The specific uptake of cloned Haemophilus DNA.

During the process of transformation $\text{Haemophilus}\text{influenzae}$ cells bind its own DNA but little or no foreign DNA. This specificity for recognition of DNA was studied by cloning Haemophilus DNA in $\text{E. coli}$. Haemophilus DNA fragments were cloned using plasmid pBR322 as a vector. The fragment cH7 cloned in pBR322 was found to be homologous to Haemophilus DNA and shown to bind irreversibly to competent Haemophilus cells. The fact that cH7 isolated from $\text{E. coli}$ lacks Haemophilus modification leads to the conclusion that modification does not play a role in the uptake mechanism. Uptake specificity is a function of recognition sequences that reside in DNA itself.     

Transposon Tn10-like tetracycline resistance determinants in Haemophilus parainfluenzae.

Tetracycline resistance (Tcr) determinants from three different strains of Haemophilus parainfluenzae expressed 10-fold higher levels of resistance when mated into Escherichia coli. No plasmid was found in any of the E. coli recipients, even in matings in which a plasmid was identified in the donor Haemophilus sp. The Tcr determinant from Haemophilus sp. caused instability of resident plasmids in the recipient E. coli: all plasmids were lost within 30 generations in antibiotic-free media. However, by serial subculture in antibiotics, stable resident plasmids were obtained which carried the Tcr determinant from Haemophilus sp. and were transferable by conjugation and transformation among E. coli strains. All Haemophilus determinants hybridized with a probe for the Tcr determinant on Tn10, which bears inducible Tcr. However, Haemophilus determinants were constitutively resistant to tetracycline in the Haemophilus donors and in the E. coli recipients. This constitutive expression was recessive to wild-type Tn10 in the same cell, indicating that the constitutive phenotype resulted from the absence of an active repressor. Restrictive enzyme analysis of various E. coli plasmid derivatives bearing a Tcr determinant from Haemophilus sp. demonstrated that the inserted DNA was of similar size (8.95 to 9.35 kilobases), close to that of Tn10. Heteroduplex analysis and DNA:DNA hybridization confirmed that the Tcr determinant from Haemophilus sp. had greater than 90% homology with the Tn10 determinant, including the DNA sequence for the repressor.     

Uptake of plasmid deoxyribonucleic acid by Haemophilus

The uptake of circular and linear plasmid RSF0885 deoxyribonucleic acids, (DNAs) obtained from Haemophilus parainfluenzae 14, in both homologous and heterologous recipients was studied and compared with that of chromosomal DNA. High concentrations of divalent cations stimulated the uptake of either circular or linear plasmid DNA in H. parainfluenzae 14 competent cells but did not affect the uptake of chromosomal DNA. The biological activity of linear plasmid DNA was similar to that of circular DNA, and the transforming efficiencies for ampicillin resistance of both molecular forms were stimulated by divalent ions. Plasmid DNA was taken up efficiently either with or without the addition of divalent ions but was not biologically active in the heterologous Haemophilus influenzae Rd recipient. Our results suggest that in H. parainfluenzae 14 some of the steps for chromosomal and plasmid DNA uptake are different.     

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.     

Comparison of transformation mechanisms of Haemophilus parainfluenzae and Haemophilus influenzae.

Transformation pathways in two closely related bacterial species, Haemophilus parainfluenzae and Haemophilus influenzae, were studied. Both organisms rapidly take up transforming DNA within minutes into specialized membranous structures on the cell surface (transformasomes). DNA within transformasomes is in a protected state, inaccessible to external DNase or internal restriction and modification enzymes. However, the subsequent processing of donor DNA differs in these two organisms. In H. influenzae, linear DNA immediately undergoes degradation from one end at a constant rate, leaving a lower-molecular-weight intermediate in the transformasome. The end undergoing degradation is searching for homologous regions of the chromosome. Once pairing is initiated, the remaining lower-molecular-weight DNA exits from the transformasome, and a single strand undergoes efficient integration. In contrast, in H. parainfluenzae little degradation of donor DNA is observed, with the majority remaining intact within the transformasomes after 1 h. Thus, whereas only 10% of donor DNA molecules leave the protected state after 1 h, portions of each molecule appear to become quantitatively integrated.     

Characterization of an ATP-dependent DNA ligase encoded by Haemophilus influenzae.

