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PubMed1995

Binding of integration host factor (IHF) to the Escherichia coli sodA gene and its role in the regulation of a sodA-lacZ fusion gene.

We used the electrophoretic mobility-shift assay to reveal specific DNA-protein interactions between DNA fragments containing the sodA promoter and proteins present in Escherichia coli cell-free extracts. We have shown specific binding of several E. coli proteins to sodA promoter sequences and identified one of these proteins as the integration host factor (IHF). Mobility-shift experiments with cell-free extracts prepared from himA (IHF-negative) mutant strains lacked a specific DNA-protein band relative to shifts made with wild-type extracts. Several potential IHF-binding sites were identified in the sodA promoter region. Purified IHF was found to bind specifically to DNA fragments containing the sodA promoter. Further evidence presented suggests that IHF binds to multiple sites in the sodA promoter. We have also investigated the transcriptional regulation of sodA by monitoring the expression of a sodA-lacZ fusion gene in an IHF-negative E. coli strain under different growth conditions. Under aerobic conditions, a deletion in himA (IHF subunit alpha) resulted in a 60% increase in sodA expression, while having no effect on induction by paraquat. The same deletion in himA did not cause derepression of sodA-lacZ during anaerobic growth, but resulted in an increased response (about twofold) to the presence of 2,2'-dipyridyl compared to the isogenic wild-type strain.

2,2'-Dipyridyl↗

PubMed1992

Catabolite gene activator protein and integration host factor act in concert to regulate tdc operon expression in Escherichia coli.

Anaerobic expression of the tdcABC operon of Escherichia coli requires cyclic AMP and the catabolite gene activator protein (CAP). Purified CAP binds to a 30-bp sequence in the tdc promoter between positions -55 and -26, and a mutant CAP site with base substitutions at positions -48, -47, and -45 failed to bind CAP and also drastically reduced the beta-galactosidase expression from a tdcB'-'lacZ fusion plasmid. Recently, we showed that efficient expression of the tdc operon also requires a functional integration host factor (IHF) and an IHF-binding site in the tdc promoter between positions -118 and -88. The levels of beta-galactosidase activity from the tdcB'-'lacZ fusion plasmids were also reduced in an IHF-deficient strain with the wild-type or mutant plasmid CAP sequence. In vitro footprinting experiments revealed that CAP and IHF occupy their specific binding sites on tdc DNA when they are present separately or together. These regulatory proteins also induced significant bending of the tdc promoter DNA. Our results suggest that CAP and IHF act in concert as positive transcription factors for tdc operon expression in vivo.

Anaerobiosis↗

PubMed1992

Integration host factor (IHF) binds to many sites in the A + T-rich b2 region of phage lambda DNA.

Computer analysis of almost the entire b2 region of lambda phage (nt 22346-27475) revealed 23 consensus-like ihf sites, with eleven pointing in one direction and twelve in the opposite direction [27 bp; Kur et al., Gene 81 (1989) 1-15]. To confirm the significance of this finding experimentally, the region was subdivided into 21 fragments and examined for integration host factor (IHF) binding by gel retardation and a variety of footprinting methods. Out of 21 fragments examined 13 were found to be retarded on gels in the presence of IHF and to contain one to three ihf sites each. All sites which differ by up to 2 bp from our 27-bp consensus ihf sequence can bind IHF in vitro. However, three of the computer-predicted sites overlap with sites of opposite orientation; therefore we could not determine at the present time which of the two antiparallel sequences binds IHF. We have compared the predictive values of various kinds of consensus sequences and show that our 27-bp consensus ihf sequence agrees best with the experimental data. It was demonstrated by Kur et al. [Virology 168 (1989) 236-244] that the IHF protein represses transcription from promoters located close to the right terminus of the b2 region, within the phage lambda attachment site. We discuss the possibility that some of the IHF-binding sites could be instrumental in repressing in vivo transcription from the A + T-rich b2 region during the lambda prophage state [Rosenvold et al., Virology 107 (1980) 476-487].

