Isolation and characterization of thermosensitive Escherichia coli mutants defective in deoxyribonucleic acid replication

Thermosensitive deoxyribonucleic acid replication-defective mutants have been isolated by using an autoradiographic selection method. The mutants have been analyzed genetically and biochemically. Some of the mutants show thermosensitivity of in vitro deoxyribonucleic acid replication. These can be classified into three groups according to their behavior in in vitro complementation assays. This classification is congruent with that obtained by genetic mapping by using cotransduction frequencies with selected markers in P1 transduction analysis.     

Chromosome structure of Escherichia coli mutants temperature sensitive for deoxyribonucleic acid replication.

Folded chromosomes were isolated from Eschericia coli thermosensitive dnaA initiation mutants incubated at the nonpermissive temperature and were analyzed by neutral sucrose density gradient centrifugation. A chromosomal structure that sedimented at approximately 1,500S accumulated when the dnaA gene product was inactive. When the cells were returned to a permissive temperature, the folded chromosomes exhibited a decrease in sedimentation velocity to 1,300S but still retained their uniform structure. Very little deoxyribonucleic acid synthesis occurred during the period in which the chromosomes exhibited the reduction in sedimentation velocity. A dnaG elongation mutant showed no unique chromosome structure when the dnaG gene product was inactive.     

Escherichia coli deoxyribonucleic acid synthesis mutants: their effect upon bacteriophage P2 and satellite bacteriophage P4 deoxyribonucleic acid synthesis.

Escherichia coli C strains containing different deoxyribonucleic acid (DNA) synthesis mutations have been tested for their support of the DNA synthesis of bacteriophage P2 and its satellite phage P4. Bacteriophage P2 requires functional dnaB, dnaE, and dnaG E. coli gene products for DNA synthesis, whereas it does not require the products of the dnaA, dnaC, or dnaH genes. In contrast, the satellite virus P4 requires functional dnaE and dnaH gene products for DNA synthesis and does not need the products of the dnaA, dnaB, dnaC, and dnaG genes. Thus the P2 and P4 genomes are replicated differently, even though they are packaged in heads made of the same protein.     

Membrane-bound deoxyribonucleic acid from Escherichia coli: effects of replication, protein synthesis, and ribonucleic acid synthesis.

The experiments presented in this paper suggest that the shift observed in sedimentation of deoxyribonucleic acid from cells of Escherichia coli subjected to amino acid starvation is related to inhibition of ribonucleic acid synthesis rather than to its release from the membrane at the termination of replication.     

Characteristics of cold-sensitive mutants of Escherichia coli K-12 defective in deoxyribonucleic acid replication.

Four cold-sensitive mutants of Escherichia coli have been isolated which show a reduced ability to synthesize deoxyribonucleic acid at low temperature. The mutants also have a reduced ability to incorporate nucleoside triphosphates into deoxyribonucleic acid at low temperature in cell preparations made permeable with toluene. All four mutations are located at or near the dnaA locus on the E. coli genetic map. They are recessive to the wild-type allele and two of them can be integratively suppressed by F episomes.     

Penicillin selection of Escherichia coli deoxyribonucleic acid repair mutants.

A rapid method has been developed for isolation of ultraviolet-sensitive mutants of Escherichia coli, by inducing delay in the growth and/or division of repair-deficienct cells with low fluences of far-ultraviolet radiation, and killing with penicillin the repair-proficient cells, which continue to grow and divide. With this technique, we have achieved about a 3,000-fold enrichment for photoreactivation less (phr) cells and have isolated and characterized three phr mutants.     

Alterations of deoxyribonucleoside triphosphate pools in Escherichia coli: effects on deoxyribonucleic acid replication and evidence for compartmentation.

