The kinetics of plasmid loss

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The kinetics of plasmid loss David K. Summers Instability of bacterial cloning vectors can present a serious problem when direct selection for plasmid-encoded phenotypes is undesirable, ineffective or impractical. Antibiotic selection may provide a satisfactory solution in enclosed fermentors but not where recombinant organisms are part of complex microbial consortia after release into the outside environment. In the past d e c ~ . ~ e r e has been significant progress towards understanding the caus,ss of plasmid loss and the iessons learned from these studies can be used in the design of a new generation of stable vectors.

Plasmid loss is evidence of either structural or segregational instability; either can mean a reduction in productivity for the biotechnologist. Structural instability involves the rearrangement or loss of plasmid DNA sequences (assodated typically with transposition or recombination) and will not be considered here. In this review, I concentrate on the causes of segregational instability, which arises from a failure to distribute plasmids to both daughters at cell division and is affected by multiple factors induding host and plasmid genotype, medium composition and growth rate.

Mechanisms ofplasmid inheritance For low-copy-number replicons, it seems inevitable that some or all plasmid molecules must be partitioned actively (Fig. 1, b and c). In the absence of active partitioning, plasmids are assumed to diffuse freely throughout the cytoplasm of the mother cell and to have an equal chance of entering either daughter. This is the basis of the randomdistribution model (Fig. la). A decade of research into the mechanism of plasmid inheritance has left little doubt that active partitioning is universal in occurrence among k)w-copy-number plasmids. Despite extensive speculation, however, there is no conclusive evidence for active partition of natural high-copy-number plasmids or the vectors derived from them, and it seems likely that their behaviour is best described by the random-distribution model.

Modelling the kinetics ofplasmid loss For randomly distributed plasmids, the probability per generation of forming a plasmid-free daughter is a function of the copy number at cell division (Box la; Fig. 2). Assuming that the molar concentration of plasmid DNA remains constant throughout the cell cycle, the copy number of dividing cells will be 1.4 times greater than experimentally determined values which represent the •mean of all cells in the population.

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Diagrammatic representation of plasmid fates at cell division. (a) Plasmids may be distributed randomly. If distribution is non-random, active partitioning may regulate the distribution of (13)a subset of plasmids or (c) all plasmids between the daughter cells. The horizontal line represents the plane of cell division,

Box 1. Modelling plasmid loss a Calculating the rate of plasmid loss The probabilitythat a plasmid molecule will not enter a specified daughter cell is 0.5. If there are n plasmids in the dividing cell, the probability that none of them will enter this daughter is (0.5)r'. Since each division produces two daughter cells, the probability (Po)that one of them will not receive any plasmids (i.e. the segregationfrequency} is given by: PO = 2(0.5) n = 2(1-n)

b How dimer distribution affects plasmid loss (1) Clonal dimer distribution If 5%of cells contain only dimers (n=20) and 95% contain only monomers (n=40) then: Po(cto.al) = 0.05 (21-20) + 0.95 (21-40)= "0.05 (21-~°) = 9.5 X 10-8 (2) Evendimerdistribution If each cell contains one dimer and 38 monomers (n=39) then: PO(even) ---- 21-39 "-

3.6 x 10 -12

po(clonai)/PO(even) 2.6 x 104 =

D. K. Summers is at the Department of Genetics, Dow~lhlg Street, Cambridge CB2 3E~, UK. ~) 1991, BsevierSciencePublishemLtd (LIK) 0167- 9430/91/$2.00

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If there is the same chance of forming a plasmid-free cell at o .o -6 each division, we would expect 10 plasmid loss to follow first-order kinetics. Figure 3 shows the rates g ,ool/ of plasmid loss predicted by this 0 10 20 30 40 simple model. Randomly distriCopy number (n) buted low-copy-number plasmids would be very unstable but Rgure 2 The relationship between dividing-cell cells with more than 20 plasmids plasmid cow number (n) and the would produce a plasmid-free frequency per generation at which daughter in less than one in a plasmid-free cells arise (Po).The theor- million cell divisions; a frequency etical basis of the relationship for which is undetectable under randomly-distributed plasmids is given normal circumstances. We might therefore expect all plasmids with in Box 1. copy numbers greater than 20 to be stable, but although this is true for naturally occurring piasmids it is not the case for the majority of cloning vectors.

