ability to bind to varoous IHF binding sites in vivo and to promote recombination of λ in vitro. DNA-binding in vivo was monitored using the challange-phage system. If IHF binds to its DNA-binding site that has been placed into the Pant region of bacteriophage P22, it acts as a repressor of the ant (antirepressor) gene, leading to the formation of lysogens of Salmonella typhimurium. If IHF cannot bind to its site, antirepressor is made leading to cell lysis. Challenge phages containing chimeras of different λ IHF binding sites were constructed to test the contribution to the binding of a dA+dT-rich region, found in the sequence of the H′ site but not in the Hl site. In one case, the binding of mutant IHF proteins was enhanced by the presence of the dA+dT-rich region, indicating that IHF may be affected by neighboring bases and local DNA structure when it binds to its site. A subset of the mutant proteins retained the ability to form a looped attL complex in vivo, representing part of a higher-order protein-DNA complex (the ‘intasome’). Additionally, this same subset of proteins also promoted the Integration and excision of bacteriophage λin vitro. Thus, these mutant proteins not only retain their DNA-bending ability but make any protein-protein contacts necessary to form a recombination-proficient intasome.

  • Integration Host Factor facilitates repression of the put operon in Salmonella typhimurium.
    Gene, 1992
    Co-Authors: Kathryn O'brien, Jeffrey F. Gardner, Gregory Deno, Paula Ostrovsky De Spicer, Stanley Maloy
    Abstract:

    Transcriptional regulation of the put operon is mediated by a unique mechanism involving autogenous regulation by the PutA protein, a membrane-associated dehydrogenase. The 420-bp put control region contains the putP and putA promoters, multiple operator sites, multiple catabolite repression protein binding sites, and several potential Integration Host Factor (IHF)-binding sites (ihf). In this study, we show that IHF facilitates repression of the put operon in vivo, and IHF binds specifically to two ihf sites in the put control region in vitro. DNA gyrase mutants that alter the degree of chromosomal supercoiling do not affect put regulation, indicating that the effect of IHF on put expression is in this case independent of supercoiling.

Hilla Giladi - One of the best experts on this subject based on the ideXlab platform.

  • Structure and function of the Pseudomonas putida Integration Host Factor.
    Journal of bacteriology, 1996
    Co-Authors: R Calb, Simi Koby, Hilla Giladi, A Davidovitch, David M. Goldenberg, Hanah Margalit, A Holtel, K N Timmis, J M Sanchez-romero, V De Lorenzo
    Abstract:

    Integration Host Factor (IHF) is a DNA-binding and -bending protein that has been found in a number of gram-negative bacteria. Here we describe the cloning, sequencing, and functional analysis of the genes coding for the two subunits of IHF from Pseudomonas putida. Both the ihfA and ihfB genes of P. putida code for 100-amino-acid-residue polypeptides that are 1 and 6 residues longer than the Escherichia coli IHF subunits, respectively. The P. putida ihfA and ihfB genes can effectively complement E. coli ihf mutants, suggesting that the P. putida IHF subunits can form functional heterodimers with the IHF subunits of E. coli. Analysis of the amino acid differences between the E. coli and P. putida protein sequences suggests that in the evolution of IHF, amino acid changes were mainly restricted to the N-terminal domains and to the extreme C termini. These changes do not interfere with dimer formation or with DNA recognition. We constructed a P. putida mutant strain carrying an ihfA gene knockout and demonstrated that IHF is essential for the expression of the P(U) promoter of the xyl operon of the upper pathway of toluene degradation. It was further shown that the ihfA P. putida mutant strain carrying the TOL plasmid was defective in the degradation of the aromatic model compound benzyl alcohol, proving the unique role of IHF in xyl operon promoter regulation.

