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.     

Genetic Integration in the Heterospecific Transformation of Haemophilus influenzae Cells by Haemophilus parainfluenzae Deoxyribonucleic Acid

The in vivo chemical linkage of Haemophilus parainfluenzae deoxyribonucleic acid (DNA) with the H. influenzae genome has been found to occur at a much higher level than is suggested by the low efficiency of the heterospecific transformation of an antibiotic resistance marker. This linkage, about 60% of the level with homospecific DNA, was found to involve alkali-stable bonding. The amount of host DNA label released (about 60%) was about the same as that released during homospecific transformation. Also, over 60% of the H. influenzae cells adsorbing H. parainfluenzae DNA could not form colonies upon plating. This lethality of the heterospecific transformation was not immediate but followed considerable metabolic activity of the host cells. These data are presented to show that the "limited-pairing" hypothesis may be only a partial explanation for the low efficiency of heterospecific transformation. Another hypothesis is presented which takes into account the lethal effect of this kind of transformation.     

Restriction enzymes do not play a significant role in Haemophilus homospecific or heterospecific transformation.

Competent Haemophilus influenzae Rd recipients, either as phage HP1 restricting (r+) or nonrestricting (r-) nonlysogens or defective lysogens, were exposed to deoxyribonucleic acids from various wild-type phage HP1 lysogenic H. influenzae serotype strains (non-encapsulated derivatives of serotypes a,b, c, d, and e), to DNA from lysogenic Haemophilus parahaemolyticus, and to DNA from modified and nonmodified phage HP1. Transformation of antibiotic resistance markers and of prophage markers in homospecific crosses was observed to be unaffected by the recipient restriction phenotype, whereas the transfection response was much reduced in r+ recipients. Heterospecific transformation of prophage markers was reduced by only 80 to 90%, whereas antibiotic resistance marker transformation was 1,000 to 10,000 times lower. Heterspecific transfection was at least 100 times lower than homospecific transfection in both r+ and r- recipients. The general conclusion is that neither class I nor class II restriction enzymes affect significantly the transformation efficiency in homospecific and heterospecific crosses. The efficiency of heterospecific transformation may depend mainly on the deoxyribonucleic acid homology in the genetic marker region.     

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.     

Heterospecific transformation in the genus Haemophilus

Summary The relationship between nine Haemophilus species and Haemophilus influenzae was studied by DNA-DNA hybridization, by transformation of H. influenzae to streptomycin resistance with heterospecific DNA, by competition of heterospecific DNA for transformation by homospecific DNA and by the lethal effect of heterospecific DNA on competent H. influenzae. H. parainfluenzae, H. parasuis, and H. aegyptius DNA transformed at more than 10% efficiency when compared to homologous transformation, but only H. aegyptius demonstrated, by hybridization, a relative binding ratio of more than 80%. H. aphrophilus and H. paraphrophilus DNA demonstrated a relative binding ratio of less than 30% and transformed H. influenzae at only 10^-5 the efficiency of homologous DNA, but they competed for H. influenzae transformation as well as or better than homospecific DNA. The data indicated that in some of the species sharing the common ecological habitat of the mammalian respiratory tract, sequences necessary for competition and efficient uptake into H. influenzae are present in large numbers in their DNAs, which nevertheless have little overall homology with H. influenzae DNA.     

Fate of heterospecific transforming DNA bound to Streptococcus sanguis.

The fate of 3H-labeled str-r fus-s DNA from Streptococcus pneumoniae, bound after a 1-min uptake to 14C-labeled str-s fus-r S. sanguis recipients, was followed by techniques previously developed for analyzing the fate of homospecific DNA. Heterospecific S. pneumoniae DNA was bound and formed complexes with recipient protein in a manner similar to that of homospecific DNA but transformed relatively poorly. The rate at which complexed heterospecific DNA becomes physically associated with recipient DNA, and at which donor markers are integrated into the chromosome, was slower than in the case of homospecific DNA. In addition, about half of the heterospecific donor counts initially bound in trichloracetic acid-insoluble form were gradually solubilized and released from the cell. The association of heterospecific DNA with the recipient chromosome was more unstable than that involving homospecific DNA, since only associations of the former type were largely dissociated by isolation and resedimentation. The donor DNA-containing material so dissociated had the same sedimentation properties as complexed heterospecific DNA before association, indicating that the complex of single-stranded donor DNA and recipient protein formed on uptake moves as a whole from its site of formation to synapse with the chromosome.     

