Analysis of a recessive plasmid copy number mutant: evidence for negative control of Col E1 replication.

The Col E1-derivative copy number mutant plasmid pOP1Δ6 has been used to investigate the control of plasmid replication. pOP1Δ6 normally exists at about 200 copies per chromosome, while the wild-type plasmid from which it was derived (pBGP120) exists at about 15 copies per chromosome. We have observed that in E. coli containing both pOP1Δ6 and pBGP120, the copy number of pOP1Δ6 is lowered to 4–6 copies per chromosome. Thus the mutation in pOP1Δ6 is recessive. The association between the two plasmids is stable in E. coli, indicating that incompatibility properties as well as replication control characteristics have been altered in pOP1Δ6. Co-residence of the unrelated plasmid pSC101 with pOP1Δ6 has no detectable effect on pOP1Δ6 copy number. These results suggest that a plasmid-specific, diffusible repressor may act negatively to control plasmid copy number, and that pOP1Δ6 produces a defective repressor or is altered in repressor synthesis. We have constructed in vitro a plasmid which is identical in size to pQP1Δ6 but contains a replication origin region derived from pBGP120. Since this plasmid, pNOP1, exists stably (like pBGP120) at 10–15 copies per chromosome, the high copy number of pOP1Δ6 is not related to its reduced size relative to pBGP120. To localize the mutation in pOP1Δ6 responsible for DNA overproduction, we have cloned fragments of pBGP120 into pOP1Δ6 and selected for plasmids with wild-type copy number. We find that a 2.0 kb region of pBGP120 DNA surrounding the origin of plasmid DNA replication is capable of suppressing the DNA overproducer phenotype of pOP1Δ6. The 2.0 kb fragment is capable of independent self-replication or can integrate into pOP1Δ6 in vivo to form a composite plasmid with two origins of replication. The overproducer phenotype of pOP1Δ6 is suppressed in either configuration.     

Detection of nonintegrated plasmid deoxyribonucleic acid in the folded chromosome of Escherichia coli: physiochemical approach to studying the unit of segregation.

Physiocochemical evidence presented indicates plasmid deoxyribonucleic acid (DNA) can associate with host chromosome without linear insertion of the former into the latter. This conclusion is based on the observation that covalently closed circular (CCC) plasmid DNA can cosediment with undegraded host chromosome in a neutral sucrose gradient. When F plus bacteria are lysed under conditions that preserve chromosome, approximately 90% of CCC F sex factor plasmid (about 1% of the total DNA) is found in folded chromosomes sedimenting at rates between 1,500 and 4,000s. The remaining 10% of the CCC F DNA sediments at the rate (80S) indicative of the free CCC plasmid form. Reconstruction experiments in which 80S, CCC F DNA is added to F plus or F minus bacteria before cell lysis show that exogenous F DNA does not associate with folded chromosomes. In F plus bacteria, F plasmid is harbored at a level of one or two copies per chromosomal equivalent. In bacteria producing colicin E1, the genetic determinant of this colicin, the Col E1 plasmid, is harbored at levels of 10 to 13 copies per chromosomal equivalent; yet, greater than 90% of these plasmids do not cosediment with the 1,800S species of folded chromosome. However, preliminary evidence suggests one or two Col E1 plasmids may associate with the 1,800S folded chromosome. Based on evidence presented in this and other papers, we postulate F plasmid can link to folded chromosome because the physicochemical structure of the plasmid resembles a supercoiled region of the chromosome and, therefore, is able to interact with the ribonucleic acid that stabilizes the folded chromosome structure. Implications of this model for F plasmid replication and segregation are discussed.     

Trans-complementation-dependent replication of a low molecular weight origin fragment from plasmid R6K

A non-self-replicating segment (1370 base pairs) of plasmid R6K was cloned in E. coli and shown to trans-complement temperature-sensitive replication mutants of this plasmid. This segment contains the gene which codes for a protein required for initiation of replication of the plasmid, and was used as a helper in a functional assay for an origin of replication in R6K derivatives. A 420 bp fragment, derived from R6K DNA, was shown to carry a functional origin since it was capable of replicating as a plasmid in E. coli cells carrying the helper segment either on the host chromosome or on a plasmid Col E1 derivative. The copy number of the origin fragment in cells carrying the helper segment on the chromosome is essentially the same as the copy number of R6K. A model for the positive regulation of plasmid R6K replication is presented.     

Intracellular location of NRl-plasmid DNA inEscherichia coli minicells

Intracellular location of plasmid NR1 (M = 58 Mg/mol, stringent control of replication, 1–2 copies perEscherichia coli chromosomal equivalent) was studied and compared with that of plasmid R6KΔ1 (M = 21 Mg/mol, relaxed control of replication, 10–15 copies perE. coli chromosomal equivalent), both inE. coli minicells. Considerable difference in relative distribution of molecules of these two plasmid DNA’s between the cytoplasm and the membrane fraction was found when components of the corresponding minicell lyzates were fractionated by sedimentation in a double-linear gradient of caesium chlorid and sucrose. Also the difference in relative numbers of NR1 DNA and R6KΔ1 DNA molecules stably associated with the membrane of minicells, determined by electron-microscopic examination of the fractions containing plasmid DNA-membrane complexes, was evaluated as statistically significant. The association of NR1 DNA molecules withE. coli minicell membrane was found to be a much more frequent event than such association of R6KΔ1 molecules. The absolute amount of plasmid DNA associated with membrane in a single minicell corresponds to one molecule for both NR1 and R6KAΔ1.     

