Role Of Integration Host Factor Research Articles

The role of integration host factor in biofilm and virulence of high-alcohol-producing Klebsiella pneumoniae.

Klebsiella pneumoniae is a well-known human nosocomial pathogen with an arsenal of virulence factors, including capsular polysaccharides (CPS), fimbriae, flagella, and lipopolysaccharides (LPS). Our previous study found that alcohol acted as an essential virulence factor for high-alcohol-producing K. pneumoniae (HiAlc Kpn). Integration host factor (IHF) is a nucleoid-associated protein that functions as a global virulence regulator in Escherichia coli. However, the regulatory role of IHF in K. pneumoniae remains unknown. In the present study, we found that deletion of ihfA or ihfB resulted in a slight defect in bacterial growth, a severe absence of biofilm formation and cytotoxicity, and a significant reduction in alcohol production. RNA sequencing differential gene expression analysis showed that compared with the wild-type control, the expression of many virulence factor genes was downregulated in ΔihfA and ΔihfB strains, such as those related to CPS (rcsA, galF, wzi, and iscR), LPS (rfbABCD), type I and type III fimbriae (fim and mrk operon), cellulose (bcs operon), iron transporter (feoABC, fhuA, fhuF, tonB, exbB, and exbD), quorum sensing (lsr operon and sdiA), type II secretion system (T2SS) and type VI secretion system (T6SS) (tssG, hcp, and gspE). Of these virulence factors, CPS, LPS, fimbriae, and cellulose are involved in biofilm formation. In addition, IHF could affect the alcohol production by regulating genes related to glucose intake (ptsG), pyruvate formate-lyase, alcohol dehydrogenase, and the tricarboxylic acid (TCA) cycle. Our data provided new insights into the importance of IHF in regulating the virulence of HiAlc Kpn. IMPORTANCE Klebsiella pneumoniae is a well-known human nosocomial pathogen that causes various infectious diseases, including urinary tract infections, hospital-acquired pneumonia, bacteremia, and liver abscesses. Our previous studies demonstrated that HiAlc Kpn mediated the development of nonalcoholic fatty liver disease by producing excess endogenous alcohol in vivo. However, the regulators regulating the expression of genes related to metabolism, biofilm formation, and virulence of HiAlc Kpn remain unclear. In this study, the regulator IHF was found to positively regulate biofilm formation and many virulence factors including CPS, LPS, type I and type III fimbriae, cellulose, iron transporter, AI-2 quorum sensing, T2SS, and T6SS in HiAlc Kpn. Furthermore, IHF positively regulated alcohol production in HiAlc Kpn. Our results suggested that IHF could be a potential drug target for treating various infectious diseases caused by K. pneumoniae. Hence, the regulation of different virulence factors by IHF in K. pneumoniae requires further investigation.

The Role of Integration Host Factor in Escherichia coli Persister Formation.

ABSTRACTPersisters represent a small subpopulation of cells that are tolerant of killing by antibiotics and are implicated in the recalcitrance of chronic infections to antibiotic therapy. One general theme has emerged regarding persisters formed by different bacterial species, namely, a state of relative dormancy characterized by diminished activity of antibiotic targets. Within this framework, a number of studies have linked persister formation to stochastic decreases in energy-generating components, leading to low ATP and target activity. In this study, we screen knockouts in the main global regulators of Escherichia coli for their effect on persisters. A knockout in integration host factor (IHF) had elevated ATP and a diminished level of persisters. This was accompanied by an overexpression of isocitrate dehydrogenase (Icd) and a downregulation of isocitrate lyase (AceA), two genes located at the bifurcation between the tricarboxylic acid (TCA) cycle and the glyoxylate bypass. Using a translational ihfA-mVenus fusion, we sort out rare bright cells, and this subpopulation is enriched in persisters. Our results suggest that noise in the expression of ihf produces rare cells with low Icd/high AceA, diverting substrates into the glyoxylate bypass, which decreases ATP, leading to antibiotic-tolerant persisters. We further examine noise in a simple model, the lac operon, and show that a knockout of the lacI repressor increases expression of the operon and decreases persister formation. Our results suggest that noise quenching by overexpression serves as a general approach to determine the nature of persister genes in a variety of bacterial species and conditions.