The effectofplasmid load The metabolic stress which a plasmid imposes is likely to reduce the growth rate of its host. This includes the sequestration of cellular resources for plasmid replication, transcription and translation and, sometimes, the toxicity of cloned gene products. The load which a plasmid imposes may vary greatly with changing environmental conditions (e.g. growth phase, nutrient availability, etc.) and is likely to be most severe with vectors such as the pUC family which have been engineered to have

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copy numbers many times greater than their naturally occurring ancestors. Although plasmid load has no effect on the rate at which plasmid-free cells arise, it can cause a dramatic increase in the rate at which they accumulate in cultured cells. Figure 4 shows the effect of load on the kinetics ofplasmid loss (Ref. 1 describes the theoretical basis of this analysis). After an initial period of slow accumulation, plasmid-free cells rapidly take over the culture. The sudden disappearance of plasmid-bearing cells (known as washout) is a common problem in chemostat culture. In serial batch culture (where cells are repeatedly grown to stationary phase and then diluted into fresh medium), the severity of phsmid load may vary with growth phase and factors such as cell density and viability in stationary phase may also be affected by the presence of a plasmid. Analysis of a series of pBR322-derived plasmids in serial batch culture2 showed that, although plasmidfree and plasmid-bearing strains had identical growth rates in exponential phase, the plasmid-free strain reached a higher final cell density. Viability in stationary phase was also reduced, particularly if the plasmid carried a tetracycline-resistance gene. These factors have a similar effect on the kinetics of plasmid loss as a difference in growth rate during exponential phase. How can the effects of load on plasmid loss be minimized? One way to avoid the consequences of excessive plasmid copy number is to use a vector which replicates at low copy number but can be induced to high copy number when required. One such vector 3 contains the pSC101 origin and a modified ColEI replication origin at which synthesis of the replication primer is repressed by the thermolabile bacteriophage lambda repressor (c1857). At 30°C, the plasmid replicates from the pSC101 origin and is maintained at approximately four copies per chromosome. At temperatures above 38°C, however, the c1857 repressor is inactive and replication from the ColE1 origin increases the copy number to over 200 per chromosome. If the main causes of plasmid load are the transcription and translation of cloned genes, it may be advantageous to delay their expression until the culture has reached a relatively high cell density. A wide range of vectors is available in which cloned genes are expressed from promoters activated either by temperature shift or by the addition of an inducer such as isopropylthiogalactoside (IPTG) (for a review of suitable vectors, see Ref. 4).

Generations Rgure 3 Kinetics of loss of randomly distributed plasmids (n copies per dividirg cell). When plasmid-containing and plasmid-free cells grow at the 8art~., rate. plasmid loss follows first-order kinetics and a constant proportior, (2i~- hi) of the remaining plasmid-containing cells is lost each generation. Under these conditions, plasmids with a copy number of 20 or more should be extremely stable. TIBTECHAUGUST199] (VOL9)

Factors which accelerate plasmid loss How far do the factors described above account for the instability of many high-copy-number plasmids? When plasmid load is taken into account, the kinetics ofplasmid loss (Fig. 4) are qualitatively similar to results obtained with plasmids such as pUC8 and pBR322, but our model is based upon a copy number (n) of only 20. This is undoubtedly

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Rgure 4 Kinetics of loss of randomly-distributedplasmids (n=20) when plasmidfree cells outgrow their plasmid-containingcounterparts. R is the ratio of generation times of plasmid-containing to plasrnid-free cells. Severe competitionmay mean that a copy number of 20 is insufficientto ensure stable maintenance.

bution of plasmids between new-born cells is corrected before the next cell division. Although the molecular basis of copy-number control is understood for an increasing number of plasmids, it is still unclear, in most cases, precisely how the number of replication events per generation is related to the copy number of the new-born cell (in other words, we understand the molecular biology, but not the physiology of replication). Although it is difficult to quantify the importance of copy-number variance on plasmid loss, modification of wild-type control functions to increase copy number is likely to compromise the efficiency of control and increase copy-number variance. The consequent detrimental effect upon stability could counteract any improvement in stability expected to accompany an increased mean copy number. Readthrough from strong promoters introduced during the construction of cloning vectors is another cause of instability through interference with the control of replication s.

Oligomerformation

Another factor which increases the rate at which plasmid-free cells arise is the formation of plasmid multimers by homologous recombination6. Cloning vectors such as pUC8, pBR322 and too low for die majority of natural high copy pACYC184 which are stable in recombinationplasmids and their derivatives. If we use n=40 (still deficient backgrounds (recF or recA) are less stable in a considerable underestimate for pUC8 and related rec+ hosts. Increased oligomer formation in recD vectors), there is no significant plasmid loss after mutants correlates with increased instability of 100 generations, even with a 25% growth advan- pSC101 and ColEl-like plasmids 7. Extreme intage for plasmid-free cells. Although plasmid load stability is seen in sbcA backgrounds which have a is undoubtedly important in influencing the ki- high occurrence of plasmid recombination and netics of plasmid loss, it is important to remember that it does not affect the rate at which new plasmid-free cells arise. We are forced to condude that plasmid-free cells arise at a significantly higher Po=2Xl0 -12 frequency than that predicted by a simple random distribution model.

