Article

Studies on the nature of hemophilus influenzae cells susceptible to heritable changes by desoxyribonucleic acids

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Abstract

In E. influenzae the highly specific desoxyribonucleic acids (DNA's) which play the role of heredity determinants of type specificity and SM resistance, have induced these traits in only a small proportion of the population exposed to their action. The evidence suggests that this small proportion, "the susceptible cells" possess a property or substance needed by the DNA in order to induce an heritable change. The size of the small proportion of susceptible cells can be influenced significantly by a number of factors; when all the factors now to be listed are operating the frequency has not exceeded 1:1000. The Type of Origin of Recipient Cells.—Type a exhibits the lowest frequency, about 1:10,000,000, and type d the highest, approximately 1 per 1000 cells exposed. This type-specific property which controls the frequency of susceptible cells is an inherited trait; repeated change to a heterologous type shows no influence on the incidence of these cells. Concentration of DNA.—Within certain limits increase in the concentration of DNA controlling streptomycin resistance can increase the size of the proportion of cells in which streptomycin resistance can be induced. However, increases in concentrations greater than 10–1 µg. per ml. have not induced streptomycin resistance in a higher proportion of cells. Phase of Growth Cycle.—Predictable fluctuations in frequency of induced heritable changes have been demonstrated in both Rb and Rd populations during growth. There is no evidence that susceptible cells reproduce their kind; they emerge in all experiments when the population reaches the end of the logarithmic period and a density of 2 to 4 x 108 cells per ml. In the early logarithmic phase it is difficult to demonstrate the presence of susceptible cells. The peak frequency of susceptible cells occurs in the early stationary phase of the growth cycle. Thereafter, the decline in frequency is a gradual one. The data suggest that in a given population the same cells are susceptible to a number of different type-specific DNA's and the DNA controlling SM resistance. Comparison of Frequency of Cells Susceptible to Different DNA's.—In a given population the frequency of cells susceptible to different type-specific DNA's and the DNA controlling streptomycin resistance is not significantly different. Competition between Type-Specific DNA's—The data suggest that DNA's of types a, b, and c compete for the same cells in Rd populations. When Rd populations are exposed simultaneously to 2 of these 3 DNA's in different concentrations the proportion which each type contributes to the total type-specific cells induced is closely correlated with the concentration of the corresponding DNA. Exclusion of DNA's.—Induction of one type specificity or streptomycin resistance can be completely prevented in a population containing susceptible cells by previous exposure for 15 minutes to a 1000-fold higher concentration of another type-specific DNA.

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... Transformation to capsular synthesis was related to the concentration of DNA (Table 2), and capsulated bacteria were never found in control preparations of recipient strain m treated with deoxyribonuclease-inactivated DNA. The response of competent bacteria to eight different concentrations of DNA was examined in experiments with DNA preparations 2 and 4. Results for both str-r and type b antigen markers corresponded to those for streptomycin resistance reported by Alexander, Leidy & Hahn (1954) and by . Capsulated bacteria were observed in tests of 0-0001 pg. ...
Article
Capsular transformation of Haemophilus influenzae strain rd was examined at the cellular level by a semi-quantitative method. Type b capsular antigen synthesized by transformants was detected by its reaction with type b antibody conjugated to fluorescein isothiocyanate. Deoxyribonucleate (DNA) preparations from type b capsular transformants of strain rd elicited somewhat higher frequencies of transformation than preparations from type b clinical isolates. Str-r markers on these DNAs conferred resistance to a concentration of 500 μg. dihydrostreptomycin sulphate/ml. For a given DNA, the frequency of transformation to streptomycin resistance was at least 100 times higher than that to capsular synthesis. The time course of expression of the two phenotypes by DNA-treated bacteria was examined in broth in which the generation time of the total population was about 50 min. Resistant transformants began to appear 25 min. after initiation of treatment and capsulated bacteria were observed after 40 min. Both properties continued to be expressed until about 120 min. Thereafter, the behaviour of recipients of the two kinds of genetic markers differed. The rate of increase of the str-r transformants became equal to that of the total population, and in a subculture diluted 1/20 they increased 20-fold during subsequent incubation for 150 min. The number of capsulated transformants, on the other hand, increased less than two-fold over the period 120-330 min.
... Anagnostopoulos and Spizizen (1961) also observed that this is the same period in which competence is acquired in Bacillus subtilis. Similarly , Haemophilus influenzae reached its highest peak of competence during the early stationary phase (Alexander, Leidy, and Hahn, 1954). However , the results obtained by observing the growth of several strains of transformable streptococci showed that optimal competence was attained in the early logarithmic phase. ...
