Transition of the R factor NR1 and Proteus mirabilis: level of drug resistance of nontransitioned and transitioned cells.

When Proteus mirabilis harboring the R factor NR1 is cultured in Penassay broth containing 100 mug of chloramphenicol (CM) per ml, there is an amplification in the number of copies of the r-determinants per cell. Under these conditions, R factors harboring multiple tandem sequences of r-determinants are formed. Autonomous poly-f-determinants consisting of multiple copies of r-determinants are also formed. This phenomenon has been referred to as the "transition". Transitioned cells have considerably higher levels of resistance to CM and streptomycin (SM), but not to tetracycline (TC), than do nontransitioned cells and grow more rapidly in medium containing either CM or SM. There is essentially no difference in growth rates between transitioned and nontransitioned cells in drug-free medium. The higher level of resistance of transitioned cells to SM has made it possible to investigate the mechanism of the transition. Using replica plating, it has been possible to isolate spontaneously occurring transitioned cells from a nontransitioned population which appear to outgrow the nontransitioned cells during growth in medium containing 100 mug of CM per ml. If transiitoned cells are subsequently cultured in drug-free medium, the cells return gradually to the nontransitioned state, which has been referred to as the "back-transition was monitored by examining the level of resisitance of the cells to SM. In both situations the cell populations were found to be heterogeneous, consisting of a mixture of nontransitioned and transitioned cells. Under the conditions of our experiments, the transition appeared to be due to the more rapid growth of a minor fraction of spontaneously occurring transitioned cells which outgrew the remainder of cells in the population. To obtain the transition, the drug resistance gene must reside on the r-determinants component of the R factor. The transition did not take place when the cells were cultured in medium containing high concentrations of TC. This indicates that the TC resistance genes reside on the resistance transfer factor component of the R factor, which is in agreement with physical studies on R factor deoxyribonucleic acid.     

Transition of the R Factor R12 in Proteus mirabilis

When Proteus mirabilis harboring the R factor R12 (a round of replication mutant of the R factor NR1) is cultured in medium containing streptomycin there can be an amplification in the number of copies of r-determinants per cell and the formation of enlarged polygenic R factors containing repeated sequences of r-determinants as well as polygenic molecules consisting of repeated sequences of r-determinants. This phenomenon has been referred to as the "transition." When transitioned cells are then cultured in drug-free medium, within a few generations two distinct density species of R factor deoxyribonucleic acid (DNA) are observed in a CsCl density gradient: a 1.712 g/ml band of covalently closed circular R factor DNA consisting of one transfer factor (RTF-TC) plus one r-determinant and a 1.718 g/ml band consisting of repeated sequences of r-determinants. The RTF-TC component of the R factor appears to control the replication of all the R factor DNA which is attached to it. In the autonomous state, however, polygenic sequences of r-determinants do not appear to replicate under the same control mechanism as when they are attached to an RTF-TC.     

Alternate forms of the resistance factor R1 in Proteus mirabilis

The resistance factor R1 may exist in either of two stable physical states in Proteus mirabilis PM-1. In one case, the R1 deoxyribonucleic acid (DNA) has a buoyant density of 1.711 g/cm(3) and replicates under stringent control. Cells harboring R1 in this form may transfer drug resistance by conjugation. In the other case, R1 DNA shows two buoyant density classes at 1.707 and 1.714 g/cm(3). The 1.714 g/cm(3) component is replicated under a degree of relaxed control, and strains carrying this form generally cannot transfer drug resistance by conjugation. Intracellular amounts of the R factor-coded enzyme, chloramphenicol acetyltransferase, did not correspond to amounts of plasmid DNA in Proteus, and the enzyme was present in lower amounts than in Escherichia coli. It is proposed that the two states of R1 in Proteus may represent stable associated and dissociated forms of the plasmid.     

Effects of chloramphenicol and rifampicin on the replication of R plasmid NR1 deoxyribonucleic acid in Escherichia coli.

The effects of inhibition of protein and ribonucleic acid (RNA) synthesis on the replication of the plasmids NR1 and F′lac in Escherichia coli were studied. When protein synthesis is inhibited, there is approximately a 25% increase in R plasmid deoxyribonucleic acid (DNA), but this newly synthesized DNA is not recoverable in the covalently closed circular (CCC) form until protein synthesis is allowed to resume. When RNA synthesis is inhibited, there is also approximately a 20% increase in R plasmid DNA, but this DNA is immediately recoverable in the CCC form. F′lac DNA, unlike R plasmid DNA, can continue to replicate for at least a generation time in the absence of protein synthesis, and this F′lac DNA is immediately recoverable in the CCC form.     

