Plasmid replication and maintenance in binary fissile microorganisms

Plasmid replication and maintenance in binary fissile microorganisms Andrew Dunn, Martin Day and Peter Randerson

The mechanisms of nonconjugative plasmid replication (in the single cell) and maintenance (at the population level) are of concern to the microbiologist and to the genetic engineer who wishes to ex- ploit their ability to express cloned genes. This article concentrates mainly on Escherichia coli as the host organism and examines the mechanisms by which both naturally occurring and genetically engineered plasmids persist in populations during periods of growth. Additional strategies to ensure high yields of recombinant product are briefly considered.

Plasmids are naturally occurring au- tonomously replicating circles of double stranded, extra-chromosomal DNA maintained in the cytoplasm of bacterial cells. These genetic elements are probably distributed throughout the bacterial kingdom [l]. They range in size from about one to several hundred kilobase pairs (kb) and are present at a regulated copy number of between one and several hundred per cell, depending on the plasmid and the host cell (table 1). Plasmids may contribute to the phenotype of the host organism (table 2) by conferring, for example, resistance to antibiotics or the ability to utilize novel substrates [I]. There are numerous types as judged by characteristics, such as replication mechanism and phenotypes. For their optimal genetic expression and maintenance they exist for most of the life cycle in a supercoiled state (figure 1). However, they are not always essential for cell via- bility and are therefore often found to be unstably maintained (lost or cured) within some populations of cells. Much of the current interest in the mechanisms of plasmid replication and maintenance is a direct result of their use as cloning vectors for the expression of a great variety of gene products and also because they provide useful models for the replication of chromosomal DNA. It is salutary to realize that our un-

Andrew Dunn, Ph.D.

Has just finished a postgraduate research pro- ject to model plasmid stabilitv.

Martin Day, Ph.D.

Has been studying aspects of plasmid stability, transfer and evolution for the last decade. His orincioal interest lies in establishina the contri- bution that plasmids make to the &olution of their individual bacterial hosts and the populations in which they exist.

Peter Randerson, Ph.D.

Is an ecologist with extensive experience in computer modelling.

Enclwvaur, New Beries, Volume 17, No. l,lBB3. 01-/93 s.lm + 0.00. 01993 Psrgsmon Fress Ltd. tinted in Great Britain.

derstanding is derived from relatively few plasmids and that the mechanisms of replication and maintenance as well as the phenotypes of many naturally occurring plasmids remain to be identified.

The host cell life cycle The life cycle of binary fissile microorganisms, such as Escherichia coli (E. coli) in- volves the processes of growth, by which an average individual doubles in volume, and division, whereby an average parent produces two daughters of equal size [2]. Consequently, there are three prerequisites for stable plasmid maintenance within a bacterial culture. First, replication must ensure a near constant plasmid number (in excess of two) at the point of each cell division. Second, each daughter cell must inherit at least one copy of the plasmid. Third, the host organism must not be at a competitive disadvantage within the population as a whole. Stable plasmids are therefore obligated to encode control mechanisms in order to ensure their persistence in the cell line. Since there is a metabolic cost to the host cell due to the presence of a plasmid, the loss of the plasmid, either spontaneously or by some posi- tive mechanism, will result in two populations of otherwise similar cells, one with and the other without a plasmid. Provided there is no selection for the plasmid phenotype(s), the population without the plasmid will be at a growth advantage due to energy savings and thus should overgrow the plasmid containing population. Figure 2 shows the relative effects on

the populations of plasmid-free and plasmid-containing cells under selection. Overgrowth by the plasmid-free strain would occur if there were an energy cost to maintaining the plasmid. For overgrowth to occur at least one cell in the population must have spontaneously lost its resident plasmid (become cured). As a consequence of energy benefit, the plasmid-free cell will grow faster and thus increase in population size (numbers) faster, that is, overgrow the plasmid-containing population. Thus this phenomenon requires plasmid instability to create plasmid-free cells. If selective pressure exists for a plasmid encoded phenotype then those cells maintaining the plasmid will be at a growth advantage and their relative numbers (proportion in the population) will rise due to overgrowth of the plasmid-bearing strain.