IHF stabilizes pathogenicity island I of uropathogenic Escherichia coli strain 536 by attenuating integrase I promoter activity

Pathogenicity islands (PAIs) represent horizontally acquired chromosomal regions and encode their cognate integrase, which mediates chromosomal integration and excision of the island. These site-specific recombination reactions have to be tightly controlled to maintain genomic stability, and their directionality depends on accessory proteins. The integration host factor (IHF) and the factor for inversion stimulation (Fis) are often involved in recombinogenic complex formation and controlling the directionality of the recombination reaction. We investigated the role of the accessory host factors IHF and Fis in controlling the stability of six PAIs in uropathogenic Escherichia coli strain 536. By comparing the loss of individual PAIs in the presence or absence of IHF or Fis, we showed that IHF specifically stabilized PAI I536 and that in particular the IHFB subunit seems to be important for this function. We employed complex genetic studies to address the role of IHF in PAI I536-encoded integrase (IntI) expression. Based on different YFP-reporter constructs and electrophoretic mobility shift assays we demonstrated that IntI acts a strong repressor of its own synthesis, and that IHF binding to the intI promoter region reduces the probability of intI promoter activation. Our results extend the current knowledge of the role of IHF in controlling directionality of site specific recombination reactions and thus PAI stability.

Integration Host Factor Is Required for RpoN-Dependent hrpL Gene Expression and Controls Motility by Positively Regulating rsmB sRNA in Erwinia amylovora.

Erwinia amylovora requires an hrp-type III secretion system (T3SS) to cause disease. It has been reported that HrpL, the master regulator of T3SS, is transcriptionally regulated by sigma factor 54 (RpoN), YhbH, and HrpS. In this study, the role of integration host factor (IHF) in regulating hrpL and T3SS gene expression was investigated. IHF is a nucleoid-associated protein that regulates gene expression by influencing nucleoid structure and DNA bending. Our results showed that both ihfA and ihfB mutants of E. amylovora did not induce necrotic lesions on pear fruits. Growth of both mutants was greatly reduced, and expression of the hrpL and T3SS genes was significantly down-regulated as compared with those of the wild type. In addition, expression of the ihfA, but not the ihfB gene, was under auto-suppression by IHF. Furthermore, both ihfA and ihfB mutants were hypermotile, due to significantly reduced expression of small RNA (sRNA) rsmB. Electrophoresis mobility shift assay further confirmed that IHF binds to the promoters of the hrpL and ihfA genes, as well as the rsmB sRNA gene. These results indicate that IHF is required for RpoN-dependent hrpL gene expression and virulence, and controls motility by positively regulating the rsmB sRNA in E. amylovora.

Integration host factor is required for the induction of acid resistance in Escherichia coli.

Integration host factor (IHF) is a heterodimeric histone-like DNA-binding protein that participates in many cellular functions. Many systems and global regulators of acid resistance (AR) under strongly acidic conditions have been reported, but the role of IHF has not been examined. In the present study, we report that IHF is necessary for the induction of AR in Escherichia coli. At acidic pH, a ∆ihfA∆ifhB-mutant strain was found to have significantly depressed levels of transcription of the arginine decarboxylase gene (adiA) and of translation of the lysine/cadaverine antiporter gene (cadB), when compared with wild-type strain. Thus, IHF induces the arginine- and lysine-dependent AR. These results indicate that in E. coli, by combined transcriptional and translational controls of gene expression, IHF activates expression of a specific set of genes required for survival at extremely acidic pH.

Targeting bacterial integration host factor to disrupt biofilms associated with cystic fibrosis

This study aims to identify whether the bacterial protein, Integration Host Factor (IHF), is present within sputum solids collected from cystic fibrosis (CF) patients and thus might contribute to the structural stability of biofilms within the lungs. The presence of IHF in sputum was determined by immunohistochemistry. The role of IHF in stabilizing biofilms within sputum was tested in vitro wherein anti-IHF was used to attempt to dissolve sputum solids. Thirty-seven of 44 sputum samples (84%) were positive for anti-IHF staining. Treatment with anti-IHF or DNase of 6 representative samples, dissolved sputum solids significantly better than treatment with normal saline in vitro, and strong synergism was observed when these agents were used in combination. IHF was detected in the majority of sputum samples from patients with CF and in vitro treatment with anti-IHF induced dissolution of sputum solids. These data support further investigation of IHF as a potential therapeutic target for patients with CF.

