Integration Host Factor Binding Research Articles

TrwC-Mediated Site-Specific Recombination Is Controlled by Host Factors Altering Local DNA Topology

R388 conjugative relaxase TrwC acts as a site-specific recombinase, promoting recombination between two cognate oriTs on double-stranded DNA substrates. The relaxosome component TrwA is also required for efficient recombination. In this work we present data on the in vivo control of this reaction by host proteins that affect local DNA topology. In the absence of TrwA, binding of integration host factor (IHF) to the oriT keeps the recombination levels low, probably by keeping the relaxosome complex, formed at recombination locus 1, in a "closed" conformation. In an IHF-deficient (IHF-) background, the formation of a transcript elongation complex at this locus still hampers recombination. A mutation abating the promoter sequence at locus 1, or repression of transcription by exposure to rifampin, lifts the inhibition imposed on recombination in an IHF- background. We also observe an increase in conjugation efficiency under these conditions. Relieving the inhibition imposed by these host factors allows efficient levels of recombination between short oriT loci in the absence of TrwA. The presence of TrwA counteracts these inhibitory effects. TrwA would then activate both recombination and conjugation by switching the conformation of the relaxosome to an "open" form that exposes single-stranded DNA at the nic site, promoting the initial TrwC nicking reaction.

P1 Partition Complex Assembly Involves Several Modes of Protein-DNA Recognition

Assembly of P1 plasmid partition complexes at the partition site, parS, is nucleated by a dimer of P1 ParB and Escherichia coli integration host factor (IHF), which promotes loading of more ParB dimers and the pairing of plasmids during the cell cycle. ParB binds several copies of two distinct recognition motifs, known as A- and B-boxes, which flank a bend in parS created by IHF binding. The recent crystal structure of ParB bound to a partial parS site revealed two relatively independent DNA-binding domains and raised the question of how a dimer of ParB recognizes its complicated arrangement of recognition motifs when it loads onto the full parS site in the presence of IHF. In this study, we addressed this question by examining ParB binding activities to parS mutants containing different combinations of the A- and B-box motifs in parS. Binding was measured to linear and supercoiled DNA in electrophoretic and filter binding assays, respectively. ParB showed preferences for certain motifs that are dependent on position and on plasmid topology. In the simplest arrangement, one motif on either side of the bend was sufficient to form a complex, although affinity differed depending on the motifs. Therefore, a ParB dimer can load onto parS in different ways, so that the initial ParB-IHF-parS complex consists of a mixture of different orientations of ParB. This arrangement supports a model in which parS motifs are available for interas well as intramolecular parS recognition.

Pressure dissociation of integration host factor-DNA complexes reveals flexibility-dependent structural variation at the protein-DNA interface

E. coli Integration host factor (IHF) condenses the bacterial nucleoid by wrapping DNA. Previously, we showed that DNA flexibility compensates for structural characteristics of the four consensus recognition elements associated with specific binding (Aeling et al., J. Biol. Chem. 281, 39236–39248, 2006). If elements are missing, high-affinity binding occurs only if DNA deformation energy is low. In contrast, if all elements are present, net binding energy is unaffected by deformation energy. We tested two hypotheses for this observation: in complexes containing all elements, (1) stiff DNA sequences are less bent upon binding IHF than flexible ones; or (2) DNA sequences with differing flexibility have interactions with IHF that compensate for unfavorable deformation energy. Time-resolved Förster resonance energy transfer (FRET) shows that global topologies are indistinguishable for three complexes with oligonucleotides of different flexibility. However, pressure perturbation shows that the volume change upon binding is smaller with increasing flexibility. We interpret these results in the context of Record and coworker's model for IHF binding (J. Mol. Biol. 310, 379–401, 2001). We propose that the volume changes reflect differences in hydration that arise from structural variation at IHF–DNA interfaces while the resulting energetic compensation maintains the same net binding energy.

Multiple co‐regulatory elements and IHF are necessary for the control of fimB expression in response to sialic acid and N‐acetylglucosamine in Escherichia coli K‐12

Expression of the FimB recombinase, and hence the OFF-to-ON switching of type 1 fimbriation in Escherichia coli, is inhibited by sialic acid (Neu(5)Ac) and by GlcNAc. NanR (Neu(5)Ac-responsive) and NagC (GlcNAc-6P-responsive) activate fimB expression by binding to operators (O(NR) and O(NC1) respectively) located more than 600 bp upstream of the fimB promoter within the large (1.4 kb) nanC-fimB intergenic region. Here it is demonstrated that NagC binding to a second site (O(NC2)), located 212 bp closer to fimB, also controls fimB expression, and that integration host factor (IHF), which binds midway between O(NC1) and O(NC2), facilitates NagC binding to its two operator sites. In contrast, IHF does not enhance the ability of NanR to activate fimB expression in the wild-type background. Neither sequences up to 820 bp upstream of O(NR), nor those 270 bp downstream of O(NC2), are required for activation by NanR and NagC. However, placing the NanR, IHF and NagC binding sites closer to the fimB promoter enhances the ability of the regulators to activate fimB expression. These results support a refined model for how two potentially key indicators of host inflammation, Neu(5)Ac and GlcNAc, regulate type 1 fimbriation.

