Structure and function of the Pseudomonas putida integration host factor.

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.     

A rapid purification procedure of recombinant integration host factor from Escherichia coli

A rapid procedure for the large-scale isolation of recombinant integration host factor (IHF) protein from Escherichia coli is presented. The protein was overproduced in the E. coli K5746 strain, whose construction has already been described. The procedure consists of a mild extraction of protein and fractionation by ammonium sulfate. A single-step affinity chromatography on heparin-Sepharose provided very pure IHF protein. A Mono-S FPLC column was used to highly concentrate the pure IHF for crystallization trials. Attempts to crystallize IHF produced small stable crystals that have a large number of molecules in the asymmetric unit and to date diffract poorly. Further attempts to crystallize IHF under other conditions as well as in a complex with the putative DNA binding site are underway.     

A bacterial protein requirement for the bacteriophage lambda terminase reaction.

The bacteriophage lambda terminase enzyme cleaves the cohesive-end sites of lambda DNA to yield the protruding 5'-termini of the mature molecule. In vitro, this endonucleolytic event requires a protein factor which has been isolated and purified from extracts of uninfected E. coli. The terminase host factor (THF) is a heat stable basic protein of M.W. approximately 22,000. The integration host factor (IHF) protein of E. coli can efficiently substitute for THF in the terminase reaction; however, THF can be demonstrated to be physically present in, and isolated with full biological activity from extracts of cells defective or deficient in IHF.     

Purification of the integration host factor homolog of Rhodobacter capsulatus: cloning and sequencing of the hip gene, which encodes the beta subunit.

We describe a method for rapid purification of the integration host factor (IHF) homolog of Rhodobacter capsulatus that has allowed us to obtain microgram quantities of highly purified protein. R. capsulatus IHF is an alpha beta heterodimer similar to IHF of Escherichia coli. We have cloned and sequenced the hip gene, which encodes the beta subunit. The deduced amino acid sequence (10.7 kDa) has 46% identity with the beta subunit of IHF from E. coli. In gel electrophoretic mobility shift DNA binding assays, R. capsulatus IHF was able to form a stable complex in a site-specific manner with a DNA fragment isolated from the promoter of the structural hupSL operon, which contains the IHF-binding site. The mutated IHF protein isolated from the Hup- mutant IR4, which is mutated in the himA gene (coding for the alpha subunit), gave a shifted band of greater mobility, and DNase I footprinting analysis has shown that the mutated IHF interacts with the DNA fragment from the hupSL promoter region differently from the way that the wild-type IHF does.     

Purification and properties of Int-h, a variant protein involved in site-specific recombination of bacteriophage lambda

Under physiological conditions, integration of lambda DNA into the Escherichia coli chromosome requires the direct participation of only two proteins, the viral int gene product and E. coli integration host factor (IHF). A variant of the int gene has been isolated that permits integrative recombination in cells mutant for one of the two subunits of IHF (Miller, H.I., Mozola, M.A., and Friedman, D.I. (1980) Cell 20, 721-729). In the present work, we have purified Int-h, the product of this variant gene. In contrast to the wild-type int gene product (Int+), which produces almost no recombinants in the absence of IHF, purified Int-h protein sponsors reduced but significant levels of integrative recombination in the absence of any E. coli supplement. This shows that the int gene encodes all the information necessary for the elementary steps in recombination and implies that IHF functions as an accessory protein. When supplemented by IHF, recombination promoted by Int-h resembles that promoted by Int+ in kinetics, stoichiometry of Int and IHF, and nature of the recombinant product. Under these conditions, Int-h uses supercoiled DNA more effectively than nonsupercoiled DNA as a substrate for recombination, as does Int+. However, in the absence of IHF, Int-h recombines supercoiled and nonsupercoiled substrates identically, indicating that IHF is an important part of the mechanism that senses the supercoiled state of the substrate DNA during recombination. A surprising difference in recombination carried out by Int-h in the presence or absence of IHF concerns the degree to which sites on the same circle recombine with one another as opposed to sites on sister molecules. In the presence of IHF, Int-h favors intramolecular recombination, as does Int+. However, in the absence of IHF, Int-h almost exclusively promotes intermolecular recombination.     

Escherichia coli integration host factor bends the DNA at the ends of IS1 and in an insertion hotspot with multiple IHF binding sites.

