Søren Molin

Low-copy-number plasmid-cloning vectors amplifiable by derepression of an inserted foreign promoter

By insertion of a DNA fragment, containing the phage lambda pR promoter and the pM-promoted cI857 allele of the lambda repressor gene, in plasmid R1 upstream of the replication control genes, cloning vectors have been constructed which are present in one copy per chromosome at temperatures below 37 degrees C, and which display uncontrolled replication at 42 degrees C. Derivatives have been made which carry the R1 par region, stabilizing the plasmid at low temperature when grown in the absence of selection pressure. Cells harbouring these plasmids stop growing after 1-2 h incubation at 42 degrees C, and at this time 50% of the total DNA in the cells is plasmid DNA corresponding to more than 1000 plasmid molecules per cell. Concomitant with plasmid amplification at the high temperature, synthesis of plasmid-coded gene products is amplified, and these vectors can therefore be utilized for obtaining greatly enhanced yields of gene products that may be detrimental to the host cell when present in large amounts.

Partitioning of plasmid R1 in Escherichia coli,: I. Kinetics of loss of plasmid derivatives deleted of the par region

The stability of inheritance of plasmid R1drd-19 was tested. The copy number of the plasmid was determined in two different ways: As the ratio between covalently closed circular DNA and chromosomal DNA, and by quantitative determination of single-cell resistance to ampicillin. In the latter case, strains carrying the R1 ampicillin transposon Tn3 on prophage lambda was used as standard. The values were transformed to copy number per cell by using the Cooper-Helmstetter model for chromosome replication as well as by determination of chromosomal DNA per cell by the diphenylamine method. The copy number was found to be five to six per cell (or about four per newborn cell). Nevertheless, plasmid R1drd-19 was found to be completely stably inherited. This stability was shown not to be due to retransfer of the plasmid by the R1 conjugation system, since transfer-negative derivatives of the plasmid were also completely stably inherited. Smaller derivatives of plasmid R1drd-19 were found to be lost at a frequency of about 1.5% per cell generation. The copy-number control was not affected in these miniplasmids, since their copy numbers were the same as that of the full size plasmid. Quantitatively, the instability of the miniplasmids was in accord with random partitioning. It is, therefore, suggested that the plasmid R1drd-19 carries genetic information for partitioning (par) of plasmid copies at cell division, and that the par mechanism is distinct from the copy number control (cop) system. Finally, the par gene maps on the resistance transfer part of the plasmid, but far away from the origin of replication and the so-called basic replicon; this is in accord with the approximate location of the repB gene (Yoshikawa, 1974, J. Bacteriol., 118, 1123-1131).

Plasmids with temperature-dependent copy number for amplification of cloned genes and their products

Miniplasmids (pKN402 and pKN410) were isolated from runaway-replication mutants of plasmid R1. At 30 degrees C these miniplasmids are present in 20--50 copies per cell of Escherichia coli, whereas at temperatures above 35 degrees C the plasmids replicate without copy number control during 2--3 h. At the end of this period plasmid DNA amounts to about 75% of the total DNA. During the gene amplification, growth and protein synthesis continue at normal rate leading to a drastic amplification of plasmid gene products. Plasmids pKN402 (4.6 Md) and pKN410 (10 Md) have single restriction sites for restriction endonucleases EcoRI and HindIII; in addition plamid pKN410 has a single BamHI site and carries ampicillin resistance. The plasmids can therefore be used as cloning vectors. Several genes were cloned into these vectors using the EcoRI sites; chromosomal as well as plasmid-coded beta-lactamase was found to be amplified up to 400-fold after thermal induction of the runaway replication. Vectors of this temperature-dependent class will be useful in the production of large quantities of genes and gene products. These plasmids have lost their mobilization capacity. Runaway replication is lethal to the host bacteria in rich media. These two properties contribute to the safe use of the plasmids as cloning vehicles.

Control of replication of bacterial plasmids: genetics, molecular biology, and physiology of the plasmid R1 system

Plasmids are autonomously replicating DNA molecules that are present in defined copy numbers in bacteria. This number may for some plasmids be very low (2-5 per average cell). In order to be stably inherited, replication and partitioning of the plasmid have to be strictly controlled. Plasmids carry genetic information for both processes. In the present paper we summarize what is known about the replication control system of one low-copy-number plasmid, R1, belonging to the FII incompatibility group. We do so because the FII group seems to be one of the best understood examples with respect to genetics, molecular biology, and physiology of the replication control system. The paper is not a classical review, but rather an essay in which we discuss the aspects of replication control that we regard as being important.

The sites of action of the two copy number control functions of plasmid R1

SummaryTwo negatively acting functions-the CopA-RNA and the CopB protein-are involved in the control of replication of plasmid R1. They both act as inhibitors of expression of a gene, repA, which seems to be positively required for autonomous plasmid replication. Here we show that the two control functions act separately and independently. The CopB protein represses initiation of transcription of the repA gene, and its target site lies within a 60 base pair region containing the repA promoter. The CopA-RNA acts downstream of the repA promoter in the leader sequence containing the copA gene itself, preceding the repA structural gene. Measurements of RepA-β-galactosidase expression from wild-type and a copA mutant fusion hybrid in the presence of extra copies of the respective copA genes show that a point mutation affecting the activity of the CopA-RNA can also affect CopA target properties. It is therefore concluded that the target site for the CopA-RNA resides within the copA gene in a small region encoding the loop of a stem-loop structure in the CopA-RNA. In addition, the data indicate a direct nucleic acid-nucleic acid interaction as the basis for the CopA inhibitor activity.