Article
Perry, Dennis (Northwestern University Medical School, Chicago, Ill.) and Hutton D. Slade. Optimal conditions for the transformation of streptococci. J. Bacteriol. 85 636–642. 1963.—A study of the properties of the streptococcal transforming system, employing streptomycin resistance as a marker, resulted in a 1,000- to 10,000-fold increase in the rate of transformation. In some cases, as high as 0.5% transformants have been obtained. Certain aspects of the system differed markedly from those of other bacterial transforming systems, particularly with regard to the time for the appearance of competence. When a group H strain was exposed to deoxyribonucleic acid after various periods of incubation, the time at which cells were most competent was about 2 hr. Similar experiments with a different group H strain and an ungroupable strain showed that competence for both occurred after 1 hr of growth. It is significant that the period of optimal competence, though attained at different times, took place at the beginning of the logarithmic phase of growth. The temperature optimum for transformation was about 37 C. Cells preincubated in sheepblood broth exhibited a higher level of transformation than cells preincubated in plain or human serum broth. That some correlation exists between the attainment of competence and growth is evident. In contrast, human serum was most effective in the transforming media. The few preparations of bovine albumin (fraction V) employed did not appreciably support transformation. Data are also presented which show that some relationship exists between transforming efficiency and serological classification, in that homologous transformation showed a 100- to 10,000-fold greater efficiency over heterologous transformation. Antibodies to deoxyribonuclease, as well as other inhibitors of deoxyribonuclease, failed to bring about transformation in group A streptococci.
... Early studies on DNA transformation in H. influenzae provided evidence that nonencapsulated variants of type d were the most efficient recipients in DNA transformation, followed by those of type b, e and a, while non-encapsulated variants of type c and f were nontransformable (Alexander et al., 1953). It was concluded that a correlation exists between frequency of transformation and serotype of origin. ...
Article
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.
Chapter
Hae.mo' phi.lus. Gr. n. haima blood; Gr. n. philos lover; M.L masc. Haemophilus blood-lover. Proteobacteria / Gammaproteobacteria / Pasteurellales / Pasteurellaceae / Haemophilus Minute to medium-sized coccobacilli or rods, generally less than 1 µm in width and variable in length, sometimes forming threads or filaments and showing marked pleomorphism. Gram negative. Nonmotile. Aerobic or facultatively anaerobic. Require preformed growth factors present in blood, particularly X factor (protoporphyrin IX or protoheme) and/or V factor (nicotinamide adenine dinucleotide [NAD] or NAD phosphate [NADP]). Even after specific growth factors have been provided, growth is best on complex media. Optimum temperature, 35–37°C. Nitrates are reduced to or beyond nitrites. Oxidase and catalase reactions vary among strains. Chemoorganotrophic. All species can attack carbohydrates fermentatively, yielding acetic, lactic, and succinic acids as end products in glucose broth. Occur as obligate parasites on the mucous membranes of humans and a variety of animal species. Several 16S rRNA sequence signatures for the family Pasteurellaceae have been demonstrated, but none of these is specific for the genus Haemophilus as presently defined (Dewhirst et al., 1992). The mol% G + C of the DNA is: 37–44. Type species: Haemophilus influenzae (Lehmann and Neumann 1896) Winslow, Broadhurst, Buchanan, Krumwiede, Rogers, and Smith 1917, 561 (Bacterium influenzae Lehmann and Neumann 1896, 187.)
Article
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A procedure has been developed for obtaining Hemophilus influenzae of such competence that 1 to 10 per cent transform to any of several genetic factors by utilizing a period of aerobic growth followed by a non-aerobic period. Differences in levels of competence were not due to differences in genetic background. Competence was due to at least one factor intrinsic to the cell or site on the cell and was not transferable to non-competent cells. Competence was affected by salt concentration, pH, and temperature. Washing competent cells reduces their ability to transform, but not their capacity to bind DNA reversibly. The irreversible step could be restored with little or no accompanying growth. These facts suggest that reversible and irreversible binding represent separate biochemical steps. DNA initiates a reaction in cells leading to a loss of competence. In the absence of DNA the cells remain competent for at least an hour. Competence correlates quantitatively with predictability of multiple transformations. The observed and calculated values of multiple transformations are in closer agreement, the higher the frequency of transformation for single markers. The correction needed to bring the two figures into agreement is a measure of the fraction of non-competent cells.
Article
Two different strains of pneumococcus show a markedly different transformation frequency for a given genetic marker (str) in a transforming preparation while showing identical frequencies for another genetic marker (ery2) in the same transforming preparation. It was concluded that the frequency of the transformation to streptomycin resistance was consistently lowered in one receptor strain.The kinetics of expression of the str marker in the affected host indicates the abrupt cessation of an otherwise normal expression of the str transformants between 40 to 50 minutes after the initial exposure to DNA, whereas in the unaffected host expression of str transformants continues for at least another 30 minutes.From the initial rate of expression of the str markers on the affected and the unaffected hosts, it was concluded that the number of incorporated transformants was identical for both hosts, and that some post-incorporation elimination of determinants was responsible for the different relative frequency on Rz.The transformation of the ability to cause a depressed transfer of the str agent has been studied, and its association with and separability from the str marker revealed.The presence of the depressor factor has been shown to greatly influence the recovery of the str marker from UV-irradiated DNA-preparations.The nature of the factor responsible for the depression of the str marker is discussed.
Article
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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.
