Accumulation of the capacity of initiation of deoxyribonucleic acid replication in Escherichia coli.

Several temperature-sensitive initiation mutants of Escherichia coli were examined for the ability to initiate more than one round of replication after being held at nonpermissive temperature for approximately 1.5 generation equivalents. The capacity for initiation was measured by residual synthesis experiments and rate experiments under conditions where protein synthesis and ribonucleic acid synthesis were inhibited. Results of the rate and density transfer experiments suggest that the cells may initiate more than one round of replication in the absence of protein or ribonucleic acid synthesis. This contrasts with the results of the residual synthesis experiments which suggest that, under these conditions, only one round of synthesis is achieved. These findings suggest that the total amount of residual synthesis achieved in the presence of an inhibitor may be both a function of the number of initiation events which occur and the effect of the inhibitor of protein or ribonucleic acid synthesis on chain elongation.     

Deoxyribonucleic acid synthesis after inhibition of initiation of rounds of replication in Escherichia coli B/r.

The theory describing the effect of inhibition of initiation of rounds of deoxyribonucleic acid (DNA) replication on the accumulation of DNA is derived, and an analysis is presented which allows the determination of the time C taken to replicate the bacterial chromosome from the kinetic changes in the accumulation of DNA. This analysis is applied to experiments in which inhibition of initiation was achieved by inhibiting protein or protein and ribonucleic acid synthesis with chloramphenicol or rifampin. The results for both antibiotics are identical and indicate that there is a delay of 6 to 11 min in the effect of the antibiotics on initiation of rounds of replication. If this delay is taken into account, then the value of the C period estimated from such experiments agrees with values obtained by other methods, whereas by conventional data evaluation of such experiments the C period would be overestimated. In the low thymine-requiring derivative of Escherichia coli B/r ATCC 12407 used here, the C period was found to be between 38 and 41 min for cultures growing with a mass doubling time of 29 min in glucose-amino acids medium, supplemented with 20 micrograms of thymine/ml.     

Deoxyribonucleic acid replication in permeable and fully viable Escherichia coli cells.

Escherichia coli cells made permeable with a hypotonic tris(hydroxymethyl)aminomethane buffer utilized exogenous deoxyribonucleoside triphosphates to perform semiconservative replication. The rate of replication was the same as in cells made permeable with toluene or sucrose.     

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.     

Gamma-ray induction of deoxyribonucleic acid synthesis in temperature-sensitive DNA initiation mutants of Escherichia coli

DNA synthesis was followed after gamma-ray irradiation of several different temperature-sensitive mutants with defects in the initiation process. The results indicate that only dnaA and dnaI mutants show induction of supplementary DNA synthesis after gamma-ray irradiation. The induction of DNA synthesis by gamma-ray irradiation was also shown to be recA+ dependent in the dna5 mutant.     

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.     

Cell wall synthesis and initiation of deoxyribonucleic acid replication in Bacillus subtilis.

We have observed a connection between cell wall synthesis and the initiation of chromosome replication in Bacillus subtilis. Initiation of chromosome replication was prevented in synchronous cultures in the presence of the cell wall synthesis inhibitor vancomycin. When vancomycin was added to the cultures after initiation of chromosome replication, one round of replication was completed but no reinitiation occurred. Similar results were obtained when cell wall synthesis was inhibited by ristocetin, cycloserine, cloxacillin, or cephaloridine. When sucrose was added to the medium, initiation of deoxyribonucleic acid replication occurred in the presence of vancomycin, to an extent which allowed replication of no more than approximately one-half of the deoxyribonucleic acid of the culture. The same was found in cultures of spheroplasts of B. subtilis. However, initiation of chromosome replication in spheroplasts was completely insensitive to cloxacillin.     

Escherichia coli DnaA interacts with HU in initiation at the E. coli replication origin

Escherichia coli HU protein is a dimer encoded by two closely related genes whose expression is growth phase-dependent. As a major component of the bacterial nucleoid, HU binds to DNA non-specifically, but acts at the chromosomal origin (oriC) during initiation by stimulating strand opening in vitro. We show that the alpha dimer of HU is more active than other forms of HU in initiation of an oriC-containing plasmid because it more effectively promotes strand opening of oriC. Other results demonstrate that HU stabilizes the DnaA oligomer bound to oriC, and that the alpha subunit of HU interacts with the N-terminal region of DnaA. These observations support a model whereby DnaA interacts with the alpha dimer or the alphabeta heterodimer, depending on their cellular abundance, to recruit the respective form of HU to oriC. The greater activity of the alpha dimer of HU at oriC may stimulate initiation during early log phase compared with the lesser activity of the alphabeta heterodimer or the beta dimer.     

Independence of deoxyribonucleic acid replication and initiation from membrane fluidity and the supply of unsaturated fatty acids in Escherichia coli.

Mutant derivatives of the unsaturated fatty acid auxotroph K1062 were employed to investigate whether the supposedly membrane-bound bacterial replication machinery requires for its replicatory functions a fluid membrane environment as is known for several membrane-associated protein functions. Temperatures Tt for fluid reversible nonfluid phase transitions of membrane phospholipids are raised from below 18 to 38 degrees C when mutant cells are supplemented with elaidate instead of with oleate. In this experimental system current or synchroneously initiated new rounds of DNA replication are shown in vivo to continue 8 degrees below Tt, provided appropriate corrections for the concurrent cellular metabolic breakdown are considered. Temperature rate profiles for in vitro deoxyribonucleic acid replication rates measured in lysates of either oleate- or elaidate-supplemented cells yield congruent Arrhenius plots without discontinuities at corresponding Tt positions. We conclude that neither the start nor the propagation of replication forks depends on a fluid membrane. The capacity for the assembly of new replication complexes was studied in replication-aligned cells either shifted from oleate to elaidate (at temperatures below Tt for newly synthesized phospholipids) or starved for oleate. Regardless of whether unsaturated fatty acids are exchanged or completely withheld, new replication complexes can be normally assembled and initiated. These results do not support the conclusions reached by Fralick and Lark (1973) that the availability of unsaturated fatty acids is a prerequisite for the assembly of a functional replication complex.     

Replication of plasmids in mutants of Escherichia coli temperature-sensitive for initiation of deoxyribonucleic acid synthesis

Plasmid deoxyribonucleic acid (DNA) replication was studied in Escherichia coli hosts carrying temperature-sensitive (ts) initiation mutations. The replication of the R plasmid NR1 continues at the nonpermissive temperature in a ts dnaA mutant host but at a decreasing rate in proportion to the residual chromosome synthesis. The replication of NR1, as well as of the F plasmid F′lac, ceases immediately at the nonpermissive temperature in a ts dnaC mutant host. The ability to reinitiate R plasmid replication in the absence of protein or ribonucleic acid synthesis is accumulated at the nonpermissive temperature in a dnaC mutant host.     

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.     

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.     

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.