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.     

Conjugation in Escherichia coli: Failure to Confirm the Transfer of Part of Sex Factor at the Leading End of the Donor Chromosome

Experiments were carried out attempting to determine whether part of sex factor is transferred at the leading end of the Hfr chromosome during conjugation. In the first experiment, an analysis was made of the donor properties of recombinant strains which had inherited the terminal but not the proximal marker from an Hfr. Secondly, recombinants integrating an extremely proximal marker from an Hfr were examined for the inheritance of a sex factor affinity locus adjacent to this marker. In the third experiment, proximal transfer of the wild-type allele of a temperature-sensitive sex factor mutation was looked for, using as recipient a temperature-sensitive Hfr strain, and as donor a wild-type Hfr isogenic with respect to the site of sex factor integration. In none of these experiments could the presence of sex factor material at the leading end be demonstrated. The results do not rule out the possibility that part of F is transferred proximally but only integrated at a very low frequency. They do, however, conflict with certain findings of other authors which, in the past, have been taken as evidence for the transfer of part of F at the leading end.     

Kinetic analysis of the products of donor deoxyribonucleate in transformed cells of Bacillus subtilis

This paper describes the major transmutations of donor deoxyribonucleic acid (DNA) after uptake by competent Bacillus subtilis cells. Kinetic experiments confirm that after exposure to competent cells, donor DNA is converted to double-stranded fragments (DSF) which can be isolated as early as 30 s from the beginning of the reaction. At this time, DSF represent the only identifiable product of donor origin. After 1 to 2 min, DSF are converted to deoxyribonuclease-resistant forms, identified as single-stranded DNA fragments (SSF). SSF are intermediates in the transformation process leading to the formation of donor-recipient complex. This component makes its appearance between 2 to 4 min from the beginning of the transformation process. All the donor-recipient complexes found at the end of the reaction can be accounted for quantitatively by the DSF and the SSF found in the initial stages of transformation. A quantitative discussion of the transformation process is included.     

Genetic exchange mechanisms in Neisseria gonorrhoeae.

There are two mechanisms for genetic exchange in Neisseria gonorrhoeae. Plasmid deoxyribonucleic acid can be transferred by conjugation, which is dependent on the presence of a 24.5-megadalton plasmid in the donor cell. We have shown that chromosomal deoxyribonucleic acid can be exchanged between all colonial variants by transformation, but not by conjugation. In the nonpiliated variants, however, this exchange was dependent on the presence of the 24.5-megadalton plasmid in the recipient cell.     

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.     

Conjugation in Agrobacterium tumefaciens in the absence of plant tissue.

A general, reliable conjugation system for Agrobacterium tumefaciens in the absence of plant tissue is described in which A. tumefaciens can serve either as the donor or recipient of plasmid deoxyribonucleic acid with reasonable efficiency. Plasmid RP4 was transferred from Escherichia coli to A. tumefaciens and from strain of A. tumefaciens. Both RP4 and the A. tumefaciens virulence-associated plasmids were detected by alkaline sucrose gradients in A. tumefaciens strains A6 and C58 after mating with E. coli J53(RP4). The pathogenicity (tumor foramtion) of strains A6 and C58 and the sensitivity of strain C58 to bacteriocin 84 were unaffected by the acquistion of RP4 by the Agrobacterium strains. Plasmid R1drd-19 was not transferred to A. tumefaciens. Transformation experiments with plasmid deoxyribonucleic acid were unsuccessful, even though, in the case of RP4, conjugation studies showed taht the deoxyribonucleic acid was compatible with that of the recipient strains.     

Inhibition of Transformation of Bacillus subtilis by Heavy Metals

Mercuric ions, as well as organomercuric ions and cadmium ions, can inhibit deoxyribonucleic acid-mediated transformation in Bacillus subtilis 168 without decreasing the viability of the total population. Differences in the inhibition of transformation by mercuric ions are identifiable on a temporal and concentration dependence basis. Sensitivity to low concentrations (9.2 x 10(-8) M) appears early in the uptake of deoxyribonucleic acid before the transformed markers have become insensitive to deoxyribonuclease. Resistance to "low concentrations" of Hg(2+) is kinetically indistinguishable from the requirement for magnesium in the transformation process. This inactivation is not reversed by the mercury-binding compound glutathione. Sensitivity to mercuric ions at a higher concentration (5.52 x 10(-7) M) occurs after the donor deoxyribonucleic acid has become insensitive to deoxyribonuclease. These complex interactions between mercuric ions and the process of transformation are discussed.     

