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Integration host factor (IHF) is a small heterodimeric DNA binding protein found in all Gram-negative bacteria and is implicated as a transcription cofactor of pilE in Neisseria gonorrhoeae (Hill, S.A., Samuels, D.S., Carlson, J.H., Wilson, J., Hogan, D., Lubke, L., Belland, R.J., 1997. Integration host factor is a transcriptional cofactor of pilE in Neisseria gonorrhoeae. Mol. Microbiol. 23, 649-656). The ihf genes (ihfA and ihfB) were cloned from N. gonorrhoeae through functional complementation of defined Escherichia coli ihf mutants for plating of phage lambda. The predicted aa sequences of each gonococcal IHF polypeptide showed extensive homology to other reported IHF polypeptide sequences. Northern blotting and primer extension analysis defined the tsp for each gene and indicated a disparity in ihfA and ihfB message levels over time, with ihfB mRNA being more abundant throughout the entire growth cycle. Furthermore, both the ihfA and ihfB message levels declined as cells entered the stationary growth phase. Overall, this study reveals several unique features of ihf transcription in the gonococcus which questions whether certain aspects if ihf transcriptional regulation are universally shared by all Gram-negative bacteria.

In the hin -mediated DNA inversion system, HU facilitates formation of the synaptic complex composed of two recombination sites spaced 996 bp apart and of the enhancer situated between them, by looping the DNA as to promote interaction of Hin invertase with the Fis enhancer factor [Johnson et al., Nature 329 (1987) 462–465]. The HU requirement for the in vivo hin -mediated inversion was demonstrated previously [Wada et al. Gene 76 (1989) 345–352; Hillyard et al. J. Bacteriol. 172 (1990) 5402–5407; Haykinson and Johnson, EMBO J. 12 (1993) 2503–2512] and in the current experiments. This HU action, however, required IHF when H-NS was present in the cell; i.e., the inversion reaction of the hin -invertible DNA fragment carried by the ptCK1202R plasmid proceeded efficiently in host cells either deficient in H-NS or in the presence of both H-NS and IHF, but not in the cells depleted for IHF alone. The level of hin mRNA in mutant cells lacking HU or IHF, in which hin inversion did not occur, was normal or slightly increased. When IHF was absent, the stimulating effect of HU on in vitro DNA circle formation of a 125-bp hin fragment between hixL and the enhancer where Fis binds was inhibited by H-NS. The present study provides an example of a multi-component interaction between HU, H-NS and IHF on the hin DNA region, which contains three characteristic sites, a d(A/T) 4 stretch and bent DNA site, and two putative IHF-binding sites.

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.

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].

Integration host factor (IHF)-mediated protection against enzymatic methylation at ihf -overlapping sites provides the basis for this novel application of the Achilles' cleavage (AC) technique [Koob., Science 241 (1998) 1084–10861 for generating rare natural cleavage sites. When applying IHF-AC to plasmid, phage λ, Escherichia coli and yeast genomes, only a few of the Eco RI, Mbo I sites (sites overlapped the ihf sites)remained cleavable after prior methylation with the cognate M· Eco RI, M· Hin fI, or Dam methyltransferases in the presence of IHF. Thus, IHF-AC essentially converted these enzymes into very rare cutters. The extent of cleavage could be controlled by varying the IHF:DNA ratio and temperature. Moreover, the method permits the genomic location and strength of the ihf sites to be determined.

The closely related Escherichia coli genes, hupA, hupB, himA and himD (hip), encode the bacterial histone-like protein subunits, HU-2, HU-1, IHFα and IHFβ, respectively. We report here that E. coli minichromosomes [plasmids (2.7–12.2 kb) with oriC] carrying the intact mioC region were unable to transform mutants deficient in both HU and integration host factor (IHF), whereas they could transform mutants deficient in either HU or IHF as efficiently as the wild-type strain. Minichromosomes carrying a deletion of the proximal part of mioC or a DnaA box just upstream from mioC could not transform cells deficient in IHF, but could transform cells deficient in HU. These results suggested that HU and IHF participate in minichromosomal replication from oriC in E. coli.

Integration host factor (IHF) is a heterodimeric, site-specific DNA-binding and DNA-bending protein from Escherichia coli. It is involved in high-precision DNA transactions where it serves as a key architectural component of specialized nucleoprotein structures (snups). We described recently a novel approach for protein engineering using a single polypeptide chain IHF, termed scIHF2, as a first example. ScIHF2 is made up of the alpha subunit of IHF which was inserted into the beta subunit at peptide bond Q39/G40 via two short linkers. The monomer behaves very similarly to the heterodimeric, parental IHF in biochemical and functional assays. Here, we describe an extension of this approach in which we shortened either one or both linkers by one amino acid, thereby generating three new variants termed scIHF1, 3, and 4. These variants exhibit distinct DNA-binding properties, different phenotypes in site-specific integrative and excisive recombination by phage lambda integrase in vitro, as well as in pSC101 replication assays in a DeltaIHF E. coli host. We also introduced a K45E substitution within the alpha domain of scIHF3 and based on electrophoretic mobility shift assays (EMSAs), argue that it significantly changes the DNA trajectory within the protein-DNA complex. Our results indicate that IHF's pleiotropic roles in DNA transactions inside E. coli require different types of high-precision DNA architectural activities. The scIHF variants described here will help to explore further how flexible these requirements are.