Inhibition of ribonucleic acid synthesis in Escherichia coli 15 TAU bar with rifampin or streptolydigin leads to large increases in the sizes of cellular ribonucleoside and deoxyribonucleoside triphosphate pools. Inhibition of protein synthesis leads to increases in the sizes of all nucleoside triphosphate pools except the guanosine triphosphate and deoxyguanosine triphosphate pools; a decrease in the size of the latter pool may be responsible for the slowing of deoxyribonucleic acid replication fork movement observed in this strain in the absence of protein synthesis. Analysis of the kinetics of incorporation of labeled precursors into deoxyribonucleic acid and into cellular pools suggests that functional compartmentation of nucleotide pools exists, allowing the incorporation of exogenously supplied precursors into deoxyribonucleic acid without prior equilibration with the cellular pools.     

Enhancement of ribosomal ribonucleic acid synthesis by deoxyribonucleic acid gyrase activity in Escherichia coli.

The effect of the deoxyribonucleic acid (DNA) gyrase inhibitors coumermycin A1, novobiocin, and oxolinic acid on ribonucleic acid (RNA) synthesis in Escherichia coli was studied in vivo and in vitro. Preferential inhibition of ribosomal RNA (rRNA) synthesis was observed. No effect of oxolinic acid and coumermycin on rRNA synthesis was seen in mutants having a DNA gyrase which is resistant to these inhibitors. In a temperature-sensitive DNA gyrase mutant rRNA synthesis was decreased at nonpermissive temperatures. Thus, a functional DNA gyrase is required for rRNA synthesis. Purified DNA gyrase had no effect on rRNA synthesis in a purified system. However, DNA gyrase does show preferential stimulation of rRNA synthesis in a system supplemented with other proteins. Apparently, DNA gyrase stimulation of rRNA synthesis requires another protein.     

Initiation of deoxyribonucleic acid replication in Escherichia coli B: uncoupling from mass/deoxyribonucleic acid ratio.

In Escherichia coli growing at different rates, the ratio of cell mass to the number of chromosome origins tended to be constant at the time of the initiation of deoxyribonucleic acid (DNA) replication. This observation led to the assumption that the initiation event is controlled in some way by cell mass, e.g., by a growth-dependent synthesis of an initiator or dilution of a repressor. We have now found that the initiation of DNA synthesis can be uncoupled from cell mass. We used a synchronous culture of newly divided cells of E. coli B which was obtained by the membrane elution technique (C.E. Helmstetter, J. Mol. Biol. 24: 417-427, 1967) and was starved for an amino acid. Upon restoration of the amino acid, the cells not only divided at a size that was smaller than normal, but also initiated DNA replication long before they could increase their masses to reach the expected ratio of mass/DNA presumably required for initiation.     

Direction of deoxyribonucleic acid replication in Escherichia coli under various conditions of cell growth.

The direction of chromosome replication in a temperature-sensitive initiation mutant of Escherichia coli (CT28) is shown autoradiographically to be bidirectional. This mode of replication persists even when the rate of replication is reduced by slow growth in succinate minimal medium or in the presence of chloramphenicol. Therefore, although the rate of replication can be affected by certain physiological stimuli, the topology of replication need not be.     

Isolation and analysis of Escherichia coli mutants that allow increased replication of bacteriophage lambda.

Escherichia coli mutants were isolated that supported the growth of a lambda Ots and, in at least one case, a lambda Bts phage at the normally nonpermissive temperature of 39 degrees C. In one such strain, Ots and Bts suppression ability appeared to be a function of the guaB gene. Ots suppression by the mutant guaB strain was prevented if high levels of guanine or xanthine were present in the medium. No other base had any effect on Ots suppression in this strain. Other strains carrying spontaneous mutations resulting in guanine or xanthine auxotrophy (guaA or guaB lesions, respectively) all allowed lambda Ots replication at 39 degrees C; Ots suppression in these strains was also abolished by addition of guanine to the medium. Thus, reduced intracellular guanine levels resulting from guaA or guaB mutations appeared to suppress the inability of lambda Ots and, at least in some cases, Bts bacteriophage to form plaques at 39 degrees C. In burst size experiments, a guaB mutant produced a larger phage yield per infected cell of both lambda Ots and lambda O+ phage at 39 degrees C than did a similar guaB+ strain. It appeared that a lower-than-normal level of guanine (or a guanine derivative) in these cells may permit unusually efficient lambda replication. The fact that O+ and lambda Ots bursts in the guaB mutant were reduced significantly by addition of exogenous guanine to the medium is consistent with this suggestion. Another strain that suppresses the Ots allele has no known auxotrophic requirements, and suppression in this strain was unaffected by addition of guanine to the medium; however, addition of cytidine to the medium specifically eliminated Ots suppression in this strain. The mutation responsible for allowing Ots replication in this strain is unknown.     