Recovery of Donor Deoxyribonucleic Acid Marker Activity from Eclipse in Pneumococcal Transformation

After the uptake of deoxyribonucleic acid (DNA), donor marker-transforming activity is temporarily lost. Restoration of the activity by annealing in vitro supports the idea that donor DNA is single-stranded at this stage. Kinetics of in vivo recovery from eclipse were examined for various markers at three temperatures. Sigmoidal recovery curves at lower temperatures indicate that the process consists of several steps. Rate of recovery was found to depend on the nature of the donor marker. Single-site markers recover much more rapidly than multisite markers corresponding to recipient deletions. Single-site markers vary somewhat in recovery rate, with rapidity of recovery inversely related to integration efficiency. Appearance of a recombinant-transforming activity lags only slightly behind recovery of its constituent donor marker.     

Molecular Events Accompanying the Fixation of Genetic Information in Haemophilus Heterospecific Transformation

Heterospecific transformation between Haemophilus influenzae and H. parainfluenzae was investigated by isopycnic analysis of deoxyribonucleic acid (DNA) extracts of (3)H-labeled transforming cells that had been exposed to (32)P-labeled, heavy transforming DNA. The density distribution of genetic markers from the resident DNA and from the donor DNA was determined by transformation assay of fractions from CsCl gradients, both species being used as recipients. About 50% of the (32)P atoms in H. parainfluenzae donor DNA taken up by H. influenzae cells were transferred to resident DNA, and only a small amount of the label was lost under conditions of little cell growth. There was less transfer in the reciprocal cross, and almost half of the donor label was lost. In both crosses, the transferred donor material transformed for the donor marker considerably more efficiently when assayed on the donor species than on the recipient species, indicating that at least some of the associated (32)P atoms are contained in relatively long stretches of donor DNA. When the transformed cultures were incubated under growth conditions, the donor marker associated with recipient DNA transformed the donor species with progressively decreasing efficiency. The data indicate that the low heterospecific transformation between H. influenzae and H. parainfluenzae may be due partly to events occurring before association of donor and resident DNA but results mostly from events that occur after the association of the two DNA preparations.     

Excretion of glutamic acid in Citrobacter intermedius C3 associated with plasmid deoxyribonucleic acid.

Several mutants of Citrobacter intermedius C3 lacking both the ability to synthesize proline and the ability to excrete glutamic acid were isolated by treatment with nitrosoguanidine. No revertants for either characteristic were obtained from these mutants. The ability to excrete glutamic acid was transferred to those mutants with very high frequencies in mating experience by using auxotropic excreting strains as donors. Moreover, the ability to synthesize proline was transferred together with the ability to excrete glutamic acid when an excreting strain was used as donor. The transconjugants showed a rapid spontaneous curing of both genetic markers. It was shown by two different methods that a band of covalently closed circular deoxyribonucleic acid is present in the cesium chloride gradients corresponding to the wild type and excretor mutants. Nonexcretor mutants described herein lacked such a band. Pro + transformants that were also excretors were obtained with plasmid deoxyribonucleic acid isolated either from wild type or from an excretor mutant. These data strongly indicate that glutamic acid excretion in C. intermedius C3 is related to the presence of extrachromosomal deoxyribonucleic acid.     

Donor deoxyribonucleic acid length and marker effect in pneumococcal transformation.

The efficiency of transformation of point mutations depends upon base pair mismatches during the recombination process. For low-efficiency markers, the genetic information carried on the donor deoxyribonucleic acid is preferentially lost. To understand this elimination process, we investigated the effect of the size of donor deoxyribonucleic acid on the relative efficiency of low-efficiency point mutations. The deoxyribonucleic acid was shortened either by mechanical shearing or by restriction enzyme treatments. The results indicate that transformation by low-efficiency markers was not affected by shortening the distance between them and the end of the molecule any more than was transformation by the other markers. Moreover, no lethal event could be detected for either cell or chromosomal marker survival. These data do not exclude the double-strand-break hypothesis that was proposed to explain the loss of genetic information for low-efficiency markers, but they offer no support for it.     

