Vegetative Replication and Transfer Replication of Deoxyribonucleic Acid in Temperature-Sensitive Mutants of Escherichia coli K-12

Crosses were carried out at 34 C and 42 C between eight pairs of isogenic strains of Escherichia coli K-12. The donor and recipient of each pair carried the same mutation for temperature-sensitive deoxyribonucleic acid (DNA) synthesis; they differed only in the presence of F-lac in the donor and a spectinomycin-resistance marker in the recipient. A different temperature-sensitive mutation was present in each of the eight pairs, the eight temperature-sensitive mutations being located in at least two different genes. In all eight pairs, the transfer of F-lac occurred at high and equal rates at 34 C and 42 C, although vegetative DNA replication at 42 C was approximately 10⁻⁴ of that at 34 C. The transfer of F-lac at 42 C was accompanied in seven of the eight crosses by an equivalent amount of DNA synthesis in excess of that observed in the unmated controls. The DNA synthesized during transfer at 42 C was characterized by equilibrium centrifugation in cesium chloride and by its sedimentation velocity in sucrose gradients. It was found to have a density and a molecular weight characteristic of F-lac DNA. A small proportion of the material labeled during transfer was recovered in the form of covalently closed DNA. It is concluded that vegetative replication of the chromosome and transfer replication of F are separate processes, the former requiring at least two gene products which are nonessential for the latter.     

Deoxyribonucleic Acid Synthesis in recA and recB Derivatives of an Escherichia coli K-12 Strain with a Temperature-Sensitive Deoxyribonucleic Acid Polymerase I

recA and recB derivatives of a strain of Escherichia coli with a temperature-sensitive deoxyribonucleic acid (DNA) polymerase I (polA12) are inviable at high temperature, but continue to incorporate (3)H-thymine into DNA for extended periods. The DNA made in pulse-chase experiments at high temperature in the polA12 parent and its double-mutant derivatives has been examined by alkaline sucrose gradient sedimentation analysis. The low-molecular-weight DNA fragments made during short pulses were joined at the same rate in each strain. Furthermore, the resulting high-molecular-weight DNA was of the same size in each case and was stable for at least 50 min. It is concluded that the inviability of the double mutants is due neither to a defect in converting low-molecular-weight DNA intermediates to high molecular weight nor to the presence of unrepaired random breaks in their DNA.     

Studies on Resistance Transfer Factor Deoxyribonucleic Acid in Escherichia coli

A variant of the derepressed R factor, R1, which does not contain any of the drug resistance markers, and represents, in large part, the resistance transfer factor (RTF) was studied in Escherichia coli. RTF deoxyribonucleic acid (DNA) was specifically labeled in a female cell after conjugation. Physical characterization of the molecule showed that RTF possessed an average molecular weight of 50 x 10(6) daltons and a buoyant density of 1.709 g/cm(3). By comparison to R1, we calculate that the region of DNA carrying the drug resistance genes is therefore about 20% of the R1 molecule and has a buoyant density of approximately 1.716 g/cm(3). These results support the hypothesis that the single species of R-factor DNA observed in E. coli represents a composite of the 1.709 and 1.716 g/cm(3) replicons seen in Proteus.     

Auxotroph Accumulation in Deoxyribonucleic Acid Polymeraseless Strains of Escherichia coli K-12

Spontaneous auxotrophs are found with high frequency in several strains of Escherichia coli K-12 deficient in Kornberg deoxyribonucleic acid polymerase. These include amino acid-, vitamin-, purine-, and pyrimidine-requiring strains. Although this was suggestive evidence that these strains might be mutators, reconstruction experiments demonstrate that auxotrophs possess a selective advantage over prototrophs in the same culture. Thus, despite the high frequency of auxotrophs in polymerase-deficient strains, it is not yet clear whether they have elevated mutation rates.     

Synthesis of Unmethylated Deoxyribonucleic Acid in a dnaB Temperature-Sensitive Mutant of Escherichia coli Starved for Methionine

Strain CRT 266, a polyauxotrophic dnaB temperature-sensitive mutant of Escherichia coli K, was investigated for residual deoxyribonucleic acid (DNA) synthesis when returned to the permissive temperature in the absence of protein synthesis. In the presence of methionine, a delayed extra-initiation occurs as well as an erratic long-lasting synthesis. In the absence of methionine, there is no evidence for extra-initiation, whereas the long-lasting synthesis is only slightly depressed. A direct role of methionine independent of protein synthesis in the extra-initiation process is postulated. The largest residual syntheses, with or without methionine, are obtained when (i) bacteria are first grown in a rich medium, (ii) bacteria are shifted to the nonpermissive temperature for 2 h in the same medium, and (iii) bacteria are then starved for aminoacid for 20 h at the permissive temperature. Under these conditions, DNA extracted from methionine-starved cells appears to be a mixture of half-methylated and unmethylated products. The possibility of the occurence of a few methyl groups on the so-called unmethylated DNA is discussed.     

