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.     

The effect of Waring Blendor treatment on transformation in Bacillus subtilis.

Treatment of competent Bacillus subtilis in a Waring Blendor for 10 s increases transformability of the culture about twofold while reducing the attachment of DNA to competent cells by 80%. The effectiveness of attached DNA in producing transformants is increased 10-fold by this treatment. The uptake of transforming DNA into a DNase-resistant state is progressively reduced by 70% during a 120-s blending treatment. Blending for 30-45 s diminishes transformability to about 10% of the original nonblended value without affecting the viable cell titer. No effect is produced by 30 s of blending on transformability if the irreversible uptake of DNA has been completed. Thus, the inhibition occurs at an early step in the transformation sequence. Treatment of the competent culture for 60 s or longer in the Waring Blendor reduces both the number of transformants obtained and the total number of viable cells.     

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.     

Alterations in Bacillus subtilis transforming DNA induced by beta-propiolactone and 1,3-propane sultone, two mutagenic and carcinogenic alkylating agents.

Transforming DNA was exposed to either beta-propiolactone or 1,3-propane sultone and then used for transformation of competent bacteria to nutritional independence from tyrosine and tryptophan (linked markers) and leucine (an unlinked marker). The ability to transform was progressively lost by the DNA during incubation with either of these two chemicals. For all three markers the inactivation curve was biphasic, with a short period of rapid inactivation followed by one characterized by a much slower rate. The overall rate of inactivation was different for all three markers and presumably was related to the size of the marker. The decrease in the transforming activity was in part due to the slower rate of penetration of alkylated DNA through the cellular membrane and its inability to enter the recipient bacteria. This decrease in the rate of cellular uptake, even for DNA eventually destined to enter the cell, began almost immediately after its exposure to the chemical and ended up with an almost complete lack of recognition of the heavily alkylated DNA by the specific surface receptors of competent cells. Such DNA attached to sites on the surface of competent bacteria which were different from receptors specific for the untreated nucleic acid. This attachment was not followed by uptake of the altered DNA. Presence of albumin during the incubation with a carcinogen further increased the degree of inactivation, indicating that the artificial nucleoproteins produced under such conditions were less efficient in the transformation assay than was the naked DNA. Cotransfomration of close markers progressively decreased, beginning immediately after the start of incubation of DNA with the chemicals. Extensively alkylated DNA fractionated by sedimentation through sucrose density gradients showed a peculiar distribution of cotransforming activity for such markers; namely, molecules larger than the bulk of DNA ("megamolecules") showed less ability to transform the second marker than did some of the apparently smaller molecules which sedimented more slowly through the gradient. An increase in cotransformation of distant markers was evident in DNA molecules after a short exposure to an alkylating agent, but cotransformation of such markers was absent in DNA treated for longer periods. The observed changes in the transforming and cotransforming activities of the alkylated DNA can be explained by what is known about the physicochemistry of such DNA and in particular about the propensity of the alkylated and broken molecules to form complexes with themselves and with other macromolecules.     

Studies on Transformations of Hemophilus influenzae : IV. Linked and unlinked transformations

Unlinked transformations were demonstrated to occur by varying the multiplicity of DNA molecules taken up by competent cells. The number of doubles was directly proportional to the product of the frequency of singles for varying concentrations of cells. The kinetics of transformation to doubles and the effect of DNA concentration on double transformations were consistent with the concept that the cell must take up two molecules of DNA in order to be doubly transformed. Linked markers, on the other hand, were a constant fraction of the single transformation for variations in DNA or cell concentration, or time. The kinetics of transformation of linked markers was the same as for the kinetics of single transforming factors. It was, therefore, concluded that linked transformations involve interaction between the cell and a molecule of DNA carrying both markers. The frequency of transformation was found to be the same from resistance to sensitivity as from sensitivity to resistance for the markers streptomycin (S) and cathomycin (C). Purified DNAs, in general, show lower levels of linkage than crude DNA preparations, and for some crude preparations all the S markers were linked to C, suggesting that some dispersion, at least, was a result of DNA preparation. The inactivation of linked markers by heat, ultraviolet, and DNAase was studied.     

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.     

Superhelical DNA in Streptococcus sanguis: role in recombination in vivo.

Summary Competent Streptococcus sanguis treated with non-lethal doses of coumermycin Al immediately before or after uptake of radioactive transforming DNA were reduced in their capacity to yield transformants. This treatment did not alter bacterial ability to bind DNA in DNase I-resistant form, nor did it prevent the single-stranded donor DNA-recipient protein complexes formed upon uptake at the surface of the bacteria from translocating to chromosomal sites. Inhibition of transformation by heterospecific DNA was greater than that by homospecific DNA. The reduction in transformant yield was not accompanied by any loss of donor counts incorporated into the recipient chromosome, but rather by a loss of genetic activity of incorporated donor material indicating a failure of genetic integration and degradation of donor DNA as a consequence of coumermycin treatment. The inhibitory effect of coumermycin on transformation was associated with in vivo loss of chromosomal DNA superhelicity. The chromosomal DNA remained intact, however, indicative of inhibition of a gyrase-like enzyme responsible for the maintenance of negative supercoiling of the S. sanguis chromosome. Upon treatment with the drug, a coumermycin-resistant mutant strain showed neither loss of chromosomal superhelicity nor any inhibitory effect on genetic integration of donor DNA. The evidence supports the idea that chromosomal superhelicity promotes genetic recombination in vivo.     