Mechanism of Action of Nalidixic Acid on Conjugating Bacteria

When nalidixic acid, a specific and effective inhibitor of cellular deoxyribonucleic acid synthesis, is added to conjugating bacteria at any time during mating, it stops genetic transfer provided the donor bacterium is sensitive to the drug. When this inhibition is released by the removal of the nalidixic acid, transfer does not resume at the point on the chromosome where it was stopped, but begins again at the transfer origin. Curves relating the effects of various low doses of nalidixic acid to the frequency of recombination reveal that several "hits" are necessary to inhibit recombination for early markers. The number of required "hits" decreases as the distance of the marker from the transfer origin increases. Transfer between drug-resistant cells may also be inhibited by nalidixic acid. The effect of high drug doses on matings between resistant cells is similar to that of low drug doses on matings with a sensitive male.     

Physical Properties and Mechanism of Transfer of R Factors in Escherichia coli

The physical properties of F-like and I-like R factors have been compared with those of the wild-type F factor in Escherichia coli K-12 unmated cells and after transfer to recipient cells by conjugation. The F-like R factor R538-1drd was found to have a molecular weight of 49 x 10(6), whereas the molecular weight of the I-like R factor R64drd11 was 76 x 10(6). The wild-type F factor, F1, had a molecular weight of 62 x 10(6). When conjugation experiments are performed by using donor strains carrying these derepressed F-like or I-like R factors, the transferred deoxyribonucleic acid can be isolated as a covalently closed circle from the recipient cells. This circular deoxyribonucleic acid was characterized by making use of the observation that the complementary strands of these R factors can be separated in a CsCl-poly (U, G) equilibrium gradient. The results of the strand-separation experiments show that only one of the complementary strands of the R factor is transferred from the donor to the recipient. With both the F-like and I-like R factors, this strand is the heavier strand in CsCl-poly (U, G). These results indicate that even though F-like and I-like R factors differ greatly in many properties (phage specificity, size, compatability, etc.), they are transferred by a similar mechanism.     

Transport of DNA from Lymphocytes to Fibroblasts in Culture

The exchange of radioactivity between lymphocytes, labelled with (³H) thymidine after stimulation with Concanavalin A, and recipient V79 fibroblasts in culture was studied. The radioactive material involved in this exchange was macromolecular deoxyribonucleic acid as well as its breakdown products. This deoxyribonucleic acid from lymphocytes localised in the nuclei of the host cells soon after contact between donor and recipients. This occurred even when the V79 fibroblasts were confluent at high cell density, and thus in a steady, non-growing state with respect to cell numbers.
The fate of the radioactive donor lymphocyte deoxyribonucleic acid, substituted with bromodeoxyuridine, was followed in the recipient cells by analysing its buoyant density in caesium chloride gradients. This deoxyribonucleic acid was found to become associated with the nuclear deoxyribonucleic acid of the host cells, involving both retention of relatively intact donor deoxyribonucleic acid as well as its breakdown and re-utilisation for host cell deoxyribonucleic acid synthesis. Nongrowing recipient cells were found to retain the donor deoxyribonucleic acid in relatively intact form for much longer periods than when the same cells were in logarithmic growth phase.     

Plasmid Replication and the Induced Synthesis of Colicins E1 and E2 in Escherichia coli

Induction of colicins E1 and E2 in Escherichia coli occurs when plasmid synthesis has been inhibited either by nalidixic acid or by lack of deoxyribonucleic acid polymerase I. Moreover, colicin E1 and E2 synthesis induced by mitomycin C and exposure to chloramphenicol is not associated with a large increase in circular plasmid deoxyribonucleic acid. The mean plasmid content of cells in populations having a low spontaneous frequency of colicin-producing cells because of growth at low temperature or because of the presence of recA(-) or crp(-) alleles, is not significantly different to that in wild-type cells grown at 37 C.     

Studies of colicin E1 plasmid functions by analysis of deletions and TnA insertions of the plasmid.

The further identification of regions of the colicin E1 plasmid that affect plasmid functions has been achieved by studying deletions and TnA insertions of the plasmid. Colicin production, colicin immunity, relaxation of plasmid deoxyribonucleic acid, and plasmid incompatibility functions have been examined. A strong correlation has been observed between the ability of colicin E1 plasmid deoxyribonucleic acid to be relaxed and the ability of that plasmid to be transferred by conjugation.     