Copynumbervariance The main determinant of the rate at which plasmid-free cells arise is the number of plasmid molecules in the dividing cell. Experimentally determined copy numbers may be misleading because they are mean values and copy number may vary among individual dividing cells in the population. Figure 5 shows hypothetical copynumber distributions for two plasmids with mean copy numbers of 40. ~ i e plasmid with low-copynumber variance will be extremely stable, but the plasmid with the high variance will be less stable because plasmid-free cells arise from the low-copynumber end of the distribution at relatively high frequency. Instab~ty is likely if only a few percent of dividing cells have a copy number much less than 20, especially if the segregants outgrow plasmidbearing cells. Copy-number variance depends, among other things, on the success with which uneven distri-

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20

40

60

80

Dividing-cell copy number Rgure 5 The effect of copy-numbervariance on the rate of plasmid loss. In a population with high copy number vadance (open curve), plasmid-freecells arise most frequentiy from the subpopulation of low-copy-number cell~ Low-copy-number cells are absent from a low variance population (hatched curve).

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Generations Rgure 6 Instability of plasmid pBR322 caused by oligomer formation. (a) The plasmid is most stable in recF (n), less stable in rec + (B) and least stable in an sbcA host (o). (b) Instability correlates with an increasing proportion of multimers (lane 1, recF; lane 2, rec+; lane 3, sbcA). Supercoiled monomer (mon) and dimer (dim) bands are indicated. Relaxed monomers run just ahead of dimer supercoils.

maintain plasmid DNA as a ladder of oligomeric forms (Fig. 6). The proportion ofplasmid oligomers in rec + cells is very variable. The cryptic plasmid p15A forms less than 10% oligomers whereas pACYCI84 (which contains the replication origin of pl5A) can be up to 95% oligomerics. pACYC184 and other high-oligomer plasmids appear to contain DNA sequences which stimulate recombination by the RecF pathway. There is a correlation between insert size and oligomer formation in pBR322 derivatives9 but it is not altogether clear whether plasmid size itself is important or whether larger inserts are simply more likely to contain recombinogenic sequences. From a practical point of view, one message is clear; cloning vectors (especially those containing large inserts) should be maintained as monomers in recombinationdeficient strains. In general, recF strains are preferable to recA as they have higher viability and their chromosomal recombination functions are unimpaired.

Oligomen havereducedcopFnumber Plasmid oligomers are less stable than monomers because oligomers are maintained at a lower copy number. There is evidence that the replication control systems of most high-copy-number plasmids regulate the number oforigins rather than the number of independent plasmid molecules; dimers of three different pBR322 derivatives are maintained at 45%, 50% and 67% ofthe monomer copy number 2. Furthermore, the reduced copy number of oligomers is due to the increase in the number of origins rather than increased size; a series of pUC8 derivatives containing tandem repeats of the origin region show a progressive drop in copy number as the number of origins increases6. The effect of TIBTECHAUGUST]991 (VOL9)

oligomerization upon stability can be inferred from Fig. 2; reducing the copy number from 40 to 20 leads to a nfillionfold increase in the frequency at which plasmid-free cells arise. In wild-type cells, only 5% of pBR322 DNA is dimeric and at first sight the detrimental effect this has upon stability is rather surprising (Fig. 6). If the dimers are distributed evenly throughout the population, each cell would contain 38 monomers and one dimer; a total of 39 plasmid molecules as opposed to 40 in a monomer-only cell. This increases P0 only twofold and is insufficient to account for the difference in stability between rec + and reef hosts. A possible explanation is that dimers may not be distributed evenly. Box lb shows a comparison of the frequency of plasmid loss from a culture in which 5% dimers are distributed evenly with one which consists of 5% dimer--only cells and 95% monomer-only cells. The total dimer content of both cultures is identical, but plasmids are lost more than 10 4 times faster from the culture containing dimer-only cells. Stability functions ofnatural high-copy-number plasmids

