Control Of Plasmid Replication Research Articles

Control of plasmid replication: theoretical considerations and practical solutions.

The life cycle of bacterial plasmids involves 2 different processes, replication (rep) and partitioning (par). Both these processes have to be controlled in order to secure stable inheritance of a plasmid in a growing population of bacteria. The crucial genetic information for (control of) replication (21) and partitioning (31,41) is present on the plasmids themselves. The 2 processes seem to be independent of each other since deletion of thepar region of a plasmid does not affect the copy-number control although it results in unstable inheritance of the plasmid (41). Furthermore, thepar region of 1 plasmid can be replaced by that of another, giving totally stable inheritance and the same copy number as that of the initial replicon (42). A third system adds to the stability of inheritance of plasmids, theccd (control ofcelldivision) system that inhibits cell division in cells containing only 1 copy of the plasmid (45).KeywordsPassive ControlPlasmid CopyControl CurveChromosome ReplicationCopy MutantThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Incompatibility and IncFII plasmid replication control.

The DNA coding for replication control and incompatibility of the plasmid NR1 serves as a template in vivo and in vitro for RNA transcription in both directions. In the rightward direction, RNA synthesis begins from 2 different promoters, one of which is regulated and the other constitutive. In vivo, each of these transcripts is more than 1,000 nucleotides long, terminating near the estimated site for the origin of replication. These transcripts serve as messenger RNA for several proteins. One protein (repA1) is required for replication and another (repA2) serves as the repressor for the regulated rightward promoter. RNA synthesis in the leftward direction is constitutive and produces a single transcript of 91 nucleotides which is complementary in sequence to the rightward transcripts. This small transcript is the incompatibility product which regulates the replication of the plasmid. When the intracellular concentration of the small transcript is experimentally varied, the rate of translation of the rightward transcripts and the rate of initiation of replication (plasmid copy number) vary inversely to its concentration. The mode of action of this inhibitor RNA is likely to be formation of an RNA-RNA duplex with the rightward transcripts, thereby inhibiting the translation which would produce the required replication protein. The probability that a rightward transcript will escape interaction with the small RNA molecules and thus allow replication to initiate can be predicted from the laws of mass action based on base-stacking free energies for the likely sequences of initial contact. The estimated intracellular RNA concentrations, based on quantitative hybridization experiments, are agreement with those predicted from the calculated equilibrium constants for duplex formation.

Interaction of small nuclear ribonucleoproteins with simian virus 40 in CV-1 cells: is U2 snRNA involved in regulating replication?

The small nuclear RNAs (snRNAs) in African Green Monkey kidney cells (CV-1 cells) were examined by polyacrylamide gel electrophoresis. Methodology was developed to improve their extraction from enriched fractions. Cellular fractionation studies and subsequent analysis of these RNAs indicate that they are tightly associated with chromatin. Treatment of cells with alpha-amanitin totally suppressed transcription of U1, U2, U4, U5, and partially suppressed transcription of U6, suggesting that these snRNAs are transcribed by RNA polymerase II. Upon infection of the cells by simian virus 40 (SV40), overall transcription of these and other cellular RNAs was stimulated. Gel filtration and formaldehyde crosslinking studies indicated that the ribonucleoproteins (snRNPs) containing snRNAs are associated with the viral minichromosome. Nucleotide sequence comparisons show extensive sequence complementarity between the 5' end of U2 RNA, the replication origin of SV40, and a prokaryotic RNA (RNA I) that is involved in control of plasmid replication. The clustered homologies between these RNAs and the association of snRNAs with the SV40 chromosome suggest that snRNAs may be evolutionarily related to small RNAs from plasmids and are consistent with an hypothesis that U2 RNA may be involved in DNA replication.

Control of ColE1 plasmid replication: the process of binding of RNA I to the primer transcript.