Article
Kinetic measurements have been made of the cumulative number of pneumococcal transformants as a function of the duration of exposure of cells to a multiply marked DNA. Transformants to one or more (linked) markers on a single particle accumulate linearly, whereas those to two unlinked markers accumulate quadratically and at a frequency very close to that expected from successive interaction of one cell with two DNA particles. Cells are not rendered less susceptible to transformation by a recent interaction with another DNA particle, even one involving a nearby region. Linkage frequency can be expressed as the ratio of the linear rate of accumulation of multiply marked to singly marked transformants.For a linked group of three markers, the linear rate of accumulation of transformants acquiring any one, two or three of these markers demonstrates the frequent occurrence of complex intramolecular events in pneumococcal transformation; under certain conditions of exposure, these can be very regular. Such an event is the type initiated by a single triply marked particle when it produces a recombinant lacking the central marker. The proportion of these recombinants suggests that there is very little interference between events occurring over the entire interval. Furthermore, the recombination is a unique event within one division cycle of the cell; and it gives rise in non-selective medium to clones of transformants which are pure and show no evidence of heterozygous cells. Redistribution of the allelic pairs between donor DNA and recipient cells seems to alter the ratio of complex to simpler intramolecular recombinations over a twofold range.
Article
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Hemophilus influenzae-transforming DNA, which has been inactivated by ultra-violet radiation, is reactivated by visible light in the presence of a cell-free extract of Escherichia coli B. The time rate of reactivation is increased by increasing the E. coli extract concentration, the temperature, and the intensity of illumination. Only DNA containing an ultraviolet-damaged genetic marker exhibits increased transforming activity after treatment with the photoreactivating system. The reactivating capacity of the extract remains in the top supernatant after centrifugation at 110,000 x g for 1 hour and is not present in the pellet. This capacity is destroyed by heating to 90 degrees C. for 1 minute. The active system of the E. coli extract is separable into dialyzable, heat-stable and non-dialyzable, heat-labile fractions. The dialyzable fraction contains at least one component which limits the maximum degree of recovery attained.
Article
Perry, Dennis (Northwestern University Medical School, Chicago, Ill.), and Hutton D. Slade. Intraspecific and interspecific transformation in streptococci. J. Bacteriol. 88 595–601. 1964.—Interspecific transformation reactions, employing streptomycin resistance as a marker, were performed with eight strains of streptococci belonging to three serological groups (F, H, and O) and one ungroupable (UG) strain. Generally, autotransformation (within the same strain) was most efficient. Homotransformation (between different strains of the same serological group), however, was sometimes as efficient or slightly better. Heterotransformation (between different serological groups) yielded the least number of transformants or none at all. The rate of transformation of different strains varied from 2.0 × 10⁻⁷ to 7.6 × 10⁻³. Group H streptococci exhibited the highest rate of autotransformation, followed by groups F and O, and strain UG. The results of heterotransformation reactions revealed that a genetic relationship exists between various strains of different serological groups. No transformation, however, occurred between F and O strains, indicating a lack of genetic homology between these serological groups of streptococci. Deoxyribonucleic acid (DNA), isolated by physical and enzymatic methods from a group O and a UG strain, failed to induce transformation. DNA from these strains, however, significantly inhibited the transforming ability of other DNA preparations. Heat and mechanical shear resulted in a marked decrease in this inhibitory property.
Article
Ultraviolet-inactivated Hemophilus influenzae transforming DNA recovers its activity when mixed with cell-free extracts of bakers' yeast and exposed to visible light. The active agent in the extract is not used up in the reaction, and purification has not separated it into more than one non-dialyzable component. It differs from the agent in Escherichia coli extract, which produces very similar photoreactivation, but which can be resolved into non-dialyzable and dialyzable components, the latter being used up during illumination. The yeast agent can be salted out of solution and recovered quantitatively; it is inactivated by crystalline trypsin and chymotrypsin and by brief heating at 60°C.—all facts suggesting that it is an enzyme for which ultraviolet lesions in the DNA serve as substrate. The kinetics of recovery are also consistent with such an assumption. This enzyme is unusual both because it is involved in a light-dependent reaction and because it has a non-destructive action on DNA outside an intact cell.
Article
The requirements and characteristics of Pasteurella novicida transformations in liquid suspensions were studied. Transformation frequencies of 0.1 to 0.3% were routinely obtained when recipient cells were harvested from 16-hr agar plates and higher than 1% when logarithmic-phase broth-grown cells were used. Calcium ions were essential for transformations. The deoxyribonucleic acid dose response curve, kinetics of transformation, and pH optimum for transformations were similar to those of other bacterial transformation systems. The genetic relatedness of P. novicida and P. tularensis was established by transforming P. novicida mutants with deoxyribonucleic acid extracted from P. tularensis.
Article
UV irradiation of transforming DNA from Haemophilus influenzae, carrying a streptomycin resistance marker (Sr), results in decreased transforming activity. At high DNA concentration the “marker survival” is lower than it is at low concentration. The transition from high to low survival occurs at concentrations ranging from 2.5×10-3 to 2.5×10-2 μg/ml; in this range the probability that transformed cells take up DNA fragments in addition to the marked one increases rapidly. A similar effect of DNA concentration on the percentage of transformants is observed for a mixture of unirradiated and irradiated DNA, where virtually all of the transformants originate from the unirradiated component. This eliminates the possible explanation that the concentration dependence of UV survival of a marker reflects increasing competition for a cellular repair system. It is concluded that the lower marker survival obtained at high DNA concentration involves lethality due to UV lesions present in the additional irradiated DNA taken up by the cell. Thus the steeper marker survival curve is due to the increasing UV dose which the additional DNa necessarily receives when a marker survival curve is being established. Intergration of UV lesions rendering a chromosomal DNA strand inviable is suggested by a slight delay in cell multiplication after uptake of irradiated and — to a lesser extent — unirradiated DNA. Acriflavine at a concentration of 0.5μg/ml enhances the effect of DNA concentration on marker survival. Similarly the number of transformants obtained with unirradiated DNA in the presence of acriflavine is more strongly decreased at high than at low DNA concentration. It is suggested that each event of DNA integration involves a small change for lethality, which is enhanced if the DNA carries UV lesions or if acriflavine is present.
