Fate of Transforming Deoxyribonucleic Acid After Uptake by Competent Bacillus subtilis: Nonrequirement of Deoxyribonucleic Acid Replication for Uptake and Integration of Transforming Deoxyribonucleic Acid

Studies on transformation of Bacillus subtilis using the inhibitor 6-(p-hydroxyphenylazo)-uracil show that deoxyribonucleic acid (DNA) replication is not required for the uptake and integration of donor DNA and genetic markers.     

Fate of Transforming Deoxyribonucleic Acid After Uptake by Competent Bacillus subtilis: Size and Distribution of the Integrated Donor Segments

A series of sheared donor-recipient complex samples were prepared from a competent Bacillus subtilis culture which had been transformed by using ³H, ²H-labeled deoxyribonucleic acid. Measurement of the molecular weights and buoyant densities of these samples led to the following conclusions. (i) The average size of the single-stranded donor segment which is integrated is 2.8 × 10⁶. (ii) These donor segments are not randomly distributed in the recipient genome, but are clustered in groups, each group containing several discrete donor segments.     

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.     

Repair of Deoxyribonucleic Acid in Haemophilus influenzae I. X-Ray Sensitivity of Ultraviolet-sensitive Mutants and Their Behavior as Hosts to Ultraviolet-irradiated Bacteriophage and Transforming Deoxyribonucleic Acid

Seven mutants of Haemophilus influenzae were isolated by the criterion of sensitivity to ultraviolet (UV) inactivation of colony formation. These mutants and the wild type were characterized with regard to X-ray inactivation of colony formation, UV induction of division inhibition, the ability of the eight strains to act as recipients to UV-irradiated H. influenzae phage and transforming deoxyribonucleic acid (DNA), and the influence of acriflavine on the survival of UV-irradiated transforming DNA with these strains as recipients. The photoreactivable sector of transforming DNA with yeast photoreactivating enzyme was measured for the most UV-sensitive mutant and was found to be greater than that of wild type. Judged by the above criteria, the order of the strains' sensitivities shows some, but by no means complete, correlation from one type of sensitivity characterization to another, indicating that a minimum of two variables is needed to explain the differences in the strains. Acriflavine increases the UV sensitivity of transforming DNA except in the most sensitive mutant. This effect is usually, but not always, more pronounced in the case of the more UV-resistant marker. The acriflavine effect is postulated to be the result of at least two factors: (i) interference with repair of transforming DNA in the host cell, and (ii) interference with the probability of recombination between transforming DNA and host DNA.     

Deoxyribonucleic Acid Synthesis in Bacteriophage SPO1-Infected Bacillus subtilis I. Bacteriophage Deoxyribonucleic Acid Synthesis and Fate of Host Deoxyribonucleic Acid in Normal and Polymerase-Deficient Strains

The effect of bacteriophage SPO1 infection of Bacillus subtilis and a deoxyribonucleic acid (DNA) polymerase-deficient (pol⁻) mutant of this microorganism on the synthesis of DNA has been examined. Soon after infection, the incorporation of deoxyribonucleoside triphosphates into acid-insoluble material by cell lysates was greatly reduced. This inhibition of host DNA synthesis was not a result of host chromosome degradation nor did it appear to be due to the induction of thymidine triphosphate nucleotidohydrolase. Examination of the host chromosome for genetic linkage throughout the lytic cycle indicated that no extensive degradation occurred. After the inhibition of host DNA synthesis, a new polymerase activity arose which directed the synthesis of phage DNA. This new activity required deoxyribonucleoside triphosphates as substrates, Mg²⁺ ions, and a sulfhydryl reducing agent, and it was stimulated in the presence of adenosine triphosphate. The phage DNA polymerase, like that of its host, was associated with a fast-sedimenting cell membrane complex. The pol⁻ mutation had no effect on the synthesis of phage DNA or production of mature phage particles.     

Competence and Deoxyribonucleic Acid Uptake in Bacillus subtilis

The distribution of uninucleate and multinucleate cells of Bacillus subtilis, fractionated by zonal centrifugation, shows that the uninucleate cells are most likely to be competent. Only the competent fraction of the population incorporates deoxyribonucleic acid.     

