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Results for “Integration Host Factors”

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PubMed2003

Complex regulation of csgD promoter activity by global regulatory proteins.

The starvation-induced csgD gene of Salmonella typhimurium encodes for the positive transcriptional regulator of extracellular matrix components curli fimbriae and cellulose. To analyse regulatory elements of csgD promoter (PcsgD) response genetic studies combined with in vitro experiments were performed. Six binding sites (D1 to D6) for OmpR, a transcriptional regulator, were identified by gel shifts and DNase I footprints. While ompR is required for PcsgD expression, binding of OmpR-P to D2 centred immediately upstream of D1 at position -70.5 is proposed to repress PcsgD activity. The elevated expression of regulated and semiconstitutive PcsgD in response to microaerophilic conditions required integration host factor (IHF). Subsequently, two IHF-binding sites were identified up- and downstream of PcsgD. IHF competes with OmpR-P for binding at its upstream site IHF1, which overlaps with D3-D6 and thereby modulates the response to microaerophilic conditions. A complex regulatory network involving IHF, H-NS and OmpR is proposed whereby the nucleo-complex composition in the csgD-csgBA intergenic region is altered in response to oxygen tension.

Aerobiosis↗

PubMed1989

Phasing of protein-induced DNA bends in a recombination complex.

Many of the structures responsible for replication, transcription initiation and recombination arise from complex sets of protein-protein interactions and the folding of DNA in three dimensions, with protein-induced bending of DNA often playing an integral role. The magnitude and orientation of DNA bending induced by various single proteins has been estimated by gel mobility shift methods and by modelling of crystallographic data. The site-specific recombination by which bacteriophage lambda (phage lambda) integrates into the chromosome of its host Escherichia coli requires a host protein, 'integration host factor' (IHF), which is known to be able to bend the DNA to which it binds. To determine the three-dimensional path of DNA within the higher order structure responsible for phage lambda site-specific recombination, we have determined the relative direction of IHF-induced bending at each of the three binding sites within the complex. IHF, which appears to bend DNA by more than 140 degrees, is a major determinant of the DNA path in the recombination complex and is also involved in a wide range of other cellular events.

Bacterial Proteins↗

PubMed1999

Hierarchy in the expression of the locus of enterocyte effacement genes of enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) elicit changes in host cell morphology and cause actin rearrangement, a phenotype that has commonly been referred to as attaching/effacing (AE) lesions. The ability of EPEC to induce AE lesions is dependent upon a type III protein secretion/translocation system that is encoded by genes clustered in a 35.6 kb DNA segment, named the locus of enterocyte effacement (LEE). We used transcriptional fusions between the green fluorescent protein (gfp) reporter gene and LEE genes rorf2, orf3, orf5, escJ, escV and eae, together with immunoblot analysis with antibodies against Tir, intimin, EspB and EspF, to analyse the genetic regulation of the LEE. The expression of all these LEE genes was strictly dependent upon the presence of a functional integration host factor (IHF). IHF binds specifically upstream from the ler (orf1) promoter and appears to activate expression of ler, orf3, orf5 and rorf2 directly. The ler-encoded Ler protein was involved in activating the expression of escJ, escV, tir, eae, espB and espF. Expression of both IHF and Ler was needed to elicit actin rearrangement associated with AE lesions. In conclusion, IHF directly activates the expression of the ler and rorf2 transcriptional units, and Ler in turn mediates the expression of the other LEE genes.

Actins↗

PubMed1996

In vitro transcriptional analysis of TyrR-mediated activation of the mtr and tyrP+3 promoters of Escherichia coli.

In order to understand the mechanism by which the TyrR protein activates transcription from the mtr and tyrP+3 promoters, we have carried out in vitro transcription experiments with supercoiled DNA templates. We have shown that addition of the histone-like protein HU or integration host factor (IHF) greatly inhibited the transcription from the mtr and tyrP+3 promoters. In the presence of phenylalanine, the wild-type TyrR protein, but not a mutant TyrR protein (activation negative), was able to relieve the HU- or IHF-mediated inhibition of transcription. In contrast, the alleviation of the HU- or IHF-mediated transcription inhibition by the wild-type TyrR protein did not occur when a mutant RNA polymerase with a C-terminally truncated alpha subunit was used to carry out the transcription reaction.

Amino Acid Transport Systems↗

PubMed1999

Transcription regulation of the colicin K cka gene reveals induction of colicin synthesis by differential responses to environmental signals.