Chapter
This chapter discusses the chemical nature of the transforming agent and mechanism of recombination occurring in transformation. The investigation of pneumococcal transformations was concerned largely with the chemical identification of the substance in the donor extract that was capable of inducing genetic transformations. A type of genetic transfer was discovered in the Salmonellae. In this type, referred to as “transduction,” a small portion of the genetic material of the donor bacterium, similar in amount to that transferred in DNA-mediated transformations, is transferred to the recipient bacterium by means of a virus that had previously reproduced in the donor and had subsequently infected the recipient. Both in conjugation and transduction DNA is the material transferred from donor to recipient. While this fact correlates the mechanisms underlying transformation, transduction, and conjugation, there are differences between the modes of transfer involved in these three processes to provide well-defined operational criteria for distinguishing them. A comparison of the mechanism of recombination occurring in these modes of transfer is presented. The chapter also discusses two classic schemes to account for “crossing-over” between homologous chromosomes in higher organisms.
Article
A mathematical study has been made of bacterial transformation with respect to the genetic and non-genetic parameters which determine transformation frequency. The phenomenon can be analysed in simplified terms by assuming that the sites of irreversible fixation of DNA in a population of competent bacteria follows a Poisson distribution. As a consequence, calculations can be made of the frequencies of single and multiple transformations as functions of the competence of the bacteria, of the nature and of the concentration of transforming DNA, as well as of two genetic parameters: the probability of integration of a marker and the genetic linkage between them. These can be defined as follows: (1)The probability of integration of a marker is inferred from the frequency with which the marker replaces its allele in the bacterial genome. Account must be taken of the penetration of the genetically marked DNA into the cell and its pairing with homologous DNA.(2)Two markers can be (a) both genetically and physically independent because they are situated on different particles; (b) physically associated on a single particle but showing no genetic linkage or (c) genetically as well as physically linked.In case (2c), genetic linkage is defined as the probability with which one of a pair of markers accompanies the other in recombination. This probability is not necessarily identical for the two markers. The relationships describing the frequency of double transformations show that the ratio of this frequency to the product of the single transformation frequencies is an insufficient criterion of genetic linkage because of other factors, e.g. the bacterial competence. The study of these two genetic parameters has been facilitated by a model of intracellular recombination based on the following considerations. The genetic integration of a sequence of the transforming molecule requires two events, e.g. two breaks in the genome: the probability of a break between two neighboring elements (recon) is constant. This probability (permitting a calculation of the average length of an integrated segment) on the one hand and the dimensions of the markers, their distances apart and their position, on the other hand determine the two genetic parameters. The probability of integration of a point mutation situated in the middle of a molecule is 0·5. It is greater if the replacement of any part of a long mutated sequence suffices for the transformation; smaller if the mutated sequence to be incorporated is long or if the marker is terminal at one of the extremities of the molecule. Under this model, the differences in reciprocal transformation can be explained only by the differences in the size of the markers. Linkage observed between two genes has been calculated for different cases. It depends generally on the linear distance between two genes, their size, and their position on the molecule. Special attention is given to the possible causes of unequal frequencies of recombination (inter and intracistronique) in reciprocal transformations. According to the model these differences are due to differences in size of the two genes and/or their positions on the molecule. Finally, the consequences of the supposition that the bacterial chromosome, composed of a single DNA filament, is broken at random upon extraction of transforming DNA, are discussed.
Article
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Article
FOR investigating the mechanism of bacterial transformation we have chosen as the experimental model the transformation of the R form of Diplococcus pneumoniae type II into the mucous form of D. pneumoniae type III, by means of crude extracts of deoxyribonucleic acid, isolated from the mucous form of D. pneumoniae type III. Systematic study was hampered by considerable variability in the results and by the low incidence of transformation in the control tubes.
Article
We did not succeed in transforming the host specificity of rhizobia reliably, in spite of the use of DNA preparations from 3 different rhizobia species (Rh. meliloti, Rh. trifolii, Rh. leguminosarum) and of 35 acceptor strains representative for 7 rhizobia species. Very few positive results could not be repeated. Rhizobia from ineffective nodules on alfalfa plants of the first inoculation test following the transformation procedure formed effective nodules during a second plant passage. Reisolates from these nodules exhibiting a rough type of growth differ from the smooth colonies of the donator strain as well as of the acceptor strains. These rough growing rhizobia agree with the donator strain but not with the acceptor strains as to the base composition of their deoxyribonucleic acids. Therefore we assume that some cells of the donator have survived the DNA preparation and the 24 h sterilization by ethanol, and being only weakened have formed at first ineffective but secondly—by way of regeneration—effective nodules.Pseudonodules of unknown origin on alfalfa roots may be very similar to ineffective bacteria root nodules. They consist mainly of parenchymatic tissue partly with one or several vascular bundles. Because these exuberances were found also on the roots of uninoculated control plants, they did not result from a transformation process.