Isolation and Characterization of Supercoiled Circular Deoxyribonucleic Acid from Beta-Hemolytic Strains of Escherichia coli

Covalently closed circular deoxyribonucleic acid (DNA) molecules were isolated by cesium chloride centrifugation in the presence of ethidium bromide from a naturally occurring beta-hemolytic Escherichia coli strain (SC52). The open circular forms have contour lengths of 2.25 ± 0.1 μm, 24.0 ± 0.3 μm, and 29.5 ± 0.5 μm. The beta-hemolytic character of E. coli SC52 can be transferred by conjugation to a nonhemolytic recipient strain. Analysis of the supercoiled DNA of the hemolytic recipient demonstrated that the two large supercoiled DNA molecules of E. coli SC52 are transferred during this event, too. A beta-hemolytic laboratory E. coli strain and several of its derivatives have been shown to contain at least one circular DNA molecule, slightly larger in size than those isolated from E. coli SC52 and its conjugant. The possible significance of these DNA molecules for hemolysin production and transfer is discussed.     

Molecular Weight of Deoxyribonucleic Acid Synthesized During Initiation of Chromosome Replication in Escherichia coli

Alkaline sucrose gradients were used to study the molecular weight of deoxyribonucleic acid (DNA) synthesized during the initiation of chromosome replication in Escherichia coli 15 TAU-bar. The experiments were conducted to determine whether newly synthesized, replication origin DNA is attached to higher-molecular-weight parental DNA. Little of the DNA synthesized after readdition of required amino acids to cells previously deprived of the amino acids was present in DNA with a molecular weight comparable to that of the parental DNA. The newly synthesized, low-molecular-weight DNA rapidly appeared in higher-molecular-weight material, but there was an upper limit to the size of this intermediate-molecular-weight DNA. This limit was not observed when exponentially growing cells converted newly synthesized DNA to higher-molecular-weight material. The size of the intermediate-molecular-weight DNA was related to the age of the replication forks, and the size increased as the replication forks moved further from the replication origin. The results indicate that the newly synthesized replication origin DNA is not attached to parental DNA, but it is rapidly attached to the growing strands that extend from the replication fork to the replication origin, or to the other replication fork if replication is bidirectional. Experiments are reported which demonstrate that the DNA investigated was from the vicinity of the replication origin and was not plasmid DNA or DNA from random positions on the chromosome.     

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.     

Deoxyribonucleic Acid-Deoxyribonucleic Acid Hybridization Assay for Replication Origin Deoxyribonucleic Acid of Escherichia coli

Deoxyribonucleic acid (DNA)-DNA hybridization on nitrocellulose filters can be used to assay for replication origin DNA from Escherichia coli if the DNA attached to the filters is enriched for the replication origin sequences. Such DNA can be readily isolated from very rapidly growing cells. When low amounts of this DNA were attached to filters, radioactively labeled DNA from the replication origin hybridized 1.7 times as well as radioactive replication terminus DNA. Under identical conditions, radioactively labeled DNA from exponentially growing cells hybridized only 1.3 times as well as radioactive replication terminus DNA. The replication origin, replication terminus, and randomly labeled DNA hybridized with similar efficiencies to filters containing DNA isolated from cells incubated in the absence of required amino acids. This DNA appeared to have all sequences present at equal frequencies. The hybridization assay was used to demonstrate that the DNA synthesized shortly after the addition of amino acids to cells previously deprived of required amino acids was primarily from the replication origin and then rapidly became similar to DNA synthesized by exponentially growing cells.     

Temperature-Sensitive Initiation of Chromosome Replication in a Mutant of Escherichia coli

The properties of Escherichia coli mutant D2-47LT indicate that it is temperature-sensitive for a protein required for the initiation of chromosome replication. The results of several different experiments are consistent with this hypothesis, and no support was found for the alternate hypotheses tested. Although the strain is usually unable to initiate replication at 42 C, some of the initiation proteins are apparently synthesized at the restrictive temperature. This can cause initiation on partially replicated, but not completed, chromosomes. It appears that the temperature-sensitive protein is required for initiation on completed chromosomes.     