Colicin-producing strains occur frequently in natural populations of Escherichia coli, and colicinogenicity seems to provide a competitive advantage in the natural habitat. A cka-lacZ fusion was used to study the regulation of expression of the colicin K structural gene. Expression is growth phase dependent, with high activity in the late stationary phase. Nutrient depletion induces the expression of cka due to an increase in ppGpp. Temperature is a strong signal for cka expression, since only basal-level activity was detected at 22 degrees C. Mitomycin C induction demonstrates that cka expression is regulated to a lesser extent by the SOS response independently of ppGpp. Increased osmolarity induces a partial increase, while the global regulator integration host factor inhibits expression in the late stationary phase. Induction of cka was demonstrated to be independent of the cyclic AMP-Crp complex, carbon source, RpoS, Lrp, H-NS, pH, and short-chain fatty acids. In contrast to colicin E1, cka expression is independent of catabolite repression and is partially affected by anaerobiosis only upon SOS induction. These results indicate that while different colicins are expressed in response to some common signals such as nutrient depletion, the expression of individual colicins could be further influenced by specific environmental cues.

Acetic Acid↗

PubMed2003

[Transduction of plasmid antibiotic resistance determinants with pseudo-T-even bacteriophages].

Transduction of antibiotic resistance determinants of the plasmid pBR322 with pseudoT-even bacteriophages RB42, RB43, and RB49 was studied. It is established that antibiotic resistance determinants of plasmid pBR322 from Escherichia coli recA(+)- and recA(-)-donor strains do not differ significantly in respect to the efficiency of transduction. Amber mutants RB43-21, RB43-33, and a double amber mutant RB43am21am33 were obtained. These mutants facilitated transduction experiments in some cases. Transduction of antibiotic resistance markers of the vector plasmid pBR325 and recombinant plasmid pVT123, containing a DNA fragment with hoc segE uvsW genes of phage T4, was studied. The frequency of appearance of transductants resistant to pseudoT-even bacteriophages used in transduction was determined, and the sensitivity of resistant transductants to 32 RB bacteriophages and also to phages lambda, T2, T4, T5, T6, T7, and BF23 was estimated. The efficiency of plating pseudoT-even bacteriophages RB42 and RB43 on strain E. coli 802 himA hip carrying mutations in genes that encode subunits of the Integration Host Factor (IHF) was shown to be higher than on isogenic strain E. coli 802. The growth of pseudoT-even bacteriophages limited in vivo by modification-restriction systems of chromosomal (EcoKI, EcoBI), phage (EcoP1I), and plasmid (EcoRI, EcoR124I, and EcoR124II) localization was analyzed. It was shown that these phages were only slightly restricted by the type I modification-restriction systems EcoBI, EcoR124I, and EcoR124II. Phage RB42 was restricted by systems EcoKI, EcoP1I, and EcoRI; phage RB43, by systems EcoKI and EcoRI; and phage RB49, by the EcoRI modification-restriction system.

DNA Helicases↗

PubMed1997

Action at a distance for glp repressor control of glpTQ transcription in Escherichia coli K-12.

The adjacent, divergently transcribed glpACB and glpTQ operons of Escherichia coli encode the anaerobic glycerol 3-phosphate dehydrogenase and glycerol 3-phosphate transporter/phosphodiesterase, respectively. These operons are negatively controlled by glp repressor binding to operators that overlap the glpA promoter elements. Using DNase I footprinting, three additional operators (OT1-3) were identified at positions +307 to +359 within the glpT coding region. To assess a potential regulatory role for these remote operators in vivo, a glpT-lacZ transcriptional fusion containing all of the glpA and glpT operators was constructed. The response of this fusion to the glp repressor was compared to fusion constructs in which OT1 and OT3 were inactivated, either by deletion or by site-directed mutagenesis. It was found that repression of glpT conferred by binding of glp repressor to glpA operators was increased about three- to fourfold upon introduction of the remote glpT operators. In addition, two integration host factor (IHF) binding sites were identified downstream of the glpT transcriptional start site at positions +15 to +51 and +193 to +227. A regulatory role for IHF was demonstrated by showing that repression of glpT mediated by GlpR was decreased about twofold in strains deficient in IHF and that mutations in IHF1 and/or IHF2 decreased repression about two- to threefold. The effect of IHF was apparent only when the remote operators were present. All of the results are consistent with a model of repression involving GlpR binding simultaneously to the glpA and remote glpT operators, with intervening DNA forming a loop.