Loss of activity of transforming deoxyribonucleic acid after uptake by Haemophilus influenzae

Voll, Mary Jane (University of Pennsylvania, Philadelphia), and Sol H. Goodgal. Loss of activity of transforming deoxyribonucleic acid after uptake by Haemophilus influenzae. J. Bacteriol. 90:873-883. 1965.-Transforming deoxyribonucleic acid (DNA) which has been irreversibly removed from solution by competent cells undergoes a progressive loss in marker activity when tested by lysis of the cells and exposure to new recipient cells. The loss of activity is limited and marker-specific, with greater inactivation of those markers with lower efficiencies of transformation. Recipient factors or donor factors which have undergone recombination, as measured by the appearance of linked markers, do not undergo inactivation. The efficiency of transformation can be correlated with the sensitivity of a marker to inactivation after DNA uptake. A mutation which affects the efficiency of transformation is found to increase sensitivity to postuptake inactivation. The rate of inactivation is temperature-dependent. At temperatures of 20 and 45 C, marker inactivation can occur without concomitant recombination. During the uptake process, DNA is retained in an acid-insoluble form, indicating that the fate of Haemophilus influenzae DNA differs from the fate of transforming DNA in pneumococcus.     

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.     

Mechanism of Inactivation of Haemophilus influenzae Transforming Deoxyribonucleic Acid by Sonic Radiation

Transforming deoxyribonucleic acid (DNA) from Haemophilus influenzae was exposed to sonic radiation of various durations. Reductions in transforming ability of the DNA, cellular DNA uptake, and integration into the genome, and single- and double-stranded molecular weights of the transforming DNA were measured and compared. We conclude that (i) sonic radiation causes DNA strand breaks (almost always double-strand breaks with relatively few alkaline-labile bonds), the number increasing with exposure until the double-stranded molecular weight is reduced to less than 10(6) daltons; and (ii) since transformation is reduced about as much as integration and much more than uptake, inactivation of transforming DNA by sonic radiation appears to be caused mostly by failure of Haemophilus cells to integrate the transforming DNA that is taken into the cells. These results are similar to those for inactivation by X radiation but differ from those for ultraviolet radiation. A strand break caused by sonic radiation, however, does not necessarily inactivate the transforming DNA, whereas in the case of ionizing radiation it may. The results may be fit by the model proposed by Cato and Guild. From our data and the equation of Lacks, the minimum active site of DNA necessary for transformation and the frequency of exchanges between donor and recipient strands upon integration of transforming DNA were estimated as 0.35 x 10(6) to 0.7 x 10(6) daltons and 0.15 to 0.4 switches per 10(6) daltons, respectively.     