Direct observation of DNA bending/unbending kinetics in complex with DNA-bending protein IHF

Regulation of gene expression involves formation of specific protein-DNA complexes in which the DNA is often bent or sharply kinked. Kinetics measurements of DNA bending when in complex with the protein are essential for understanding the molecular mechanism that leads to precise recognition of specific DNA-binding sites. Previous kinetics measurements on several DNA-bending proteins used stopped-flow techniques that have limited time resolution of few milliseconds. Here we use a nanosecond laser temperature-jump apparatus to probe, with submillisecond time resolution, the kinetics of bending/unbending of a DNA substrate bound to integration host factor (IHF), an architectural protein from Escherichia coli. The kinetics are monitored with time-resolved FRET, with the DNA substrates end-labeled with a FRET pair. The temperature-jump measurements, in combination with stopped-flow measurements, demonstrate that the binding of IHF to its cognate DNA site involves an intermediate state with straight or, possibly, partially bent DNA. The DNA bending rates range from approximately 2 ms(-1) at approximately 37 degrees C to approximately 40 ms(-1) at approximately 10 degrees C and correspond to an activation energy of approximately 14 +/- 3 kcal/mol. These rates and activation energy are similar to those of a single A:T base pair opening inside duplex DNA. Thus, our results suggest that spontaneous thermal disruption in base-paring, nucleated at an A:T site, may be sufficient to overcome the free energy barrier needed to partially bend/kink DNA before forming a tight complex with IHF.

Indirect Recognition in Sequence-specific DNA Binding by Escherichia coli Integration Host Factor: THE ROLE OF DNA DEFORMATION ENERGY

Integration host factor (IHF) is a bacterial histone-like protein whose primary biological role is to condense the bacterial nucleoid and to constrain DNA supercoils. It does so by binding in a sequence-independent manner throughout the genome. However, unlike other structurally related bacterial histone-like proteins, IHF has evolved a sequence-dependent, high affinity DNA-binding motif. The high affinity binding sites are important for the regulation of a wide range of cellular processes. A remarkable feature of IHF is that it employs an indirect readout mechanism to bind and wrap DNA at both the nonspecific and high affinity (sequence-dependent) DNA sites. In this study we assessed the contributions of pre-formed and protein-induced DNA conformations to the energetics of IHF binding. Binding energies determined experimentally were compared with energies predicted for the IHF-induced deformation of the DNA helix (DNA deformation energy) in the IHF-DNA complex. Combinatorial sets of de novo DNA sequences were designed to systematically evaluate the influence of sequence-dependent structural characteristics of the conserved IHF recognition elements of the consensus DNA sequence. We show that IHF recognizes pre-formed conformational characteristics of the consensus DNA sequence at high affinity sites, whereas at all other sites relative affinity is determined by the deformational energy required for nearest-neighbor base pairs to adopt the DNA structure of the bound DNA-IHF complex.

TheEscherichia coliK-12 NarL and NarP Proteins Insulate thenrfPromoter from the Effects of Integration Host Factor

The Escherichia coli K-12 nrf operon promoter can be activated fully by the FNR protein (regulator of fumarate and nitrate reduction) binding to a site centered at position -41.5. FNR-dependent transcription is suppressed by integration host factor (IHF) binding at position -54, and this suppression is counteracted by binding of the NarL or NarP response regulator at position -74.5. The E. coli acs gene is transcribed from a divergent promoter upstream from the nrf operon promoter. Transcription from the major acsP2 promoter is dependent on the cyclic AMP receptor protein and is modulated by IHF and Fis binding at multiple sites. We show that IHF binding to one of these sites, located at position -127 with respect to the nrf promoter, has a positive effect on nrf promoter activity. This activation is dependent on the face of the DNA helix, independent of IHF binding at other locations, and found only when NarL/NarP are not bound at position -74.5. Binding of NarL/NarP appears to insulate the nrf promoter from the effects of IHF. The acs-nrf regulatory region is conserved in other pathogenic E. coli strains and related enteric bacteria but differs in Salmonella enterica serovar Typhimurium.