Plasmid replication The smallest section of plasmid DNA required for replication is known as the basic replicon. The replicon consists of sequences which specify the origin of replication and allow it to function in a regulated manner. The size of this region is dependent upon the complexity of the replication control mechanism and ranges from about 1 kb in many high copy number plasmids (greater than 20 per cell) to about 2-2.5 kb in low copy number types [3]. There are three functional units [4] common to every basic replicon. These are an origin (ori) from which DNA replication may proceed uni- or bi-directionally, a gene-encoding function essential for the

TABLE 1 COPY NUMBER AND SIZES OF SOME PLASMIDS

Plasmid Size (kb) Copy number

ColEl 6.4 15-20

F 100 l-2

RI 100 1-2

pTl81 4.4 20-25

RK2 60 4-7

R6K 38 15-30

21

TABLE 2 EXAMPLES OF PHENOTYPES ENCODED ON PLASMIDS AND THEIR SIZES

Phenotype

Catabolic

Resistance

Biosynthetic

Bacterial

interactions

Miscellaneous

Camphor

Cellulolysis

Nopalene

Sucrose

Urease

Ampicillin

Arsenate

Borate

Hexachlorophene

Mercury

Silver

Streptomycin

Antibiotics

Auxotrophy

Gas vacuoles

Nitrogen fixation

Bacteriocin

Enterotoxinogenicity

Haemolysin

Phage

Loss of motility

Restriction/modification

Transfer/conjugation

Symbiosis

Plasmid

CAM

NIC

Scr

RPl

R773

Rms163

pMG1

FP2

R300B

SCPl

Nif

ColEl

Ent

COIV

Pl

pUMS

RPI

F

Size (kb)

148

47-315

168

56

226

94

230

500

97

8.9

560

160

100-160

6.4

95

94

94

100

241

Host species

Pseudomonas

Acetobacter

Arthrobacter

Escherichia

Providencia

Pseudomonas

Escherichia

Pseudomonas

Pseudomonas

Pseudomonas

Enterobacter

Pseudomonas

Streptom yces

Erwinia

Halobacterium

Klebsiella

Escherichia

Escherichia

Escherichia

Escherichia

Salmonella

Escherichia

Escherichia

Rhizobium

Host range

Narrow

Narrow

Broad

Narrow

Narrow

Narrow

Narrow

Broad

Narrow

Narrow

Narrow

Narrow

Narrow

Broad

Narrow

Narrow

initiation of replication (rep) and one or more genes controlling the rate of initiation from the origin (cop). It should be noted that, although at least one origin is required, there are many examples of plasmids which contain more than this number. For example, the naturally occurring E. coEi plasmids F [5] and Pl [6] are known to replicate via at least three different independent origins.

The inhibitor dilution theorem [7] pro- vides the basis for all models of plasmid replication control during the bacterial cell cycle. This predicts the existence of an unstable, continually expressed, cytoplasmic agent (a cop gene product) which inhibits the activity of a replication initiator (a rep gene product). The cellular concentration of this inhibitor is therefore proportional to both the dosage of the cop gene(s) and its (their) level of expression and hence deter- mines the number of plasmids per unit volume of cytoplasm. On average, the number

Figure 1 An electron micrograph of plasmid R300B, showing supercoiled and relaxed forms of a small streptomycin-resistance plasmid.