The Xq28 chromosomal band represents a C + G-rich region onto which several genes have been mapped. In most cases, the exact relationship between the mapped genes has not yet been established, and neither the regulatory nor the spacer regions between the various transcription units have been defined. In the region around the L1CAM gene (encoding L1 cell adhesion molecule), the transcription units appear, from preliminary analyses, to be quite compact. By sequencing the region at the 3' end of the recently found host cell factor 1-encoding gene (HCFC1), we report that the renin-binding protein-encoding gene (RBP) major transcription start point lies 2763 bp downstream from the 3′ end of HCFC1 and that both are transcribed in the same direction from the telomere to the centromere.

Sequence inspection identified several potential IHF binding sites adjacent to the Rhizobium leguminosarum dctA promoter. IHF protected the -30 to -76 region from DNase I digestion, but systematic error in quantitative assays suggested that this protein DNA interaction is complex. IHF stimulated DctD-mediated transcriptional activation from the R. leguminosarum dctA promoter both in vivo and in vitro. In contrast to R. leguminosarum dctA, the Sinorhizobium meliloti dctA promoter region was found to have a much weaker match to the consensus IHF binding site and a low affinity for IHF. Moreover, IHF had no effect on transcriptional activation from the S. meliloti dctA promoter in vitro. A base substitution was introduced into the IHF binding site of R. leguminosarum dtA that reduced the affinity of the promoter regulatory region for IHF by approximately 30-fold and resulted in an eight-fold decrease in transcriptional activation in both R. leguminosarum and S. meliloti. These data suggest that both rhizobial species have an IHF homolog that stimulates DctD-mediated transcriptional activation from the R. leguminosarum dctA promoter. Consistent with this hypothesis, a 12.5 kDa protein was identified from R. leguminosarum as a putative homolog of IHF subunit beta by immunoblotting and N-terminal sequence analysis.

We have shown that the B. stearothermophilus argCJBD genes form a single operon. In B. stearothermophilus, a specific repressor governs operon expression by binding to the argCo operator site overlapping the Parg promoter sequence ( Dion et al., 1997 ). Therefore, the enzymatic and transcriptional analyses performed in this work did not reflect the potential strength of Parg in the native host. For evaluation of the Parg promoter strength, E. coli was used as a host since its own ArgR repressor does not interact with the B. stearothermophilus heterologous operator. Parg-promoted argC gene expression dramatically increased, reaching up to 38% of the total protein in E. coli cells. An AT-rich sequence upstream of a −35 site of Parg was found to be indispensable for the promoter strength. Plasmids carrying the B. stearothermophilus argCJBD operon linked with its Parg/argCo region were unstable in E. coli. Stabilization of plasmids was achieved by repression of B. stearothermophilus arg genes through the action of the B. subtilis AhrC repressor.

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).

The Escherichia coli nucleoid-associated DNA-binding proteins HU and IHF are required for numerous biological processes, including phage growth (e.g., λ, o80, Mu and f1) and DNA replication. Here, we show that growth of T4 phage is inhibited both in hupA hupB and himA himD double mutants. The growth profile of triple mutants ( hupA hupB himA and hupA hupB himD ) suggests that HimD subunits can form homodimers, which are functionally competent for supporting in vivo growth of phage T4.

Histone-like proteins, such as HU and the integration host factor (IHF), are small, dimeric, DNA-bending proteins which play a role in maintaining constrained DNA structures and hence in regulating gene expression. Two different strategies were used to isolate the genes coding for Pseudomonas aeruginosa (Pa) HU and IHF, two proteins that we have previously isolated from a mucoid strain. By use of a PCR-based technique with oligodeoxyribonucleotides (oligos) designed from the N-terminal amino acid (aa) sequences of HU and the β-subunit of IHF, and Southern blot analyses, hupB and himD , encoding HU and IHFβ, respectively, have been cloned. The himA gene of Pa , encoding the α-subunit of IHF, was isolated using himA of Escherichia coli (Ec) as a probe in Southern blot analyses. The deduced hupB product (90 aa, 9 kDa) is 79% identical to HUβ and 61% to HUα of Ec . The predicted products of himA (100 aa, 11.5 kDa) and of himD (94 aa, 10.6 kDa) share 77 and 70% identity with IHFα and IHFβ of Ec , respectively. The promoter region of himD contains an IHF consensus sequence, as is the case for Ec himD

The integration host factor (IHF) participates in a diverse array of DNA transactions such as replication, recombination and gene expression. We describe a fast and very efficient isolation procedure which yields highly purified IHF in one Chromatographic step.