Molecular Characterization of Pantoea stewartii subsp. stewartii HrpY, a Conserved Response Regulator of the Hrp Type III Secretion System, and its Interaction with the hrpS Promoter

Pantoea stewartii subsp. stewartii is a bacterial pathogen of corn. Its pathogenicity depends on the translocation of effector proteins into host cells by the Hrp type III secretion system. We previously showed by genetic analysis that the HrpX sensor kinase and the HrpY response regulator are at the head of a complex cascade of regulators controlling hrp/hrc secretion and wts effector genes. This cascade also includes the HrpS response regulator and the HrpL alternative sigma factor. These regulators are shared among many important plant pathogens in the genera Pantoea, Erwinia, and Pseudomonas. In this study, we dissect the regulatory elements in the hrpS promoter region, using genetic and biochemical approaches, and show how it integrates various environmental signals, only some of which are dependent on phosphorylation of HrpY. Primer extension located the transcriptional start site of hrpS at a sigma70 promoter 601 bp upstream of the open reading frame. Electrophoretic mobility shift assays and DNase I footprinting analysis demonstrated that HrpY binds to conserved regulatory elements immediately adjacent to this promoter, and its binding affinity was increased by phosphorylation at D57. A consensus sequence for the two direct repeats bound by HrpY is proposed. Deletion analysis of the promoter region revealed that both the HrpY binding site and additional sequences farther upstream, including a putative integration host factor binding site, are required for hrpS expression. This finding suggests that other unknown regulatory proteins may act cooperatively with HrpY.

Regulatory components at thecsgDpromoter – additional roles for OmpR and integration host factor and role of the 5′ untranslated region

CsgD is a master regulator of multicellular behaviour in Salmonella enterica serovar Typhimurium. Expression of CsgD is highly regulated on the transcriptional level. A nucleo-protein complex had been defined where the global regulators OmpR and integration host factor (IHF) bind up- and downstream of the csgD core promoter. In this study, the nucleo-protein complex of PcsgD was extended through characterization of additional OmpR and IHF binding sites that influence the transcriptional activity of the csgD promoter. Furthermore, the role of the 174 bp long 5'-untranslated region on transcriptional activity was defined.

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.

Structural and Genetic Analyses Reveal a Key Role in Prophage Excision for the TorI Response Regulator Inhibitor

TorI (Tor inhibition protein) has been identified in Escherichia coli as a protein inhibitor acting through protein-protein interaction with the TorR response regulator. This interaction, which does not interfere with TorR DNA binding activity, probably prevents the recruitment of RNA polymerase to the torC promoter. In this study we have solved the solution structure of TorI, which adopts a prokaryotic winged-helix arrangement. Despite no primary sequence similarity, the three-dimensional structure of TorI is highly homologous to the (lambda)Xis, Mu bacteriophage repressor (MuR-DBD), and transposase (MuA-DBD) structures. We propose that the TorI protein is the structural missing link between the (lambda)Xis and MuR proteins. Moreover, in vivo assays demonstrated that TorI plays an essential role in prophage excision. Heteronuclear NMR experiments and site-directed mutagenesis studies have pinpointed out key residues involved in the DNA binding activity of TorI. Our findings suggest that TorI-related proteins identified in various pathogenic bacterial genomes define a new family of atypical excisionases.

Dual Role of Response Regulator StyR in Styrene Catabolism Regulation

In Pseudomonas fluorescens ST, the promoter of the styrene catabolic operon, PstyA, is induced by styrene and repressed by the addition of preferred carbon sources. PstyA is regulated by the StyS/StyR two-component system. The integration host factor (IHF) also plays a positive role in PstyA regulation. Three distinct StyR binding sites, which have different affinities for this response regulator, have been characterized on PstyA. The high-affinity StyR binding site (STY2) is necessary for promoter activity. The DNA region upstream of STY2 contains a lower-affinity StyR binding site, STY1, that partially overlaps the IHF binding site. Deletion of this region, designated URE (upstream regulatory element), has a dual effect on the PstyA promoter, decreasing the styrene-dependent activity and partially relieving the glucose repression. The lowest-affinity StyR binding site (STY3) is located downstream of the transcription start point. Deletion of the URE region and inactivation of the STY3 site completely abolished glucose-mediated repression of PstyA. In the proposed model StyR can act either as an activator or as a repressor, depending on which sites it occupies in the different growth conditions. We suggest that the cellular levels of phosphorylated StyR, as determined by StyS sensor kinase activity, and the interplay of this molecule with IHF modulate the activity of the promoter in different growth conditions.