A transcript (RNA II) from the ColE1 primer promoter forms a persistent hybrid with the template DNA near the replication origin. The hybridized RNA II is cleaved by RNAase H to form the primer. Binding of RNA I to RNA II inhibits the hybridization and thus prevents primer formation. The binding is demonstrated by identification of the product. Analyses of the product with RNAase T1 revealed that pairing initiates at or near the 5' end of RNA I and propagates toward its 3' end. Removal of five nucleotides from the 5' end of RNA I greatly reduces its binding activity. Prior to initiation of pairing, RNA I interacts with RNA II at their single-stranded loops. This interaction is transient but facilitates complete pairing along the entire length of RNA I that initiates from the 5'-end region.

The repA2 gene of the plasmid R100.1 encodes a represser of plasmid replication

We have constructed two miniplasmids, derived from the resistance plasmid R100.1. In one of these plasmids 400 bp of R100.1 DNA have been replaced by DNA from the transposon Tn1000 (gamma-delta). This substitution removes the amino-terminal end of the repA2 coding sequence of R100.1 and results in an increased copy number of the plasmid carrying the substitution. The copy number of the substituted plasmid is reduced to normal levels in the presence of R100.1. The repA2 gene thus encodes a trans-acting repressor function involved in the control of plasmid replication.

Control of plasmid replication: a synthesis occasioned by the recent EMBO Workshop "Replication of Prokaryotic DNA," held at de Eemhof, The Netherlands, May 1982 (organizers: Veltkamp and Weisbeek).

Control of plasmid replication: a synthesis occasioned by the recent EMBO Workshop "Replication of Prokaryotic DNA," held at de Eemhof, The Netherlands, May 1982 (organizers: Veltkamp and Weisbeek).

Plasmid replication functions. VII. Electron microscopic localization of RNA polymerase binding sites in the replication control region of plasmid R6-5.

RNA polymerase binding sites on the R6-5 miniplasmid derivative, plasmid pKT401, were mapped by electron microscopy of DNA:RNA polymerase complexes formed with both circular-supercoiled and restriction endonuclease-linearized plasmid DNA molecules. Of eight specific binding sites on pKT401 that were identified, three were found to be in the P-6 fragment of the plasmid replication region, three in the Tn3 element, and two in other parts of the plasmid molecule. Binding sites 1 and 3 in the P-6 fragment are most probably the promoters of the copB and copA/incA plasmid replication control genes, respectively, whereas site 2 in this fragment appears to be the promoter of the essential replication gene, repA. The location of these promoters in relation to the site of action of the plasmid replication control elements, copT, and the origin of replication, oriV, suggests that replication control may be effected by regulation of transcription events initiated at site 2, or of the activity of transcripts initiated from this site, i.e., by regulation of the expression of the repA gene or another function dependent upon these events.

Control of plasmid R6K copy numbers in isogenic rep+ and rep Escherichia coli strains.

We have analysed as a function of cell doubling times the control of R6K plasmid replication in rep+ and rep strains of Escherichia coli. The rep mutation results in an alteration or loss of an enzyme that unwinds helical DNA. We found in rep+ bacteria that R6K relative dosage (plasmids per genome equivalent) remained nearly constant as growth rates increased. From this we concluded that the average plasmid concentration (plasmids per unit cell mass or volume) fell relative to the average concentration of chromosome origins when growth rates increased. In this context, the control of R6K replication is similar to that of other plasmids as seen by different workers. We also found that the relative dosage of R6K in rep mutants is greater than in rep+ bacteria when both strains were grown at fast growth rates. This finding was expected since at fast growth rates the number of genome equivalents per unit mass is expected to be lower in rep mutants. Unexpectedly, however, we found the effect of the rep mutation on R6K relative dosage had occurred in a step-like manner at a slow growth rate of about 120 min per generation. This implies that both the relative dosage and concentration of R6K had increased in a step-like manner. We also found that the effect of the rep mutation on R6K concentration was lost at fast growth rates while the effect of the mutation on R6K relative dosage was not lost.

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.