Mutants of Escherichia coli with Cold-Sensitive Deoxyribonucleic Acid Synthesis

Ten cold-sensitive mutants defective in deoxyribonucleic acid (DNA) synthesis at 20 C have been identified among 218 cold-sensitive mutants isolated from a mutagenized population of Escherichia coli K-12. Four of the ten mutant alleles, dna-339 dna-340, dna-341, and dna-342, cotransduce with serB(+) and hence may be dnaC mutants. Two of these, dna-340 and dna-341, are recessive to their wild-type allele. The gene product of their wild-type allele is trans acting. Complementation tests have demonstrated that dna-340 and dna-341 are in the same cistron. The mapping of the remaining six mutations is in progress. In an attempt to determine whether LW4 and LW21 were initiator mutants, cultures of these strains were starved of an essential amino acid at 37 C and then incubated at 15 C with the essential amino acid. The amount of DNA synthesis observed under these circumstances was insignificant. These data are consistent with the idea that LW4 and LW21 are initiator mutants. However, attempts to integratively suppress LW4 and LW21 with F' factors were unsuccessful. To resolve the question of whether or not LW4 and LW21 are initiator mutants, more specific tests and criteria are required. Cultures of LW4 and LW21 were toluene treated and used to measure in vitro DNA synthesis. If the cells were incubated either at 15 or 20 C before toluene treatment, they were capable of markedly less DNA synthesis than if preincubation had not occurred. The amount of in vitro DNA synthesis is directly proportional to the amount of DNA synthesis occurring during preincubation in vivo; i.e., more DNA synthesis is observed at 20 than at 15 C. The fact that the cold-sensitive mutants are unable to synthesize DNA when supplied with deoxyribonucleoside triphosphates, DNA precursors, is evidence they are not defective in precursor synthesis.     

R Factor-Controlled Restriction and Modification of Deoxyribonucleic Acid: Restriction Mutants

Restriction mutants of two different R factor-controlled host specificities (RI and RII) were isolated. All of the restriction mutants examined had a normal modification phenotype. No complementation was observed between the RI and RII host specificities. It is concluded that for each host specificity no protein subunit is shared by the restriction endonuclease and modification methylase.     

Electron Microscope and Autoradiographic Study of Ultrastructural Aspects of Competence and Deoxyribonucleic Acid Absorption in Bacillus subtilis: Localization of Uptake and of Transport of Transforming Deoxyribonucleic Acid in Competent Cells

With the aid of serial-section electron microscopy two types of mesosomes can be distinguished in cells of competent cultures of Bacillus subtilis: (i) mesosomes connected to the plasma membrane only (plasma membrane mesosomes) and (ii) mesosomes which extend from the plasma membrane into the nuclear bodies (nuclear mesosomes). Contrary to plasma membrane mesosomes, nuclear mesosomes are absent from the tip zones. Electron microscopic autoradiography of sections of Bacillus subtilis cells exposed to [(3)H]thymidine-labeled transforming deoxyribonucleic acid (DNA) for a short period of time shows that the DNA becomes associated with mesosomes. As a function of time the DNA migrates towards the nucleoids. Transport of DNA is completed within 15 to 60 min after termination of DNA uptake. During its migration the DNA continues to be associated with mesosomes, presumably with nuclear mesosomes. DNA initially associated with plasma membrane mesosomes of the tip zones is probably transported first towards the middle zones peripherally and from there towards the nucleoids.     

Fate of Recipient Deoxyribonucleic Acid During Transformation in Haemophilus influenzae

During genetic transformation of Haemophilus influenzae, segments of the host deoxyribonucleic acid (DNA) corresponding to the integrating donor DNA were degraded and liberated into the medium. This degradation was detected by the release of the radioactive label from host DNA during a time period matching the time of development of maximal linkage between donor and host markers. The host label released above that released from nontransformed, control cultures was equivalent to about 2% of the host genome or 16 x 10(6) daltons of DNA. The released, labeled material was acid-soluble and dialyzable. The label release from control cultures was unaffected at 30 C; at this temperature, the recombination-specific release from transformed cells was suppressed. High molecular weight fragments of host DNA corresponding in size to the donor fragments could not be found free within the cell, weakly bound to other host DNA, or bound to non-integrated donor DNA by a reciprocal cross mechanism.     