We report that Haemophilus influenzae encodes a 268 amino acid ATP-dependent DNA ligase. The specificity of Haemophilus DNA ligase was investigated using recombinant protein produced in Escherichia coli. The enzyme catalyzed efficient strand joining on a singly nicked DNA in the presence of magnesium and ATP (Km = 0.2 microM). Other nucleoside triphosphates or deoxynucleoside triphosphates could not substitute for ATP. Haemophilus ligase reacted with ATP in the absence of DNA substrate to form a covalent ligase-adenylate intermediate. This nucleotidyl transferase reaction required a divalent cation and was specific for ATP. The Haemophilus enzyme is the first example of an ATP-dependent DNA ligase encoded by a eubacterial genome. It is also the smallest member of the covalent nucleotidyl transferase superfamily, which includes the bacteriophage and eukaryotic ATP-dependent polynucleotide ligases and the GTP-dependent RNA capping enzymes.     

Introduction of transposon Tn916 DNA into Haemophilus influenzae and Haemophilus parainfluenzae.

Enterococcus (Streptococcus) faecalis transposon Tn916 was introduced into Haemophilus influenzae Rd and Haemophilus parainfluenzae by transformation and demonstrated to transpose efficiently. Haemophilus transformants resistant to tetracycline were observed at a frequency of approximately 3 x 10(2) to 5 x 10(3)/micrograms of either pAM120 (pGL101::Tn916) or pAM180 (pAM81::Tn916) plasmid DNAs, which are incapable of autonomous replication in this host. Restriction enzyme analysis and Southern blot hybridization revealed that (i) Tn916 integrates into many different sites in the H. influenzae and H. parainfluenzae genomes; (ii) only the 16.4-kilobase-pair Tn916 DNA integrates, and no vector DNA was detected; and (iii) the Tetr phenotype was stable in the absence of selective pressure. Second-generation Tn916 transformants occurred at the high frequency of chromosomal markers and retained their original chromosomal locations. Similar results were obtained with H. influenzae Rd BC200 rec-1 as the recipient strain, which suggests host rec functions are not required in Tn916 integrative transposition. Transposition with Tn916 is an important procedure for mutagenesis of Haemophilus species.     

Functional characterization of UvrD helicases from Haemophilus influenzae and Helicobacter pylori

Haemophilus influenzae and Helicobacter pylori are major bacterial pathogens that face high levels of genotoxic stress within their host. UvrD, a ubiquitous bacterial helicase that plays important roles in multiple DNA metabolic pathways, is essential for genome stability and might, therefore, be crucial in bacterial physiology and pathogenesis. In this study, the functional characterization of UvrD helicase from Haemophilus influenzae and Helicobacter pylori is reported. UvrD from Haemophilus influenzae (HiUvrD) and Helicobacter pylori (HpUvrD) exhibit strong single-stranded DNA-specific ATPase and 3'-5' helicase activities. Mutation of highly conserved arginine (R288) in HiUvrD and glutamate (E206) in HpUvrD abrogated their activities. Both the proteins were able to bind and unwind a variety of DNA structures including duplexes with strand discontinuities and branches, three- and four-way junctions that underpin their role in DNA replication, repair and recombination. HiUvrD required a minimum of 12 nucleotides, whereas HpUvrD preferred 20 or more nucleotides of 3'-single-stranded DNA tail for efficient unwinding of duplex DNA. Interestingly, HpUvrD was able to hydrolyze and utilize GTP for its helicase activity although not as effectively as ATP, which has not been reported to date for UvrD characterized from other organisms. HiUvrD and HpUvrD were found to exist predominantly as monomers in solution together with multimeric forms. Noticeably, deletion of distal C-terminal 48 amino acid residues disrupted the oligomerization of HiUvrD, whereas deletion of 63 amino acids from C-terminus of HpUvrD had no effect on its oligomerization. This study presents the characteristic features and comparative analysis of Haemophilus influenzae and Helicobacter pylori UvrD, and constitutes the basis for understanding the role of UvrD in the biology and virulence of these pathogens.     

DNA-binding vesicles released from the surface of a competence-deficient mutant of Haemophilus influenzae

Haemophilus influenzae com-51, a mutant deficient in DNA uptake, produces an extracellular DNA-binding activity. The activity was specific for Haemophilus DNA and was isolated from cell-free competence medium after incubation for 100 to 130 min. Initial steps in the purification procedure resulted in the loss of detectable binding activity, but activity was restored by the addition of a nonionic detergent. The active fractions contained vesicles derived from the outer membrane of the cells. The vesicles were produced only under conditions that normally lead to competence development. The lack of competence of com-51 cells was not due to loss of protein synthesis in M-IV competence medium or to competition of extracellular protein for exogenous DNA. Results suggest that the inability of cells to bind DNA was due in part to the loss of DNA receptors that are released into the medium in membrane fragments.     

