Functional characterization of the DNA mismatch binding protein MutS from Haemophilus influenzae

This investigation demonstrates DNA mismatch repair activity in Haemophilus influenzae cell free extracts. The mutS gene as well as purified protein of H. influenzae restored repair activity in complementation assays performed with mutS deficient Escherichia coli strain. The difference in affinity for GT and AC mismatched bases by H. influenzae MutS was reflected in the efficiency with which these DNA heteroduplexes were repaired in vitro, with GT being repaired well and AC the least. Unlike E. coli MutS, the H. influenzae homolog failed to give protein-DNA complex with homoduplex DNA. Interestingly, MutS was found to bind single-stranded DNA but with lesser affinity as compared to heteroduplex DNA. Apart from the nucleotide- and DNA-mediated conformational transitions, as monitored by circular dichroism and limited proteolysis, our data suggest a functional role when H. influenzae MutS encounters single-stranded DNA during exonucleolytic step of DNA repair process. We propose that, conformational changes in H. influenzae MutS not only modulate mismatch recognition but also trigger some of the down stream processes involved in the DNA mismatch repair process.     

Specificity in Deoxyribonucleic Acid Uptake by Transformable Haemophilus influenzae

Cells of Haemophilus influenzae strain Rd competent for genetic transformation irreversibly bound approximately five molecular fragments of H. influenzae deoxyribonucleic acid (DNA) per cell; under identical conditions, DNA derived from Escherichia coli B was not taken up (<1 molecule per 50 cells). Similarly, DNA from Xenopus laevis was not taken up by competent H. influenzae. Of the heterologous DNAs tested, only DNA from H. parainfluenzae interfered with the uptake of H. influenzae DNA, as judged by competition experiments employing either DNA binding or genetic transformation as the test system. The extracellular heterologous DNA did not suffer either single- or double-strand breakage upon exposure to competent H. influenzae.     

Action of Haemophilus Endodeoxyribonuclease on Biologically Active Deoxyribonucleic Acid

An enzyme similar to that described by Smith and Wilcox (15) for Haemophilus influenzae which attacks foreign deoxyribonucleic acid (DNA) but not its own has been isolated and purified from H. parainfluenzae. The enzyme degrades foreign DNA to limited sizes and can destroy the transforming activity of H. influenzae and Bacillus subtilis DNA. The enzyme can also destroy the biological activity of H. influenzae phage and prophage DNA. On the other hand, the H. influenzae endodeoxyribonuclease can destroy the transforming activity of H. parainfluenzae DNA but not its own DNA. It also attacks B. subtilis DNA and its transforming activity.     

Cloning and expression in Escherichia coli of Haemophilus influenzae fimbrial genes establishes adherence to oropharyngeal epithelial cells.

In this report the first example of functional expression of a fimbrial gene cluster of a non-enteric human pathogen in Escherichia coli is described. This is shown for Haemophilus influenzae fimbriae which mediate adherence to oropharyngeal epithelial cells. A genomic library of H.influenzae type b, strain 770235f+bo, was constructed using a cosmid vector and screened with a synthetic oligonucleotide probe derived from the N-terminal sequence of the fimbrial subunit of H.influenzae. Four cosmid clones were found which hybridized to this oligonucleotide probe. Escherichia coli strains harbouring these clones expressed the H.influenzae fimbriae at their cell surface, as was demonstrated in a whole-cell ELISA and by immunogold electron microscopy using a monoclonal antibody specific for the H.influenzae fimbriae. Surface expression could be maintained during subcloning until a minimal H.influenzae DNA insert of approximately 8.1 kb was obtained. Escherichia coli strains harbouring the 8.1 kb H. influenzae DNA were able to cause a mannose-resistant adherence to oropharyngeal epithelial cells and a mannose-resistant haemagglutination of human AnWj-positive erythrocytes. The nucleotide sequence of hifA, the gene encoding the major fimbrial subunit, was determined. The predicted amino acid sequence shows a significant homology with a number of E.coli fimbrial subunits.     