Heterospecific transformation of Pneumococcus and Streptococcus

Summary Transformations of two linked ribosomal loci (str and ery) were carried out between the SIII-1 strain of pneumococcus and the Challis and SBE strains of group H streptococcus. Transfer of markers between the Challis and SBE strains is as efficient as in the corresponding intrastrain transformations. Transfer between either of these strains and the pneumococcus, however, is less efficient than in the corresponding intrastrain transformation, and is referred to as heterospecific transformation. The inefficiency of the heterospecific transformation is due neither to specific lethality nor reduced uptake of heterologous DNA.When DNA was extracted from the hybrid resulting from a heterospecific cross and used to transform the original donor and recipient species, we found: (a) no donor material in the hybrid DNA responsible for the markedly low efficiency of integration into the recipient species; (b) donor material, in addition to the transforming marker itself, detectable by the higher efficiency with which hybrid DNA transforms the original donor species than does DNA from the original recipient species.DNA was extracted from each of 36 independently derived, doubly marked transformants resulting from the cross: Challis str-s ery-sxSIII-1 str-r53 ery-r2 DNA. Variability was observed between the different hybrid DNAs when the integration efficiency of the str marker in each DNA was compared with that of the ery marker. Variability of as great a magnitude was not observed when the same hybrid DNA was tested in repeated experiments, or when different DNA preparations were extracted from the same hybrid strain, or when several DNA preparations were obtained from a number of independent homospecific transformants. It is concluded that different kinds of donor material are present in the various hybrids, and that the nature of this extra-marker material affects the integration of the marker.Linkage of the str and ery markers was reduced in heterospecific transformations. The kind of donor DNA in the hybrid genome did not affect the linkage reduction observed when the str and ery markers were transferred back to the donor species in which they originated. Indeed, this linkage reduction was the same as that observed when the markers were originally transferred from the SIII-1 to the Challis strain. Specific factors reducing linkage in heterologous crosses must, therefore, be distinct from other factors which affect integration efficiency. The former, however, may be primarily responsible for the inefficiency of heterospecific transformation.One of the hybrid DNAs was used to obtain a second generation of hybrids by passing it through each of the original parental strains. Tests of the DNAs extracted from 24 independently produced, second-generation hybrids showed that hybrid DNA is subject to further alteration by a second integration involving some heterologous confrontation. The probability of such alteration appears to be increased if the second integration is accompanied by linkage reduction.Supported by NIH grant AI-00917.     

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.     

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.     

Action of restriction endonucleases on transforming DNA of Haemophilus influenzae.

Cleavage of DNA from Haemophilus influenzae with restriction endonucleases caused inactivation of transforming ability to an extent that depended on the genetic marker and the enzyme. The rate of inactivation, but not the final level of survival, depended on the concentration of enzyme in the restriction digest. In general, the greatest extent of inactivation of transforming activity was obtained with endonucleases that are known to produce the shortest fragments. We electrophoresed restriction digests of H. influenzae DNA in agarose gels and assayed transforming activity of DNA extracted from gel slices. In this way, we determined the lengths of restriction fragments that contain genetic markers of H. influenzae. For the marker that we studied most thoroughly (nov), the shortest restriction fragment that possessed detectable transforming activity was a 0.9-kilobase pair fragment produced by endonuclease R . PstI. The shortest marker-bearing restriction fragment that retained substantial transforming activity (50% of value for undigested DNA) was a 2.1-kilobase pair EcoRI fragment bearing the kan marker. Among marker-bearing restriction fragments 1 to 4 kilobase pairs in length, survival of transforming activity varied 10,000-fold. We relate these observations to the recent findings by Sisco and Smith (Proc. Natl. Acad. Sci. U.S.A. 76:972-976, 1979) that efficient entry of DNA into competent H. influenzae cells appears to require the presence of a recognition sequence that is scattered throughout the Haemophilus genome in many more copies than in unrelated genomes.     

Relationship Between Prophage Induction and Transformation in Haemophilus influenzae

The interaction between transformation and prophages of HP1c1, S2, and a defective phage of Haemophilus influenzae has been investigated by measurement of (i) the effect of prophage on transformation frequency and (ii) the effect of transformation on phage induction. The presence of any of the prophages does not appreciably alter transformation frequencies in various Rec(+) and Rec(-) strains. However, exposure of competent lysogens to transforming deoxyribonucleic acid (DNA) may induce phage but only in Rec(+) strains, which are able to integrate transforming DNA into their genome. Transformation of Rec(+) lysogens with DNA irradiated with ultraviolet (UV) light causes the production of even more phage than results from unirradiated DNA, but this indirect UV induction is not as effective as direct induction by UV irradiation of lysogens. Both types of UV induction are influenced by the repair capacity of the host. Wild-type cells contain a prophage and can be induced by transformation to produce a defective phage, which kills a small fraction of the cells. Defective phage in wild-type cells are also induced by H. parainfluenzae DNA, and a much larger fraction of the cells is killed. Strain BC200, which is highly transformable but is not inducible for defective phage, is not killed by H. parainfluenzae DNA, suggesting that wild-type cells are killed by killed by this DNA because of phage induction. A minicell-producing mutant, LB11, has been isolated. Some phage induction occurs in this strain when the cells are made competent, unlike the wild type. A large majority of LB11 cells surviving the competence regime are killed by exposure to transforming DNA.     