Ultraviolet inactivation and excision-repair in Bacillus subtilis. I. Construction and characterization of a transformable eightfold auxotrophic strain and two ultraviolet-sensitive derivatives

An eightfold auxotrophic strain of Bacillus subtilis was constructed. It was about equally well transformable for all markers. When this strain was used as recipient in transformation, single marker transformation frequencies of 0.5–2.0% were obtained. The markers were located relatively to each other using marker frequency analysis. Two UV-sensitive derivatives, equally well transformable as the parental multiple auxotroph, were isolated. One was highly sensitive to UV irradiation, was host cell reactivation-negative and did not show DNA breakdown or recovery of DNA synthesis after exposure to UV. Using UV-inactivated transforming DNA, this strain's transformability was strongly reduced as compared with both the UV-resistant parental strain and the other, moderately UV-sensitive, strain.     

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.     

Recognition of Streptococcal DNA by a Mutant Pneumococcus Unable to Discriminate among Markers in Pneumococcal DNA

A mutant strain of pneumococcus which fails to discriminate against low-efficiency markers during transformation by homospecific pneumococcal donor DNA retains the wild-type capacity to discriminate against heterospecific (streptococcal) donor DNA. We conclude that discrimination against heterospecific DNA must differ from that against low-efficiency markers by the kind or number of elements being recognized.     

Integration Efficiency in DNA-Induced Transformation of Pneumococcus. I. a Method of Transformation in Solid Medium and Its Use for Isolation of Transformation-Deficient and Recombination-Modified Mutants

A method of transformation on solid medium especially adapted for pneumococcus has been developed. Under specific conditions, all colonies that are allowed to grow in the presence of transforming DNA for six hours give rise to transformed bacteria. Combined with replica plating this technique has been used to isolate mutants modified with regard to recombination. Most of the mutants found are transformation-defective and show a large diversity in their response to ultraviolet light. Some of these mutants have lost their ability to take up transforming DNA. One shows a reduced yield of transformants for a given quantity of DNA taken up. Mutants that manifest altered behavior with regard to marker efficiencies have also been isolated. One of these exhibits a decrease in the transformation efficiency of only the high efficiency markers and two mutants show a decrease in the transformation efficiency of the low efficiency markers.     

Transformability of Streptomycin-resistant Group H Streptococci

Several resistant mutants of a transformable group H streptococcus, strain Challis, were isolated from media containing high concentrations of streptomycin. Mutants SR5a and SR5 exhibited high and low transformability, respectively, when exposed to deoxyribonucleic acid (DNA) from a novobiocin-resistant Challis strain. With similar exposure, mutant SR30 exhibited loss of transformability. The mutants further differed from the parent strain in time of appearance of optimal competence, and, in the case of SR5 and SR30, total growth was somewhat less than that of the parent. The rapidity with which transformants appeared upon initial exposure to DNA was approximately the same in the mutants and the parent strain. The decrease or loss of transformability of mutants SR5 and SR30 was found to be due to an alteration in capacity to take up DNA. Mutant SR5a (highly transformable) was further differentiated from mutants SR5 and SR30 in that it was somewhat more sensitive to high concentrations of streptomycin. Transformants obtained by treating strain Challis with the three types of mutant DNA, on the other hand, exhibited similar degrees of resistance to increasing concentrations of streptomycin. The additional decrease in transforming ability of mutant SR5a and the loss of transforming ability of mutant SR5 after a second exposure to streptomycin may indicate a stepwise process in the change from transformability to nontransformability. Although streptomycin resistance may not be directly related to inability to transform, results indicate that streptomycin greatly increases the chances of selecting these mutants and also can be of value in serving as a marker in studies of this nature.     

Plasmid uptake by bacteria: a comparison of methods and efficiencies

The ability to introduce individual molecules of plasmid DNA into cells by transformation has been of central importance to the recent rapid advancement of plasmid biology and to the development of DNA cloning methods. Molecular genetic manipulation of bacteria requires the development of plasmid-mediated transformation systems that include (1) chemical transformation, (2) electro-transformation, (3) biolistic transformation, and (4) sonic transformation, leading to the introduction of exogenous plasmid DNA into bacterial cells. In this review, the manipulation properties and transformation efficiencies of these techniques are described. In addition to these methods, a conceptually novel transformation technique, namely the hydrogel exposure method, was developed. The hydrogel exposure method, based on the Yoshida effect, provides a significant advance over chemical means for transforming many strains of Escherichia coli and a variety of other bacterial species. The new term “tribos transformation” has been proposed for this novel technique. We also determined that, compared to conventional methods, the hydrogel exposure method is a novel and convenient method by which to transform bacteria.     