Electroporation of Haemophilus influenzae is effective for transformation of plasmid but not chromosomal DNA.

Electroporation of plasmid and chromosomal DNAs were tested in Haemophilus influenzae because of an interest in introducing DNA into mutants that are deficient in competence for transformation. The initial experiments were designed to investigate and optimize conditions for electroporation of H. influenzae. Plasmid DNA was introduced into the competence proficient strain Rd and its competence-deficient uptake mutants com-52, com-59, and com-88, and the recombination deficient mutant rec1. Plasmid DNA could also be electroporated into the non-transforming strains Ra, Rc, Re and Rf. Plasmid DNA without sequences that are involved in tight binding (uptake) of DNA by competent cells of H. influenzae Rd was electroporated into both competent and non-competent cells. Competent cells were several orders of magnitude less efficient than non-competent cells for electroporation of plasmid DNAs. Electroporation of H. influenzae chromosomal DNA was not successful. Low levels of integration of chromosomal markers were observed following electroporation and these could be ascribed to transformation. The treatment of cells with DNasel following electroporation separated the effects due to electroporation from those due to transformation. The DNasel treatment did not affect the efficiency of plasmid incorporation, but severely restricted effects due to natural DNA transformation.     

Application of PCR to Haemophilus influenzae typing. I. PCR standardization for bexA gene fragment detection and for detection of DNA fragment specific for H.influenzae type B

PCR standardization was performed in order to detect a fragment of bexA gene, which is presented in all capsulate H. influenzae isolates, and a DNA fragment specific for H. influenzae type b. Implementing of such PCR into H. influenzae typing may be very useful in cases of isolates from clinical material for which serotyping alone gives unclear results. Standardization of PCR detecting DNA fragments specific for all capsular types will enable to perform complete typing of H. influenzae isolates.     

Mobilization of Haemophilus influenzae chromosomal markers by an Escherichia coli F'' factor.

Filter matings between E. coli K-12 strains carrying an F'::Tn5,Tn9 factor with H. influenzae Rd strains gave rise to kanamycin-chloramphenicol-resistant H. influenzae strains at a frequency of approximately 10(-6). Transfer of the F' factor to H. influenzae was verified by expression of unselected markers in H. influenzae (lac+ or cotransfer of the nonselected antibiotic resistance), physical presence of a high-molecular-weight plasmid in recipient H. influenzae cells, and detection by Southern hybridization analysis of DNA sequences specific for the F' factor replication and partition functions in recipient H. influenzae cells. H. influenzae (F' Tn5,Tn9) strains were capable of transferring kanamycin and chloramphenicol resistances to other H. influenzae strains and were capable of mobilizing H. influenzae chromosomal markers at a low frequency. Insertion of a Tn5 element in the H. influenzae genome near the novobiocin resistance gene increased the frequency of transfer of novobiocin resistance about 30-fold. Transfer of other chromosomal markers also increased, although to a lesser extent, and ordered transfer of chromosomal markers could be demonstrated. Gene transfer was insensitive to DNase I, and transfer of chromosomal (but not F' factor) markers was dependent on the H. influenzae rec-1 and rec-2 gene functions.     

Size and physical map of the chromosome of Haemophilus influenzae.

A variation of pulse-field electrophoresis, field-inversion gel electrophoresis, was used to determine the size and physical map of the chromosome of Haemophilus influenzae. The DNA of H. influenzae had a low G + C content (39%) and no restriction sites for the enzymes NotI or SfiI. However, a number of restriction enzymes (SmaI, ApaI, NaeI, and SacII) that recognized 6-base-pair sequences containing only G and C nucleotides were found to generate a reasonable number of DNA fragments that were separable in agarose gels by field-inversion gel electrophoresis. The sizes of the DNA fragments were calibrated with a lambda DNA ladder and lambda DNA restriction fragments. The sum of fragment sizes obtained with restriction digests yielded a value for the chromosome of 1,980 kilobase pairs. Hybridization of a labeled fragment with two or more fragments from a digest with a different restriction enzyme provided the information needed to construct a circular map of the H. influenzae chromosome.     