F plasmid incompatibility and copy number genes: Their map locations and interactions

The genes coding for vegetative F plasmid replication, replication control, and incompatibility are known to map between the kilobase coordinates 40.3 and 49.3 (abbreviated 40.3–49.3F). We have subdivided this region of the F genome by a combination of in vivo and in vitro genetic techniques and have constructed F:pSC101 hybrid plasmids which contain the F DNA sequences having the approximate coordinates 41–43, 43–46, and 46–49F. We find that hybrids with regions 43–46 and 46–49F are incompatible with an F′ lac + plasmid while the hybrid with the region 41–43F is compatible. We have also constructed similar F:pSC101 hybrid plasmids with the regions 43–46 and 46–49F derived from mini-F plasmid copy number mutants. We find that hybrids made from three independent F copy number mutants show a loss of the incompatibility function associated with the 43–46F region and retention of the incompatibility function associated with 46–49F region. Moreover, spontaneous revertants, selected for regain of the 43–46F incompatibility function, have also regained normal control over their copy numbers. We also find that copy number mutations map in the 43–46F region. From our results we conclude (i) that F contains at least two inc + loci, designated incA + (46–49F) and incB + (43–46F), and (ii) that gene(s) regulating F copy number may be related to the incB + gene(s).

Plasmid incompatibility: cloning analysis of an incFII determinant of R6-5.

SalI and PstI restriction endonuclease-generated DNA fragments that specify an FII-type incompatibility function (incFII) of the low copy number antibiotic resistance plasmid R6-5 have been cloned in the high copy number pBR322 plasmid vector. A 1-kilobase DNA sequence that contains this incFII determinant has been identified and is shown to have coordinates of 95.5 and 96.5 kilobases on the R6-5 plasmid physical map. Expression of incompatibility by the cloned PstI fragment depends on its orientation within the vector molecule. The behaviour of pBR322-incFII hybrid plasmids suggests that plasmid replication control is not the only mechanism that can cause incompatibility between two plasmids.

Properties of two conjugative plasmids mediating tetracycline resistance, raffinose catabolism and hydrogen sulfide production in Escherichia coli

The tetracycline resistance (Tc) and raffi nose-hydrogen sulfide (Raf-Hys) characters of Escherichia coli D1021 are located on two compatible, conjugative fi− plasmids named pRSDI and pRSD2, respectively. These were transferred together or separately to the isogenic background of E. coli K12 and isolated as transconjugants. Plasmid molecules isolated as covalently closed circular DNA have been analysed by buoyant density centrifugation, sucrose gradient sedimentation and electron microscopy. pRSDI (Tc) showed a buoyant density of 1.716 g/cm3 (56% GC), the open circular (OC) form had a sedimentation coefficient of 37s. If grown without tetracycline prior to isolation the contour length of most pRSDI molecules was 14.6 (±0.1) μm (30.3 × 106 daltons) with a minor species of 13.9 (± 0.1) μm owing to a small deletion. Pronounced length variation of pRSDI by growth in the presence of tetracycline owing to gene amplification is subject of a subsequent paper. In contrast, pRSD2 (Raf-Hys) molecules are stable under all conditions tested. The buoyant density was 1.713 g/cm3 (53% GC), the sedimentation coefficient of OGDNA was 58s and the contour length was 40.2 ± 0.6 μm (83 × 106 daltons). During exponential growth one copy of pRSD2 per chromosome was found indicating stringent control of plasmid replication. At the onset of stationary growth the copy number rose from one to four per host chromosome indicating relaxation of the replication control. The level of plasmid-coded α-galactosidase increases with copy numbers and has been used to test the gene dosage.

Indepencence of plasmid incompatibility and replication control functions in Staphylococcus aureus.

THERE are two basic requirements for the stable maintenance of an autonomous plasmid replicon, namely replication at a constant rate with respect to the cell division cycle and distribution of replicas to daughter cells.

Control of plasmid replication in Escherichia coli: correlation of the membrane site of DNA replication with the bacterial segregation unit.

The fate of parental lambda genomes after superinfection of homoimmune lysogenic cells was studied. The data confirm a previous observation that in the presence of the lambda repressor, superinfecting lambda DNA does not become associated with replication sites on the bacterial cell membrane. Under these conditions, the nonreplicating, superinfecting phage genomes do not become associated with the bacterial segregation unit. These results support the concept that the attachment of DNA to the bacterial membrane at specific sites is involved in the control of both chromosome replication and segregation, as predicted by the replicon hypothesis.