Temperature-Sensitive Divisionless Mutant of Bacillus subtilis Defective in the Initiation of Septation

A temperature-sensitive divisionless mutant of Bacillus subtilis 168, tms-12, is shown to be defective in an early step in septum formation at the restrictive temperature. The nature of this defect has been studied by comparing the growth and composition of mutant and wild-type (tms-12(+)) cells at the restrictive (48 C) and permissive (34 C) temperatures. At 48 C, tms-12 cells grow as nonseptate, multinucleate filaments. Filamentation does not appear to be a result of alterations in properties of the cell wall, since the ratio of mucopeptide to teichoic acid, the autolytic activity, and the ability of the walls to protect cells against osmotic shock are comparable in tms-12 filaments and tms-12(+) bacilli grown at 48 C. Synthesis of deoxyribonucleic acid and the segregation of nucleoids also proceed normally during filamentation. The synthesis of membrane, however, is delayed during filamentation of tms-12. No gross alterations were observed in the protein or lipid composition of membranes isolated from mutant filaments. Septum formation resumes when filaments are returned to 34 C and appears to be associated with an increased synthesis of membrane. The occurrence of septa was monitored both by microscopic observation of cross walls and by assays of the number of viable protoplasts released from bacillary filaments upon removal of the cell wall. Septation recovery can be blocked by inhibitors of ribonucleic acid and protein synthesis added during, but not after, the first 7 min of recovery at 34 C. By contrast, inhibition of deoxyribonucleic synthesis does not block recovery.     

Studies on Escherichia coli Sex Factors: Evidence That Covalent Circles Exist Within Cells and the General Problem of Isolation of Covalent Circles

We examined in detail conditions necessary for making reproducible and for maximizing the amount of deoxyribonucleic acid obtained from a sex factor-containing cell as covalent circles. The results argue that under optimal conditions covalent circles are neither created nor lost during the isolation procedure. The causes of the culture-to-culture variation in recovery of covalent circular deoxyribonucleic acid were investigated but an understanding of this is not yet at hand. Some commonly used conditions which drastically reduce the recovery of covalent circles are described.     

Ultraviolet-sensitive Targets in the Enzyme-synthesizing Apparatus of Escherichia coli

Inhibition by ultraviolet light of beta-galactosidase and alkaline phosphatase synthesis was investigated in both ultraviolet (UV)-sensitive and UV-resistant (wild-type) Escherichia coli, with the objective of determining the sensitivity of various targets. Kinetics of enzyme formation by unmated bacteria and in mating systems, in which the donor provided the specific genetic material and the recipient the cytoplasm, permit the following conclusions regarding the sensitivity of various targets. Catabolite repression resulting from UV damage causes most of the inhibition of beta-galactosidase formation. When it is largely eliminated by a step-down in nutrition, the principal target in UV-sensitive bacteria appears to be the structural gene (lacZ(+)), but damage to the cytoplasm is also important. Transitory inhibition by inactivation of messenger ribonucleic acid is also observed. In wild-type bacteria, repair reduces the importance of lesions in deoxyribonucleic acid sufficiently that cytoplasmic damage appears to be at least as important. Repair occurs within 10 min, as shown by recovery of enzyme-synthesizing ability. Caffeine and proflavine prevent recovery. Newly mated bacteria respond to irradiation very differently than do unmated bacteria. The beta-galactosidase or alkaline phosphatase structural gene (lacZ(+) or phoP(+)) is much more inhibited after it is transferred than it is in unmated bacteria. This sensitivity seems to depend on a sensitive state of the injected material, rather than on a different physiological condition of the entire zygote. Irradiation of recipient uvr(+) bacteria much more strongly inhibited expression of injected genes than if the F(-) was uvr(s). Studies on mating systems are not very useful for learning about the function of unmated bacteria.     

Entry of double-stranded deoxyribonucleic acid during transformation of Neisseria gonorrhoeae.

The state of donor deoxyribonucleic acid after entry into competent cells was examined by assaying the transformed cell lysates for donor-marker transforming activity and density of donor deoxyribonucleic acid in CsCl gradients. The experiments showed that deoxyribonucleic acid entered in native, double-stranded form.