  • Genetic and biochemical analysis of the Integration Host Factor of Escherichia coli
    Journal of molecular biology, 1993
    Co-Authors: Galina Mengeritsky, Itai Mendelson, Hilla Giladi, Daniel Goldenberg, Amos B. Oppenheim
    Abstract:

    Abstract Integration Host Factor (IHF) is a small, heterodimeric DNA-binding protein of Escherichia coli composed of two subunits, α and β, encoded by the himA and hip genes, respectively. IHF binds to the minor groove at a consensus sequence and bends DNA. We mutagenized the hip gene and studied the activity of the mutant IHF proteins in vivo and in vitro. Substitutions at the C-terminal α-helix (α-helix 3) reduced IHF activity and relaxed the specificity to DNA without abolishing the ability of IHF to bend DNA. These results indicate that the C-terminal region of Hip participates in determining IHF specificity. Alanine substitutions in β-strands 2 and 3 generally had no effect on IHF activity in vivo suggesting that individually, many of these residues make only small contributions to the binding of IHF to DNA. Replacing the single amino acid of Hip that differs from HU in a highly conserved region of the arm did not affect IHF activity. This finding led us to conclude that this region of Hip does not contribute to specific DNA recognition by IHF. The binding of IHF to DNA is probably not restricted to one domain, but requires the co-operative participation of a number of regions of the protein.

  • Stimulation of the phage λ pL promoter by Integration Host Factor requires the carboxy terminus of the α-subunit of RNA polymerase
    Journal of molecular biology, 1992
    Co-Authors: Hilla Giladi, Kazuhiko Igarashi, Akira Ishihama, Amos B. Oppenheim
    Abstract:

    Abstract Escherichia coli Integration Host Factor (IHF) binds with high affinity to two tandem IHF consensus sequences located upstream from the pL promoter of bacteriophage lambda. IHF was shown to stimulate transcription initiation from the pL promoter by increasing close complex formation (KB). We show here, by the use of reconstituted mutant RNA polymerases, that the C-terminal portion of the α subunit of RNA polymerase plays an essential role in the stimulation of transcription by IHF. Our results are in agreement with the hypothesis that IHF, like the cAMP-CRP activator, increases the affinity of RNA polymerase to the promoter by protein-protein interaction.

  • Genes coding for Integration Host Factor are conserved in gram-negative bacteria.
    Journal of bacteriology, 1991
    Co-Authors: H Haluzi, Simi Koby, D Goitein, Itai Mendelson, Dinah Teff, Galina Mengeritsky, Hilla Giladi, Amos B. Oppenheim
    Abstract:

    A genetic system for the selection of clones coding for Integration Host Factor and HU homologs is described. We demonstrate that the himA and hip genes of Serratia marcescens and Aeromonas proteolytica can substitute for the Escherichia coli genes in a variety of biological assays. We find that the sequence and genetic organization of the himA and hip genes of S. marcescens are highly conserved.

Ute Römling - One of the best experts on this subject based on the ideXlab platform.

Matthew K. Waldor - One of the best experts on this subject based on the ideXlab platform.

  • Requirement for Vibrio cholerae Integration Host Factor in conjugative DNA transfer.
    Journal of bacteriology, 2006
    Co-Authors: Sarah M. Mcleod, Vincent Burrus, Matthew K. Waldor
    Abstract:

    The requirement for Host Factors in the transmission of integrative and conjugative elements (ICEs) has not been extensively explored. Here we tested whether Integration Host Factor (IHF) or Fis, two Host-encoded nucleoid proteins, are required for transfer of SXT, a Vibrio cholerae-derived ICE that can be transmitted to many gram-negative species. Fis did not influence the transfer of SXT to or from V. cholerae. In contrast, IHF proved to be required for V. cholerae to act as an SXT donor. In the absence of IHF, V. cholerae displayed a modest defect for serving as an SXT recipient. Surprisingly, SXT Integration into or excision from the V. cholerae chromosome, which requires an SXT-encoded integrase related to λ integrase, did not require IHF. Therefore, the defect in SXT transmission in the V. cholerae IHF mutant is probably not related to IHF's ability to promote DNA recombination. The V. cholerae IHF mutant was also highly impaired as a donor of RP4, a broad-Host-range conjugative plasmid. Thus, the V. cholerae IHF mutant appears to have a general defect in conjugation. Escherichia coli IHF mutants were not impaired as donors or recipients of SXT or RP4, indicating that IHF is a V. cholerae-specific conjugation Factor.