Plasmid R1drd-19 Research Articles

Extension of a Chromosome Linkage Group of Proteus mirabilis

Mating procedures for detection of mobilization of the Proteus mirabilis chromosome were re-investigated. The chromosome was mobilized by plasmid D, the previously used hybrid between plasmids P-lac and R1drd19. About a 40-fold increase in recombinant recovery correlated with the absence of swarming during mating and a lower temperature of incubation. The modification introduced was that conjugation was allowed to proceed on a non-selective supplemented minimal medium at 30 degrees C before washing and plating on selective media. Final incubation was also at 30 degrees C. This technique enabled eight additional chromosomal markers to be mapped. Polarized transfer of the chromosome was shown by gradient of transmission experiments using a previously described marker as reference, by linkage analysis with reference to proximal and distal markers and (less successfully) by interrupted mating on solid medium. Markers of plasmid D transferred at high frequency to all recombinants. The plasmid was stable in recombinants and could transfer itself and chromosomal markers of the new hosts in further matings. Resulting recombination of markers occurred at usual frequencies. The marker order, his-1, ser-2, ura-2, pyrB1, trp-3, cysA1, ade-2, ilv-2, cysG1, gly-1, cysC1, argA2, metF2, nalA1, thr-1, leuB2, did not resemble the order of these markers in Escherichia coli.

Selection and timing of replication of plasmids R1 drd-19 and F′ lac in Escherichia coli

The selection and timing of plasmid replication was studied in exponentially growing cultures of Escherichia coli K-12 carrying the plasmid R1 drd-19 and E. coli strains B/r A and B/r F carrying the plasmid F′ lac. In all cases plasmid replication was studied by analysis of covalently closed circular (CCC) DNA. The turnover time of replicating plasmid DNA into CCC-DNA was found to be less than 4 min. Density shift experiments (from 15NH 4 +, D 2O to 14NH 4 +, H 2O) showed that plasmids R1 drd-19 and F′ lac are selected randomly for replication. This means that one of the plasmid copies in a cell is selected and replicated. There is no further plasmid replication in the cell until all plasmid copies, including the newly formed ones, have the same probability of being selected for replication. The early kinetics of the appearance of light plasmid DNA after the density shift showed that the time interval between successive replications of plasmids R1 drd-19 and F′ lac is τ n , where τ is the generation time and n is the average number of plasmid replications per cell and cell cycle. In a second type of experiment, exponentially growing cells were separated into a series of size classes by low-speed centrifugation in sucrose step gradients. Replication of plasmids R1 drd-19 and F′ lac was equally frequent in all size classes. This result is in accordance with the results of the density shift experiment. It can therefore be concluded that replication of plasmids R1 drd-19 and F′ lac is evenly spread over the whole cell cycle, which means that one plasmid replication occurs every time the cell volume has increased by one initiation mass.

Temperature-dependent and amber copy mutants of plasmid R1 drd-19 in Escherichia coli

The isolation of conditional mutants with an altered copy number of the R plasmid R1 drd-19 is described. Temperature-dependent as well as amber-suppressible mutants were found. These mutant plasmids have been named pKN301 and pKN303, respectively. Both types of mutations reside on the R plasmid. No difference in molecular weight could be detected by neutral sucrose gradient centrifugation for any of the mutant plasmids when compared with the wild-type plasmid. The number of copies of the plasmids was determined by measurement of the specific activity of the R plasmid-mediated β-lactamase and by measurement of covalently closed circular (CCC) DNA in alkaline sucrose gradients and dye-CsCl density gradients. Below 34 °C the temperature-dependent mutant, pKN301, had the same copy number as the wild type, while this was four times that of the wild type above 37 °C. The amber mutant pKN303 had a copy number indistinguishable from that of the wild-type plasmid in a strain containing a strong amber suppressor and a copy number about five times that of the wild-type plasmid in a strain lacking an amber suppressor. In a strain containing a temperature-sensitive amber suppressor, the amber mutant's copy number increased with the decrease in amber suppressor activity. Thus, the existence of the temperature-dependent and the amber-suppressible R-plasmid copy mutants indicates that the system that controls the replication of plasmid R1 drd-19 contains an element with a negative function and that this element is a protein.

