Molecular Basis for the Transformation Defects in Mutants of Haemophilus influenzae

To determine the molecular basis of transformation defects in Haemophilus influenzae, the fate of genetically marked, (32)P-labeled, heavy deoxyribonucleic acid (DNA) was examined in three mutant strains (rec(1) (-), rec(2) (-), and KB6) and in wild type having (3)H-labeled DNA and a second genetic marker. Transforming cells upon lysis with digitonin followed by low-speed centrifugation are separable into the supernatant fraction, containing mainly the unintegrated donor DNA, and the pellet, containing most of the resident DNA along with integrated donor DNA. Electron micrographs of digitonin-treated cells also indicate that the resident DNA is trapped inside a cellular structure but that cytoplasmic elements such as ribosomes are extensively released. DNA synthesis in digitonin-treated cells is immediately blocked, as is any further integration of donor DNA into the resident genome. Isopycnic and sedimentation analysis of supernatant fluids and pellets revealed that in strains rec(2) (-) and KB6 there is little or no association between donor and resident DNA, and thus there is negligible transfer of donor DNA genetic information. In these strains, the donor DNA is not broken into pieces of lower molecular weight as it is in strain rec(1) (-) and in the wild type, both of which show association between donor and recipient DNA. In strain rec(1) (-), although some donor DNA atoms become covalently linked to resident DNA, the incorporated material does not have the donor DNA transforming activity.     

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.     

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.     

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.     

Plasmid marker rescue transformation proceeds by breakage-reunion in Bacillus subtilis.

Bacillus subtilis carrying a plasmid which replicates with a copy number of about 1 was transformed with linearized homologous plasmid DNA labeled with the heavy isotopes 2H and 15N, in the presence of 32Pi and 6-(p-hydroxyphenylazo)-uracil to inhibit DNA replication. Plasmid DNA was isolated from the transformed culture and fractionated in cesium chloride density gradients. The distribution of total and donor plasmid DNA was examined, using specific hybridization probes. The synthesis of new DNA, associated with the integration of donor moiety, was also monitored. Donor-specific sequences were present at a density intermediate between that of light and hybrid DNA. This recombinant DNA represented 1.4% of total plasmid DNA. The latter value corresponded well with the transforming activity (1.7%) obtained for the donor marker. Newly synthesized material associated with plasmid DNA at the recombinant density amounted to a minor portion of the recombinant plasmid DNA. These data suggest that, like chromosomal transformation, plasmid marker rescue transformation does not require replication for the integration of donor markers and, also like chromosomal transformation, proceeds by a breakage-reunion mechanism. The extent of donor DNA replacement of recipient DNA per plasmid molecule of 54 kilobases (27 kilobase pairs) was estimated as 16 kilobases.     

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.     

Fate of Donor Deoxyribonucleic Acid in a Highly Transformation-Deficient Strain of Haemophilus influenzae

A transformation-deficient strain of Haemophilus influenzae (efficiency of transformation 10⁴-fold less than that of the wild type), designated TD24, was isolated by selection for sensitivity to mitomycin C. In its properties the mutant was equivalent to recA type mutants of Escherichia coli. The TD24 mutation was linked with the str-r marker (about 30%) and only weakly linked with the nov-r2.5 marker. The uptake of donor deoxyribonucleic acid (DNA) was normal in the TD24 strain, but no molecules with recombinant-type activity (molecules carrying both the donor and the resident marker) were formed. In the mutant the intracellular presynaptic fate of the donor DNA was the same as that in the transformation-proficient (wild-type) strain, and the radioactive label of the donor DNA associated covalently with the recipient chromosome in about the same quantity as in the wild type. However, many fewer donor atoms were associated with segments of the mutant's recipient chromosome as compared with segments of the wild-type chromosome. In the mutant the association was accompanied by complete loss of donor marker activity. The lack of donor marker activity of the donor-recipient complex of DNA isolated from the mutant was not due to lack of uptake of the complex by the second recipient and its inability to associate with the second recipient's chromosome. Because the number of donor-atom-carrying resident molecules was higher than could be accounted for by the lengths of presynaptic donor molecules, we favor the idea that the association of donor DNA atoms with the mutant chromosome results from local DNA synthesis rather than from dispersive integration of donor DNA by recombination.     