RNase H and replication of ColE1 DNA in Escherichia coli

Amber mutations within the rnh (RNase H) gene of Escherichia coli K-12 were isolated by selecting for bacteria capable of replicating in a sup+ background replication-defective cer-6 mutant of the ColE1 replicon. The cer-6 mutation is an alteration of one base pair located 160 nucleotides upstream of the unique replication origin of this plasmid. Subsequently, we determined the DNA alterations present within these mutants. ColE1 DNA replicated in rnh(Am) recA cells, indicating that (i) RNase H, which has been shown to be absolutely required for in vitro initiation of ColE1 DNA replication, is dispensable in vivo, and (ii) ColE1 replication in the absence of RNase H is not dependent on "stable DNA replication," which has been reported to be an alternative mode of chromosomal DNA replication. Another class of bacterial mutations was also isolated. These mutations, named herB, suppressed cer-6 replication in rnh+ bacteria. herB mutations mapped close to the polA gene on the E. coli chromosome and increased the activity of DNA polymerase I. These findings suggest that when the DNA polymerase I has an opportunity to initiate DNA synthesis before RNase H acts, the replication-defective cer-6 mutant or the wild-type ColE1 replicates in E. coli.     

Nature of Col E1 Plasmid Replication in Escherichia coli in the Presence of Chloramphenicol

The colicinogenic factor E(1) (Col E(1)) in Escherichia coli continues to replicate by a semiconservative mechanism in the presence of chloramphenicol (CAP) for 10 to 15 hr, long after chromosomal deoxyribonucleic acid (DNA) synthesis has terminated. Following CAP addition, the rate of synthesis of plasmid DNA gradually increases to an extent dependent on the medium employed. Within 2 to 4 hr after the addition of CAP, replication in a glucose-Casamino Acids medium approaches a maximum rate representing approximately eight times an average rate which would be required for a net doubling of DNA per cell in one generation. The number of copies of Col E(1) DNA molecules that accumulate under these conditions approaches about 3,000 copies per cell, representing a 125-fold increase over the normal level of 24 copies per cell. The system is particularly convenient for studying the mechanism of DNA replication.     

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.     

Polyadenylation can regulate ColE1 type plasmid copy number independently of any effect on RNAI decay by decreasing the interaction of antisense RNAI with its RNAII target

Replication of ColE1-type plasmids is regulated by RNAI, an antisense RNA that interacts with the replication pre-primer, RNAII. Exonucleolytic attack at the 3' end of RNAI is impeded in pcnB mutant bacteria, which lack poly(A) polymerase I-the principal RNA polyadenylase of E. coli; this leads to accumulation of an RNAI decay intermediate (RNAI(-5)) and dramatic reduction of the plasmid copy number. Here, we report that polyadenylation can also affect RNAI-mediated control of plasmid DNA replication by inhibiting interaction of RNAI(-5) with RNAII. We show that mutation of the host pcnB gene profoundly affects the plasmid copy number, even under experimental conditions that limit the effects of polyadenylation on RNAI(-5) decay. Moreover, poly(A) tails interfere with RNAI/RNAII interaction in vitro without producing any detectable alteration of RNAI secondary structure. Our results establish the existence of a previously undetected mechanism by which RNA polyadenylation can control plasmid copy number.     

Effects of rifampicin, streptolydigin and actinomycin D on the replication of Col E1 plasmid DNA in Escherichia coli

We recently reported (Clewell et al., 1972) on an inhibitory effect of rifampicin on Col E1 plasmid replication. The present study represents a further characterization of this phenomenon as well as a study of the effects of two other known inhibitors of RNA synthesis, Streptolydigin and actinomycin D.During treatment of cells with chloramphenicol the colicinogenic factor E₁ (Col E1) continues to replicate for more than ton hours. During this time 4 to 5 S RNA is also synthesized. When varying concentrations of rifampicin were included during chloramphenicol treatment, inhibition of plasmid DNA synthesis correlated very closely with inhibition of cellular RNA synthesis. Similar experiments testing the effects of Streptolydigin and actinomycin D (during chloramphenicol treatment) showed that cellular RNA synthesis was at least 100 times more sensitive to these drugs than was plasmid DNA synthesis.When actively growing cells (i.e. cells not treated with chloramphenicol) were treated with a high concentration of rifampicin (250 μg/ml), chromosomal DNA synthesis continued to an extent representing about a 50% increase in DNA, while plasmid DNA synthesis appeared to stop abruptly.