Role of Integration Host Factor in the Transcriptional Activation of Flagellar Gene Expression in Caulobacter crescentus

In the Caulobacter crescentus predivisional cell, class III and IV flagellar genes, encoding the extracytoplasmic components of the flagellum, are transcribed in the nascent swarmer compartment. This asymmetric expression pattern is attributable to the compartmentalized activity of the sigma54-dependent transcriptional activator FlbD. Additionally, these temporally transcribed flagellar promoters possess a consensus sequence for the DNA-binding protein integration host factor (IHF), located between the upstream FlbD binding site and the promoter sequences. Here, we deleted the C. crescentus gene encoding the beta-subunit of the IHF, ihfB (himD), and examined the effect on flagellar gene expression. The DeltaihfB strain exhibited a mild defect in cell morphology and impaired motility. Using flagellar promoter reporter fusions, we observed that expression levels of a subset of class III flagellar promoters were decreased by the loss of IHF. However, one of these promoters, fliK-lacZ, exhibited a wild-type cell cycle-regulated pattern of expression in the absence of IHF. Thus, IHF is required for maximal transcription of several late flagellar genes. The DeltaihfB strain was found to express significantly reduced amounts of the class IV flagellin, FljL, as a consequence of reduced transcriptional activity. Our results indicate that the motility defect exhibited by the DeltaihfB strain is most likely attributable to its failure to accumulate the class IV-encoded 27-kDa flagellin subunit, FljL.

A role for the leucine-responsive regulatory protein and integration host factor in the regulation of the Salmonella plasmid virulence (spv ) locus in Salmonella typhimurium.

The Salmonella plasmid virulence (spv ) genes of Salmonella typhimurium are activated at the level of transcription as the bacteria enter stationary phase in vitro or in response to signals received during intracellular growth. Activation requires the LysR-like transcription factor SpvR and the alternative sigma factor RpoS. In this report, we show by biochemical and genetic analyses that two chromosomally encoded DNA-binding proteins contribute to the control of spv expression. These are the integration host factor (IHF), which binds to DNA sequences upstream of the spvR regulatory gene, and the leucine-responsive regulatory protein (Lrp), which binds to sequences upstream of the spvABCD operon. Under all conditions tested, inactivation of IHF expression reduces the level of spvR transcription by twofold. It also alters the response of the spv regulon to loss of DNA gyrase activity, consistent with a role for IHF in organizing DNA structure in the vicinity of the spvR promoter. Lrp represses spvA gene expression by up to fivefold and Lrp-mediated repression is antagonized by leucine. The Lrp binding site upstream of the spvA gene overlaps one of the binding sites for the positive regulator SpvR, suggesting a mechanism by which Lrp repression is exerted. This is a first demonstration of a role for Lrp in controlling genes that are also subject to intracellular regulation. These data show that the spv virulence genes belong simultaneously to several regulons in the cell, raising the possibility that spv expression can be fine-tuned in response to multiple environmental inputs.

Active recruitment of sigma54-RNA polymerase to the Pu promoter of Pseudomonas putida: role of IHF and alphaCTD.

The sequence elements determining the binding of the sigma54-containing RNA polymerase (sigma54-RNAP) to the Pu promoter of Pseudomonas putida have been examined. Contrary to previous results in related systems, we show that the integration host factor (IHF) binding stimulates the recruitment of the enzyme to the -12/-24 sequence motifs. Such a recruitment, which is fully independent of the activator of the system, XylR, requires the interaction of the C-terminal domain of the alpha subunit of RNAP with specific DNA sequences upstream of the IHF site which are reminiscent of the UP elements in sigma70 promoters. Our data show that this interaction is mainly brought about by the distinct geometry of the promoter region caused by IHF binding and the ensuing DNA bending. These results support the view that binding of sigma54-RNAP to a promoter is a step that can be subjected to regulation by factors (e.g. IHF) other than the sole intrinsic affinity of sigma54-RNAP for the -12/-24 site.

Isolation of an IHF-deficient mutant of a Pseudomonas aeruginosa mucoid isolate and evaluation of the role of IHF in algD gene expression.