Integration host factor (IHF) is known to be required for the expression of early genes and formation of the transpososome of mutator phage Mu. Prophage Mucts62 was stably maintained at 30 degrees C and proliferated effectively after thermal induction at 42 degrees C in an Escherichia coli mutant defective in the histone-like H-NS and IHF proteins. No IHF activity was detected in cells lacking H-NS and IHF; cells could not be transformed with plasmid pCL1920, which is based on the pSC101 replicon whose replication requires IHF. No difference in the superhelical densities of the reporter plasmid was detected in the H-NS, IHF null mutant and parental cells. From these results it is concluded that IHF is not essential for Mu development. These results also suggest that H-NS may function as a silencer for Pe operon expression and that IHF overcomes the inhibitory effect of H-NS.

The gene hns of Escherichia coli K-12, which encodes the histone-like protein H-NS, was inactivated by insertion of a DNA (gene hph ) encoding hygromycin phosphotransferase. The growth rates of two mutants lacking either one or the other of the histone-like proteins, HU and IHF, were not affected by introduction of the hns mutation. However, cells depleted of HU, IHF and H-NS simultaneously, could not be constructed by P1 phage-mediated transduction. These results, together with our previous finding that cells deficient in both HU and IHF are viable at 30–37°C [Kano and Imamoto, Gene 89 (1990) 133–137] showed that E. coli cells deficient in any two of these three histone-like proteins are viable at 30–37°C, and suggested that simultaneous deficiency of all three of the proteins is lethal. There were no detectable differences in the levels of superhelicity of the reporter plasmids isolated from cells deficient in either IHF or H-NS, or from wild-type cells, but about 15% decrease in negative superhelicity was detected for the reporter plasmid isolated from cells lacking HU and lacking both HU and H-NS. However, the cryptic bgl operon, whose expression was reported to be regulated through topological changes of cellular DNA, could not be activated in cells depleted of HU or IHF. The bgl operon was expressed in cells depleted of both HU and H-NS as well as in cells depleted of H-NS. These results indicate that expression of the bgl operon is not affected by a change in superhelical density of the cellular DNA and suggest that its expression depends on some physiological processes other than topological alteration of DNA.

Escherichia coli mutants deficient in histone-like protein, HU, and integration host factor (IHF) were constructed and their growth characteristics were examined. Mutants deficient in both HU and IHF grew slowly and were filamentous at both 30 degrees C and 37 degrees C. These mutants scarcely grew in LB broth at 42 degrees C. They formed minute colonies on LB plates at 42 degrees C and no colonies at 46 degrees C, indicating temperature-sensitive (ts) growth. On the contrary, mutants deficient in either HU or IHF were not ts for growth. These results indicate that IHF compensates for the absence of HU and permits normal cell growth; this suggests functional similarity between HU and IHF.

The integration host factor (IHF) binds to a site (ihf) that overlaps the -35 region of the phage lambda late rightward promoter (p'R). This interaction represses p'R-promoted transcription, both in vivo and in vitro. In vivo repression was observed when a plasmid carrying both p'R and the galK reporter gene was transfected into IHF+ or IHF- hosts. In vitro repression of transcription by IHF was observed only with linear, but not with supercoiled wild-type p'R templates. When binding to ihf, IHF imposes a strong bend on the DNA and protects this site from cleavage by neocarzinostatin, pancreatic DNase I, and hydroxyl radicals, as assessed by footprinting experiments. Both the functional and nonfunctional p'R mutants, in which the upstream part of the -35 region was replaced by an EcoRI linker, show modified behavior toward IHF. Some are more sensitive to IHF-mediated repression, even in the supercoiled form, while others have lost their affinity for IHF. We conclude that IHF binding depends not only on the consensus ihf sequence, but also on a suitable combination of the sequences of both ihf and neighboring regions, together with the DNA conformation, which includes both natural and imposed bends in DNA and the degree of supercoiling. Based on most of the present data, it is difficult to predict the relationship between the ihf sequence and IHF interaction, since two very different sequences (less than 50% homology) show strong IHF binding, whereas very similar sequences (80-87% homology) show a very different behavior. However, the hydroxylradical footprinting data show that three A + T-rich sequences are protected by IHF: the central sequence, which overlaps the -35 region of p'R, and two flanking sequences removed by one helix turn. All three sequences are located on the same face of the helix, and the amino acid side chains of IHF seem to occupy the narrow minor groove. A novel consensus sequence is proposed.