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PubMed1994

Integration host factor and sequences downstream of the Pseudomonas aeruginosa algD transcription start site are required for expression.

Pseudomonas aeruginosa is an extremely important opportunistic pathogen in immunocompromised individuals. Strains of P. aeruginosa isolated from chronic lung infections in patients with the genetic disease cystic fibrosis have a mucoid colony morphology. This phenotype is due to overproduction of the exopolysaccharide alginate, which is believed to confer a selective advantage on P. aeruginosa in cystic fibrosis lungs. Alginate biosynthesis is controlled by a complex regulatory mechanism. Genes located in the 34-min region of the P. aeruginosa chromosome form an operon which encodes most of the biosynthetic enzymes necessary for alginate production. algD, the first gene in the operon and a critical point for the transcriptional regulation of alginate biosynthesis, is controlled by several trans, cis, and environmental factors. In this study, the involvement of the histone-like protein integration host factor (IHF) in algD expression was examined. Sequences with similarity to consensus IHF-binding sites of Escherichia coli were identified 75 bp upstream (site 1) and 90 bp downstream (site 2) of the start of algD transcription. In gel band mobility shift assays, DNA fragments containing either site bind IHF but site 2 has an approximately 90-fold higher affinity for IHF. Mutations in each of the elements were generated, and they resulted in the reduction or loss of in vitro IHF binding and a three- to fourfold decrease in algD-cat expression. This indicates that IHF binding is necessary for high-level algD transcription. The presence of a high-affinity IHF-binding site located 3' of the algD transcription start site suggested that sequences further downstream of this element are involved in algD expression. When a fragment located downstream of site 2 and upstream of the promoterless cat gene (+110 to +835) was deleted, algD-cat expression was reduced 10-fold supporting the notion that 3' enhancer elements are required for algD transcription. This is the first direct evidence of a 3' element involved in the control of a P. aeruginosa gene. It is postulated that IHF mediates the formation of a higher-order looped structure which is necessary for efficient algD transcription.

Bacterial Proteins↗

PubMed1999

Isolation of a putative tobacco host factor interacting with cucumber mosaic virus-encoded 2b protein by yeast two-hybrid screening.

The cucumber mosaic virus (CMV)-encoded 2b protein has been implicated to play a role in long distance movement of the virus through the plant's transport system. It is unknown, however, how it mediates virus movement and whether any intrinsic components of plant cells also participate in this process. To isolate a host factor that interacts with 2b, the yeast two-hybrid system was used. First, it was found that the 2b protein per se could function as a transcriptional activator in yeast. However, its two carboxyl terminal deletion mutants, 2bdelta98 and 2bdelta95, which lacked 12 and 15 amino acids from the carboxyl terminus respectively, showed complete absence of transcriptional activation in yeast. A tobacco cDNA library expressing the GAL4 activation domain fusion proteins was screened using 2bdelta98 as a bait. A clone named 2bip (2b-interacting protein) was isolated whose translation product apparently interacted with 2b. Consistent with this observation, bacterially expressed GST-2bip fusion protein bound tightly to 2bdelta95 and 2bdelta98 polypeptides in vitro, as well as to the unmodified 2b protein. Nucleotide sequencing and database searches revealed that the amino acid sequence deduced from it was similar to a prokaryotic LytB protein and an unknown protein of Arabidopsis. DNA and RNA gel blot analyses showed that 2bip-related sequences were present in the tobacco genome and that transcripts corresponding to 2bip were expressed constitutively in various plant organs and in response to CMV infection. These results suggest 2bip as a novel host factor that is capable of interacting with CMV2b.

Amino Acid Sequence↗

PubMed1991

Genes coding for integration host factor are conserved in gram-negative bacteria.

A genetic system for the selection of clones coding for integration host factor and HU homologs is described. We demonstrate that the himA and hip genes of Serratia marcescens and Aeromonas proteolytica can substitute for the Escherichia coli genes in a variety of biological assays. We find that the sequence and genetic organization of the himA and hip genes of S. marcescens are highly conserved.

Amino Acid Sequence↗

PubMed1991

Integration host factor binds specifically to multiple sites in the ompB promoter of Escherichia coli and inhibits transcription.