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A 28-year-old female in Denver was found in early 1974 to have frontal sinusitis, osteomyelitis, and bacteremia due to Haemophilus influenzae, type B. The minimal inhibitory concentration of ampicillin for this organism was 100 mug/ml and the minimal bactericidal concentration was >100 mug/ml. It was inhibited by chloramphenicol at 0.4 mug/ml. Further studies demonstrated that ampicillin and methicillin were synergistic against this organism. It was shown to produce a diffusible beta-lactamase. Transferase of resistance from this organism to a susceptible Haemophilus parainfluenzae and a reciprocal transfer were accomplished. A test for transformation was negative as was a test for reversal of resistance by ethylenediaminetetraacetic acid.
Article
A feature of many bacteria of diverse genera is the production of extracellular acidic polysaccharides. These polysaccharides may be organised into distinct structures termed capsules, or may be excreted as an extracellular slime. However, this distinction is arbitrary and in practice may be of no functional significance.
Article
Rhizobium japonicum, capable of binding high-molecular-weight donor (32)P-labeled deoxyribonucleic acid (DNA) during late log phase in a competence medium, was transformed for streptomycin resistance with a frequency of transformation ranging between 0.02 and 0.08%. Eight to 10% of the homologous native (32)P-labeled input DNA was bound irreversibly in a temperature-dependent manner. Homologous denatured (32)P-labeled DNA was incapable of binding to the recipient under similar conditions. CsCl density gradient banding of the donor and recipient DNA indicated homology. The low frequency of transformation could be due to one or more steps that occur between DNA uptake and integration.
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Article
A relatively competent state of Mycobacterium smegmatis for infection with deoxyribonucleic acid (DNA) extracted from phage B1 was found in the late log phase of bacterial growth. This state of the culture was used in quantitative studies on the infectivity of the DNA. The buoyant density of B1 DNA was 1.728 g/cc in CsCl, and 1 mug of the DNA produced 84 infective centers, the phage equivalent of which was 1.5 x 10(-8). The infectivity was destroyed by catalytic amounts of deoxyribonuclease but not by specific B1 antiserum. Tween 80, which prevents phage adsorption, did not prevent DNA infection. The response of plaque-forming ability to DNA concentration suggested that two or more molecules are required to initiate an infective center. The low efficiency of DNA infection in mycobacteria was considered to be caused by a limiting population of competent cells in the culture employed; in this experiment less than 10(-5) of the cells were infected with DNA. A typical cycle of infection was observed, although the latent period was prolonged and the burst size reduced after DNA infection. The transition of B1 DNA infection to deoxyribonuclease insensitivity had a lag period of about 10 min, and increased linearly with a velocity of about 0.24 infective centers per min per mug of DNA. Half of the infective titer was inactivated by heating at 92 C for 15 min. The melting temperature was about 96 C. Species barriers were not crossed by B1 DNA; however, the DNA was infectious for a B1-resistant mutant of the host.
Article
Quantitative transformation of Acinetobacter calcoaceticus BD4-Ss exposed to autologous DNA in a liquid system was examined with streptomycin resistance as a marker. The exposure to DNA was terminated by addition of DNase at set intervals. The variables tested were concentrations of autologous Sr-DNA, exposure time, growth phase, recipient cell density, and the pH of the liquid transformation system. The ratio of transformants to total number of recipient cells (T/R-ratio) was proportional to the DNA concentration up to a level of about 10 micrograms/ml. There was a constant increase of transformant numbers upon time of exposure. The T/R ratio was highest at the early growth phase, but the total number of transformed cells per ml culture aliquot was only raised by a factor approximately 10 from the earlier to the later growth phases and non increase was observed in recipients grown for more than 3 hours. There was a marked relationship between the relative number of transformants per total number of recipients in that the T/R-ratio was markedly higher with fewer cells per ml in the transformation broth system where exposure to DNA occurred. The transformation was higher at a pH of 7.3 than 7.0. The results enabled definition of the optimal test system for quantitative transformation of A. calcoaceticus with the strain BD4 as recipient.