Replication of the R Factor Rts1 in Proteus mirabilis

The replication of the R factor Rts1 in Proteus mirabilis was examined by using the technique of CsCl density-gradient centrifugation. The proportion of Rts1 deoxyribonucleic acid (DNA) relative to the host chromosomal DNA (% R-DNA) was 7% in both exponential and stationary growth phases in Penassay Broth and supplemented M9 minimal medium at 30 C. The chromosomal DNA content per cell varied over a threefold range in the different growth media. In agreement with previous genetic observations, the replication of Rts1 was found to be temperature-sensitive and Rts1 DNA was diluted from the cells during exponential growth at 42 C. (14)N-(15)N medium transfer experiments have shown that individual copies of Rts1 are selected at random for replication during the duplication of the multicopy episome pool.     

Denaturation mapping of R factor deoxyribonucleic acid.

The R factor NR1 consists of two components: a resistance transfer factor which harbors the tetracycline resistance genes (RTF-TC) and the r-determinants component which harbors the other drug resistance genes. Using partial denaturation mapping it is possible to distinguish the RTF-TC region from the r-determinants region of the composite R factor NR1 DNA which has a contour length of 37 mum and a density of 1.712 g/ml. The r-determinants region was a relatively undenatured 8.5-mum segment of the molecule when the deoxyribonucleic acid was partially denatured at pH 10.7. An RTF-TC genetic segregant of NR1 which had lost the r-determinants component had a contour length of 28.7 mum and a density of 1.710 g/ml. Characterization of an RTF-TC using partial denaturation mapping at pH 10.7 confirmed that the relatively undenatured 8.5-mum r-determinants segment of the composite R factor had been deleted. Circular, transitioned NR1 DNA molecules (1.716 to 1.718 g/ml), whose contour lengths were consistent with an RTF-TC plus an integral number of tandem copies of r-determinants, were also characterized by denaturation mapping. The relatively undenatured region in these molecules had a length equal to an integral number of copies of r-determinants and was located at the same site in the partially denatured RTF-TC as the single copy of r-determinants in the 37-mum composite NR1. This indicates that there is a unique integration site for r-determinants in the RTF-TC component. The R factor UCR122, a TC deletion mutant of NR1, was also characterized by denaturation mapping. The translocation of the TC resistance gene(s) on the denaturation map permitted the alignment of the denaturation map with the heteroduplex map of Sharp et al. (u073). Linear and circular monomeric and presumed multimeric r-determinants DNA molecules (p = 1.718 g/ml) were partially denatured at a higher pH (11.10). The r-determinants multimers showed a repeating 8.3-mum (monomeric) partial denaturation pattern indicating a head-to-tail arrangement of monomers in these poly-r-determinant molecules.     

Direction of deoxyribonucleic acid transfer and replication in a derivative of plasmid R100-1.

The site of integration and the molecular orientation of a prophage Mu integrated within the resistance transfer factor component of plasmid R100-1 have been determined on the physical map of the plasmid. This allowed us (i) to determine the direction of deoxyribonucleic acid transfer from oriT during conjugation and (ii) to demonstrate the unidirectionality of replication in conditions of exponential growth (by determining the strand preference of Mu-specific Okazaki fragments).     

Mobilization of the Proteus mirabilis chromosome by R plasmid R772.

The P-1 incompatibility group plasmid R772 can mobilize the chromosome of Proteus mirabilis strain PM5006. The decreasing gradient of recombinant recovery frequencies found for markers which were increasingly distal to 0 min with plasmid D donors was not found with R772. Instead, it produced recombinants for all markers at frequencies of about 5 X 10(-5) per donor. This is about 10-fold lower than the plasmid transfer frequency. Recombinants were stable and recombination was only detected over short segments of the chromosome which corresponded to about 10 min on the D plasmid map of the chromosome. All recombinants had inherited R772 and expressed all properties of the plasmid. Attempts to isolate variant plasmids with increased frequencies of recombinant formation were unsuccessful.     