Filamentous Bacterial Viruses IV. Fate of the Infecting Parental Single-Stranded Deoxyribonucleic Acid

Some of the infecting fd single-stranded viral deoxyribonucleic acid (DNA) molecules were found to be degraded after having initiated infection. The degradation products were reused for synthesis of DNA, primarily bacterial. Degradation was most extensive when viral DNA was replicating.     

Mechanism of Inactivation of Transforming Deoxyribonucleic Acid by X Rays

Transforming deoxyribonucleic acid (DNA) from Haemophilus influenzae was exposed to X rays either in phosphate buffer or in 10% yeast extract. Relations between determinations of biological inactivation, DNA uptake by competent H. influenzae, integration of DNA into the competent cell genome, and induced single-and double-strand breaks indicate that transforming DNA is inactivated by the direct and the indirect effect of X radiation primarily because integration of DNA is prevented as a result of the production of double-strand breaks.     

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.     

Adenosine Triphosphate-Dependent Deoxyribonuclease from Diplococcus pneumoniae: Fate of Transforming Deoxyribonucleic Acid in a Strain Deficient in the Enzymatic Activity

The adenosine triphosphate-dependent deoxyribonuclease is required for wild-type levels of deoxyribonucleic acid (DNA) repair and genetic recombination. In an attempt to determine the physiological function of this enzyme in pneumococcal transformation, the fate of transforming DNA was followed in a wild-type strain and in a strain lacking the enzymatic activity. The qualitative and quantitative findings were closely comparable in the two strains through the step of physical association of a single strand of donor DNA with the recipient chromosome. These results are interpreted to mean that the enzyme may be involved in the subsequent hypothetical removal of excess polynucleotide sequences during conversion of the presumed hydrogen-bonded intermediate into a covalently linked recombinant structure.     

Nonphotoreactivating Repair of Ultraviolet Light-Damaged Transforming Deoxyribonucleic Acid by Micrococcus lysodeikticus Extracts.

Elder, Robert L. (Johns Hopkins University, Baltimore, Md.), and Roland F. Beers, Jr. Nonphotoreactivating repair of ultraviolet light-damaged transforming deoxyribonucleic acid by Micrococcus lysodeikticus extracts. J. Bacteriol. 90:681-686. 1965.-Extracts from Micrococcus lysodeikticus repair Haemophilus influenzae transforming deoxyribonucleic acid (DNA) damaged by ultraviolet light radiation. The repair is demonstrable over a wide dose range, with a constant dose reduction factor for a given concentration of DNA. The active component in the crude extract may be separated into a heat-stable dialyzable and a heat-labile nondialyzable component. The dialyzable fraction contains at least one component which appears to limit the maximal level of repair. Mg(2+) ions are required for the repair process.     

Serological Analysis of the Deoxyribonucleic Acid Polymerase of Avian Oncornaviruses II. Comparison of Avian Deoxyribonucleic Acid Polymerases

Monospecific antiserum prepared against the isolated deoxyribonucleic acid (DNA) polymerase of avian myeloblastosis virus (AMV) neutralized the endogenous ribonucleic acid-instructed DNA polymerase activity of detergent-disrupted virus. The viral polymerase was serologically unrelated to the seven major structural polypeptides of AMV. Furthermore, the viral enzyme was distinguished from normal cellular DNA polymerases by serological criteria; thus, antiserum against the viral enzyme neutralized its homologous antigen but not normal cellular DNA polymerases. Neutralization by antibody of viral DNA polymerase activity was observed with all avian leukemia-sarcoma viruses tested, irrespective of viral antigenic subtype. The DNA polymerase activity of avian reticuloendotheliosis virus, and of a variety of mammalian oncornaviruses, was not neutralized by antisera against the AMV polymerase. Immunological analysis of the RSValpha(O) mutant, which is deficient in DNA polymerase activity, shows this mutant to lack demonstrable polymerase antigen. Viral polymerase was identified by immunofluorescence as a cytoplasmic constituent in virus-producing chicken cells; polymerase antigen was not detected in uninfected (gs(-)) chicken cells.     