Naturally occurring NAD-independent Haemophilus parainfluenzae

Four, NAD-independent, clinical isolates of Haemophilus parainfluenzae were recovered from a genital ulcer, a purulent skin lesion, a sputum specimen and a throat swab respectively. With the exception of NAD requirement, the strains exhibited the biochemical characteristics of H. parainfluenzae biotype II. The genetic relationship between these isolates and a standard strain of H. parainfluenzae was determined by testing transforming activities of two chromosomal markers, streptomycin resistance and nalidixic acid resistance. The clinical isolates were efficient donors and recipients in transformation. In addition, we demonstrated transfer of the genes conferring NAD independence to typical, NAD-requiring H. parainfluenzae and Haemophilus influenzae strains.     

Relation of Macromolecular Synthesis in Streptococci to Efficiency of Transformation by Markers of Homospecific and Heterospecific Origin

In previous studies with Streptococcus sanguis and S. pneumoniae as recipients and donors of transforming deoxyribonucleic acid (DNA), it was found that heating recipients just prior to exposure to DNA caused an increase in the number of transformants induced by heterospecific DNA relative to that induced by homospecific DNA. In the present studies, S. sanguis recipients were found to recover from this effect of heat (48 C, 15 min) when incubated at 37 C before exposure to DNA. Inhibitors of nucleic acid synthesis, such as rifampin, 5-fluorodeoxyuridine, actinomycin, and p-hydroxyphenylazo-uracil, but not inhibitors of protein synthesis, such as chloramphenicol and erythromycin, prevented recovery from the effect of heat. Inhibitors of nucleic acid synthesis caused changes in unheated cells similar to those observed with heat treatment; these changes included increased transformability by genetically hybrid DNA and by low-efficiency markers in homospecific DNA. The effect of a combination of heat and inhibitors on transformation by heterospecific DNA was greater than when single treatments were used. The most effective inhibitor used alone was rifampin: in treated recipient cells, the yield of transformants produced by a given amount of irreversibly bound heterospecific DNA was increased without a significant change in the yield of transformants produced by bound homospecific DNA. A cell being doubly transformed by homospecific and heterospecific DNA was enhanced specifically in its transformability with the latter as a consequence of rifampin treatment. Treatment with rifampin also increased co-transformation by linked heterospecific markers. The period during which recipient cells were sensitive to the effects induced by rifampin and fluorodeoxyuridine lasted from 10 to 20 min after DNA uptake.     

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.     

Genetic Hybridization at the Unlinked THY and STR Loci of Streptococcus

The sanguis and pneumoniae species of Streptococcus were used as recipients in transformations from str+ to str-r and from thy- to thy+. The str-r mutations in the two species had been previously shown to be allelic. Homology of the thy- mutations in the two species was demonstrated in the similar phenotypic properties they conferred (death in the absence of thymidine, lack of thymidylate synthetase). The str and thy loci are unlinked in each species.--- When the two species are transformed by both homospecific and heterospecific DNA, the efficiency is always lower in the heterospecific cross. The efficiency of heterospecific transformation is considerably lower at the thy than at the str locus. DNA was extracted from recipients that had integrated markers of heterospecific origin. When such hybrid DNA is tested on the original recipient species, the heterospecific markers are usually as efficient as homospecific markers. When tested on the original donor species, however, the hybrid DNA is usually more efficient than heterospecific DNA. This is true for both thy and str transformation. -- -- Forty independent thy+ hybrids were obtained in the cross of sanguis thy- recipients with pneumoniae thy+ DNA. These hybrids fall into a number of classes based upon the relative efficiency with which their extracted DNA's are able to transfer the thy+ marker into pneumoniae thy- cells. The most efficient of these DNA's exhibits about 20% of the efficiency of homospecific pneumoniae thy+ DNA and three orders of magnitude greater efficiency than heterospecific sanguis thy+ DNA. Thus, very little of the inefficiency of heterospecific transformation of the thy locus is ascribable to a classic restriction mechanism. Rather, the wild-type thy+ loci in the two species appear to differ at multiple sites, and independent heterospecific transfers result in differential extents of integration of these sites. On this basis, the thy+ loci of the two species differ at a greater number of sites than do the respective str+ loci.     

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.     

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.