Intracellular forms of lambda deoxyribonucleic acid in Escherichia coli infected with clear or virulent mutants of bacteriophage lambda

Weissbach, Arthur (National Institutes of Health, Bethesda, Md.), Allan Lipton, and Arnold Lisio. Intracellular forms of lambda deoxyribonucleic acid in Escherichia coli infected with clear or virulent mutants of bacteriophage lambda. J. Bacteriol. 91:1489-1493. 1966.-Infection of either the sensitive or lysogenic strain of Escherichia coli K-112S by lambda(+) leads to the formation of a new phage deoxyribonucleic acid (DNA) species having the properties of a twisted circular DNA duplex. This new phage DNA species is also seen in cells infected with clear or virulent mutants of lambda which cannot lysogenize, or do so at a low frequency. The sedimentation rate of circular lambda DNA duplex at various pH values and its lability were examined.     

Impaired incision of ultraviolet-irradiated deoxyribonucleic acid in uvrC mutants of Escherichia coli.

The production of single-strand breaks in the deoxyribonucleic acid of irradiated uvrC mutants of Escherichia coli K-12 was studied both in vivo and in vitro. In vivo, uvrC mutants displayed a slow accumulation of breaks after irradiation, and in this respect appeared different from uvrA mutants, in which very few breaks could be detected. The breakage observed in uvrC mutants differed from that observed in wild-type strains in both the slow rate of break accumulation and the very limited dose response. The behavior of the uvrC lig-7(Ts) double mutant was shown not to be consistent with the suggestion of ligase reversal as the explanation for the lower rate and limited dose response of break formation observed in ultraviolet-irradiated uvrC mutants in vivo. Rather, there appeared to be a real defect in incision. In toluene-treated cells, we studied the effect of the ligase inhibitor nicotinamide mononucleotide on strand incision. Whereas uvrC mutants displayed more strand breakage in the presence of this inhibitor, the same amount of breakage was seen in uvrA mutants, and as such the breakage could be judged as not due to the main excision repair pathway. Experiments using a cell-free system comprising the partially purified uvr+ gene products demonstrated clearly that there is a requirement for the uvrC+ gene product for strand incision. We suggest that in vivo in the absence of the uvrC+ gene product, a partial analog of this protein may allow some abnormal incision.     

The effects of acridine orange on deoxyribonucleic acid in Escherichia coli.

1. Acridine Orange inhibits growth of Escherichia coli K12 when incubated at pH 7.9, but not at pH 7.4.2. At a non-permissive temperature for DNA polymerase I, Acridine Orange inhibits growth of a temperature-sensitive strain and also increases the rate of elimination of the F'-Lac plasmid. 3. DNA isolated from cells treated with Acridine Orange under conditions that inhibit growth contains material of low molecular weight, which is absent from DNA isolated from cells treated under conditions in which growth is not impaired. 4. Cells incubated with Acridine Orange at both pH 7.4 and 7.9 suffer degradation of DNA, as shown by loss of labelled DNA from the acid-insoluble fraction, which is not observed with untreated cells at either pH. 5. The results suggest that elimination of the F'-Lac plasmid by Acridine Orange requires inactivation of repair processes.     