.A---•- l -•

”

Figure 2 Relative effects of a plasmid on the growth rate of the host cell population, compared to a population of the same strain without the plasmid, in the absence and presence of selection pressure. A = growth rate of p+ > p-due to selection of plasmid phenotype: B = growth rate of p+ = p-; C = growth rate of p+ < p- due to the increased energy demand required by the presence of the plasmid.

of plasmids per new daughter cell immedi- ately after division is half that present in the parental cell. This means that as cell volume increases the plasmids initiate replication. Consequently dilution of the inhibitor, due to an increase in internal cell volume by growth, enables further rounds of replication to take place. The choice of the plasmid on which to initiate replication has been shown to occur on a random selection basis [8], whereby all origins in the cytoplasmic pool have an equal probability of initiating replication. In the steady-state condition, where new born cells inherit about half of the parent copy number, this model therefore predicts a constant cytoplasmic plasmid concentration (molecules per unit volume, although the numbers per cell will increase), throughout the cell cycle. Experimental evidence [9] shows this to be the case for a number of plasmids even though they have diverse copy number control mechanisms. Further evidence for the existence of a cytoplasmic agent is provided by the phenomenon of replication-mediated incompatibility, where two or more different (in terms of their phenotype) coresident plasmids are not stably maintained as a result of sharing a common feature of replication control [lo]. Because each plasmid type interferes with the replication of the other it means that at cell division daughter cells have the correct total number of plasmids. But as their individual copy numbers are reduced by half, the cell has a lowered probability of acquiring both plasmids. Thus because of their relatively lowered copy numbers there will be a tendency for the plasmids to segregate independently.

Probably the best understood, and possi- bly one of the simpler basic replicons, is that of the ColEl plasmid ([ 1 I], (figure 3)

which is maintained at a copy number of about 40-60 per average cell in E.coli [12]. In this case, two cop (RNA I and rop) and one rep (RNA II) gene products physi- cally interact to control the rate of initiation of replication at the origin (ori). Replication of the plasmid is preceded by the synthesis of these three gene products (RNA I, RNA II and the rop protein). The primer transcript RNA II is initiated from a promoter Pr2 located 555 base pairs away, upstream of the origin, with respect to the direction of DNA replication, and termi- nates a short distance downstream of it at a point where DNA synthesis takes over. The transcript RNA I, a 108-nucleotide strand with a half life 0.55 min, is initiated at promoter PC1 located 445 base pairs upstream of ori and proceeds in the opposite

direction (away from the origin) and uses the opposite DNA strand, to RNA II synthesis, as the template. RNA II is produced from promoter PR and, as figure 3 shows, is complementary for part of its length with RNA I. One model proposes that RNA molecules interact to form a duplex and that the rom gene product enhances the rate of formation of this tertiary structure between the two RNA molecules. The hybrid formed by the two transcripts is un- able to initiate replication at the origin. The 66 amino acid polypeptide termed rop, which has a long half life of 69 min, and which increases the affinity of RNA I, for RNA II, thus regulates the rate of replication. Only if RNA I (half life 0.55 min) or rom are in short supply, when the copy number is low, is RNA II able to complete its synthesis and DNA replication is then initiated. RNA II synthesis proceeds just past the origin and the RNA forms the primer for unidirectional DNA synthesis via DNA polymerases I and III. The DNA dependent polymerase takes over synthesis at the promoter Pd and the primer RNA is released by the action of RNAase H. Of those RNA II molecules which escape hy- bridization, about half go on to initiate replication [ 111.

Plasmid segregation at cell division For plasmids which are distributed randomly throughout the cytoplasm of the host cell, the probability (PO) that a dividing cell containing n plasmid molecules will give rise to a plasmid-free daughter is given by the zero class of the binomial distribution [13]. If cell division is exactly symmetrical, then we may write: P, = 2l-“. This relationship predicts that high copy number plasmids (n > 20 per dividing cell) may well rely upon random segregation at cell division and yet be stably maintained in the population (figure 4). Deviation from this predicted ‘ideal behaviour’ means that this is often not the case. The most significant source of deviation

I - - - - IDNA PT2 P’

1 RNA II RNA rom

I

L t 0

rop protein

Figure 3 The spatial organization of the genes involved in the initiation of ColEl replication. The thick arrows denote the direction of the expression from the promoters for RNA (Prf and the origin of DNA (Pd) synthesis. The filled ellipse is the repressor protein (rap).