The role of integration host factor (IHF) in the regulation of alginate synthesis was investigated in a mucoid strain of Pseudomonas aeruginosa (strain CHA) isolated from a cystic fibrosis patient. Escherichia coli strain BL21(DE3) was made IHF-deficient by inactivation of its chromosomal IHF genes, himA and himD, then used as host strain to overproduce P. aeruginosa IHF. The purified recombinant IHF protein was used to determine the affinity of IHF for the two IHF binding sites in the algD promoter. The Kd values were determined to be 130 nM for algD IHF site 2 and about 2 microM for algD IHF site 1. Two IHF-deficient mutants of P. aeruginosa strain CHA were constructed by insertional inactivation of the himA gene, and the activity of the algD promoter was determined using transcriptional fusion with xylE as reporter gene. The expression of algD, the structural gene for GDP-mannose dehydrogenase, was decreased three- to fourfold in the himA mutants under conditions of high salinity and nitrogen limitation. Assays of alginate production by cultures grown on agar plates indicated that the IHF-deficient mutants synthesized 50% less polymer than the mucoid parental strain. These results demonstrate clearly that although IHF is dispensable for alginate production, himA expression is required for full activation of algD expression.

Glucose starvation stimulon of Escherichia coli: role of integration host factor in starvation survival and growth phase-dependent protein synthesis.

The DNA-binding protein IHF was found to be required for starvation survival and for the induction of 14 proteins of the glucose starvation stimulon. Many of these proteins have been shown previously to be general responders to diverse stress conditions. Overexpression of IHF during balanced growth was not sufficient to induce these proteins, but it resulted in an increased synthesis of rpoH-dependent heat shock proteins.

Integration host factor suppresses promiscuous activation of the sigma 54-dependent promoter Pu of Pseudomonas putida.

In the presence of m-xylene, the Pu promoter of the TOL plasmid of Pseudomonas putida is activated by the prokaryotic enhancer-binding protein XylR. The intervening DNA segment between the upstream activating sequences (UASs) and those for RNA polymerase binding contains an integration host factor (IHF) attachment site that is required for full transcriptional activity. In the absence of IHF, the Pu promoter can be cross-activated by other members of the sigma 54-dependent family of regulatory proteins. Such illegitimate activation does not require the binding of the heterologous regulators to DNA and it is suppressed by bent DNA structures, either static or protein induced, between the promoter core elements (UAS and RNA polymerase recognition sequence). The role of IHF in some sigma 54 promoters is, therefore, not only a structural aid for assembling a correct promoter geometry but also that of an active suppressor (restrictor) of promiscuous activation by heterologous regulators for increased promoter specificity.

Geometry of the process of transcription activation at the sigma 54-dependent nifH promoter of Klebsiella pneumoniae.

The Klebsiella pneumoniae nifH promoter is very strictly controlled by nitrogen availability and highly dependent on sigma 54 and integration host factor (IHF) for expression. This promoter region has been used to examine the role of IHF in the activation of transcription from sigma 54-dependent promoters and to analyze the positional restrictions which may exist for an activation mechanism from distant sites such as this one. By functionally replacing the binding site of IHF by sequence-directed curved DNA fragments, it has been shown that the role of IHF in stimulating transcription is structural; it brings the molecules directly involved in the process into close proximity. Unlike other promoter regions with an activation mechanism at a distance, this IHF-dependent promoter requires a precise geometry for efficient transcription. In this sense, it resembles an activation mechanism from near sites. However, alternative functional structures which are very different from the native one can be isolated.

Co-regulation by bent DNA. Functional substitutions of the integration host factor site at sigma 54-dependent promoter Pu of the upper-TOL operon by intrinsically curved sequences.

The role of integration host factor (IHF) in the regulation of the sigma 54-dependent promoter Pu of the TOL plasmid of Pseudomonas putida has been examined. We have selected in vivo insertions of intrinsically curved DNA that restore the responsiveness of an IHF-binding site deletion variant of Pu to the cognate activator of the system, XylR. We found five Pu derivatives which had inserted a core sequence with 6 phased [A]6 tracts, flanked by different lengths of DNA at the location of the former IHF site. They displayed 40-100% of the activity of Pu, were independent of IHF, and maintained the overall geometry of the wild-type promoter. The induction patterns of Pu, compared to those of hybrid promoters, were virtually indistinguishable. This supports the notion that, in native conditions, IHF co-regulates the system by providing a structural aid for promoter architecture and not by interacting directly with the RNA polymerase, as it has been suggested with other known IHF-dependent promoters.