Vertical dye-buoyant density gradients for rapid analysis and preparation of plasmid DNA

Abstract A rapid and easy method for the isolation of plasmid DNA, both in analytical and preparative scale, is described. Using dye-buoyant density-gradient centrifugation in a vertical rotor, separation of covalently closed, supercoiled plasmid DNA from relaxed circular and linear DNA is completed within 1 to 2 h.

Partitioning of plasmid R1 in Escherichia coli: II. Incompatibility properties of the partitioning system

Abstract A theoretical as well as an experimental study of the effect of the partitioning system on plasmid R1drd-19 incompatibility was performed. The theoretical numerical analysis (by computer) was based upon the following assumptions: (i) The partitioning (par) mechanism is independent of the replication (rep) and replication control (cop) mechanism. (ii) A par mutation causes random (binomial) distribution of plasmid copies between the daughter cells at cell division. (iii) In the par+ case, the plasmid copies are equipartitioned and selected randomly for partitioning. (iv) Selection of plasmid copies for replication is random. (v) Two different replication control systems were considered: Model 1 assumes that the plasmid copy number is set to exactly 2n before cell division, whereas in Model 2 exactly n copies are synthesized per cell per cell cycle. Numerical analysis was performed for the n values 2–8. The result was that in all cases (par+/par+, par+/par, par/par), steady states with respect to copy number distribution within the heteroplasmid population were rapidly (within five or six generations) established, giving constant loss rates. The rate of loss was slightly higher in the par/par case than in the other two. The two replication control models gave almost identical loss rates. In the par+/par case, the par+ plasmid had an advantage over the par plasmid. The experimental approach was to create heteroplasmid populations of Escherichia coli by introducing two genetically marked derivatives of plasmid R1drd-19 and then follow the reduction in the relative size of this population during exponential growth in LB medium. The loss rate was essentially the same in the par+/par+ and par+/par combination and slightly higher in the par/par case, suggesting that plasmid incompatibility mainly is caused by the replication and copy number control system. In the par+/par situation, the par+ plasmid had a pronounced advantage over the par plasmid. The par region of plasmid R1 (without the basic replicon) was cloned onto the vector pSF2124. A par (deletion) mutation was not complemented by par+. Plasmid pSF2124, which does not seem to carry a par system of its own, could use the R1 par system, adding to the conclusion that par is independent of rep and cop. Plasmids pSF2124 and R1drd-19 are completely compatible, whereas plasmid pSF2124 carrying the R1 par system and plasmid R1drd-19 showed a weak incompatibility although the copy numbers of the two plasmids were not affected in the heteroplasmid cells. Hence, the partitioning system causes incompatibility, but the effect is weak compared to that of the cop system. The result is consistent with some sort of assortment of plasmids before partitioning.

Isolation and characterization of new copy mutants of plasmid R1, and identification of a polypeptide involved in copy number control

SummarySite-specific deletions and insertions in the replication region of plasmid R1 have generated a new class of copy mutants that are present in the cell with 10–15-fold increased copy number. All mutations described inactivate a copy number control gene which is distinct from another cop inc gene that was identified previously (Molin and Nordström 1980). Insertion of the lac operon lacking the normal lac promoter has been used to determine the direction of transcription of this cop gene. The mutants may all be complemented by wild-type plasmid derivatives and are thus recessive. In incompatibility tests with wild-type R1 plasmids, these mutants are indistinguishable from the wild-type plasmid. It therefore seems that this cop function does not play an important role for the incompatibility function. A polypeptide, molecular weight 11,000, has been identified as being the product of this cop gene.

Replication control functions of plasmid R1 act as inhibitors of expression of a gene required for replication

SummaryConstruction of translational fusions betwen the repA gene of plasmid R 1 (required for replication) and the lacZ gene has allowed a quantitative analysis of expression of this gene. It is suggested that the replication of R 1 is controlled by two replication control functions acting as inhibitors of repA expression.

The nucleotide sequence of the replication control region of the resistance plasmid R1drd-19

SummaryThe region of plasmid R1 containing the replication control genes has been sequenced using the Maxam-Gilbert method. The nucleotide sequence of two small PstI restriction fragments (a total of about 1,000 base pairs) was determined for the wildtype R1 plasmid as well as for two different copy mutants. It was found that one copy mutant has a single base substitution in the fragment which was recently shown to harbor an important inc/cop gene (Molin and Nordström 1980). Furthermore, the sequence indicates the presence of a structural gene that codes for a polypeptide of size 10,500 daltons. Possible gene products predicted from the nucleotide sequences and their role in replication control are discussed.