23

Parental cell Daughter cells Plasmld number

I-LIIII l

ccl-F-ICI 2

1

0.5

I 30 2 x 10-g

Figure 4 Plasmid segregation at cell division. The probability that all plasmids in the parental cell assort into one of the two daughter cells leaving the second daughter cell plasmid free (a cured cell) is dependant upon plasmid number. Probabilities are given by the zero class of the binomial distribution, that is, random segregation is assumed.

can be attributed to the formation of con- catenanes (circular multimers joined together like links in a chain) and concatamers (figure 5). Concatamer formation occurs when several plasmids become covalently linked in a tandem array (repeated in a single ring). It occurs as a result of recombination across the common sequence of the homologous plasmid molecules in the cell. Recombination is a normal cellular function acting on relatively long repeated DNA sequences and is enzy- mologically controlled. It allows for the in- version or removal of common DNA sequences from within a plasmid or for their exchange when they occur in two separate DNA molecules (that is plasmid and chromosome). In both instances each cluster, containing a number of individual plasmid molecules, is effectively reduced to the one molecule which is free to segregate randomly into either daughter at cell division. The replication mechanism is un- able to correct for this because it titres (measures) only the number of origins and not the number of independent plasmid molecules. Thus concatamers and cocate-

nanes both assort as one unit, despite the number of plasmids associated together and it is easy to see the effect that this re- duction in segregating units has on their relative stability (loss) at cell division. Many plasmids have evolved and encode sites which are highly site specific for their recombinational reversal (resolution) from the multimer forms. The specificity is derived from the presence of unique DNA sequences in the cointegrated plasmid molecules which are recognized by a plasmid or chromosomally encoded enzyme. The presence of two or more of these sites greatly increases the rate of recombination (dissociation) between duplicated sites in the mummer and hence promotes (drives) the resolution of the plasmid complex to its monomeric components. The cell does have a normal recombination enzyme but its specificity/activity for this reaction is so low that its efficiency is several orders of magnitude lower than that of a site-specific enzyme system. For example, the ColEl plasmid encodes the cer recombination site (a small DNA sequence) which is recognized by (therefore requires) a specific

multimer reversible recombinational reaction monomers

concatemer

0 ‘( o+o+o+o+o

monomer

concatenane

IQJ ’ ’ o+o+o+o+o

Figure 5 The relationship between concatanenes, concatemers and monomers.

24

host-mediated recombinase enzyme known as Xer [ 141. The normal recombination enzyme rarely recognizes this site and thus does separate the plasmid molecules with an efficiency which allows efficient assort- ment at cell division. A similar system is used by the plasmid Pl to resolve multimers formed in its synthesis, except that the recombination site (lox) and the recombinase enzyme (Cre) are plasmid encoded WI.

Further deviation from ideal behaviour arises because the replication mechanism cannot ensure that every dividing cell contains exactly n copies of the plasmid. Although most new-born cells inherit half the parent copy number (n), the binomial distribution, implying random segregation, predicts that a significant proportion will contain numbers other than this. It may therefore take several generations of cell growth before the equilibrium concentration is re-established, resulting in a distribution of plasmid numbers at less than (hence in some more than) and equal to the ‘average’ copy number at cell division which will tend to increase the rate at which segregants appear, especially in cases where copy numbers are low (table 3). This distribution is further widened by the cell division process, since individual members of the population do not all di- vide at exactly the same volume or into exactly equal halves [ 161. Unfortunately, current cytometric techniques are not sensitive enough to measure accurately the distribution of plasmid DNA in the cell population [17], and so in this respect we must rely upon the predictions of mathe- matical models.