Effect of adenosine 5''-triphosphate-dependent deoxyribonuclease deficiency on properties and transformation of Haemophilus influenzae strains.

A transformation-deficient strain of Haemophilus influenzae, lacking adenosine 5'-triphosphate-dependent deoxyribonuclease activity, was isolated by selection for sensitivity to mitomycin. The mutant, designated JK57, possibily showed a moderate sensitivity to ultraviolet (UV) irradiation and treatment with methyl methane sulfonate. Contrary to the wild type, the mutant degraded chromosomal deoxyribonucleic acid (DNA) to some extent. However, after UV irradiation to the mutant degraded considerably less DNA than the wild type and the TD24 mutant of H. influenzae, the latter being equivalent to a recA mutant of Escherichia coli. A TD2457 double mutant, constructed by transferring the TD24 mutation into the JK57 strain, was as sensitive to deleterious agents and as deficient in transformation as the TD24 single mutant; in the double mutant, however, after UV irradiation chromosomal DNA was degraded to the same extent as in the JK57 mutant. The number of transformants per unit of radioactive donor DNA taken up by JK57 recipient cells was approximately 10-fold smaller than in the wild type. Presynaptically, the fate of donor DNA in the adenosine 5'-triphosphate-dependent deoxyribonuclease-deficient mutants was not different from that in the wild type. In contrast to TD24 and the TD2457 double mutant, in the JK57 mutant, recombinant-type activities (molecules carrying both the donor and recipient markers) were formed almost as well as in the wild type. After integration into the JK57 recipient genome, the rate of replication of the donor marker was equal to that of the recipient marker during a number of generations, which suggests that the donor DNA is normally integrated into the JK57 chromosome. It is suggested that transformed JK57 cells pass with a high frequency into a type of cells that can replicate their chromosomes many times but have lost the ability to form visible colonies after plating.     

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.     

Effect of 4,5'',8-trimethylpsoralen interstrand cross-links present in recipient Bacillus subtilis on the integration of transforming DNA.

When recipient Bacillus subtilis carrying chromosomal trimethylpsoralen cross-links were transformed, the donor marker activity decreased with the extent of cross-linking. Additional donor marker activity was lost upon incubation of the reextracted DNA with nuclease S1, particularly at higher levels of cross-linking. Physical analysis of the reextracted DNA showed that the donor DNA was progressively excluded from heteroduplex formation as the frequency of cross-links in the recipient DNA increased. In the donor-recipient complexes still being formed, increasing amounts of donor DNA became susceptible to nuclease S1 digestion under these conditions. These results suggest that resident interstrand cross-links interfere both with initiation of recombination and with the completion of heteroduplex formation.     

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.     

Integration and Repair of Ultraviolet-Irradiated Transforming Deoxyribonucleic Acid in Haemophilus influenzae

The extent of association between donor transforming deoxyribonucleic acid (DNA) and recipient DNA in Haemophilus influenzae as a function of ultraviolet (UV) dose to the transforming DNA has been measured by isopycnic analysis of lysates of (3)H-labeled recipient cells exposed to DNA labeled with (32)P and heavy isotopes. Except for doses above 15,000 ergs/mm(2), the results of these measurements are in good agreement with previous estimates made by another technique. Experiments with a mutant temperature sensitive for DNA synthesis and another mutant defective in excision of pyrimidine dimers suggest that the discrepancy between the methods of high doses results from DNA synthesis, in which portions of the associated donor DNA containing pyrimidine dimers are excised and broken down, and the components are reutilized for synthesis.Repair of UV-irradiated, transforming DNA during incubation of recipient cells is observed as an increase in transforming ability when fractions from CsCl gradients of cell lysates are assayed on excision-deficient cells. When transforming DNA containing markers of different UV sensitivities is used, repair of the UV-resistant nov marker by excision proficient cells takes place exclusively in the donor DNA that is associated with recipient DNA, and this repair is observed even in the absence of DNA synthesis. However, no repair is observed in the case of the more UV-sensitive str marker, possibly because excision events may remove a large fraction of the integrated str markers in addition to repairing a small fraction of the integrated DNA containing this marker.     

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 fate of DNA in a strain of Bacillus subtilis which is impaired in genetic transformation.