Article
Streptomycin resistance of a high degree has been induced in sensitive populations of Hemophilus influenzae and Hemophilus parainfluenzae by desoxyribonucleic acids (DNA's) derived from streptomycin (SM)-resistant cells of at least one heterologous species of Hemophilus. The specificity of the DNA which controls SM resistance has been studied within and among species of Hemophilus by comparing, in a given population, the proportion of cells transformed to SM-resistant by DNA's derived from highly resistant cells of heterologous type or species with the proportion changed by the DNA derived from SM-resistant cells of the homologous type or species. The ratio resulting from this comparison correlates in general with the degree of kinship between recipient and donor cells suggested by accepted methods of bacteriologic classification. The numerical value of the ratio is much lower when the species of the recipient population and donor of the DNA differ than when they are of the same species. The data suggest that this ratio is of value as an index of degree of kinship of recipient and donor cells. Comparison of the activity of heterologous and homologous DNA's shows differences within species and degrees of differences among species not brought out by other available methods. The data suggest that H. influenzae is more closely related to H. parainfluenzae than to H. suis and that the relationship between H. parainfluenzae and H. suis is remote. Within the species H. influenzae and H. parainfluenzae the ratio of hetero-specific transformants to homospecific transformants appears to be relatively constant for a given recipient population. This ratio also appears to be independent of the type or group source of the heterologous species SM resistance DNA. The low proportion of cells in H. influenzae populations which are transformed to SM-resistant by DNA's derived from SM-resistant H. parainfluenzae and vice versa has been increased 4- to 15-fold by the replication of the heterologous species SM resistance DNA in the heterologous species. An alteration of the heterologous DNA by the host is suggested.
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The typically recovered quantity of nontypeable Haemophilus influenzae (NTHi) bacteria in an ex vivo middle ear (ME) aspirate from the chinchilla model of experimental otitis media is insufficient for direct analysis of gene expression by microarray or of lipopolysaccharide glycoforms by mass spectrometry. This prompted us to investigate a strategy of multiple consecutive lavage samplings to increase ex vivo bacterial recovery. As multiple consecutive lavage samples significantly increased the total number of bacterial CFU collected during nasopharyngeal colonization or ME infection, this led us to evaluate whether bacteria sequentially acquired from consecutive lavages were similar. Comparative observation of complete ex vivo sample series by microscopy initially revealed ME inflammatory fluid consisting solely of planktonic-phase NTHi. In contrast, subsequent lavage samplings of the same infected ear revealed the existence of bacteria in two additional growth states, filamentous and biofilm encased. Gene expression analysis of such ex vivo samples was in accord with different bacterial growth phases in sequential lavage specimens. The existence of morphologically distinct NTHi subpopulations with varying levels of gene expression indicates that the pooling of specimens requires caution until methods for their separation are developed. This study based on multiple consecutive lavages is consistent with prior reports that NTHi forms a biofilm in vivo, describes the means to directly acquire ex vivo biofilm samples without sacrificing the animal, and has broad applicability for a study of mucosal infections. Moreover, this approach revealed that the actual burden of bacteria in experimental otitis media is significantly greater than was previously reported. Such findings may have direct implications for antibiotic treatment and vaccine development against NTHi.
Article
The transforming principles of Hemophilus influenzae have been purified by a new method including fractional extraction. The active molecule behaves in these extractions like the bulk of the DNA preparation. The minimal amount of DNA necessary for transformation appeared to be of the same order of magnitude as the amount of DNA in a single cell. Quantitative study has been made of the resistance of transforming activity to various agents. When subjected to heat, the temperature at which the activity starts to decrease corresponds rather closely to the temperature at which the viscosity of the bulk of the DNA preparations starts to decrease. Similar correspondence was found when the transforming principle was subjected to pH changes. This is further evidence that the behavior of the active molecules is similar to the behavior of the average DNA molecule of the preparation. The activity is reduced by exposure to low ionic strength and by dehydration. Desoxyribonuclease in concentrations less than 10–4 γ/cc. is able to destroy the activity; a lag period during which the activity but not the viscosity decreases has been observed. NaNO2 at pH 5.3, HCHO and 10–5 M Fe++ reduce or destroy the activity; the importance of intact amino groups in the DNA molecule for the activity is discussed. Several protein-denaturing, sterilizing, and mutagenic agents have been found to have no effect on the transforming activity.
Article
Heritable type-specific traits have been induced in meningococcus populations by exposure to desoxyribonucleic acid (DNA)-containing extracts derived from meningococcus cells of the type desired. The DNA has been shown to be an essential component of the transforming extract. As in the H. influenzae system, the reaction between the susceptible cell and the DNA responsible for the heritable change requires less than 15 minutes. Only a minute proportion of the total cells exposed to the DNA extracts for a short time are susceptible to the change; but in the growth and reproduction of bacteria susceptible cells appear with fairly predictable frequency. Type I specific traits have been induced in populations of RIIa and SIIa cells. Type IIa specificity has been induced in RIIa cells.
Article
1. From Type III pneumococci a biologically active fraction has been isolated in highly purified form which in exceedingly minute amounts is capable under appropriate cultural conditions of inducing the transformation of unencapsulated R variants of Pneumococcus Type II into fully encapsulated cells of the same specific type as that of the heat-killed microorganisms from which the inducing material was recovered. 2. Methods for the isolation and purification of the active transforming material are described. 3. The data obtained by chemical, enzymatic, and serological analyses together with the results of preliminary studies by electrophoresis, ultracentrifugation, and ultraviolet spectroscopy indicate that, within the limits of the methods, the active fraction contains no demonstrable protein, unbound lipid, or serologically reactive polysaccharide and consists principally, if not solely, of a highly polymerized, viscous form of desoxyribonucleic acid. 4. Evidence is presented that the chemically induced alterations in cellular structure and function are predictable, type-specific, and transmissible in series. The various hypotheses that have been advanced concerning the nature of these changes are reviewed.