Bacterial Proteins↗

PubMed1999

Cross-induction of glc and ace operons of Escherichia coli attributable to pathway intersection. Characterization of the glc promoter.

The metabolic pathways specified by the glc and ace operons in Escherichia coli yield glyoxylate as a common intermediate, which is acted on by two malate synthase isoenzymes: one encoded by glcB and the other by aceB. Null mutations in either gene exhibit no phenotype, because of cross-induction of the ace operon by glycolate and the glc operon by acetate. In this study, the regulation of the glc operon, comprising the structural genes glcDEFGB, was analyzed at the molecular level. This operon, activated by growth on glycolate, is transcribed as a single message and is under the positive control of GlcC encoded by a divergent gene. Expression of the glc operon is strongly dependent on the integration host factor (IHF) and is repressed by the global respiratory regulator ArcA-P. In vitro gel-shift experiments demonstrated direct binding of the promoter DNA to IHF and ArcA-P. Mutant analysis indicated that cross-induction of the glc operon by acetate is mediated by the GlcC protein that recognizes the compound as an alternative effector. The similar pattern of regulation of the Glc and Ace systems by IHF and ArcA-P ensures their effective cross-induction.

Acetic Acid↗

PubMed1994

Two genes for carbohydrate catabolism are divergently transcribed from a region of DNA containing the hexC locus in Pseudomonas aeruginosa PAO1.

The hexC locus of Pseudomonas aeruginosa PAO1 was localized to a 247-bp segment of chromosomal DNA on the multicopy broad-host-range vector pRO1614. The presence of this plasmid (pPZ196) in strain PAO1 produced the so-called "hexC effect," a two- to ninefold increase in the activities of four carbohydrate catabolism enzymes, glucokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase. The extent of the hexC effect was restricted, since three independently regulated metabolic enzymes were not affected by the presence of the hexC plasmid. Furthermore, the hexC-containing plasmid did not suppress catabolite repression control. Nucleotide sequence analysis of the segment of DNA encompassing hexC revealed a 128-bp region rich in adenosine-plus-thymine (AT) content separating two divergent open reading frames (ORFs). Transcriptional start sites for these two genes were mapped to the intergenic region, demonstrating that this sequence contained overlapping divergent promoters. The intergenic region contained potential regulatory sequences such as dyad symmetry motifs, polydeoxyadenosine tracts, and a sequence matching the integration host factor recognition site in Escherichia coli. One of the ORFs encoded a 610-amino-acid protein with 55 to 60% identity to 6-phosphogluconate dehydratase from E. coli and Zymomonas mobilis. The second ORF coded for a protein of 335 amino acids that displayed 45 to 60% identity to the NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAP) family of enzymes. The NAD-dependent GAP gene on the P. aeruginosa chromosome was previously unmapped. GAP was found to exhibit the hexC-dependent increase in its basal activity, establishing it as a fifth catabolic enzyme in the multioperonic hex regulon.

Amino Acid Sequence↗

PubMed1993

Specific interaction of IHF with RIBs, a class of bacterial repetitive DNA elements located at the 3' end of transcription units.

The prokaryotic REP (repetitive extragenic palindromes) or PU (palindromic units) sequences are often associated with other repetitive elements, forming arrangements which have been called 'BIMEs' (bacterial interspersed mosaic elements). It is estimated that the Escherichia coli chromosome carries approximately 300-500 BIMEs, whose biological role is at present unknown. We have identified a family of BIMEs consisting of two converging REP sequences flanking a 35 bp conserved segment which carries a static DNA bend and a binding site for IHF, the integration host factor of E.coli. We estimate that the E.coli genome harbors approximately 100 copies of this module, which we name 'RIB' (reiterative ihf BIME). We have analyzed by gel retardation and by footprinting the in vitro interaction of IHF with individual RIBs, and shown that the protein binds strongly and specifically to their center. We have also demonstrated binding of IHF to the chromosomal population of RIBs, using a new approach which combines two-dimensional bandshift and Southern blotting. RIB elements are at the end of transcription units, and thus define a new class of ihf sites. Possible implications for genome structure and DNA topology are discussed.

Bacterial Proteins↗

PubMed2006

The effect of IHF on sigmaS selectivity of the phoA and pst promoters of Escherichia coli.