The integration host factor of Escherichia coli (IHF) is a small, histone-like protein which participates in the integration of bacteriophage lambda into the E. coli chromosome and in a number of regulatory processes. Our recent footprinting analysis has shown that IHF binds specifically to the ends of the transposable element IS1, as well as to several sites within a short segment of the plasmid pBR322. We have extended our studies of the binding of the IHF molecule to these sites in vitro using a gel retardation assay. We report here that IHF bends the DNA upon binding, as judged from the strong cyclic dependence of the protein-induced mobility shift on the position of the binding site. Using cloned, synthetic ends of IS1 as substrates, we have found that some mutations within the conserved bases of the IHF consensus binding sequence abolish binding, and that alterations of the flanking sequences can greatly reduce IHF binding. The presence of multiple IHF sites on a single DNA fragment increases binding very little, indicating that IHF does not bind cooperatively in this complex. We discuss the possibility that DNA bending is related to the role IHF plays in forming and stabilizing nucleoprotein complexes, and suggest that bending at the IHF sites may be important to its diverse effects in the cell.     

Bending of the origin of replication of E. coli by binding of IHF at a specific site

In studies of DNA replication in Escherichia coli, an important question concerns the role of the initiator protein DnaA. This protein is known to bind to a specific 9-bp sequence in the origin of replication, but it is not understood how it can recognize another, relatively distant, 13-bp sequence that has no homology to the binding site but is where the DnaA protein serves its catalytic function in the initiation of DNA replication. This effect of DnaA might be achieved by bending of DNA in this region. I have searched for putative binding sites for integration host factor (IHF), a protein known to bend DNA. Here I report the finding of an IHF binding site in the E. coli origin and present direct evidence that IHF binds and causes DNA bending in this region. On the basis of these results I propose a model wherein formation of a higher-order nucleoprotein structure would facilitate the action of DnaA protein in the initiation events.     

The expression of nifB gene from Herbaspirillum seropedicae is dependent upon the NifA and RpoN proteins

The putative nifB promoter region of Herbaspirillum seropedicae contained two sequences homologous to NifA-binding site and a -24/-12 type promoter. A nifB::lacZ fusion was assayed in the backgrounds of both Escherichia coli and H. seropedicae. In E. coli, the expression of nifB::lacZ occurred only in the presence of functional rpoN and Klebsiella pneumoniae nifA genes. In addition, the integration host factor (IHF) stimulated the expression of the nifB::lacZ fusion in this background. In H. seropedicae, nifB expression occurred only in the absence of ammonium and under low levels of oxygen, and it was shown to be strictly dependent on NifA. DNA band shift experiments showed that purified K. pneumoniae RpoN and E. coli IHF proteins were capable of binding to the nifB promoter region, and in vivo dimethylsulfate footprinting showed that NifA binds to both NifA-binding sites. These results strongly suggest that the expression of the nifB promoter of H. seropedicae is dependent on the NifA and RpoN proteins and that the IHF protein stimulates NifA activation of nifB promoter.     

Role of architectural elements in combinatorial regulation of initiation of DNA replication in Escherichia coli

Bending of DNA is a prerequisite of site-specific recombination and gene expression in many regulatory systems involving the assembly of specific nucleoprotein complexes. We have investigated how the uniquely clustered Dam methylase sites, GATCs, in the origin of Escherichia coli replication ( oriC  ) and their methylation status modulate the geometry of oriC and its interaction with architectural proteins, such as integration host factor (IHF), factor for inversion stimulation (Fis) and DnaA initiator protein. We note that 3 of the 11 GATC sites at oriC are strategically positioned within the IHF protected region. Methylation of the GATCs enhances IHF binding and alters the IHF-induced bend at oriC . GATC motifs also contribute to intrinsic DNA curvature at oriC and the degree of bending is modulated by methylation. The IHF-induced bend at oriC is further modified by Fis protein and IHF affinity for its binding site may be impaired by protein(s) binding to GATCs within the IHF site. Thus, GATC sites at oriC affect the DNA conformation and GATCs, in conjunction with the protein-induced bends, are critical cis -acting elements in specifying proper juxtapositioning of initiation factors in the early steps of DNA replication.     

Integration host factor is required for positive regulation of the tdc operon of Escherichia coli.

A 14-bp segment in the promoter region of the tdcABC operon of Escherichia coli shows sequence identity with the consensus binding site for the E. coli integration host factor (IHF). In an himA (IHF-deficient) strain, expression of beta-galactosidase from a tdcB'-'lacZ protein fusion plasmid was about 10% of that seen with an isogenic himA+ strain. Threonine dehydratase activity from the chromosomal tdcB gene in the himA mutant was also about 10% of the wild-type enzyme level. Two different mutations introduced into the putative IHF-binding site in the fusion plasmid greatly reduced the plasmid-coded beta-galactosidase activity in cells containing IHF. In vitro gel retardation and DNase I footprinting analyses showed binding of purified IHF to the wild-type but not to the mutant promoter. IHF protected a 31-bp region between -118 and -88 encompassing the conserved IHF consensus sequence. These results suggest that efficient expression of the tdc operon in vivo requires a functional IHF and an IHF-binding site in the tdc promoter.