Modulation of the mtrCDE‐encoded efflux pump gene complex of Neisseria meningitidis due to a Correia element insertion sequence

The mtr (multiple transferable resistance) gene complex in Neisseria gonorrhoeae encodes an energy-dependent efflux pump system that is responsible for export of anti-bacterial hydrophobic agents. Expression of the mtrCDE operon in gonococci is negatively regulated by the MtrR protein. Hydrophobic agent resistance mediated by the mtr system is also inducible, which results from an AraC-like protein termed MtrA. In this work, we identified and characterized a pump similar to the gonococcal mtr system in various strains of Neisseria meningitidis. Unlike the situation with gonococci, the mtr system in meningococci is not subject to the MtrR or MtrA regulatory schemes. An analysis of the promoter region of the mtrCDE operon in a panel of meningococcal strains revealed the presence of one or two classes of insertion sequence elements. A 155-159 bp insertion sequence element known as the Correia element, previously identified elsewhere in the gonococcal and meningococcal genomes, was present in the mtrCDE promoter region of all meningococcal strains tested. In addition to the Correia element, a minority of strains had a tandemly linked, intact copy of IS1301. As described previously, a binding site for the integration host factor (IHF) was present at the centre of the Correia element upstream of mtrCDE genes. IHF was found to bind specifically to this site and deletion of the IHF binding site enhanced mtrC transcription. We also identified a post-transcriptional regulation of the mtrCDE transcript by cleavage in the inverted repeat of the Correia element, as previously described by Mazzone et al. [Gene278: 211-222 (2001)] and De Gregorio et al. [Biochim Biophys Acta 1576: 39-44 (2002)]for other Correia element. We conclude that the mtr efflux system in meningococci is subject to transcriptional regulation by IHF and post-transcriptional regulation by cleavage in the inverted repeat of the Correia element.

Integration host factor is involved in transcriptional regulation of theBrucella abortus virBoperon

Type IV secretion systems (T4SSs) are multicomponent machineries that play an essential role in pathogenicity of many facultative intracellular bacteria. The virB operon of Brucella abortus codes for a T4SS essential for virulence and intracellular multiplication. Here, virB expression analyses carried out using lacZ transcriptional fusions showed that virB promoter (PvirB) is temporally activated within J774 cells. Primer extension experiments revealed that virB transcription starts at 27 bp upstream of the first gene of the virB operon. Structural analyses showed that PvirB and regulatory sequences involved in intracellular regulation span 430 bp upstream of the transcription start site. A protein able to bind PvirB was isolated and identified. This protein, homologue to integration host factor (IHF), specifically interacts with PvirB and induces a DNA bending with an angle of 50.36 degrees . DNAse I footprinting experiments showed that IHF protects a 51 bp region that contains two overlapped IHF binding consensus motifs. VirB expression experiments carried out with PvirB-lacZ fusions showed that in B. abortus IHF participates in the regulation of PvirB activity during the intracellular and vegetative growth in different media. A mutant strain with a 20 bp IHF binding site replacement failed to turn on the virB operon during the initial stages of macrophage infection and displayed severe intracellular multiplication defects. These data indicate that IHF plays a key role during intracellular virB operon expression being required for the biogenesis of the endoplasmic reticulum-derived replicative vacuole.

IHF is the limiting host factor in transposition of Pseudomonas putida transposon Tn4652 in stationary phase.

Transpositional activity of mobile elements is not constant. Conditional regulation of host factors involved in transposition may severely change the activity of mobile elements. We have demonstrated previously that transposition of Tn4652 in Pseudomonas putida is a stationary phase-specific event, which requires functional sigma S (Ilves et al., 2001, J Bacteriol 183: 5445-5448). We hypothesized that integration host factor (IHF), the concentration of which is increased in starving P. putida, might contribute to the transposition of Tn4652 as well. Here, we demonstrate that transposition of Tn4652 in stationary phase P. putida is essentially limited by the amount of IHF. No transposition of Tn4652 occurs in a P. putida ihfA-defective strain. Moreover, overexpression of IHF results in significant enhancement of transposition compared with the wild-type strain. This indicates that the amount of IHF is a bottleneck in Tn4652 transposition. Gel mobility shift and DNase I footprinting studies revealed that IHF is necessary for the binding of transposase to both transposon ends. In vitro, transposase can bind to inverted repeats of transposon only after the binding of IHF. The results obtained in this study indicate that, besides sigma S, IHF is another host factor that is implicated in the elevation of transposition in stationary phase.