Article
1. From Type III pneumococci a biologically active fraction has been isolated in highly purified form which in exceedingly minute amounts is capable under appropriate cultural conditions of inducing the transformation of unencapsulated R variants of Pneumococcus Type II into fully encapsulated cells of the same specific type as that of the heat-killed microorganisms from which the inducing material was recovered. 2. Methods for the isolation and purification of the active transforming material are described. 3. The data obtained by chemical, enzymatic, and serological analyses together with the results of preliminary studies by electrophoresis, ultracentrifugation, and ultraviolet spectroscopy indicate that, within the limits of the methods, the active fraction contains no demonstrable protein, unbound lipid, or serologically reactive polysaccharide and consists principally, if not solely, of a highly polymerized, viscous form of desoxyribonucleic acid. 4. Evidence is presented that the chemically induced alterations in cellular structure and function are predictable, type-specific, and transmissible in series. The various hypotheses that have been advanced concerning the nature of these changes are reviewed.
Article
1. Osmotic shock disrupts particles of phage T2 into material containing nearly all the phage sulfur in a form precipitable by antiphage serum, and capable of specific adsorption to bacteria. It releases into solution nearly all the phage DNA in a form not precipitable by antiserum and not adsorbable to bacteria. The sulfur-containing protein of the phage particle evidently makes up a membrane that protects the phage DNA from DNase, comprises the sole or principal antigenic material, and is responsible for attachment of the virus to bacteria. 2. Adsorption of T2 to heat-killed bacteria, and heating or alternate freezing and thawing of infected cells, sensitize the DNA of the adsorbed phage to DNase. These treatments have little or no sensitizing effect on unadsorbed phage. Neither heating nor freezing and thawing releases the phage DNA from infected cells, although other cell constituents can be extracted by these methods. These facts suggest that the phage DNA forms part of an organized intracellular structure throughout the period of phage growth. 3. Adsorption of phage T2 to bacterial debris causes part of the phage DNA to appear in solution, leaving the phage sulfur attached to the debris. Another part of the phage DNA, corresponding roughly to the remaining half of the DNA of the inactivated phage, remains attached to the debris but can be separated from it by DNase. Phage T4 behaves similarly, although the two phages can be shown to attach to different combining sites. The inactivation of phage by bacterial debris is evidently accompanied by the rupture of the viral membrane. 4. Suspensions of infected cells agitated in a Waring blendor release 75 per cent of the phage sulfur and only 15 per cent of the phage phosphorus to the solution as a result of the applied shearing force. The cells remain capable of yielding phage progeny. 5. The facts stated show that most of the phage sulfur remains at the cell surface and most of the phage DNA enters the cell on infection. Whether sulfur-free material other than DNA enters the cell has not been determined. The properties of the sulfur-containing residue identify it as essentially unchanged membranes of the phage particles. All types of evidence show that the passage of phage DNA into the cell occurs in non-nutrient medium under conditions in which other known steps in viral growth do not occur. 6. The phage progeny yielded by bacteria infected with phage labeled with radioactive sulfur contain less than 1 per cent of the parental radioactivity. The progeny of phage particles labeled with radioactive phosphorus contain 30 per cent or more of the parental phosphorus. 7. Phage inactivated by dilute formaldehyde is capable of adsorbing to bacteria, but does not release its DNA to the cell. This shows that the interaction between phage and bacterium resulting in release of the phage DNA from its protective membrane depends on labile components of the phage particle. By contrast, the components of the bacterium essential to this interaction are remarkably stable. The nature of the interaction is otherwise unknown. 8. The sulfur-containing protein of resting phage particles is confined to a protective coat that is responsible for the adsorption to bacteria, and functions as an instrument for the injection of the phage DNA into the cell. This protein probably has no function in the growth of intracellular phage. The DNA has some function. Further chemical inferences should not be drawn from the experiments presented.
Article
The purification of DNA preparations of Hemophilus influenzae, types b and c, endowed with transforming activity is described. The mild procedure makes use of electrophoretic separation of the DNA from contaminating PNA and polysaccharides. The final DNA preparations, free of detectable amounts of these contaminants and of proteins, were active in transformation in concentrations of less than 0.0004 μg. and 0.01 μg. DNA/ml. for types b and c, respectively.
Article
Resistance to streptomycin, of a degree exceeding 1000 µg. per ml., has been induced in sensitive strains of Hemophilus influenzae by exposure for 10 minutes to desoxyribonucleic acid-containing extracts isolated from a strain of type b Hemophilus influenzae which had emerged resistant to 1000 µg. of streptomycin per ml. DNA is essential for the process which brings out this change; the reaction can be prevented by destruction of the DNA with crystalline desoxyribonuclease.The resistant trait which is created in this way is heritable. The nature of the process which induces resistance is similar in all respects to the reaction which induces heritable changes in type specificity of H. influenzae. These results offer another example of the gene-like action of highly specific DNA's. The pattern of resistance brought out in a bacterial population exposed to the DNA-containing, resistance-inducing extract, is similar to that which occurs when emergence of resistance of H. influenzae to streptomycin follows the selection by streptomycin of spontaneously occurring resistant mutants. The change in a bacterial cell from average susceptibility to streptomycin to resistance to 1000 µg. of streptomycin per ml. can occur in a single step.