Conditional Lethality of recA and recB Derivatives of a Strain of Escherichia coli K-12 with a Temperature-Sensitive Deoxyribonucleic Acid Polymerase I

We have isolated a strain of Escherichia coli K-12 carrying a mutation, polA12, that results in the synthesis of a temperature-sensitive deoxyribonucleic acid (DNA) polymerase I. The double mutants polA12 recA56 and polA12 recB21, constructed at 30 C, are inviable at 42 C. About 90% of the cells of both double mutants die after 2 hr of incubation at 42 C. Both double mutants filament at 42 C and show a dependence on high cell density for growth at 30 C. In polA12 recB21 cells at 42 C, DNA and protein synthesis gradually stop in parallel. In polA12 recA56 cells, DNA synthesis continues for at least 1 hr at 42 C, and there is extensive DNA degradation. The results suggest that the primary lesion in these double mutants is not in DNA replication per se.     

Survival, Deoxyribonucleic Acid Breakdown, and Synthesis in Salmonella typhimurium as Compared with Escherichia coli B Strains

Salmonella typhimurium LT-2 was compared with radioresistant (B/r) and radiosensitive (B(s-2)) strains of Escherichia coli in respect to the survival, deoxyribonucleic acid (DNA) breakdown, and DNA synthesis after X irradiation. It is shown that S. typhimurium LT-2 is about four times more sensitive than E. coli B/r but less sensitive than B(s-2). The DNA breakdown is in S. typhimurium LT-2 lower than the postirradiation breakdown of DNA in both E. coli strains and DNA synthesis proceeds in this bacterium in spite of a much lower survival, as in the radioresistant E. coli B/r.     

Evidence for a Relationship Between Deoxyribonucleic Acid Metabolism and Septum Formation in Escherichia coli

Septum formation is a key step in bacterial division, but the mechanism which controls periodic septum formation is unknown. In an attempt to understand this mechanism, lon(-) mutants, in which septum formation is blocked by very doses of ultraviolet light (UV), were investigated. UV must act on some part of the apparatus of cytokinesis; thus, identification of the UV target would identify part of this apparatus. As likely possibilities, UV might damage the septum-forming site or it might damage deoxyribonucleic acid (DNA), since DNA replication is normally coordinated with septum formation. To distinguish between these possibilities, DNA was specifically sensitized by incorporating bromodeoxyuridine into lon(-) bacteria. These bacteria were strongly sensitized to longer wavelength UV (2,900 to 3,100 A) so that they failed to form septa, grew into filaments which lysed, and did not form colonies. Various control experiments supported the conclusion that UV inhibits septum formation as a result of alterations in DNA metabolism. A relationship thus exists between DNA metabolism and septum formation.     

Control of Deoxyribonucleic Acid and Ribonucleic Acid Synthesis in Pyrimidine-Limited Escherichia coli

The effects of pyrimidine limitation on chromosome replication and the control of ribosomal and transfer ribonucleic acid syntheses were investigated. Chromosome replication was studied by autoradiography of (3)H-thymine pulse-labeled cells. Pyrimidine limitation did not affect the fraction of cells incorporating radioactive thymine during a short pulse, indicating that when growth is limited by the supply of pyrimidine, the time required for chromosome duplication increases in proportion to the time required for cell duplication. Control of ribosomal RNA and transfer RNA syntheses was examined by chromatographing cell extracts on methylated albumin kieselguhr columns. When growth was controlled by carbon-nitrogen limitation, the ratio of tRNA to total RNA remained roughly constant at growth rates above 0.5 doublings per hour. During pyrimidine limitation, however, the control of rRNA synthesis was apparently dissociated from the control of tRNA synthesis: the ratio of tRNA to total RNA increased as the growth rate decreased.     