Increased transformation ability of Escherichia coli strains carrying the plasmid pLD4

The effect of resident plasmid pLD4, a derivative of plasmid Hly241, on transformability of the host bacteria cells has been studied. Plasmid pLD4 was transferred into the different strains of E. coli subsequently transformed by the DNA of plasmids pBR322, pBR325, pAL-R2, pMB9. The majority of strains harbouring pLD4 obtain the increased ability to be transformed as compared with the ability of isogenic plasmidless strains. The similar but less expressed effect was conferred by the plasmid Hly241. Another hemolytic plasmid Hly195 and its derivatives, carrying the different transposons, as well as plasmid F' tet Hly did not increase the transformability of host bacteria. The optimal parameters for transformation of the strains harbouring pLD4 and plasmidless strains coincide, but the number of competent cells is considerably higher for plasmid containing strains, due to preliminary results.     

Genetic transformation in encapsulated clinical isolates of Haemophilus influenzae type b

Haemophilus influenzae type b strains isolated from children with meningitis, septicaemia and pharyngitis were studied for their ability to undergo genetic transformation by two chromosomal markers, streptomycin resistance and nalidixic acid resistance. Fifty-eight percent of the strains were non-transformable while the remaining 42% showed considerable strain variation with regard to their transformation frequencies, which ranged from 8 x 10(-4) to 1 x 10(-6). The effect of type b capsule on competence development and transformation activity was studied by comparing encapsulated strains with their non-encapsulated variants. Type b capsule did not inhibit either competence development or transforming efficiency. The lack of transformability in the majority of strains was not due to the presence of a capsule.     

The extracellular nuclease Dns and its role in natural transformation of Vibrio cholerae

Free extracellular DNA is abundant in many aquatic environments. While much of this DNA will be degraded by nucleases secreted by the surrounding microbial community, some is available as transforming material that can be taken up by naturally competent bacteria. One such species is Vibrio cholerae, an autochthonous member of estuarine, riverine, and marine habitats and the causative agent of cholera, whose competence program is induced after colonization of chitin surfaces. In this study, we investigate how Vibrio cholerae's two extracellular nucleases, Xds and Dns, influence its natural transformability. We show that in the absence of Dns, transformation frequencies are significantly higher than in its presence. During growth on a chitin surface, an increase in transformation efficiency was found to correspond in time with increasing cell density and the repression of dns expression by the quorum-sensing regulator HapR. In contrast, at low cell density, the absence of HapR relieves dns repression, leading to the degradation of free DNA and to the abrogation of the transformation phenotype. Thus, as cell density increases, Vibrio cholerae undergoes a switch from nuclease-mediated degradation of extracellular DNA to the uptake of DNA by bacteria induced to a state of competence by chitin. Taken together, these results suggest the following model: nuclease production by low-density populations of V. cholerae might foster rapid growth by providing a source of nucleotides for the repletion of nucleotide pools. In contrast, the termination of nuclease production by static, high-density populations allows the uptake of intact DNA and coincides with a phase of potential genome diversification.     

Bacterial gene transfer by natural genetic transformation in the environment.

Natural genetic transformation is the active uptake of free DNA by bacterial cells and the heritable incorporation of its genetic information. Since the famous discovery of transformation in Streptococcus pneumoniae by Griffith in 1928 and the demonstration of DNA as the transforming principle by Avery and coworkers in 1944, cellular processes involved in transformation have been studied extensively by in vitro experimentation with a few transformable species. Only more recently has it been considered that transformation may be a powerful mechanism of horizontal gene transfer in natural bacterial populations. In this review the current understanding of the biology of transformation is summarized to provide the platform on which aspects of bacterial transformation in water, soil, and sediments and the habitat of pathogens are discussed. Direct and indirect evidence for gene transfer routes by transformation within species and between different species will be presented, along with data suggesting that plasmids as well as chromosomal DNA are subject to genetic exchange via transformation. Experiments exploring the prerequisites for transformation in the environment, including the production and persistence of free DNA and factors important for the uptake of DNA by cells, will be compiled, as well as possible natural barriers to transformation. The efficiency of gene transfer by transformation in bacterial habitats is possibly genetically adjusted to submaximal levels. The fact that natural transformation has been detected among bacteria from all trophic and taxonomic groups including archaebacteria suggests that transformability evolved early in phylogeny. Probable functions of DNA uptake other than gene acquisition will be discussed. The body of information presently available suggests that transformation has a great impact on bacterial population dynamics as well as on bacterial evolution and speciation.     

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.