Isolation of Haemophilus influenzae genes that suppress Escherichia coli polA mutations

Haemophilus influenzae was found to produce a DNA polymerase that was similar to polymerase I of Escherichia coli. E. coli polA mutants were used as backgrounds for the selection of H. influenzae polA suppressor genes. Six different H. influenzae fragments were isolated that could suppress E. coli polA mutations. None of the suppressors appeared to encode the H. influenzae equivalent of the E. coli polA gene. One type of clone, represented by pGW41, caused a polymerase I activity to appear in a suppressed polA1 mutant. Plasmids from the pGW41 class contained two genes (pol-2 and pol-3) that were both required for polA suppression. Mutated nonsuppressing derivatives of the pGW41 class were used to create H. influenzae mutants that were deficient in polymerase I.     

Uptake of heterologous DNA by Haemophilus influenzae.

With the use of highly competent Haemophilus influenzae cells, it was possible to demonstrate the uptake of heterologous DNAs. However, these DNAs, as expected, were only 1% or less as effective when competing for uptake with Haemophilus DNA. Escherichia coli DNA was removed from solution by competent cells to the extent expected if all the E. coli DNA particles contained at least one uptake recognition signal. The data were consistent with a model in which there was one uptake signal per 20 X 10(6) to 30 X 10(6) daltons of E. coli DNA. Since H. influenzae DNA has many more recognition signals, approximately one per 2 X 10(6) daltons (Danner et al., Gene 77:311-318, 1980; K. Vogt and S. H. Goodgal, submitted for publication), it has been suggested that the slower rate of E. coli DNA binding and the so-called specificity of Haemophilus DNA binding are due to the number of recognition signals per molecule of DNA as well as the nature of the DNA receptor (Vogt and Goodgal, submitted for publication). The specificity of native H. influenzae DNA binding does not apply to the uptake of denatured DNA in the transforming system (low pH) for denatured DNA.     

Killing of Haemophilus influenzae Cells by Integrated Ultraviolet-Induced Lesions from Transforming Deoxyribonucleic Acid

Highly competent cultures of Haemophilus influenzae are inactivated by exposure to transforming deoxyribonucleic acid (DNA) irradiated with ultraviolet light (UV). As a function of UV dose to the DNA, the killing goes to a maximum and then decreases. The killing of H. influenzae by unirradiated H. parainfluenzae DNA, reported by other workers, is enhanced by low doses of UV, but drops off at high doses. Since there are no such lethal effects in a strain of H. influenzae that takes up DNA normally but does not integrate it, it is concluded that the killing is associated with integrated UV lesions. All the killing of wild-type cells due to irradiated DNA is eliminated by photoreactivation of the DNA. The killing of an excisionless strain of H. influenzae, however, is not eliminated by maximal photoreactivation of the irradiated transforming DNA. The nonphotoreactivable fraction of killing in the excisionless strain increases with increasing dose. The kinetics of the killing-dose curves may be explained only partially in terms of UV-induced loss of integration. It is postulated that the number of pyrimidine dimers relative to other DNA components integrated decreases at higher UV doses.     

Methylase activities from Haemophilus influenzae that protect Haemophilus parainfluenzae transforming deoxyribonucleic acid from inactivation by Haemophilus influenzae endonuclease R.