Amino Acid Sequence↗

PubMed1989

Action at a distance in Mu DNA transposition: an enhancer-like element is the site of action of supercoiling relief activity by integration host factor (IHF).

The first committed step in the in vitro strand transfer reaction of a mini-Mu donor molecule is the formation of a Type 1 complex in which the Mu ends are held together in a non-covalent protein-DNA complex. Efficient formation of this complex at high levels of donor supercoiling (sigma approximately -0.06) requires the Mu A and Escherichia coli HU proteins. At in vivo levels of supercoiling, efficient reaction also requires E. coli integration host factor (IHF). We demonstrate that this supercoiling relief activity of IHF is mediated through an IHF binding site in the Mu early promoter region. This site is part of a larger enhancer-like element which includes operator 1 (01) and part of operator 2 (02) with the IHF site in between. The enhancer-like element stimulates the initial rate of the in vitro reaction 100-fold and acts in a distance-independent fashion. Inversion of the orientation of the element results in a total loss of enhancer activity in the absence of IHF. However, a 10-fold stimulation in the initial rate of reaction is induced by the addition of IHF. Furthermore, correct helical phasing between 01 and 02 is required for maximal activity. The results indicate that a specific geometrical configuration of the enhancer-like element, which includes a sharp bend between 01 and 02, is required for optimal induction of synapsis.

Bacterial Proteins↗

PubMed1993

Complex transcriptional control of the sigma s-dependent stationary-phase-induced and osmotically regulated osmY (csi-5) gene suggests novel roles for Lrp, cyclic AMP (cAMP) receptor protein-cAMP complex, and integration host factor in the stationary-phase response of Escherichia coli.

osmY (csi-5) is a representative of a large group of sigma s-dependent genes in Escherichia coli that exhibit both stationary-phase induction and osmotic regulation. A chromosomal transcriptional lacZ fusion (csi-5::lacZ) was used to study the regulation of osmY. We show here that in addition to sigma s, the global regulators Lrp, cyclic AMP (cAMP) receptor protein-cAMP complex (cAMP-CRP), and integration host factor (IHF) are involved in the control of osmY. All three regulators negatively modulate the expression of osmY, and they act independently from sigma s. Stationary-phase induction of osmY in minimal medium can be explained by stimulation by sigma s combined with a relief of Lrp repression. Stationary-phase induction of osmY in rich medium is mediated by the combined action of sigma s, Lrp, cAMP-CRP, and IHF, with the latter three proteins acting as transition state regulators. The transcriptional start site of osmY was determined and revealed an mRNA with an unusual long nontranslated leader of 244 nucleotides. The regulatory region is characterized by a sigma 70-like -10 promoter region and contains potential binding sites for Lrp, CRP, and IHF. Whereas sigma s, Lrp, CRP, and IHF are clearly involved in stationary-phase induction, none of these regulators is essential for osmotic regulation of osmY.

Amino Acid Sequence↗

PubMed1976

Host factor requirements and some properties of phiXtB. An evolutionary aspect of phiX-type phages.

Replication of phiXtB, a capsid mutant of bacteriophage phiX174, depends on the host functions directed by the E.coli genes dnaE, dnaF, dnaG, dnaZ, lig and rep. The cellular products of dnaA, dnaB, dnaC(D), dnaI, dnaP, polA, polB and xth genes are, however, dispensable for the viral growth. In these host factor requirements, phiXtB resembles phages phiK and St-1 rather than phiX174. Host ranges of phiXtB, St-1 and phiK overlap considerably, and growth temperature of the three phages is somewhat higher than that of phiX174. Furthermore, phiXtB is, like phiK, inactivated by antiserum against St-1. phiXtB may thus fill an evolutionary gap between the phiX174 group and the St-1 group.

Biological Evolution↗

PubMed1993

Integration host factor binds to a unique class of complex repetitive extragenic DNA sequences in Escherichia coli.

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.

Bacterial Proteins↗

PubMed1993

Integration host factor is required for anaerobic pyruvate induction of pfl operon expression in Escherichia coli.