It is clear in the case of low copy number plasmids that a random segregation would predict a high spontaneous loss (curing rate) and thus it alone cannot pos- sibly meet the requirements for stable maintenance. If a large plasmid (> 100 kb) had a copy number of over 25 then this would increase the genome size by 65 per cent and significantly increase the metabolic load to the cell. Hence some sort of

TABLE 3 THE RELATIONSHIP BETWEEN COPY NUMBER AND THE PROBABILITY OF RANDOM SEGREGATION (SPONTANEOUS LOSS) OF A PLASMID AT CELL DIVISION

Plasmid copy Probable curing number frequency

5 6x10-*

10 2x103

15 6xlod

20 2x10-3

25 6~10~

30 2x10-9

35 6 x IO-”

40 2 x 1o-‘2

TABLE 4 EXAMPLES OF SOME PRODUCTS SYNTHESISED FROM CLONING VECTORS

Host expression system Protein

E. coli Human insulin

E. co/i Somatotrophin

E. co/i Antitrypsin

E. coli Interleukin-2

Mammalian cells Factor VIII

Mammalian cells Erythropoietin

Clinical symptoms Comments

Diabetes Two peptides 26 and 30 amino acids long

Dwarfism 191 amino acids

Emphasemia 394 amino acids

Cancer therapy, renal failure 133 amino acids

Haemophilia 2332 amino acids

Anaemia 166 amino acids, glycosylated

active partition mechanism is necessary for larger plasmids to offset such penalising effects [ 181. Several stability functions (designated par, sop or stb) have been characterized in the E. coli plasmids F, Pl and Rl. In each case, the partitioning (par) region contains open reading frames for two proteins, one of which binds to a cis- acting site within the parDNA sequence. Although the mode of action has not yet been elucidated, theoretical models [ 18, 191 are similar in nature to the proposed mechanism for chromosomal segregation in E. coli [20]. These models predict that plasmid molecules exist freely in the cytoplasm during most of the cell cycle. During cell division, however, plasmid copies bind to each other in pairs and become attached to sites on a particular host structure which pulls or pushes each member of the pair towards opposing poles of the dividing cell. It is not known whether there might be an unlimited number of such binding sites in the host cell (the equipartition model), or just one site (the exclusive-or pair-site model). Possible candidates for the location of the host binding sites are the cell membrane [21] or the chromosome [5].

A function implicated in partition is also found in plasmid pSClO1 [22] which is stably maintained at a copy number of about 15 per dividing cell in E. coli [23]. An 81 base pair section of DNA, having the potential to form a hairpin loop structure, appears to be the only requirement for partition activity [23] and no gene products are thought to be expressed [24]. Although the par sequence of pSClO1 is able to sta- bilize the unrelated and normally unstable replicon pACYC184 [22], it does not pro- mote stable inheritance when joined to minichromosomes in E. coli [25]. More recent work shows that the hairpin loop structure is a specific binding site for DNA gyrase, the enzyme which introduces su- percoils into DNA, and so the segregational abnormalities (spontaneous changes in copy number) of the par mutant could be due to a defect in DNA topology. In the light of such evidence [19], it has been suggested that it may act by assisting the replication mechanism to narrow the distribution of copy number in dividing cells. Similar stability functions have also been described in the Staphylococcus plasmid

pT181 [26] and in several Bacillus subtilis plasmids, for example pC194 [27].

Mechanism preventing survival of plasmid-free segregants In addition to the par function described above, plasmid Rl also specifies a maintenance determinant known as the hok-sok system [28]. The hok gene product is relatively stable messenger RNA coding for a protein which is toxic to the host cell, whereas sok encodes an unstable, antisense RNA molecule which blocks translation of the hok mRNA. In those cells which do not inherit the plasmid, the sok RNA quickly decays, allowing translation of the toxic protein. The ccd mechanism of plasmid F also works on a similar poison and anti- dote principle, although it is not clear whether this acts by killing plasmid-free cells [29] or by delaying cell division in cells with too few plasmid copies [30]. An analogous system is found in the Co1 plasmids [31], except in this case the toxic agent (colicin) is released into the medium supporting cell growth.