On the Role of H-NS in the Organization of Bacterial Chromatin: From Bulk to Single Molecules and Back…

The chromosomal DNA in the bacterium Escherichia coli is thought to be organized and compacted at least in part as a consequence of the interaction with so-called histone-like or nucleoid-associated proteins. The groups of Stavans and Oppenheim have recently embarked on an ambitious project which aims to quantify the compactive effects of the various members of this group of proteins using magnetic tweezers (Ali et al., 2001). Eventually this could lead to a better understanding of how these proteins work together in the formation of a compact nucleoid.

A dual binding site for integration host factor and the response regulator CtrA inside the Caulobacter crescentus replication origin.

The response regulator CtrA controls chromosome replication by binding to five sites, a, b, c, d, and e, inside the Caulobacter crescentus replication origin (Cori). In this study, we demonstrate that integration host factor (IHF) binds Cori over the central CtrA binding site c. Surprisingly, IHF and CtrA share DNA recognition sequences. Rather than promoting cooperative binding, IHF binding hinders CtrA binding to site c and nearby site d. Unlike other CtrA binding sites, DNA mutations in the CtrA c/IHF site uniquely impair autonomous Cori plasmid replication. These mutations also alter transcription from distant promoters more than 100 bp away. When the CtrA c/IHF site was deleted from the chromosome, these cells grew slowly and became selectively intolerant to a CtrA phosphor-mimic allele (D51E). Since CtrA protein concentration decreases during the cell cycle as IHF protein concentration increases, we propose a model in which IHF displaces CtrA in order to bend Cori and promote efficient chromosome replication.

Recruitment of σ54-RNA Polymerase to the Pu Promoter of Pseudomonas putida through Integration Host Factor-mediated Positioning Switch of α Subunit Carboxyl-terminal Domain on an UP-like Element

The interactions between the sigma54-containing RNA polymerase (sigma54-RNAP) and the region of the Pseudomonas putida Pu promoter spanning from the enhancer to the binding site for the integration host factor (IHF) were analyzed both by DNase I and hydroxyl radical footprinting. A short Pu region centered at position -104 was found to be involved in the interaction with sigma54-RNAP, both in the absence and in the presence of IHF protein. Deletion or scrambling of the -104 region strongly reduced promoter affinity in vitro and promoter activity in vivo, respectively. The reduction in promoter affinity coincided with the loss of IHF-mediated recruitment of the sigma54-RNAP in vitro. The experiments with oriented-alpha sigma54-RNAP derivatives containing bound chemical nuclease revealed interchangeable positioning of only one of the two alpha subunit carboxyl-terminal domains (alphaCTDs) both at the -104 region and in the surroundings of position -78. The addition of IHF resulted in perfect position symmetry of the two alphaCTDs. These results indicate that, in the absence of IHF, the sigma54-RNAP asymmetrically uses only one alphaCTD subunit to establish productive contacts with upstream sequences of the Pu promoter. In the presence of IHF-induced curvature, the closer proximity of the upstream DNA to the body of the sigma54-RNAP can allow the other alphaCTD to be engaged in and thus favor closed complex formation.

IHF and HU stimulate assembly of pre-replication complexes at Escherichia coli oriC by two different mechanisms.

Pre-replication complexes (pre-RC) assemble on replication origins and unwind DNA in the presence of chromatin proteins. As components of Escherichia coli pre-RC, two histone-like proteins HU and IHF (integration host factor), stimulate initiator DnaA-catalysed unwinding of the chromosomal replication origin, oriC. Using in vivo footprint analysis just before DNA synthesis initiates, we detect IHF binding coincident with a shift of DnaA to weaker central oriC sites. Integration host factor redistributed pre-bound DnaA to identical sites in vitro. HU did not redistribute DnaA, but suppressed binding specifically at I3. These results suggest that different pathways mediated by bacterial chromatin proteins exist to regulate pre-RC assembly and unwind oriC.

Transposon-like Correia elements: structure, distribution and genetic exchange between pathogenic Neisseria sp.

Correia elements are a prominent feature of all four Neisseria genome sequences. We report an in silico analysis of the structure and genomic distribution of these elements and some preliminary biochemical data. Correia elements fall into four major families, distinguished by a 50 bp internal deletion and five point mutations. The elements resemble a transposon with 25 bp inverted repeats and a TA duplication at the target site. Within the element there is a functional integration host factor binding site. The genomic distribution of Correia elements is essentially random except for some small Correia-less regions apparently acquired by horizontal transfer. Phylogenetic analysis suggests that their presence predates the divergence of Neisseria meningitidis and Neisseria gonorrhoeae.