Specific methylases that have the properties of deoxyribonucleic acid (DNA) modification enzymes have been isolated from Haemophilus influenzae strain Rd. Two activities ((Methylase IIa and methylase III) were found to protect transforming DNA of H. parainfluenzae from the action of H. influenzae restriction enzymes. To determine the specificty of the protection, a procedure based on biological activity was developed for the separation and purification of the restriction endonucleases from H. influenzae strain Rd. Two endonuclease R activities presumably corresponding to Hind II and Hind III (P. H. Roy and H. O. Smith, 1973; H. O. Smith and K. W. Wilcox, 1970) were characterized by differences in their chromatographic properties, ability to attack T7 DNA, and inactivation of the transforming activity of different markers of H. parainfluenzae DNA. One endonuclease R enzyme (Hind II) attacked T7 DNA and was found to inactivate the dalacin resistance marker (smaller than 0.01% activity remaining) with only a slight effect on the streptomycin resistance marker (83% activity remaining). Methylase IIa treatment protected 40% of the dalacin resistance marker of H. parainfluenzae DNA from inactivation by Hind II. The other restriction activity (Hind III) was inert towards T7 DNA and inactivated the streptomycin resistance marker of H. parainfluenzae DNA (smaller than 0.01% activity remaining) without any effect on the dalacin resistance marker. The methylation of H. parainfluenzae DNA accomplished by methylase III protected 60% of the transforming activity of the streptomycin resistance marker of H. parainfluenzae DNA from the action of Hind III.     

Mechanism of Haemophilus influenzae transfection by single and double prophage deoxyribonucleic acid.

Whole phages HP1 and HP3, vegetative-phage deoxyribonucleic acid (DNA), and single and tandem double prophage DNA were exposed to ultraviolet radiation and then assayed on a wild-type (DNA repair-proficient) Haemophilus influenzae Rd strain and on a repair-deficient uvr-1 strain. Host cell reactivation (DNA repair) was observed for whole-phage and vegetative-phage DNA but not for single and double prophage DNA. Competent (phage-resistant) Haemophilus parainfluenzae cells were normally transfected with H. influenzae-grown phage DNA and with tandem double prophage DNA but not at all with single prophage DNA. CaCl2-treated H. influenzae suspensions could be transfected with vegetative phage DNA and with double prophage DNA but not with single prophage DNA. These observations support the hypothesis that transfection with single prophage DNA occurs through prophage DNA single-strand insertion into the recipient chromosome (at the bacterial att site) followed by DNA replication and then prophage induction.     

Determination of genome size of Haemophilus influenzae Sb: analysis of DNA restriction fragments.

Genomic DNA size was measured in clinical isolates of Haemophilus influenzae by Pulsed-Field Gel Electrophoresis of DNA restriction fragments. Because of the high (64%) A+T content of H. influenzae DNA, restriction enzymes that recognize sequences with at least four GC base pairs were expected to be rare cutters. Five enzymes that produced fragments greater than 200 kb in size were used to digest intact chromosomes and fragments resolved by TAFE and/or FIGE: ApaI (GGGCCC), EagI (CGGCCG), NotI (GCGGCCGC), RsrI (CGGA/TCCG), and SmaI (CCCGGG). All five had recognition sequences with at least six GC base pairs. The genomic DNA size of H. influenzae serotype b, estimated with ApaI, EagI, NotI, RsrII, and SmaI, is 1,950 kb.     

Peracetic acid stress-induced genetic rearrangements in Escherichia coli H10407 detected by RAPD and RFLP analyses

The discriminatory powers of random amplified polymorphic DNA (RAPD) analysis and restriction fragment length polymorphism (RFLP) were assessed for the detection and comparison of DNA modifications caused by an oxidative stress. DNA extracted from peracetic acid (PAA)-treated Escherichia coli H10407 was randomly amplified with the 10-mer primer OPZ14, which generated one stress-induced fragment. RFLP and RAPD profiles were hybridized by Southern blotting with the digoxigenin-labelled RAPD product. Untreated and PAA-treated cells had difference band profiles. The results indicate that RAPD analysis could be used as a discriminatory tool for investigating genetic rearrangements in E. coli caused by oxidative stress and that RFLP analysis could be used to confirm the rearrangements.     