Escherichia coli integration host factor (IHF) is a DNA-binding protein that participates in gene regulation, site-specific recombination, and other processes in E. coli and some of its bacteriophages and plasmids. In the present study, we showed that IHF is a direct negative effector of the ompB operon of E. coli. Gel retardation experiments and DNase I footprinting studies revealed that IHF binds to three sites in the ompB promoter region. In vitro transcription from ompB promoter fragments was specifically blocked by IHF. In vivo experiments showed that IHF is a negative effector of ompB expression in growing cells. Analysis of IHF binding site mutations strongly suggested that IHF binding in the ompB promoter region is necessary for the negative effects seen in vivo.

Bacterial Outer Membrane Proteins↗

PubMed1987

The integration host factor of Escherichia coli binds to bent DNA at the origin of replication of the plasmid pSC101.

The integration host factor (IHF) of Escherichia coli is necessary for maintenance of pSC101. The protein binds specifically to the replication origin of the plasmid, in the AT-rich region located immediately adjacent to the left, weak binding site for the plasmid-encoded initiator protein. DNAase I and OH- radical footprinting experiments showed that IHF protects 49 bp of the DNA at the origin region. Methylation protection analyses revealed that IHF contacts purine residues in both the major and minor grooves of the DNA. Electrophoretic analyses showed that IHF binds to bent DNA, and the protein binding further enhances the degree of DNA bending. Site-directed mutagenesis of three of the contact points not only abolished binding of the protein to the DNA but also inactivated the replication origin. Therefore, binding of IHF to the ori sequence most probably is necessary for initiation of plasmid replication.

Bacterial Proteins↗

PubMed2005

A genomic-scale search for regulatory binding sites in the integration host factor regulon of Escherichia coli K12.

We examined general aspects of the DNA-protein interaction between the integration host factor (IHF) global regulator and its regulatory binding sites in the Escherichia coli K12 genome. Two models were developed with distinct weight matrices for the regulatory binding sites recognized by IHF. Using these matrices we performed a genome scale scan and built a set of computationally predicted binding sites for each of the models. The sites found by the model associated with repetitive sequences had a higher score in the sequence to matrix alignment. They were also more rare than the other sites. The sites not associated with repeats rapidly tended to become undistinguishable from the background as statistical stringency was relaxed. We compared our results to the known sites documented in RegulonDB and found new members of the IHF Regulon. The two models exhibit clearly distinct affinity patterns (scores in the sequence to matrix alignments and in the number of regulatory sites), as we vary the stringency of the statistical confidence parameters. We suggest that these differences may play an important role in the dynamics of the network. We concluded that IHF may regulate two genes encoding ATP-dependent RNA helicases. This interaction is not described in RegulonDB, even as a computational prediction. IHF may also regulate RNA modification processes.

Binding Sites↗

PubMed2000

Host factor Hfq of Escherichia coli stimulates elongation of poly(A) tails by poly(A) polymerase I.

Current evidence suggests that the length of poly(A) tails of bacterial mRNAs result from a competition between poly(A) polymerase and exoribonucleases that attack the 3' ends of RNAs. Here, we show that host factor Hfq is also involved in poly(A) tail metabolism. Inactivation of the hfq gene reduces the length of poly(A) tails synthesized at the 3' end of the rpsO mRNA by poly(A) polymerase I in vivo. In vitro, Hfq stimulates synthesis of long tails by poly(A) polymerase I. The strong binding of Hfq to oligoadenylated RNA probably explains why it stimulates elongation of primers that already harbor tails of 20-35 A. Polyadenylation becomes processive in the presence of Hfq. The similar properties of Hfq and the PABPII poly(A) binding protein, which stimulates poly(A) tail elongation in mammals, indicates that similar mechanisms control poly(A) tail synthesis in prokaryotes and eukaryotes.

Carrier Proteins↗

PubMed1975

Host factor for coliphage Qbeta RNA replication is present in Pseudomonas putida.