Reinitiation of deoxyribonucleic acid synthesis by deoxyribonucleic acid initiation mutants of Escherichia coli: role of ribonucleic acid synthesis, protein synthesis, and cell division.

The dnaA and dnaC genes are thought to code for two proteins required for the initiation of chromosomal deoxyribonucleic acid replication in Escherichia coli. When a strain carrying a mutation in either of these genes is shifted from a permissive to a restrictive temperature, chromosome replication ceases after a period of residual synthesis. When the strains are reincubated at the permissive temperature, replication again resumes after a short lag. This reinitiation does not require either protein synthesis (as measured by resistance to chloramphenicol) or ribonucleic acid synthesis (as measured by resistance to rifampin). Thus, if there is a requirement for the synthesis of a specific ribonucleic acid to initiate deoxyribonucleic acid replication, this ribonucleic acid can be synthesized prior to the time of initiation and is relatively stable. Furthermore, the synthesis of this hypothetical ribonucleic acid does not require either the dnaA of dnaC gene products. The buildup at the restrictive temperature of the potential to reinitiate deoxyribonucleic acid synthesis at the permissive temperature shows rather complex kinetics the buildup roughly parallels the rate of mass increase of the culture for at least the first mass doubling at the restrictive temperature. At later times there appears to be a gradual loss of initiation potential despite a continued increase in mass. Under optimal conditions the increase in initiation potential can equal, but not exceed, the increase in cell division at the restrictive temperature. These results are most easily interpreted according to models that postulate a relationship between the initiation of deoxyribonucleic acid synthesis and the processes leading to cell division.     

An Escherichia coli mutant thermosensitive in the B subunit of DNA gyrase: effect on the structure and replication of the colicin E1 plasmid in vitro

Summary An E. coli strain which carries a mutation conferring clorobiocin resistance and temperature sensitivity for growth has recently been described and evidence has been presented suggesting that the mutation is located in the gyrB gene (Orr et al. 1979). The replication of the ColE1 plasmid was analysed in cell-free extracts from this thermosensitive strain. These extracts were totally deficient in the replication of exogenous plasmid DNA and were unable to maintain the superhelical structure of the plasmid DNA. Both defects could be fully complemented by addition of purified gyrB protein.     

Methylated bases in the host-modified deoxyribonucleic acid of Escherichia coli and bacteriophage lambda

Gough, Michael (Brown University, Providence, R.I.), and Seymour Lederberg. Methylated bases in the host-modified deoxyribonucleic acid of Escherichia coli and bacteriophage lambda. J. Bacteriol. 91:1460-1468. 1966.-The deoxyribonucleic acid (DNA) from strains of Escherichia coli and phage lambda was examined to determine whether the types or amounts of methionine-derived methylated bases present correlated with the host-specific modification of that DNA. The DNA of strain C600 (which has K-12 modification specificity) and of a modificationless mutant of C600 are similar in their content of 5-methylcytosine and 6-methylaminopurine. Strains Bc251 and its P1-lysogen differ in P1-controlled specificity, but they have the same content of 6-methylaminopurine, and both lack 5-methylcytosine in their DNA. Phage lambda contains the same methylated bases as its host of origin, but in reduced amounts and in different proportions. Although minor amounts of these methylated bases may have importance as a result of their location, the presence of the majority of these methylated bases is irrelevant to the specificity of host modification of DNA.     

Repair of x-ray-induced deoxyribonucleic acid single-strand breaks in xth mutants of Escherichia coli.

An exonuclease III-deficient strain of Escherichia coli K-12, BW2001 (xthA11), was unable to perform rapid repair of X-ray-induced deoxyribonucleic acid single-strand breaks and appeared to have a defect in the priming of the 3'-termini necessary for initiation of repair synthesis at the breaks. This defect cannot be explained solely by the lack of exonuclease III activity, because other xth mutants tested, including a deletion mutant, repaired radiation-induced strand breaks at close to the normal rate.