Susceptibility of Haemophilus aegyptius to trooleandomycin: lack of taxonomic value

Two hundred and nine strains of Haemophilus influenzae and Haemophilus aegyptius were screened for trooleandomycin susceptibility. Four strains were shown to be sensitive to the drug. Of these four, two were Haemophilus aegyptius (ATCC 11116, NCTC 8134), and the other two were Haemophilus influenzae biotype I (1-605) and IV (80-212. One strain of Haemophilus aegyptius (NCTC 8135) was resistant to trooleandomycin. Restriction enzyme assays and DNA homology were carried out to establish relationships between the strains. It is concluded that trooleandomycin susceptibility has no taxonomic value to differentiate between Haemophilus aegyptius and biotype III Haemophilus influenzae.     

Effect of glycerol on Haemophilus influenzae transfection.

Competent Haemophilus influenzae bacteria were exposed to purified phage HP1 DNA and then plated for transfectants (PFU). When 32% (final concentration) glycerol was added before plating, between 10- and 100-fold more transfectants were observed. Glycerol had no significant effect on transfection with DNA from single or tandem double lysogens. It also had little effect on transformation with chromosomal DNA or on transformation of defective HP1 lysogens with phage HP1 DNA. It was concluded that glycerol induced the release of adsorbed linear double-stranded DNA into the interior of the cells.     

A second specific endonuclease from Haemophilus aegyptius.

A second restriction-like endonuclease has been partially purified from Haemophilus aegyptius. This enzyme cleaves bacteriophage λ DNA and adenovirus 2 DNA at many sites, but cleaves simian virus 40 DNA at only one site.     

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.     

Characterization of Haemophilus parainfluenzae strains with low-Mr or ladder-like lipopolysaccharides

Twenty-five Haemophilus parainfluenzae strains were characterized for lipopolysaccharide (LPS) profiles, outer membrane protein profiles, serum sensitivity, plasmid profiles and DNA homology. Seventeen strains produced low-Mr LPS that did not contain O-sidechains, while the remaining eight strains contained ladder-like LPS suggestive of O-repeated units. This is the first time in the genus Haemophilus that LPS with O-repeated groups has been described. The strains producing the different types of LPS could not be distinguished from each other in outer membrane protein profiles or the other characteristics examined.     

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.     

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.     

Genetic Transformation in Haemophilus parainfluenzae Clinical Isolates

Haemophilus parainfluenzae isolates recovered from patients with respiratory diseases were studied for their ability to undergo genetic transformation by isogenic DNA. Two chromosomal markers, streptomycin resistance and nalidixic acid resistance, were tested for transformation efficiencies in H. parainfluenzae recipients from three biotypes. Most efficient in transformation was biotype II, followed by biotype I, while biotype III was nontransformable. Lack of transformation was not owing to poor donor activity of DNA, but to inability of the cells to develop competence. Strains that formed clumps in liquid media were nontransformable. Since the transformable biotype II is one of the prevalent biotypes world wide, one can speculate that DNA transformation probably plays a major role in the spread of drug resistance in H. parainfluenzae. Received: 9 December 1997 / Accepted: 26 February 1998     

Cloning of the Dam methyltransferase gene from Haemophilus influenzae bacteriophage HP1

The putative product of orf13 from the genome of Haemophilus influenzae HP1 bacteriophage shows homology only to bacteriophage T1 Dam methyltransferase, and a weak similarity to the conserved amino acids sequence motifs characteristic of m6A-methyltransferases. Especially interesting is lack of characteristic motif I responsible for binding of S-adenosylmethionine. Despite this fact, a DNA sequence of HP1 bacteriophage of Haemophilus influenzae encoding methyltransferase activity was cloned and expressed in Escherichia coli using pMPMT4 omega expression vector. The cloned methyltransferase recognizes the sequence 5'-GATC-3' and methylates an adenine residue. The enzyme methylates both double- and single-stranded DNA substrates.     

Fragment Maps of φX-174 Replicative DNA Produced by Restriction Enzymes from Haemophilus aphirophilus and Haemophilus influenzae H-I

phiX-174 replicative form (RF) DNA was cleaved by restriction enzymes from Haemophilus aphirophilus (Hap) and H. influenzae (H-I strain Osaka) (HinH). Five fragments were produced by Hap and eight by HinH. The positions of all of the Hap fragments and six of the HinH fragments were mapped on the genome by heteroduplex transfection and DNA-DNA hybridization methods. In addition, fragment sizes of phiX-174 RF DNA and S13 RF DNA by Hap and HinH were compared. Considerable differences of the size of the fragments produced from these closely related phage genomes were observed.