Host Factor (HF)1, is a 12000 molecular weight polypeptide that is found in uninfected Escherichia coli and is required as a hexamer along with Qbeta replicase for in vitro replication of Qbeta phage RNA. It has recently been found to be associated with ribosomes and to bind tightly to poly(A). We report here the identification and purification of HF from Pseudomonas putida. HF can be detected in crude extracts by both functional activity in the Qbeta RNA replication assay and by immunodiffusion with antibody made against E. coli HF. HF from E. coli and P. putida chromatograph similarly on DEAE-cellulose and phosphocellulose. They have similar but not identical molecular weights as judged by SES-polyacrylamide gel electrophoresis. Like E. coli HF, P. putida HF was found to be associated with ribosomes and to bind tightly to poly(A). Furthermore, the pure protein from P. putida has full funcitonal activity in the in vitro Qbeta RNA replication assay. The findings that HF has been conserved during evolution, is associated with ribosomes, and binds poly(A), suggest that HF may be an important translational element in uninfected cells and that its role involves an interaction with RNA.

Coliphages↗

PubMed1993

Larger axillary metastases in obese women and smokers with breast cancer--an influence by host factors on early tumor behavior.

To better define the influence by host factors on very early breast cancer behavior, we retrospectively analyzed nodal status, diameter of the largest axillary metastasis (M), diameter of the primary tumor (P), the M/P ratio, tumor estrogen receptor status, age, obesity, and smoking habits in 176 women with node-positive breast cancer. Both M/P ratios and M were larger in the 72 obese women and in the 40 nonobese smokers than in the 64 nonobese nonsmokers after control for other factors. Step-wise regression analysis demonstrated independent associations between M/P ratios and obesity (p = 0.0002), larger primary tumors (p < 0.0001), more positive nodes (p < 0.0001), and smoking (p = 0.0268), as well as between M and obesity (p = 0.0201), larger primary tumors (p = 0.0093), and more positive nodes (p = 0.0001). Among the 104 nonobese women, smoking was associated both with larger M (p = 0.0257) and larger M/P (p = 0.0055). Our observations suggest more rapid growth by metastases in obese women and smokers with breast cancer, as well as earlier metastasis from their primary tumors.

Adult↗

PubMed1979

An epidemiologic approach to host factors in the etiology of cancer.

We examined the contribution of host factors in carcinogenesis by evaluating the frequency distributions and secular trends in available epidemiologic data of mortality and morbidity around the world. In most developed countries, the age-adjusted death rates of cancer of all sites have levelled off in recent years and the intercountry difference in mortality is small. Deaths from cancer account for approximately 20% of total deaths in these countries. It is believed that the difference in cancer mortality of all sites between the developed and developing countries can be explained by the different levels of mortality from infectious and parasitic diseases, including deaths from tuberculosis. A certain upper limit for the number of cancer deaths in each population is strongly suggested by the unaltered cumulative mortality rate from cancer and tuberculosis up to 85 years of age in a defined birth cohort in the developed countries. The apparent age dependency in cancer mortality as well as some other essential epidemiologic implications in carcinogenesis, the commonly observed proportion of cancer deaths of around 20% of total deaths, and the well-known etiologic importance of environmental factors which determines the site-specific cancer mortality and morbidity lead one to consider the definite existence of "susceptibles to cancer" in each population. However, the concept of susceptibles to cancer is different from the usual one of hereditary predisposition to so-called "intractable diseases," which have low incidence rates.

Adolescent↗

PubMed1996

Integration host factor amplifies the induction of the aceBAK operon of Escherichia coli by relieving IclR repression.

A binding site for integration host factor (IHF) was identified upstream of the aceBAK promoter. Under inducing conditions, IHF activates aceB::lacZ expression by opposing IclR repression. In contrast, IHF has little effect on aceB::lacZ expression under repressing conditions. The ability of IHF to relieve repression under inducing but not repressing conditions allows this protein to amplify the induction of aceBAK.

Acetates↗

PubMed1990

Searching for and predicting the activity of sites for DNA binding proteins: compilation and analysis of the binding sites for Escherichia coli integration host factor (IHF).