Molecular Events Accompanying the Fixation of Genetic Information in Haemophilus Heterospecific Transformation

Heterospecific transformation between Haemophilus influenzae and H. parainfluenzae was investigated by isopycnic analysis of deoxyribonucleic acid (DNA) extracts of (3)H-labeled transforming cells that had been exposed to (32)P-labeled, heavy transforming DNA. The density distribution of genetic markers from the resident DNA and from the donor DNA was determined by transformation assay of fractions from CsCl gradients, both species being used as recipients. About 50% of the (32)P atoms in H. parainfluenzae donor DNA taken up by H. influenzae cells were transferred to resident DNA, and only a small amount of the label was lost under conditions of little cell growth. There was less transfer in the reciprocal cross, and almost half of the donor label was lost. In both crosses, the transferred donor material transformed for the donor marker considerably more efficiently when assayed on the donor species than on the recipient species, indicating that at least some of the associated (32)P atoms are contained in relatively long stretches of donor DNA. When the transformed cultures were incubated under growth conditions, the donor marker associated with recipient DNA transformed the donor species with progressively decreasing efficiency. The data indicate that the low heterospecific transformation between H. influenzae and H. parainfluenzae may be due partly to events occurring before association of donor and resident DNA but results mostly from events that occur after the association of the two DNA preparations.     

RAPD-marking of genomes of representative Hordeum species

Random amplification of polymorphic DNA (RAPD) was used to analyze six species, three populations, and seven regional cultivars of barley. A unique pattern of amplified DNA products was obtained for each species of the genus Hordeum. High polymorphism of barley species was revealed. Specific fragments were found in most RAPD patterns; the fragments can be used as molecular markers of corresponding species and subspecies. Several other DNA fragments were shown to serve as molecular markers of the H genome. Specific RAPD patterns were obtained for each population and each cultivar of H. vulgare sensu lato. In total, variation between the populations and between the cultivars was substantially lower than between species. Cluster analysis (UPGMA) was used to estimate genetic distances between the Hordeum species, between the H. spontaneum populations, and between regional H. vulgare cultivars and a dendrogram was constructed.     

DNA sequence and characterization of Haemophilus influenzae dprA+, a gene required for chromosomal but not plasmid DNA transformation.

Natural genetic transformation in Haemophilus influenzae involves DNA binding, uptake, translocation, and recombination. In this study, we cloned and sequenced a 3.8-kbp H. influenzae DNA segment capable of complementing in trans the transformation defect of an H. influenzae strain carrying the tfo-37 mutation. We used subcloning, deletion analysis, and in vivo protein labeling experiments to more precisely define the gene required for efficient DNA transformation on the cloned DNA. A novel gene, which we called dprA+, was shown to encode a 41.6-kDa polypeptide that was required for efficient chromosomal but not plasmid DNA transformation. Analysis of the deduced amino acid sequence of DprA suggested that it may be an inner membrane protein, which is consistent with its apparent role in DNA processing during transformation. Four other open reading frames (ORFs) on the cloned DNA segment were identified. Two ORFs were homologous to the phosphofructokinase A (pfkA) and alpha-isopropyl malate synthase (leuA) genes of Escherichia coli and Salmonella typhimurium, respectively. Homologs for the two other ORFs could not be identified.     

A peroxide/ascorbate-inducible catalase from Haemophilus influenzae is homologous to the Escherichia coli katE gene product.

Bacterial catalases are induced by exposure to peroxide (e.g., Escherichia coli katG) or entry into stationary phase (e.g., E. coli katE). To study regulatory systems in Haemophilus influenzae, we complemented an E. coli rpoS mutant, which is unable to induce katE in stationary phase, with a plasmid library of H. influenzae Rd- chromosomal DNA. Nineteen complementing clones with a catalase-positive phenotype were obtained and characterized after screening about 10(5) transformants. All carried the same structural gene for an H. influenzae catalase. The DNA sequence of this gene, called hktE, encodes a 508-amino-acid polypeptide with strong homology to eukaryotic catalases and E. coli katE. However, hktE is regulated like E. coli katG, with catalase activity increasing 10-fold and hktE mRNA levels increasing 4-fold upon exposure to ascorbic acid, which serves to generate hydrogen peroxide. Mutations in the known global regulatory genes of H. influenzae--crp, cya, and sxy--do not affect the inducibility of hktE. The hktE gene maps to a 225-kb segment of the H. influenzae chromosome in a region encoding resistance to spectinomycin.