Effect of Iododeoxyuridine upon Conjugation and the Fate of Transferred Deoxyribonucleic Acid in Escherichia coli K-12

The incorporation of 5-iododeoxyuridine (IUdR) into Escherichia coli K-12 deoxyribonucleic acid (DNA) has been found to decrease significantly the viability of female strains A288 and JC411(r) but to have only minor effect upon their ability to act as conjugational recipients and to perform recombination after conjugation. In contrast, IUdR incorporation into male strain HfrC appears to interfere with both chromosome transfer and genetic recombination. By using IUdR to densitylabel female DNA, and carrying out large-scale matings with (3)H-thymidine-labeled male cells, we examined the fate of transferred DNA. After a 30-min mating, the T6-sensitive male cells were lysed, and the DNA of the merozygotes and remaining female cells was isolated. Initial centrifugation of this DNA in a CsCl gradient showed that the male and female DNA species were associated. The nature of this association of the parental DNA species was determined by formaldehyde denaturation followed by CsCl centrifugation. Denaturation of DNA isolated immediately after T6 lysis gave a peak of radioactivity banding at the density of light single-stranded DNA. However, denaturation of DNA isolated after T6 lysis and dilution of the cells into fresh medium, exhibited peaks of radioactivity banding at positions corresponding to single-stranded, density-labeled DNA. The results indicate that recombination after conjugation in E. coli takes place by a breakage-and-reunion mechanism. The process of recombination can be separated into two stages. In the first stage, the donor and recipient DNA molecules become associated. The second stage consists of the formation of phosphodiester bonds between the donor and recipient segments comprising the recombinant molecule.     

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.     

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.     

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.     

Origin of Escherichia coli K-12 Hfr B7.

Several F' plasmids encoding resistance to tetracycline have been derived from a trg::Tn10 Hfr B7 strain of Escherichia coli K-12. One of these plasmids, JGF312, was analyzed by restriction endonuclease digestion and Southern blot hybridization to cloned chromosomal fragments. This analysis revealed that JGF312 was formed by Tn10-promoted deletion from the Tn10 insertion (31.4 min) to within the prophage rac at 30.1 min. Hfr B7 was shown to result from recombination between IS2 of F delta (33-43) and a chromosomal IS2 located within the rac-man region at 30.9 min on the genetic map.     

Conjugal Deoxyribonucleic Acid Replication by Escherichia coli K-12: Effect of Nalidixic Acid

During the conjugal transfer of the R64-11 plasmid at 42 C from donor cells thermosensitive for vegetative deoxyribonucleic acid (DNA) synthesis to recipient minicells, the plasmids are conjugally replicated in the donor cells. This conjugal replication is inhibited by nalidixic acid, and the degree of inhibition is comparable to the reduction in the amount of plasmid DNA transferred to the recipient minicells in the presence of the drug. In addition, the size of DNA transferred to the minicells and the fraction of conjugally replicated DNA in the donor cells that can be isolated as closed-circular plasmid DNA under alkaline conditions are both reduced by nalidixic acid. When the drug is added to a mating that is underway, the rate of conjugal replication is immediately reduced. This change is accompanied by a reduction in the amount of conjugally replicated DNA in the donor cells that can be isolated as closed-circular plasmid DNA. Furthermore, conjugally replicated plasmid DNA that is not associated with the donor cell membrane becomes membrane bound after the addition of nalidixic acid.     

Effect of Thymine Starvation on Deoxyribonucleic Acid Repair Systems of Escherichia coli K-12

Thymine starvation of Escherichia coli K-12 results in greatly increased sensitivity to ultraviolet light (UV). Our studies, using isogenic strains carrying rec and uvr mutations, have shown the following. (i) Common to all strains tested is a change from multihit to single-hit kinetics of survival to UV after 60 min of thymine starvation. However, the limiting slope of UV survival curves decreases in the rec(+)uvr(+) strain and changes very little in several rec mutant strains and one uvrB mutant strain. Thus, when either the rec or uvr system is functioning alone, the limiting slopes of the UV survival curves are relatively unaffected by thymine starvation. (ii) Thymine starvation does not significantly inhibit repair processes carried out by either repair system alone; i.e., host cell reactivation of irradiated phage (carried out by the uvr system), excision of thymine dimers (uvr), or X-ray repair (rec). (iii) In a rec(+)uvr(+) strain, repair appears to be a synergistic rather than additive function of the two systems. However, after thymine starvation, repair capacity is reduced to about the sum of the repair capacities of the independent systems. (iv) The kinetics of thymineless death are not changed by rec and uvr mutations. This indicates that the lesions responsible for thymineless death are not repaired by rec or uvr systems. (v) Withholding thymine from thy rec(+)uvr(+) bacteria not undergoing thymineless death has no effect on UV sensitivity. Under these conditions one sees higher than normal UV resistance in the presence or absence of thymine. This is due to increased repair carried out by the uvr system. To explain these results we postulate that thymine starvation does not inhibit either the rec or uvr repair pathway directly. Rather it appears that thymine starvation results in increased UV sensitivity in part by inhibiting a function which normally carries out efficient coordination of rec and uvr pathways.     

Membrane Association of Conjugally Transferred Deoxyribonucleic Acid in Escherichia coli Minicells

The conjugally acquired deoxyribonucleic acid (DNA) of small, anucleate cells ("minicells") of a mutant strain of Escherichia coli K-12 was found to be predominantly associated with the bacterial membrane. Evidence from X-irradiation studies in vivo shows that there is no decrease in DNA-membrane association under conditions which reduce the DNA to one-sixth its original size and suggests the possibility of multiple DNA-membrane association sites. Preliminary enzymatic studies indicate the involvement of protein, DNA, and lipids in the membrane association of the DNA.     