An analysis of the sequence information contained in a compilation of published binding sites for E. coli integration host factor (IHF) was performed. The sequences of twenty-seven IHF sites were aligned; the base occurrences at each position, the information content, and an extended consensus sequence were obtained for the IHF site. The base occurrences at each position of the IHF site were used with a program written for the Apple Macintosh computers in order to determine the similarity scores for published IHF sites. A linear correlation was found to exist between the logarithm of IHF binding and functional data (relative free energies) and similarity scores for two groups of IHF sites. The MacTargsearch program and its potential usefulness in searching for other sites and predicting their relative activities is discussed.

Bacterial Proteins↗

PubMed1989

The interaction of E. coli integration host factor and lambda cos DNA: multiple complex formation and protein-induced bending.

The interaction of E. coli's integration Host Factor (IHF) with fragments of lambda DNA containing the cos site has been studied by gel-mobility retardation and electron microscopy. The cos fragment used in the mobility assays is 398 bp and spans a region from 48,298 to 194 on the lambda chromosome. Several different complexes of IHF with this fragment can be distinguished by their differential mobility on polyacrylamide gels. Relative band intensities indicate that the formation of a complex between IHF and this DNA fragment has an equilibrium binding constant of the same magnitude as DNA fragments containing lambda's attP site. Gel-mobility retardation and electron microscopy have been employed to show that IHF sharply bends DNA near cos and to map the bending site. The protein-induced bend is near an intrinsic bend due to DNA sequence. The position of the bend suggests that IHF's role in lambda DNA packaging may be the enhancement of terminase binding/cos cutting by manipulating DNA structure.

Bacterial Proteins↗

PubMed1991

A compact nucleoprotein structure is produced by binding of Escherichia coli integration host factor (IHF) to the replication origin of plasmid R6K.

Understanding the role of Escherichia coli histone-like protein integration host factor (IHF) in replication of R6K plasmid (Dellis, S., and Filutowicz, M. (1991) J. Bacteriol. 173, 1279-1286) requires detailed analyses of the interaction of IHF protein with the plasmid's replication origin (gamma ori). We describe an electron microscopic analysis which shows that a compact structure can be formed in the presence of IHF, in which, on average, a 102-base pair (bp) ori segment is involved. IHF.gamma ori complexes also undergo a two-step conformational change in an IHF concentration-dependent manner when analysed by band shift assay. We believe that the DNA is bent at low IHF concentrations, but folded at high IHF concentrations. This idea is supported by the fact that electrophoretic mobility of the IHF.gamma ori complexes is faster at higher concentrations of IHF. Furthermore, it is shown that the formation of a compact nucleoprotein structure depends on the two regions flanking the AT-rich segment; the iterons to the right and the 106-bp ori domain to the left. Finally we show that IHF protects the entire AT-rich segment of the ori against nuclease cleavage. In addition to the protection, an altered cleavage pattern by DNase I, in the presence of high levels of IHF, was observed within the iterons but not within the 106-bp domain of the ori. Implications of the IHF-mediated gamma ori folding as a possible mechanism protecting the ori from replication inhibition by R6K initiator protein tau are discussed.