The expression of the pyruvate formate-lyase gene (pfl) is induced by anaerobic growth, and this is increased further by growth in pyruvate. Previous work has shown that anaerobic induction is strongly dependent on the activator FNR and partially dependent on a second transcription factor, ArcA, while pyruvate induction only required FNR. Anaerobic and pyruvate regulation both require the presence of a 5' nontranslated regulatory sequence which spans approximately 500 bp of DNA. A mobility shift assay was developed to identify proteins that bind to this regulatory region. Several binding activities were separated by heparin agarose chromatography, and one of these activities was characterized and shown to be integration host factor (IHF). Mobility shift and DNase I footprinting experiments defined a single IHF binding site in the pfl promoter-regulatory region. With pfl-lacZ fusions, it could be shown that introduction of a himD mutation abolished pyruvate-dependent induction of anaerobic expression in vivo. The same result was observed when the pfl IHF binding site was mutated. In addition, the partial anaerobic induction of expression found in an fnr strain was completely blocked in an fnr himD double mutant and in an fnr IHF binding site double mutant. Taken together, these data suggest that IHF is necessary for both pyruvate induction and the anaerobic induction mediated by ArcA.

Acetyltransferases↗

PubMed1994

Specific photocrosslinking of DNA-protein complexes: identification of contacts between integration host factor and its target DNA.

Azide moieties have been specifically placed in the backbone of DNA by chemical coupling between azidophenacyl bromide and uniquely positioned phosphorothioate residues. The derivatized DNA forms specific complexes with a DNA-binding protein and, following irradiation with 302-nm light, makes specific crosslinks to the protein. Isolation of this covalent complex, followed by tryptic digestion and Edman degradation of the resulting crosslinked peptide, identifies the portion of the protein that is near the derivatized segment of the target DNA. We use this method to probe the interaction between a specific DNA sequence and integration host factor (IHF) protein. A single IHF heterodimer is known to contact > 25 bp of DNA and thereby introduce a sharp bend. Two segments of a typical IHF site were derivatized with aryl azide. Although the segments were separated by only 5 bp, they crosslinked to different subunits of IHF. The locations of the crosslinks support our current view for the way IHF protein binds to and bends its specific targets.

Amino Acid Sequence↗

PubMed1998

Integration host factor and cyclic AMP receptor protein are required for TyrR-mediated activation of tpl in Citrobacter freundii.

The tpl gene of Citrobacter freundii encodes an enzyme that catalyzes the conversion of L-tyrosine to phenol, pyruvate, and ammonia. This gene is known to be positively regulated by TyrR. The amplitude of regulation attributable to this transcription factor is at least 20-fold. Three TyrR binding sites, designated boxes A, B, and C, centered at coordinates -272.5, -158.5, and -49.5, respectively, were identified in the upstream region of the tpl promoter. The results of mutational experiments suggest that TyrR binds in cooperative fashion to these sites. The nonavailability of any TyrR site impairs transcription. Full TyrR-mediated activation of tpl required integration host factor (IHF) and the cAMP receptor protein (CRP). By DNase I footprinting, it was shown that the IHF binding site is centered at coordinate -85 and that there are CRP binding sites centered at coordinates -220 and -250. Mutational alteration of the IHF binding site reduced the efficiency of the tpl promoter by at least eightfold. The proposed roles of CRP and IHF are to introduce bends into tpl promoter DNA between boxes A and B or B and C. Multimeric TyrR dimers were demonstrated by a chemical cross-linking method. The formation of hexameric TyrR increased when tpl DNA was present. The participation of both IHF and CRP in the activation of the tpl promoter suggests that molecular mechanisms quite different from those that affect other TyrR-activated promoters apply to this system. A model wherein TyrR, IHF, and CRP collaborate to regulate the expression of the tpl promoter is presented.

Adenosine Triphosphate↗

PubMedDate not supplied

Influence of host factors on survival in dogs with malignant mammary gland tumors.