Restriction map of the antibiotic resistance plasmid R1 drd-19 and its derivatives pKN102 (R1drd-19B2) and R1drd-16 for the enzymes BamHI, HindIII, EcoRI and SalI

The conjugative R plasmid R1drd-19, mediating antibiotic resistance to ampicillin (Ap), chloramphenicol (Cm), kanamycin (Km), streptomycin (Sm) and sulfonamides (Su) was mapped using the restriction endonucleases BamHI, HindIII, EcoRI and SalI. BamHI generates 5 fragments (A-E) with molecular weights between 46 x 10(6) 0.25 x 10(6) dalton, and HindIII 8(A-H) between 42 x 10(6) dalton (representing mainly the RTF) and 0.25 x 10(6) dalton (representing the main part of the RTF) and 0.1 x 10(6) dalton. EcoRI recognises 17 sites and produces fragments (A-Q) with molecular weights between 11.7 and 0.1 x 10(6) dalton. SalI yields 7 fragments (A-G) of 16.5 to 2.0 x 10(6) dalton. A physical map was constructed from fragments obtained by partial digestion of R1drd-19 with one restriction enzyme, by double and triple digestion of the DNA with two or three enzymes with and without isolation of individual bands from preparative gels. In addition the restriction patterns of several mutants of R1drd-19 were compared with it. Evidence is presented which indicates that the derivatives of R1 investigated are generated by extended deletions, namely the copy mutant pKN102 which has lost the Km resistance, R1drd-16, which has lost all resistances other than Km and the Kms derivative of R1drd-16, which represents the pure RTF. The map of R1drd-19 is remarkably different from those of R100 and R6-5. Its molecular weight was estimated to be 62.5 Md. The circular fragment order for BamHI is: A-C-B-D-E, for HindIII: A-D-C-B-F-H-E-G, for EcoRI: A-C-K-B-F-J-O-D-H-L-G-P-Q-N-I-E-M- and for SalI A-B-C-D-G-F-E.

A runaway-replication mutant of plasmid R1drd-19: Temperature-dependent loss of copy number control

A two-step mutant plasmid, originating from the resistance plasmid R1drd-19, was shown to replicate without control of the plasmid copy number when the host bacteria were grown at temperatures above 35°C. The uncontrolled plasmid replication, so called runaway-replication, eventually inhibits the host cell growth and results in a conditionally lethal phenotype. As much as 75% of the total DNA in the cells was found to be covalently closed circular plasmid DNA at the stage when the cell growth stopped. At 30°C the mutant plasmid had a strictly controlled copy number and the onset and kinetics of runaway-replication was studied by temperatureshift experiments. The synthesis of plasmid DNA after a temperature shift from 30°C to 40°C (or 37°C) was found to be exponential with a doubling time of about half the doubling time of the host bacteria, in broth medium as well as in minimal salt-glucose medium. In broth medium the plasmid population had a doubling time of about 12 min which is longer than expected if it is assumed that the rate of elongation during the DNA polymerization is the only limitation on the uncontrolled replication. The results indicate that the minimum time required between successive replications of a plasmid copy is determined by some rate-limiting step(s) presumably after the duplication process. The uncontrolled plasmid replication was shown to be inhibited by the protein synthesis inhibitor chloramphenicol. The plasmid synthesis stopped within 20 min after the addition of the drug and the plasmid DNA increment was less than 50%, suggesting that the uncontrolled plasmid replication requires de novo protein synthesis. Similarly, the RNA polymerase inhibitor rifampicin did inhibit the plasmid replication. The mutational alteration could be reversed, and the copy number control restored, by lowering the temperature before the host cell growth was inhibited. This type of plasmid mutant offers new possibilities in studies of host cell-plasmid interactions.