Bacterial Proteins↗

PubMed1993

Randomized double-blinded trial of rifampin with either novobiocin or trimethoprim-sulfamethoxazole against methicillin-resistant Staphylococcus aureus colonization: prevention of antimicrobial resistance and effect of host factors on outcome.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major pathogen in hospitals. Current antimicrobial regimens for eradicating colonizing strains are not well defined and are often complicated by the emergence of resistance. The combination of novobiocin plus rifampin in vitro and in vivo was found to prevent the emergence of resistant populations of initially susceptible strains of MRSA, particularly resistance to rifampin. We therefore studied, in a randomized, double-blind, multicenter comparative trial, the combination of novobiocin plus rifampin versus trimethoprim-sulfamethoxazole (T/S) plus rifampin in order to determine the efficacy of each regimen in eradicating MRSA colonization and to further characterize the host factors involved in the response to this antimicrobial therapy. Among the 126 individuals enrolled in the study, 94 (80 patients; 14 hospital personnel) were evaluable. Among the 94 evaluable subjects, no significant demographic or medical differences existed between the two treatment groups. Successful clearance of the colonizing MRSA strains was achieved in 30 of 45 (67%) subjects receiving novobiocin plus rifampin, whereas successful clearance was achieved in 26 of 49 (53%) subjects treated with T/S plus rifampin (P = 0.18). The emergence of resistance to rifampin developed more frequently in 14% (7 of 49) of subjects treated with T/S plus rifampin than in 2% (1 of 45) of subjects treated with novobiocin plus rifampin (P = 0.04). Restriction endonuclease studies of large plasmid DNA demonstrated that the same strain was present at pretherapy and posttherapy in most refractory cases (24 of 29 [83%] subjects). Among the 56 successfully treated subjects, clearance of MRSA was age dependent: 29 of 36 (80%) subjects in the 18- to 49-year-old age group, 19 of 35 (54%) subjects in the 50- to 69-year-old age group, and 8 of 23 (35%) in the 70- to 94-year-old age group (P < 0.01). Clearance was also site dependent; culture-positive samples from wounds were related to a successful outcome in only 22 (48%) of 46 subjects, whereas culture-positive samples from sites other than wounds (e.g., nares, rectum, and sputum) were associated with a success rate of 34 of 48 (71%) subjects (P = 0.02). Foreign bodies in wounds did not prevent the eradication of MRSA by either regimen. T/S plus rifampin was less effective in clearing both pressure and other wounds, whereas novobiocin plus rifampin was equally effective in clearing both pressure and other wounds. There were no significant differences in toxicity between the two regimens. Thus, the combination of novobiocin plus rifampin, in comparison with T/S plus rifampin, was more effective in preventing the emergence of resistance to rifampin and demonstrated a trend toward greater activity in clearing the MRSA carrier state. The response to either combination depended on host factors, particularly age and the site of MRSA colonization.

Adolescent↗

PubMed1990

Integration host factor is necessary for lysogenization of Escherichia coli by bacteriophage P2.

Whether infection by bacteriophage P2 results in lysogenization of the host or vegetative growth of the phage depends upon a race between transcription from the repressor promoter Pc and the early promoter Pe; transcription from these promoters is mutually exclusive, since the Pc repressor Cox is formed from the Pe transcript and the Pe repressor C from the Pc transcript. The involvement of integration host factor (IHF) in the lysogenization of Escherichia coli K12 by P2 was tested by comparing wild-type and IHF-deficient (himA and himD) mutants. No lysogenic clones were formed following infection of the mutant bacteria. A switch plasmid that contains Pc-C-cat and Pe-cox-kan was used to test the choice for expression of Pc versus Pe. In the wild-type K12 bacteria, 20% of the clones expressed Pe transcription and 80% Pc transcription, whereas all transformed IHF-defective clones expressed transcription from Pe only. The effects of IHF on the in vivo expression of the Pe and Pc promoters were only marginal. The IHF protein was found to bind upstream of the Pe promoter, where a potential ihf sequence is located.

Bacterial Proteins↗

PubMed1988

Integration host factor interacts with the DNA replication enhancer of filamentous phage f1.

We present data which show that the Escherichia coli integration host factor (IHF) is an activator of phage f1 DNA replication. Phage f1 poorly infects bacterial strains lacking IHF because IHF is required for efficient expression of F-pili, the receptor for f1 phage. However, when F- strains are transfected with f1 DNA the phage replicates in IHF mutants (himA, himD, or himA himD) at a rate of only 3% of that in wild-type bacteria. A plasmid dependent on the f1 replicon fails to transform IHF mutants. By gel retardation analysis, we show that IHF specifically binds to the origin of replication. DNase I "footprinting" experiments demonstrate that IHF binds to multiple sites within the replication enhancer sequence, a cis-acting, A + T-rich sequence that potentiates f1 DNA replication. Moreover, the effect of IHF mutation on f1 growth is suppressed by initiator protein (f1 gene II) mutations that restore efficient replication from origins that lack a functional replication enhancer sequence. This genetic evidence supports the conclusion that the replication enhancer sequence is the site of action of IHF.

Bacterial Proteins↗

PubMed1987

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.

Bacterial Proteins↗