Discontinuous replication of colicin E1 plasmid deoxyribonucleic acid.

The plasmid pML 21, which was found to contain approximately 49% of the Col E1 genome was used to determine the template origin of single-stranded deoxyribonucleic acid (DNA) fragments (4 to 32% of the Col E1 units length) associated with Col E1 dna replicative intermediates. The results of DNA hybridization competition experiments indicate that the single-stranded fragments derive from the full length of the Col E1 DNA template as expected for Okazaki fragments and the plasmid pML 21 contains the replication origin of Col E1 DNA.     

Properties of the relaxation complex of supercoiled deoxyribonucleic acid and protein of R plasmid NR1 in Escherichia coli.

Some properties of the supercoiled deoxyribonucleic acid (DNA)-protein relaxation complex of the R plasmid NR1, which contains more than one origin for DNA replication, were examined. The percentage of complexed NR1 molecules that can be converted to the relaxed (nicked) form appeared to be unaffected by the conditions under which the host cells were cultured. However, the percentage of supercoiled NR1 DNA that can be relaxed was highly dependent on the method used to prepare the DNA and the agents used to induce relaxation. Our data suggest that 100% of NR1 molecules may exist in situ as DNA-protein relaxation complexes. An RTF-Tc segregant of NR1, which has deleted the r-determinants component of the NR1 and therefore does not contain the two origins of replication located in the r-determinants, has indistinguishable relaxation properties in comparison with NR1 itself.     

Patterns of mobilization of the Proteus mirabilis chromosome by R plasmids.

R plasmids R40a, Rip69, R447b, R769 belonging to incompatibility groups A-C, M, N, V, respectively, were investigated for chromosomal mobilizing ability in Proteus mirabilis. Plasmids R40a, Rip69 and R447b mediated polarized transfer of markers in a clockwise direction from origins near tyr-1, metF and ser-2, respectively, on the linkage map. The recovery frequency per donor cell of proximal markers approached 1 x 10(-4) for these three plasmids and the efficiency of chromosomal transfer was higher than that of the previously studied plasmid D. The plasmid-guided chromosomal trajectories overlap and it was possible to complement results obtained with plasmid D to assemble a time-of-entry chromosomal map and directly establish the circularity of the linkage group. The map comprises a length of 93 min in terms of transfer time. Plasmid R769 had a different pattern of chromosome transfer. This plasmid produced recombinants for all markers at frequencies of about 4 x 10(-6) per donor. It effected multiple and more or less simultaneous entry of markers and produced recombination over lengths of chromosome rarely corresponding to more than 10 min on the linkage map.     

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.     

Frequency of replication from alternative origins in the composite R plasmid NR1.

The relative frequency of initiation of DNA replication within the RTF-Tc and r-determinant components of the composite drug resistance plasmid NR1 in Proteus mirabilis was evaluated. Using fractionated radioactively labeled plasmid DNA, analytical procedures that distinguished between the two components of the composite plasmid were carried out. A mixture of uniformly 14C-labeled and 3H-pulse-labeled plasmid DNA (pulse-labeled origin[s] of replication) was used in each of three experiments. First, shear products of the DNA were analyzed using CsCl density gradient centrifugation. Second, fragmented DNA was hybridized to nonradioactive RTF-Tc and r-determinant DNAs immobilized on nitrocellulose filters. Third, the radioactive plasmid DNAs immobilized on nitrocellulose filters. Third, the radioactive plasmid DNA was digested with restriction enzyme (EcoRI), producing a set of RTF-Tc and r-determinant fragments with differing 3H/14C isotpe ratios. The three experiments suggested that under the conditions used to accumulate replicating plasmid DNA molecules (DNA substrate limitation), the r-determinant origin of replication was preferentially utilized in the composite plasmid.     

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.     

Base composition of deoxyribonucleic acid of the temperature-sensitive kanamycin-resistant R factor, Rts1

The buoyant density of deoxyribonucleic acid (DNA) from the temperature-sensitive R factor, Rts1, was determined by CsCl density-gradient centrifugation. Rts1 was found to consist of a single species of DNA of density 1.705 g/cm(3), which corresponds to a base composition of 45% guanine plus cytosine. This value is distinct from the densities previously reported for other R factors, suggesting that Rts1 represents a new molecular class of R factors.