The purpose of our study was to determine if specific host factors, such as age at diagnosis, obesity, and hormone status, influence the prognosis of canine mammary gland carcinomas and to confirm if previously reported risk factors (ie, histologic subtype, tumor size, and World Health Organization [WHO] stage) were important in a large series of affected dogs. Ninety-nine female dogs with mammary gland carcinomas, no previous therapy, an excisional biopsy, and known cause of death were studied. No significant association with survival was noted for age at diagnosis (chronologic or physiologic), obesity, or hormone status (ie, spayed versus intact, regardless of time of being spayed). Of the tumor factors analyzed, the histologic subtype anaplastic carcinoma (P = .02), WHO stage I (P = .01), evidence of metastasis at the time of diagnosis (P = .004), and tumor size of 3 cm or smaller (P = .005) all significantly influenced survival. Dogs that were classified as having tumor-related mortality had a shorter postoperative survival compared to dogs that died of other causes (14 months versus 23 months; P = .03). In conclusion, histologic subtype, WHO stage, and tumor size remain important prognostic factors in canine mammary gland tumors. Further study of other prognostic factors is needed to determine which tumors are adequately addressed with local therapy only and which dogs may require adjuvant treatment with chemotherapy.

Aging↗

PubMed1992

Role of integration host factor in stimulating transcription from the sigma 54-dependent nifH promoter.

In a wide variety of nitrogen-fixing organisms among the Purple Bacteria (large division of Gram-negative bacteria) the nitrogen fixation (nif) operons are transcribed by an alternative holoenzyme form of RNA polymerase, sigma 54-holoenzyme. Transcription depends on the activator protein NIFA (nitrogen fixation protein A), which catalyzes isomerization of closed complexes between this polymerase and a promoter to transcriptionally productive open complexes. NIFA-mediated activation of transcription from the nifH promoter of Klebsiella pneumoniae is greatly stimulated by the integration host factor IHF, which binds to a site between the upstream binding site for NIFA and the promoter, and bends the DNA. IHF fails to stimulate activation of transcription from this promoter by another activator of sigma 54-holoenzyme, NTRC (nitrogen regulatory protein C), which lacks a specific binding site in the nifH promoter region. As predicted, if the IHF-induced bend facilitates interaction between NIFA and sigma 54-holoenzyme, substitution of an NTRC-binding site for the NIFA-binding site allowed IHF to stimulate NTRC-mediated activation of transcription from the nifH promoter. The stimulation was of the same order of magnitude as that for NIFA in the native configuration of the promoter-regulatory region (up to 20-fold). With purified NTRC and the substitution construct we could demonstrate that stimulation by IHF in a purified transcription system was comparable to that in a crude coupled transcription-translation system, indicating that the stimulation in the crude system could be accounted for by IHF. The IHF stimulation was observed on linear as well as supercoiled templates, indicating that the geometric requirements are relatively simple. We have attempted to visualize the arrangement of proteins on DNA fragments carrying the nifH promoter-regulatory region of K. pneumoniae by electron microscopy. IHF stimulated NIFA-mediated activation of transcription from the nifH and nifD promoters of Bradyrhizobium japonicum and less so from the nifH promoters of Rhizobium meliloti and Thiobacillus ferrooxidans, consistent with previous observations that stimulation is greatest at promoters that are weak binding sites for sigma 54-holoenzyme in closed complexes.

Bacterial Proteins↗

PubMed1990

Characterization of the integration host factor binding site in the ilvPG1 promoter region of the ilvGMEDA operon of Escherichia coli.