Effect of Deoxyribonucleic Acid Ligands on Deoxyribonucleases and Deoxyribonucleic Acid Polymerase I of Escherichia coli K-12

Endonuclease I, exonuclease I, and exonuclease II-deoxyribonucleic acid (DNA) polymerase I activities are not vital functions in Escherichia coli, although the latter two enzymes have been indirectly shown to be involved in DNA repair processes. Acridines such as acridine orange and proflavine interfere with repair in vivo, and we find that such compounds inhibit the in vitro activity of exonuclease I and DNA polymerase I but stimulate endonuclease I activity and hydrolysis of p-nitrophenyl thymidine-5′-phosphate by exonuclease II. Another acridine, 10-methylacridinium chloride, binds strongly to DNA but is relatively inert both in vivo and in vitro. These experiments suggest that acridines affect enzyme activity by interacting with the enzyme directly as well as with DNA. Resulting conformational changes in the DNA-dependent enzymes might explain why similar acridines which form similar DNA complexes have such a wide range of physiological effects. Differential sensitivity of exonuclease I and DNA polymerase I to acridine inhibition relative to other DNA-dependent enzymes may contribute to the acridine sensitivity of DNA repair.     

Single-Strand Breaks in Deoxyribonucleic Acid and Viability Loss During Deoxyribonucleic Acid Synthesis Inhibition in Escherichia coli

The effects of deoxyribonucleic acid (DNA) synthesis inhibition brought about in four different ways-thymidine starvation, nalidixic acid, hydroxyurea, and dnaB mutation-were examined in isogenic strains of Escherichia coli K-12. Three parameters were examined to determine whether there are strict correlations among them: (i) the extent of DNA synthesis inhibition; (ii) cell survival; and (iii) the rate of breakage of DNA molecules. There was no significant correlation between the extent of DNA synthesis inhibition and the rate of viability loss caused by the four DNA synthesis inhibitors, nor was there a strict correlation between the rate of occurrence of single-strand breaks in DNA and loss of viability. During treatment with hydroxyurea (0.1 M), no viability loss was observed and little, if any, single-strand breakage of DNA occurred. Both thymidine starvation and nalidixic-acid (20 mug/ml) treatment resulted in viability loss and breakage of DNA. For these latter two inhibitors, the two events appeared to be associated because greater rates of both viability loss and DNA breakage were observed for nalidixic acid compared with thymidine starvation. However, viability loss need not be associated with extensive breakage of DNA as demonstrated with a temperature-sensitive DNA synthesis mutant; at 39 C, viability loss occurred at a high rate without significant DNA breakage. With the other agents, the amount of DNA breakage accumulated when a cell population has sustained an average of one lethal hit was estimated to be about 30 single-strand breaks per genome. Differences in chromosomal and episomal breakage rates were observed.     

Conditional Lethality of Deletions Which Include uvrB in Strains of Escherichia coli Lacking Deoxyribonucleic Acid Polymerase I

Deletions of the uvrB gene were not obtained in polA1 strains of Escherichia coli either by selecting for spontaneous deletions or by transduction from strains carrying such deletions. A strain forming a temperature-sensitive deoxyribonucleic acid polymerase I and carrying a deletion of the uvrB gene is inviable at the nonpermissive temperature.     

Replication of Deoxyribonucleic Acid in Escherichia coli C Mutants Temperature Sensitive in the Initiation of Chromosome Replication

An Escherichia coli HF4704S mutant temperature sensitive in deoxyribonucleic acid (DNA) synthesis and different from any previously characterized mutant was isolated. The mutated gene in this strain was designated dnaH. The mutant could grow normally at 27 C but not at 43 C, and DNA synthesis continued for an hour at a decreasing rate and then ceased. After temperature shift-up, the increased amount of DNA was 40 to 50%. When the culture was incubated at 43 C for 70 min and then transferred to 27 C, DNA synthesis resumed after about 50 min, initiating synchronously at a fixed region on the bacterial chromosome. The initiation step in DNA replication sensitive to 30 mug of chloramphenicol per ml occurs synchronously before the resumption of DNA replication after the temperature shift-down, being completed about 30 min before the start of DNA replication. When the cells incubated at 27 C in the presence of 30 mug of chloramphenicol per ml after the temperature shift-down to 27 C were transferred to 43 C with simultaneous removal of the antibiotic, no resumption of DNA replication was observed. When the culture was returned to 43 C after being released from high-temperature inhibition at 30 min before the start of DNA replication, no recovery replication was observed; whereas at 20 min, the recovery of replication was observed. These results indicated that HF4704S was temperature sensitive in the initiation of DNA replication. Analysis of HF4704S, by an interrupted conjugation experiment, indicated that gene dnaH was located at about 64 min on the E. coli C linkage map. In E. coli S1814 (a K-12 derivative), which was a dnaH(ts) transductant from HF4704S (C strain) with phage P1, the mutated gene (dnaH) was demonstrated to be closely linked to the thyA marker by conjugation and P1 transduction experiments and to be distinct from genes dnaA through dnaG.