Hfr formation by I pilus-determining plasmids in Escherichia coli K-12.

R483, an atypical, I pilus-determining plasmid, and also R144, a typical one, were shown to suppress the DnaA phenotype by integration into the Escherichia coli chromosome.     

Gross Map Distances and Hfr Transfer Times in Escherichia coli K-12

Hfr strains B4 and B8 transfer the Escherichia coli chromosome in opposite directions, each transferring lac⁺ as the last known marker. They were mated in concurrent crosses with the proA leu metE lys trp purE lac strain χ462. Analysis of the time of entry values for these markers showed that Hfr strain B8 transfers the whole chromosome more rapidly than does Hfr strain B4. In both crosses, the rate of transfer observed decelerates. If deceleration occurs as a function of the amount of chromosome transferred, the data are consistent with the markers examined being very accurately placed on the Taylor-Trotter map of the E. coli K-12 genome.     

Isolation of Circular Deoxyribonucleic Acid from Salmonella typhosa Hybrids Obtained from Matings with Escherichia coli Hfr Donors

Heterozygous, partial diploid Salmonella typhosa hybrids obtained from matings with Escherichia coli K-12 Hfr strains were observed to contain supercoiled, circular deoxyribonucleic acid (DNA) when examined by the dye-buoyant density method. Examination of one such S. typhosa hybrid after its loss, by segregation, of the inherited E. coli genetic markers revealed a concurrent loss of its supercoiled circular DNA. Subsequent remating of this segregant with various E. coli Hfr strains resulted in the reappearance of the circular DNA. Molecular weight determinations of circular DNA molecules isolated from a number of S. typhosa partial diploid hybrids were made by sucrose density gradient ultracentrifugation and electron microscopy. These studies revealed a range of molecular sizes among the various hybrids examined, but each hybrid exhibited only a single characteristic size for its contained circular DNA. The range of size is consistent with the presence in each hybrid of a different length of E. coli chromosome. It was concluded that the E. coli Hfr genetic segments transferred to these S. typhosa hybrids were conserved, in the diploid state, in the form of supercoiled, circular DNA molecules.     

Conjugal Deoxyribonucleic Acid Replication by Escherichia coli K-12: Effect of Chloramphenicol and Rifampin

Conjugal replication of R64-11 deoxyribonucleic acid (DNA) and the concomitant transfer of R64-11 DNA to DNA-deficient minicells are dependent upon processes that are inhibited by rifampin and chloramphenicol. The rifampin-sensitive product is not present in vegetatively growing cells and is needed to initiate both conjugal DNA replication in donor cells and DNA transfer to recipient minicells. If the rifampin-sensitive product is a ribonucleic acid (RNA) molecule (rather than RNA polymerase itself), our data indicate that this RNA species required for initiation of conjugal activity does not need to be translated into a protein product. The chloramphenicol-sensitive product(s) is present in vegetatively growing cells in sufficient quantity to permit most donor cells to carry out one round of plasmid conjugal replication and transfer. The initiation of second and subsequent rounds of conjugal replication and transfer are dependent on the synthesis of both the rifampin-sensitive and chloramphenicol-sensitive products. Our results demonstrate a correspondence between the amount of conjugal DNA replication in the donor and the amount of DNA transferred to recipient minicells under all conditions, and therefore suggest but do not prove that plasmid transfer is dependent on conjugal DNA replication. The results also add additional proof that R64-11 transfer to minicells is discontinuous. All of these results are discussed in regard to further refinements of old models for the mechanism of conjugal transfer as well as a more radical departure from current dogma.     

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.     

Endonuclease from Dictyostelium discoideum That Attacks Ultraviolet-Irradiated Deoxyribonucleic Acid

An endonuclease activity yielding single-strand breaks in ultraviolet light-irradiated phiX-174 RF I DNA in vitro has been detected in homogenates of the amoebae of the cellular slime mold, Dictyostelium discoideum.     

Acid response of exponentially growing Escherichia coli K-12

Induction of acid tolerance response (ATR) of exponential-phase Escherichia coli K-12 cells grown and adapted at different conditions was examined. The highest level of protection against pH 2.5 challenges was obtained after adaptation at pH 4.5-4.9 for 60 min. To study the genetic systems, which could be involved in the development of log-phase ATR, we investigated the acid response of E. coli acid resistance (AR) mutants. The activity of the glutamate-dependent system was observed in exponential cells grown at pH 7.0 and acid adapted at pH 4.5 in minimal medium. Importantly, log-phase cells exhibited significant AR when grown in minimal medium pH 7.0 and challenged at pH 2.5 for 2 h without adaptation. This AR required the glutamate-dependent AR system. Acid protection was largely dependent on RpoS in unadapted and adapted cells grown in minimal medium. RpoS-dependent oxidative, glutamate and arginine-dependent decarboxylase AR systems were not involved in triggering log-phase ATR in cells grown in rich medium. Cells adapted at pH 4.5 in rich medium showed a higher proton accumulation rate than unadapted cells as determined by proton flux assay. It is clear from our study that highly efficient mechanisms of protection are induced, operate and play the main role during log-phase ATR.     

Conjugation in Escherichia coli: Recombination Events in Terminal Regions of Transferred Deoxyribonucleic Acid

An investigation of recombination events occurring in zygotes formed during conjugation has been carried out. The frequencies with which donor markers situated close to the origin are recovered indicates an obligatory interaction between the recipient chromosome and a region on the donor chromosome adjacent to the leading end. The partial exclusion from recombinants of some of the most proximal markers studied, however, indicates that this interaction does not occur at the free extremity of the transferred deoxyribonucleic acid. Alternate models to explain these facts are presented.