The pst operon, a member of the PHO regulon of Escherichia coli, encodes a high-affinity phosphate transport system whose expression is induced when the cells enter a phase of phosphate starvation. The expression of pst is stimulated by the integration host factor (IHF). Transcription of the PHO regulon genes is initiated by the RNA polymerase complexed with sigma (D) (Esigma (D)). Owing to a cytosine residue at position -13 of the pst promoter its transcription can also be initiated by Esigma (S). Here, we show that inactivation of IHF in vivo abolishes the sigma (S)-dependent transcription initiation of the pst operon, indicating that both -13C residue and IHF are required to confer on pst the ability to be transcribed by Esigma (S). Introduction of a -13C residue in the promoter region of phoA, another PHO regulon gene that is not directly affected by IHF, did not affect its exclusive transcription initiation by Esigma (D).

Alkaline Phosphatase↗

PubMed1991

An upstream XylR- and IHF-induced nucleoprotein complex regulates the sigma 54-dependent Pu promoter of TOL plasmid.

Transcription from promoter Pu of the upper catabolic operon of the Pseudomonas putida TOL plasmid which specifies conversion of toluene/xylenes to benzoate/toluates is activated by the TOL-encoded regulator XylR protein in the presence of substrates of the catabolic pathway and in conjunction with the sigma 54(NtrA)-containing form of RNA polymerase. This regulatory circuit was faithfully reproduced in Escherichia coli in single copy gene dosage by integrating the corresponding controlling determinants into the chromosomes of several K12 derivatives by means of specialized transposons. In vivo monitoring of the activity of a Pu-lacZ fusion in E. coli strains with different genetic backgrounds demonstrated that integration host factor (IHF) is involved in Pu regulation and that hyperproduction of the XylR protein leads to a decrease of Pu activity in a manner in which deletion of the putative DNA-binding domain of the XylR does not impair its inhibitory effect when hyperproduced. One discrete IHF binding site and two potential XylR sites (consensus sequence 5'-TTGANCAAATC-3'), bracketted by short stretches of DNase I-hypersensitive bonds, were detected upstream of the transcription initiation site. A model accounting for the features found is proposed which includes the IHF-promoted looping of upstream XylR-DNA complexes so that they contact the sigma 54(NtrA)-RNA polymerase bound at -12/-24 positions.

Bacterial Proteins↗

PubMed1996

Transcriptional pattern of Escherichia coli ihfB (himD) gene expression.

Integration host factor (IHF) is a small heterodimer containing subunits encoded by the unlinked ihfA (himA) and ihfB (himD, hip) genes. The transcriptional pattern of ihfB expression in the logarithmic and stationary growth phases was investigated. The ihfB gene is expressed as both monocistronic and polycistronic (hybridizing also to an internal rpsA probe) transcript. The intensity of the polycistronic transcripts, initiated upstream of rpsA, decreased sharply upon growth cessation. In contrast, expression of the monocistronic ihfB transcript strongly increased when cells entered stationary growth phase. The observed growth rate-dependent regulation of the transcription of these transcripts is in agreement with the previously published data about the regulation of the rpsA and ihfB promoters (Pedersen et al., 1984; Aviv et al., 1994).

Bacterial Proteins↗

PubMed2005

Cyclic changes in the affinity of protein-DNA interactions drive the progression and regulate the outcome of the Tn10 transposition reaction.

The Tn10 transpososome is a DNA processing machine in which two transposon ends, a transposase dimer and the host protein integration host factor (IHF), are united in an asymmetrical complex. The transitions that occur during one transposition cycle are not limited to chemical cleavage events at the transposon ends, but also involve a reorganization of the protein and DNA components. Here, we demonstrate multiple pathways for Tn10 transposition. We show that one series of events is favored over all others and involves cyclic changes in the affinity of IHF for its binding site. During transpososome assembly, IHF is bound with high affinity. However, the affinity for IHF drops dramatically after cleavage of the first transposon end, leading to IHF ejection and unfolding of the complex. The ejection of IHF promotes cleavage of the second end, which is followed by restoration of the high affinity state which in turn regulates target interactions.

DNA↗

PubMed1994

Effect of heme and oxygen availability on hemA gene expression in Escherichia coli: role of the fnr, arcA, and himA gene products.