A mutation in Bacillus subtilis call recC4 which results in an impairment of genetic transformation was transferred to a new strain using the closely linked marker mit-2 (mitomycin C-resistance) for selection. This derived strain was in turn impaired in transformation but showed normal levels of sensitivity to ultraviolet irradiation and methyl methane sulfonate. The genetic and molecular fate of transforming DNA in the recC4 strain was studied. Normal amounts of DNA were taken up by the cells and this DNA or parts of it became associated with recipient DNA. Linkage between genes on donor and recipient molecules was, however, not established and transformants were not generated. The recC4 mutation therefore affects a step in the recombination pathway during transformation. Either the association between donor and recipient DNA molecules is abnormal or the cells are deficient in the further processing of the associated complex.     

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.     

Plasmid-mediated NAD independence in Haemophilus parainfluenzae.

The location of the genes coding for NAD independence in four unusual clinical isolates of Haemophilus parainfluenzae was determined by transferring these genes to plasmid-free Haemophilus influenzae Rd by transformation and analysing transformants for the presence of plasmids by agarose gel electrophoresis. All NAD-independent transformants were found to carry a single plasmid species. The plasmids, originally harboured by the four H. parainfluenzae isolates recovered from unrelated sources, were of the same size (5.25 kb). Spontaneous reversion to NAD dependence occurred with a low frequency (0.1 to 0.2% of the progeny of a single clone) in both H. parainfluenzae and H. influenzae Rd. The revertants had lost this small plasmid. Mitomycin C exhibited a plasmid 'curing' effect with a frequency of 'curing' of between 1 and 6% of the surviving clones. It was concluded that the genes conferring NAD independence were located on the small 5.25 kb plasmid.     

Genetic transformation of Streptococcus pneumoniae by DNA cloned into the single-stranded bacteriophage f1.

A Staphylococcus aureus plasmid derivative, pFB9, coding for erythromycin and chloramphenicol resistance was cloned into the filamentous Escherichia coli phage f1. Recombinant phage-plasmid hybrids, designated plasmids, were isolated from E. coli and purified by transformation into Streptococcus pneumoniae. Single-stranded DNA was prepared from E. coli cells infected with two different plasmids, fBB101 and fBB103. Introduction of fully or partially single-stranded DNA into Streptococcus pneumoniae was studied, using a recipient strain containing an inducible resident plasmid. Such a strain could rescue the donor DNA marker. Under these marker rescue conditions, single-stranded fBB101 DNA gave a 1% transformation frequency, whereas the double-stranded form gave about a 31% frequency. Transformation of single-stranded fBB101 DNA was inhibited by competing double-stranded DNA and vice versa, indicating that single-stranded DNA interacts with the pneumococcus via the same binding site as used by double-stranded DNA. Heteroduplexed DNA containing the marker within a 70- or 800-base single-stranded region showed only slightly greater transforming activity than pure single-stranded DNA. In the absence of marker rescue, both strands of such imperfectly heteroduplexed DNA demonstrated transforming activity. Pure single-stranded DNA demonstrated low but significant transforming activity into a plasmid-free recipient pneumococcus.     

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.     

Transformation of natural genetic variation into Haemophilus influenzae genomes

Many bacteria are able to efficiently bind and take up double-stranded DNA fragments, and the resulting natural transformation shapes bacterial genomes, transmits antibiotic resistance, and allows escape from immune surveillance. The genomes of many competent pathogens show evidence of extensive historical recombination between lineages, but the actual recombination events have not been well characterized. We used DNA from a clinical isolate of Haemophilus influenzae to transform competent cells of a laboratory strain. To identify which of the ~40,000 polymorphic differences had recombined into the genomes of four transformed clones, their genomes and their donor and recipient parents were deep sequenced to high coverage. Each clone was found to contain ~1000 donor polymorphisms in 3-6 contiguous runs (8.1±4.5 kb in length) that collectively comprised ~1-3% of each transformed chromosome. Seven donor-specific insertions and deletions were also acquired as parts of larger donor segments, but the presence of other structural variation flanking 12 of 32 recombination breakpoints suggested that these often disrupt the progress of recombination events. This is the first genome-wide analysis of chromosomes directly transformed with DNA from a divergent genotype, connecting experimental studies of transformation with the high levels of natural genetic variation found in isolates of the same species.