Isolation of Deoxyribonucleic Acid Methylase Mutants of Escherichia coli K-12

Fourteen deoxyribonucleic acid (DNA) and 10 ribonucleic acid (RNA) methylation mutants were isolated from Escherichia coli K-12 by examining the ability of nucleic acids prepared from clones of unselected mutagenized cells to accept methyl groups from wild-type crude extract. Eleven of the DNA methylation mutants were deficient in 5-methylcytosine (5-MeC) and were designated Dcm. Three DNA methylation mutants were deficient in N(6)-methyladenine (N(6)-MeA) and were designated Dam. Extracts of the mutants were tested for DNA-cytosine:S-adenosylmethionine and DNA-adenine:S-adenosylmethionine methyltransferase activities. With one exception, all of the mutants had reduced or absent activity. A representative Dcm mutation was located at 36 to 37 min and a representative Dam mutation was located in the 60-to 66-min region on the genetic map. The Dcm mutants had no obvious associated phenotypic abnormality. The Dam mutants were defective in their ability to restrict lambda. None of the mutations had the effect of being lethal.     

Effect of cis-Platinum(II)Diamminodichloride on Wild Type and Deoxyribonucleic Acid Repair-Deficient Mutants of Escherichia coli

The anti-tumor drug cis-platinum(II)diamminodichloride (PDD) induced extensive filamentation in wild-type Escherichia coli and in mutants lacking certain deoxyribonucleic acid (DNA) repair functions (uvrA, recB, recC, and polA); viability of repair-deficient mutants treated with PDD was significantly less than that of wild-type cells. PDD was highly toxic to lex1, lex1 uvrA6 (where its effect was cummulative), and recA13 mutants, all of which were killed without formation of filaments. ³H-thymine incorporated into DNA of cells subsequently treated with PDD became trichloroacetic acid-soluble at rates similar to those observed after exposure to comparable doses of ultraviolet light (UV) or mitomycin C. PDD, like UV, induced extensive degradation of DNA in recA organisms. After a 30-min lag, PDD inhibited significantly the synthesis of DNA but not of ribonucleic acid or protein in E. coli. However, the relative differences between rates of DNA synthesis observed in PDD-treated and control cells decreased substantially when the duration of pulses (³H-thymine) was prolonged from 2 to 5 min. These observations suggest that PDD-induced damage to DNA is reversible, possibly by defined mechanisms of excision and recombination repair.     

Early Energy-Dependent Step in the Entry of Transforming Deoxyribonucleic Acid

It was found that cyanide can partially reverse as well as arrest the transformation of a newly transformed deoxyribonuclease-treated culture for 6 to 8 min after the addition of the enzyme. These findings strongly suggest that deoxyribonucleic acid which attained deoxyribonuclease-insensitivity is not necessarily in the cell, but stored in some region peripheral to the membrane, and that a rate-limiting, energy-dependent step is required to transport this deoxyribonucleic acid into the cell.     

Endonuclease from Micrococcus luteus Which Has Activity Toward Ultraviolet-Irradiated Deoxyribonucleic Acid: Its Action on Transforming Deoxyribonucleic Acid

An endonuclease purified from Micrococcus luteus makes single-strand breaks in ultraviolet (UV)-irradiated, native deoxyribonucleic acid (DNA). The purified endonuclease is able to reactivate UV-inactivated transforming DNA of Haemophilus influenzae, especially when the DNA is assayed on a UV-sensitive mutant of H. influenzae. After extensive endonuclease action, there is a loss of transforming DNA when assayed on both UV-sensitive and -resistant cells. The endonuclease does not affect unirradiated DNA. The results indicate that the endonuclease function is involved in the repair of biological damage resulting from UV irradiation and that the UV-sensitive mutant is deficient in this step. We interpret the data as indicating that the various steps in the repair of DNA must be well coordinated if repair is to be effective.