While many organisms synthesize delta-aminolevulinate, the precursor of heme, by condensing succinyl-coenzyme A and glycine, others use a glutamate-dependent pathway in which glutamyl-tRNA dehydrogenase catalyzes the rate-determining step. The hemeA gene that encodes this latter enzyme in Escherichia coli has been cloned and sequenced. To examine how its expression is regulated, we constructed hemA-lacZ operon and gene fusions and inserted them into the chromosome in single copy. The effect of aerobic and anaerobic growth conditions and the availability of electron acceptors and various carbon substrates were documented. Use of different types of cell culture medium resulted in a fivefold variation in hemA-lacZ expression during aerobic cell growth. Anaerobic growth resulted in 2.5-fold-higher hemA-lacZ expression than aerobic growth. This control is mediated by the fnr and arcA gene products. Fnr functions as a repressor of hemA transcription during anaerobic cell growth only, whereas the arcA gene product activates hemA gene expression under both aerobic and anaerobic conditions. Integration host factor protein was also shown to be required for control of hemA gene regulation. To determine whether an intermediate or a product of the heme biosynthetic pathway is involved in hemA regulation, hemA-lacZ expression was analyzed in a hemA mutant. Expression was elevated by 20-fold compared with that in a wild-type strain, while the addition of the heme pathway intermediate delta-aminolevulinate to the culture medium restored expression to wild-type levels. These results suggest that the heme pathway is feedback regulated at the level of hemA gene expression, to supply heme as it is required during different modes of cell growth.

Aerobiosis↗

PubMed1996

Crystal structure of an IHF-DNA complex: a protein-induced DNA U-turn.

Integration host factor (IHF) is a small heterodimeric protein that specifically binds to DNA and functions as an architectural factor in many cellular processes in prokaryotes. Here, we report the crystal structure of IHF complexed with 35 bp of DNA. The DNA is wrapped around the protein and bent by >160 degrees, thus reversing the direction of the helix axis within a very short distance. Much of the bending occurs at two large kinks where the base stacking is interrupted by intercalation of a proline residue. IHF contacts the DNA exclusively via the phosphodiester backbone and the minor groove and relies heavily on indirect readout to recognize its binding sequence. One such readout involves a six-base A tract, providing evidence for the importance of a narrow minor groove.

Amino Acid Sequence↗

PubMed1997

The RIB element in the goaG-pspF intergenic region of Escherichia coli.

The sequence (2,700 bp) between the aldH and pspF genes of Escherichia coli was determined. The pspF gene encodes a sigma54 transcriptional activator of the phage shock protein (psp) operon (pspA to pspE). Downstream of the pspF transcribed region are two open reading frames (ORFs), ordL and goaG, convergently oriented with respect to pspF. These two ORFs, together with the adjacent aldH gene, may constitute a novel operon (aldH-ordL-goaG). The goaG-pspF intergenic region contains a complex extragenic mosaic element, RIB. The structure of this RIB element, which belongs to the BIME-1 family, is Y(REP1) > 16 < Z1(REP2), where Y and Z1 are palindromic units and the central 16 bases contain an L motif with an ihf consensus sequence. DNA fragments containing the L motif of the psp RIB element effectively bind integration host factor (IHF), while the Y palindromic unit (REP1) of the same RIB element binds DNA gyrase weakly. Computer prediction of the pspF mRNA secondary structure suggested that the transcribed stem-loop structures formed by the 3'-flanking region of the pspF transcript containing the RIB element can stabilize and protect pspF mRNA. Analysis of pspF steady-state mRNA levels showed that transcripts with an intact RIB element are much more abundant than those truncated at the 3' end by deletion of either the entire RIB element or a single Z1 sequence (REP2). Thus, the pspF 3'-flanking region containing the RIB element has an important role in the stabilization of the pspF transcript.

Antigens, Bacterial↗

PubMed1996

Replication of plasmid R6K gamma origin in vivo and in vitro: dependence on IHF binding to the ihf1 site.

The gamma origin of plasmid R6K requires the specific initiator protein pi for initiation of replication. However, increased pi concentrations inhibit replication. The host-encoded integration host factor (IHF) protein permits gamma origin replication at otherwise inhibitory pi levels. IHF is thought to mediate this positive effect by directly binding to the gamma origin. In this study we demonstrate that IHF binding to one IHF site in the gama origin, ihf1, but not to the other side, ihf2, is necessary for the gamma origin to replicate at high pi protein levels. We also show that in vitro replication of the gamma origin plasmid requires IHF binding to the ihf1 site. Finally, we demonstrate both in vivo and in vitro that, when mutant pi proteins (hyperactive) are provided instead of wild-type pi, gamma origin plasmids can replicate in the absence of IHF. This supports a previously proposed hypothesis that the pi mutants can bypass the IHF requirement for gamma origin replication.

Bacterial Proteins↗