Isolation and characterization of the minimal fragment required for autonomous replication (“Basic replicon”) of a copy mutant (pKN102) of the antibiotic resistance factor R1

The mini plasmids deriving from pKN102, a copy mutant of the antibiotic resistance factor R1drd-19 of E. coli, share a common DNA sequence of 2.6 kb, which carries the minimal functions for autonomous replication. By cloning of two PstI fragments of this region it could be demonstrated that the "basic replicon" is a DNA segment not larger than 1.8 kb, which carries the orgin of replication and the genetic information for at least two proteins. Protein F (NW=11.000 dalton) seems to be synthesed in larger amounts in minicells of E. coli than protein C (20.000). Plasmids containing this isolated replicon of R1 are completely compatible with the parental plasmid R1drd-19.

R plasmid gene dosage effects in Escherichia coli K-12: Copy mutants of the R plasmid R1 drd-19

Copy mutants of the R plasmid R1 drd-19 were used to study gene dosage effects in Escherichia coli K-12. The specific activity of β-lactamase, chloramphenicol acetyltransferase, and streptomycin adenylylase, as well as ampicillin resistance, increased linearly with the gene dosage up to a level at least tenfold higher than that of the wild-type plasmid. This makes it possible to use ampicillin resistance to determine plasmid copy number and also to select for plasmid copy mutants with defined copy number. Chloramphenicol resistance, despite the increase in enzyme activity, reached a plateau level at a gene dosage less than twice that of the wild-type plasmid, presumably due to the high energy demand on the cells during inactivation of the antibiotic by acetylation with acetyl-coenzyme A. Similarly, resistance to streptomycin plateaued at a gene dosage about three times that of the wild-type plasmid, presumably because of a decreased efficiency of the cells' outer penetration barriers when carrying the R plasmid. The susceptibility of the cells to rifampicin was increased by the presence of plasmid copy mutants.

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.

Isolation and genetic characterizaion of Escherichia coli K-12 mutations affecting bacteriophage T5 restriction by the ColIb plasmid.

A mutant derivative of Escherichia coli K-12 has been isolated which is permissive for bacteriophage T5 infection even when harboring a wild-type ColIb plasmid. The fully permissive phenotype was the result of two mutations that are located near the rpsL-rpsE region on the E. coli chromosome and are recessive to the wild-type alleles. These mutations had little or no effect on induction of colicin synthesis and did not affect the expression of antibiotic resistance by the resistance plasmids R64drd11 or R1drd19. Cells harboring the mutant alleles grew more slowly than isogenic wild-type derivatives in either minimal or complete media.

Inhibition of cell division in Escherichia coli K-12 by the R-factor R1 and copy mutants of R1.

The effect of the copy number of plasmid R1drd-19 on cell division of Escherichia coli K-12 was studied in populations growing as steady-state cultures at different growth rates, the growth rate being varied by use of different carbon sources. The plasmid copy number was also varied by using copy mutants of the R-factor. The mean cell size was larger in populations carrying an R-factor than in R-factorless populations, an effect that was more pronounced at low growth rates and in populations carrying R-factor copy mutants. The increased cell size was due to formation of elongated cells in a fraction of the population and to an increase in the diameter of all cells. The majority of the cells divided at a normal cell length, but the presence of an R-factor caused some cells to elongate, probably by the uncoupling of chromosome replication and cell division. This can be explained as a competition between the chromosome and plasmid replicons for some replication factor(s), presumably acting on both initiation and elongation of replication. The formation of elongated cells was a reversible process, but occasionally some of the elongated cells reached lengths 20 times that of newborn cells. If cell division did not occur at the normal cell size, the septum was not formed until the cell size was four times that of a newborn cell. When an elongated cell divided, it usually formed a polar septum, thus producing a newborn cell of normal cell length. The ability of plasmid-containing cells to omit one cell division but to retain the capacity of dividing one mass doubling later is compatible with a mechanical model for septum formation and cell division.