The ilvGMEDA operon of Escherichia coli, which encodes four of the five enzyme activities required for the biosynthesis of isoleucine and valine, is preceded by tandem promoters ilvPG1 and ilvPG2 which are separated by 72 base pairs. While both of these promoters are transcriptionally active in vitro, only the operon proximal promoter, ilvPG2, is transcriptionally active in vivo, and upstream DNA sequences encoding the ilvPG1 promoter region enhance the in vivo transcriptional activity of the ilvPG2 promoter 60-fold. The binding of the integration host factor protein (IHF) to this upstream region (Tsui, P., and Freundlich, M. (1989) J. Mol. Biol. 203, 817-820) has been shown to repress transcription from the ilvPG1 promoter both in vivo and in vitro (Pereira, R. F., Ortuno, M. J., and Lawther, R. P. (1988) Nucleic Acids Res. 16, 5972-5989). Furthermore, E. coli strains deficient for IHF are compromised for isoleucine and valine biosynthesis (Friden, P., Voelkel, K., Sternglantz, R., and Freundlich, M. (1984) J. Mol. Biol. 172, 573-579). Therefore, in order to further understand this repressor/activator role of IHF, we have undertaken a detailed analysis of the interaction of IHF with the DNA sequences in the ilvPG1 promoter region. The results of hydroxyl radical footprinting, dimethyl sulfate protection, and ethylation interference experiments show that IHF binds to a target site that overlaps the ilvPG1 promoter region. The results of these experiments also demonstrate that IHF interacts primarily with the minor groove of the DNA helix and that the IHF target site in the ilvPG1 promoter region shares a high degree of DNA sequence identity with other high affinity IHF target sites involved in DNA replication and site-specific recombination.

Bacterial Proteins↗

PubMed1992

In vivo requirement of integration host factor for nar (nitrate reductase) operon expression in Escherichia coli K-12.

The nitrate reductase operon (narGHJI) of Escherichia coli encodes an anaerobic respiratory enzyme. Previous work has identified two cis-acting sites in the nar operon control region: a proximal site required for anaerobic induction mediated by the activator Fnr and a remote upstream site required for nitrate induction mediated by the activator NarL [Li, S. & DeMoss, J. A. (1988) J. Biol. Chem. 263, 13700-13705]. Our search for nar regulatory mutants yielded one strain with a mutation in himD, the structural gene for one of the subunits of integration host factor (IHF). Strains carrying null alleles of the IHF structural genes, himD and himA, had severe defects in nitrate induction of the nar operon but were normal for nitrate induction of the coordinately regulated fdn operon. Anaerobic expression of both operons was normal in him mutants. Gel-mobility-shift and DNase I protection experiments revealed a single IHF binding site in the nar operon control region, located midway between the upstream activation site and the promoter. We conclude that an IHF-mediated DNA bend is essential for efficient nitrate induction of the sigma 70-dependent nar operon promoter. This requirement of IHF for transcriptional activation had been noted for several sigma 54-dependent promoters.

Bacterial Proteins↗

PubMed2002

The Pseudomonas aeruginosa hemA promoter is regulated by Anr, Dnr, NarL and Integration Host Factor.

Pseudomonas aeruginosa forms most of its heme under anaerobic denitrifying conditions. To study the regulation of the hemA gene, which codes for the first enzyme of heme biosynthesis in P. aeruginosa, a lacZ reporter gene fusion was constructed. Expression of lacZ under the control of the hemA promoter was found to be increased by 2.8-fold under anaerobic conditions in the presence of the alternative electron acceptor nitrate, relative to the level observed under aerobic growth conditions. Anaerobic fermentative growth or the presence of nitrite did not affect the lacZ expression. The genes encoding the oxygen sensor protein Anr, the redox regulator Dnr, the nitrate regulator NarL and the DNA-bending Integration Host Factor (IHF) are all required for the cooperative anaerobic induction of the hemA promoter hemAp (1). Potential binding sites for these regulatory proteins were identified by site-directed mutagenesis of the promoter fused to the reporter gene. The mode of regulation of P. aeruginosa hemA differs significantly from that described for the hemA gene of Escherichia coli K-12.

Aldehyde Oxidoreductases↗

PubMed1997

Regulation of the ndh gene of Escherichia coli by integration host factor and a novel regulator, Arr.