The regulation of nitrogen utilization in enteric bacteria

The regulation of nitrogen utilization in enteric bacteria

Role of integration host factor in stimulating transcription from the σ54-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, σ 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 σ 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 σ 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 σ 54-holoenzyme in closed complexes.

The role of integration host factor in gene expression in Escherichia coli.

Integration host factor is a sequence-specific, histone-like, multifunctional DNA-binding and -bending protein of Escherichia coli. The characterization and functional analysis of this protein has been done mainly in bacteriophage lambda and other mobile genetic elements. Less is known concerning the role of integration host factor (IHF) in E. coli, although it has been implicated in a number of processes in this organism including DNA replication, site-specific recombination, and gene expression. This review presents recent work which suggests that IHF alters the activity of an unusually large number of operons in E. coli. We discuss the possible physiological relevance of the involvement of IHF in gene expression and the hypothesis that IHF is a member of a class of functionally redundant proteins that participate in chromosome structure and multiple processes involving DNA.

Conformational changes induced by integration host factor at origin gamma of R6K and copy number control

We have investigated the role of integration host factor (IHF) in the replication of plasmid R6K by studying the maintainance of the plasmid in a strain of Escherichia coli that lacks both subunits of IHF and in an isogenic wild type strain and found that all three origins, alpha, beta, and gamma, were functional in the absence of IHF; however, loss of IHF reduced the copy number of those replicons initiating solely from ori gamma by 5-fold. Concomitant loss of direct repeats within the origin that bind the R6K replication initiator protein, Pi, resulted in a further reduction in copy number. Using gel mobility shift analysis, we showed that IHF bound specifically only to one site within the A/T rich region of the minimal origin adjacent to the Pi binding sites. The origin region possessed no intrinsic DNA curvature although IHF induced a strong bend upon binding. Combination footprinting with different orders of addition of Pi and IHF suggested that there was no cooperativity between the two proteins with regard to DNA binding. Hydroxyl-radical footprinting revealed hypersensitive asymmetric periodic cleavage sites within the origin region in the presence of IHF that extended over 200 base pairs and a localized perturbation of cleavage chemistry. The presence of periodic cleavages was dependent upon the presence of the wild type R6K origin sequence and was not observed when the IHF binding site was positioned adjacent to a heterologous sequence. We observed that the conformational changes induced by IHF upon binding to the R6K origin were negatively correlated with the observed decrease in copy number, and therefore, origin conformation altered by protein-DNA interaction may play an important role in the regulation of replication initiation.

Role of integration host factor in the regulation of the glnHp2 promoter of Escherichia coli.

The glnHPQ operon of Escherichia coli encodes components of the high-affinity glutamine transport system. One of the two promoters of this operon, glnHp2, is responsible for expression of the operon under nitrogen-limiting conditions. The general nitrogen regulatory protein (NRI) binds to two overlapping sites centered at -109 and -122 from the transcription start site and, when phosphorylated, activates transcription of glnHp2 by catalyzing isomerization of the closed sigma 54-RNA polymerase promoter complex to an open complex. The DNA-bending protein integration host factor (IHF) binds to a site immediately upstream of glnHp2 and enhances the activation of open complex formation by NRI phosphate. The NRI-binding sites can be moved several hundred base pairs further upstream without altering the ability of NRI phosphate to activate open complex formation. We propose that the IHF-induced bend can facilitate or obstruct the interaction between NRI phosphate and the closed complex depending on the relative positions of NRI phosphate and sigma 54-RNA polymerase on the DNA.

Integration host factor binds specifically to sites in the ilvGMEDA operon in Escherichia coli.

Integration host factor (IHF) of Escherichia coli is a histone-like protein that is involved both in site-specific recombination and in regulating the expression of a number of phage and bacterial genes. We have shown previously that transcription of the ilvGMEDA operon in E. coli is greatly reduced in IHF mutants. We report here that IHF specifically protects two sites within the ilvGMEDA promoter-regulatory region against DNase I digestion. These sites are located upstream from the promoter and in the leader region just prior to the sequence that specifies the attenuator. The footprinting experiments and gel retardation assays show that these sites have strong affinity for IHF. These data and results with ilvGMEDA-lac promoter fusions suggest a direct role for IHF in expression of the ilvGMEDA operon.