The host cell growth rate Plasmids are dependent upon the host-mediated supply of functional components normally associated with chromosomal replication and gene expression. Such functions include the DNA and RNA polymerase enzymes, transfer RNA and ribo- somes. Consequently plasmids are often found to reduce the growth rate of the host cell, especially in those cases where the plasmid is very large [32], at high copy number or is expressing large amounts of a particular gene product [33]. Plasmid-free cells, which arise at cell division due to unequal partition (see previous section) or structural instability of the plasmid DNA [34], may therefore be at a competitive advantage and quickly displace the plasmid-

bearing cell fraction of the population. The effect of cellular growth rate differ-

ence is of particular importance to the genetic engineer. Table 4 shows some of the heterologous (novel cloned gene) products synthesized in fermentations. To maximize the expression of plasmid-encoded genes by use of strong transcriptional promoters requires them to be repressed during cell growth to produce biomass. This is because strong promoters will direct cellular resources into product, reducing the over- all cell biomass; thus optimum product yield will not be achieved. A possible solution to this problem is to select positively for plasmid-bearing cells (and thus against plasmid-free ones), often by use of an antibiotic to which the plasmid confers resistance. However, this method has been shown to be inefficient since the mechanism of antibiotic resistance itself imposes a significant metabolic load on the cell [35]. A more satisfactory solution for the biologist/engineer running the fermenta- tion is to regulate the expression rate of the cloned gene using an externally controllable promoter (table 5), such as hpL which uses a temperature shift to induce/repress expression [36]. The opera- tional strategy used to obtain gene expression in genetically engineered organisms, is to establish a large plasmid-bearing population in which the expression of the cloned gene is in the repressed condition. Once the optimal cell biomass (population) is reached, the transcriptional promoter is derepressed and expression is induced from the cloned gene. The gene dosage can also be controlled by using a plasmid which is temperature-sensitive for replication; shifting the host cells to a permissive temperature allows the plasmid copy number to rise. This in turn results in more gene copies of the cloned gene and pro- vides for a higher potential level of expres-

tABLE 5 SOME CONTROLLABLE PROMOTERS FOR THE EXPRESSION OF CLONED GENES

Promoter

LPL

lac

phoA

bla

Induction/expression

>37C

IPTG

Excess phosphate

B-lactam

Repression

30°C

None

Low phosphate

None

25

sion and hence yield of product. The other examples of specifically inducible (hence controllable) promoters are Provided by lac (induced by the lactose analogue, a gratuitous inducer isopropylthiogalacto- side), phoA induced by excess phosphate and bla by p-lactams. Gratuitous inducers can be of advantage as they are not substrates for the enzyme system they induce and thus maintain its expression. In other instances, for example with the p-lactams, the inducer is continually degraded by the enzyme. Hence the level of expression falls coincidently with the removal of the inducer.

Thus plasmid replication and maintenance systems are not simple, but show a coordination which implies a selective process over a considerable evolutionary period for efficient host/plasmid interactions. From an analysis of relatively few well-established bacterial plasmids it is ap- parent that various mechanisms have arisen for plasmid persistence during cell growth and in their respective populations. It indicates that parallel evolution of several highly varied biological mechanisms has occurred to answer the demand for stability. It remains to be seen whether these are representative of the general mechanisms in bacterial systems, or whether further studies will reveal plasmids from diverse microbial groups which provide other novel mechanisms.

Acknowledgement Much of the work was done under the aegis of a University of Wales studentship.

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[W

1111

[121

[I31

[I41

[I51

[I61

1171

WI

[I91

PO1

WI

PI

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~241

LW

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P81

PI

[301

[311

[321

[331

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[35] Bentley, W. E., Mirjalili, N., Andersen, D. C., Davis, R. H. and Kompala, D. S. Plasmid encoded protein: the principal factor in the ‘metabolic burden’ associated with recombinant bacteria. Biorechnol. Bioengng, 35,668-681, 1990.

[36] Betenbaugh. M. J., diPasquantonio, V. M. and Dhurjati, P. Growth kinetics of Escherichia coli containing temperature- sensitive plasmid pOU140. Biorechnol. Bioengng, 24, 1164-l 172,1987. [23] Tucker, W. T., Miller, C. A. and Cohen, S.

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Plasmid replication and maintenance in binary fissile microorganisms