The ndh gene of Escherichia coli encodes the non-proton-translocating NADH dehydrogenase II. Expression of the ndh gene is subject to a complex network of regulatory controls at the transcriptional level. Under anaerobic conditions ndh is repressed by the regulator of fumarate and nitrate reduction (FNR). However, in the absence of FNR, ndh expression is activated by the amino acid response regulator (Arr) during anaerobic growth in rich medium. Expression of the ndh gene varies during the growth cycle in response to the intracellular concentration of the heat-stable DNA-binding protein, Fis. In this work two additional heat-stable proteins, integration host factor (IHF) and the histone-like protein HU were found to interact with the ndh promoter. IHF was shown to bind at three sites centred at +26, -17 and -58 in the ndh promoter (Kd = 10(-8) M), to prevent open-complex formation and to repress ndh transcription in vitro. Studies with an ndh-lacZ fusion confirmed that IHF represses ndh expression in vivo. Two putative binding sites for Arr, which overlap the two FNR boxes in the ndh promoter, were identified. Studies with the FNR-activated and amino-acid-inducible asparaginase II gene (ansB) showed that IHF and a component of the Arr-containing fraction (but not HU) interact with the corresponding ansB promoter.

Amino Acid Sequence↗

PubMed2006

Integration Host Factor is required for FarR repression of the farAB-encoded efflux pump of Neisseria gonorrhoeae.

The farAB operon encodes an efflux pump system that mediates the resistance of Neisseria gonorrhoeae to antimicrobial long-chain fatty acids. We previously observed that expression of farAB is negatively regulated by the FarR repressor. In this study, we examined the molecular mechanism by which FarR represses expression of farAB. DNase I footprinting analysis, coupled with a deletion analysis of the farAB promoter region, indicated that FarR binds to three sites (termed sites A, B and C) within the DNA sequence upstream of farA; genetic analysis revealed, however, that site B is not required for FarR repression of farAB. This repression also required the presence of Integration Host Factor (IHF), which was found to bind to sequences located between FarR binding sites A and C. We determined that IHF binding to the farAB promoter region could inhibit transcription in vitro and that such binding induced a bending of the target DNA, which we propose to be important in regulating this operon. IHF binding to the promoter region was found to stabilize the binding of FarR to its binding sites A and C and as a consequence, enhanced repression of farAB expression mediated by FarR. We propose a model in which expression of the farAB-encoded efflux pump in N. gonorrhoeae is modulated by the DNA binding activities of FarR and IHF.

Base Pairing↗

PubMed2006

Mutational analysis of intervening sequences connecting the binding sites for integration host factor, PepA, PurR, and RNA polymerase in the control region of the Escherichia coli carAB operon, encoding carbamoylphosphate synthase.

Transcription of the carAB operon encoding the unique carbamoylphosphate synthase of Escherichia coli reflects the dual function of carbamoylphosphate in the biosynthesis of arginine and pyrimidine nucleotides. The tandem pair of promoters is regulated by various mechanisms depending on the needs of both pathways and the maintenance of a pyrimidine/purine nucleotide balance. Here we focus on the linker regions that impose the distribution of target sites for DNA-binding proteins involved in pyrimidine- and purine-specific repression of the upstream promoter P1. We introduced deletions and insertions, and combinations thereof, in four linkers connecting the binding sites for integration host factor (IHF), PepA, PurR, and RNA polymerase and studied the importance of phasing and spacing of the targets and the importance of the nucleotide sequence of the linkers. The two PepA binding sites must be properly aligned and separated with respect to each other and to the promoter for both pyrimidine- and purine-mediated repression. Similarly, the phasing and spacing of the IHF and PEPA2 sites are strictly constrained but only for pyrimidine-specific repression. The IHF target is even dispensable for purine-mediated regulation. Thus, a correct localization of PepA within the higher-order nucleoprotein complex is a prerequisite for the establishment of pyrimidine-mediated repression and for the coupling between purine- and pyrimidine-dependent regulation. Our data also suggest the existence of a novel cis-acting pyrimidine-specific regulatory target located around position -60. Finally, the analysis of a P1 derivative devoid of its control region has led to a reappraisal of the effect of excess adenine on P1 and has revealed that P1 has no need for a UP element.

Base Sequence↗