Article
Two new types of Hemophilus influenzae, Sab and Sad have been produced in vitro. Each exhibits the presence of the type specific polysaccharides of 2 types of E. influenzae within the same cell. In Sab the polysaccharides of types a and b have been demonstrated and in Sad those which characterize types a and d. The Sab and Sad traits are inherited. Sab was produced by the action of DNA-containing extract isolated from type a on either type b cells or Rb cells (non-encapsulated non-type-specific cells derived from type b). Sad cells were formed as a result of the action of the DNA-containing extract isolated from type d on cells intermediate between Rab and Sab cells. DNA-containing extracts isolated from Sab cells have induced the Sab trait in Rd cells with predictable regularity. Evidence has been presented that the hereditary determinant of Sab cells is a new genetic substance with new functions. Therefore, the interaction of the DNA-containing substance from cells of one genetic type with living cells of a genetically different type has produced what appears to be a new individual which differs from each of the cells contributing the differing genetic traits but has at least one trait in common with each. Sab cells derived presumably from a single cell show the appearance of type b cells sometime during the first 7 generations.
Article
Heritable type-specific traits have been induced in meningococcus populations by exposure to desoxyribonucleic acid (DNA)-containing extracts derived from meningococcus cells of the type desired. The DNA has been shown to be an essential component of the transforming extract. As in the H. influenzae system, the reaction between the susceptible cell and the DNA responsible for the heritable change requires less than 15 minutes. Only a minute proportion of the total cells exposed to the DNA extracts for a short time are susceptible to the change; but in the growth and reproduction of bacteria susceptible cells appear with fairly predictable frequency. Type I specific traits have been induced in populations of RIIa and SIIa cells. Type IIa specificity has been induced in RIIa cells.
Article
The transforming principles of Hemophilus influenzae have been purified by a new method including fractional extraction. The active molecule behaves in these extractions like the bulk of the DNA preparation. The minimal amount of DNA necessary for transformation appeared to be of the same order of magnitude as the amount of DNA in a single cell. Quantitative study has been made of the resistance of transforming activity to various agents. When subjected to heat, the temperature at which the activity starts to decrease corresponds rather closely to the temperature at which the viscosity of the bulk of the DNA preparations starts to decrease. Similar correspondence was found when the transforming principle was subjected to pH changes. This is further evidence that the behavior of the active molecules is similar to the behavior of the average DNA molecule of the preparation. The activity is reduced by exposure to low ionic strength and by dehydration. Desoxyribonuclease in concentrations less than 10(-4) gamma/cc. is able to destroy the activity; a lag period during which the activity but not the viscosity decreases has been observed. NaNO(2) at pH 5.3, HCHO and 10(-5)M Fe(++) reduce or destroy the activity; the importance of intact amino groups in the DNA molecule for the activity is discussed. Several protein-denaturing, sterilizing, and mutagenic agents have been found to have no effect on the transforming activity.
Article
Change of non-typable (R) strains of H. influenzae into the specific type of origin or new types (S) has been effected in vitro in a predictable fashion within a single 24 hour growth period, by a desoxyribonucleic acid-containing fraction isolated from type-specific cells of the type desired. Only a small proportion of the population of R cells are susceptible to the change induced in inherited characteristics by the desoxyribonucleic acid fraction. The data suggest that the number of susceptible cells present in any given population size varies with the specific type of origin of the R cells; a lesser degree of variation in different independent cultures of the same strain and population size has been demonstrated. The results suggest that the R H. influenzae cells which are susceptible to transformation arise as the result of mutation. It has been demonstrated that the reaction necessary for transformation takes place virtually immediately if susceptible cells are present. Furthermore it has been shown that the change which enables an R cell to form a colony of type-specific organisms has been completed within 15 minutes in an environment which does not permit cell multiplication.
Article
1. Normal strains of types b and d H. influenzae have been directly transformed to new types of H. influenzae; type b was changed to types a, c and d, and type d into types a, b and c. 2. The rate of occurrence in type d populations of cells susceptible of transformation has been shown to be not significantly different from that found earlier in R cell populations derived from type d. Comparisons in the presence of more potent anti d antibody have been made subsequently between populations of Rd and type d for frequency of cells susceptible (initially or during growth for 24 hours) to transformation to type c. No real differences could be demonstrated when this more efficient selective agent was used.
Article
Excerpt Two discoveries were reported in 1927 and 1928 which indicated that the hereditary elements of living organisms were more open to modification by experimental intervention than had previously been supposed by geneticists. On the one hand, Muller, working with animal material, and Stadler, working with plants, demonstrated conclusively that X-rays could induce genes to mutate. On the other hand, Griffith (1928) showed that un encapsulated pneumo cocci could be made to synthesize a capsule of a given type, under the action of some material present in encapsulated pneumo cocci. The induced change was permanent and inherited by the descendants of the transformed bacteria. While the experiments with X-rays oriented genetical research for the succeeding decade, Griffith's discovery exerted virtually no influence on biological thought until nearly twenty years later, for the absence of sexual reproduction in bacteria was sufficient to discourage any geneticist from studying these induced transformations. Fortunately, Griffith's...
Cold Spring Harbor Syrup
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