In the absence of convincing evidence to the contrary, we have assumed that natm'ally occurring high-copy-number plasmids such as ColE1 are distributed randomly at cell division. Despite this, they are extremely stable, even in strains with a high occurrence of plasmid recombination. The instability of the majority of common cloning vectors (which were derived from these stable p!asmids) implies that functions which influence copy number at cell division (and hence the rate of plasmid loss) may have been lost or inactivated during vector construction. Oligomer

resolutionfunctions

The stability of ColE1 and other natural highcopy-number plasmids is independent of the recombination proficiency of the host. However, deletion derivatives of ColE1 which lack a 240 bp cis-acting site (cer) behave like the majority of cloning vectors; they form oligomers and are unstable in recombination-proficient strains, cer is a substrate for intramolccular site-specific recombination6 and oligomers ofcer + plasmids formed by homologous recombination are converted rapidly to monomers by recombination between directly repeated cer sites. Insertion of cer into unstable vectors such as pAT153 simultaneously prevents the accumulation of oligomers and restores stability6. Oligomer resolution sites are widespread and related sites have been identified in many high copy plasmids including CIoDF13 t°, pMB1, ColK, CoIN and ColA. An unusual feature of these systems is that all proteins involved in recombination are encoded by the host chromosome; mutations in argR II , p e p A 12 and x e r C 13 completely abolish oligomer resolution.

277

reviews Bacteriocins select again¢t plasmid,fr¢¢ cells Plasmid load causes plasmid-containing cells to grow more slowly than plasmid-free segregants, with an adverse effect upon stability. Sometimes, however, plasmid-free segregants grow more slowly and do not accumulate in the culture. Many natural high-copy-number plasmids encode colicins or related polypeptides which are released into the external environment and kill cells which do not produce the plasmid-encoded immunity protein 14. Although this has no effect upon the frequency at which plasmid-free cells arise, it prevents their accumulation and greatly increases stability. The widespread occurrence of colicin production in nature suggests that it may be an important factor promoting plasmid proliferation and maintenance in natural populations. Strategiesfor the constructionofstable cloningvectors What can our studies of natural high-copynumber plasmids teach us about the construction of stable cloning vectors? High copy-number variance is likely to be a cause of instability and attempts to increase copy number may often be at the root of the problem. Add to this the replication load imposed by excessive copy number and it is clear that the wise biotechnologist will keep the original control circuits intact and, if necessary, achieve high product yield by activating an inducible promoter as late as possible during the growth of the culture. The problems of oligomer formation in recombination-proficient hosts can be overcome by using a plasmid such as pKS450 ts, a derivative ofpUC9 containing the ColE1 multimer-resohtion site. Multimer-resolution systems represent a very good deal in molecular terms because the plasmid need carry only the small recombination site; the proteins required for multimer resolution are supplied by the host.

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Plane of cell division Rgure 7 Active partitioning the pre-pairing model. (a) Individual plasmids bind protein monomers. (b) DNA--protein complexes form dimers. (c) The dimeric complex binds a membrane recognition site in the plane of cell division. (d) After division, each daughter cell contains one member of the plasmid pair.

protoplasts, plasmids are not distributed randomly but may be sequestered in a cellular compartment analogous to the nucleoid TM. If this is true during normal growth, some form of active partitioning may be important even for high-copy-number plasmids in this organism.

Stability functions oflow-copy-number plasmids

Stable maintenance of low-copy-number plasmids requires the presence in cis of an active partitioning system. Current understanding of such systems is reviewed in Refs 19 and 20. The Plasmid loss in other bacteria Surprisingly little attention has been paid to the best-characterized partition loci are those of plascauses of plasmid instability in Gram-positive mid F and the Pl prophage. Their genetic organizorganisms such as Staphylococcus aureus and Bacillus ation is similar; each encodes two transacting subtilis. Progress has sometimes been hampered by proteins and a cis-acfmg site. One protein binds to an incomplete understanding of the mechanism of the plasmid site while the other may form part of a replication. Thus a supposed partition site from a membrane-recognition complex. cryptic Bacillus subtilis plasmid pLS1116 subsequently proved to contain the lagging-strand rep- The mechanismofactivepartition The detailed mechanism of active partkion is not lication origin. Although our understanding of factors affecting plasmid stability has come mostly clear but the pre-pairing model 19 provides a plausfrom studies of E. coli, the basic principles should ible hypothesis (Fig. 7). The interaction of a apply to randomly distributed plasmids in other monomeric protein with a specific site on the bacterial species. There is evidence that multicopy plasmid is followed by protein dimerization, thus plasmids may be distributed randomly in B. sub- pairing the associated plasmids. The dimeric comtilis 17; DNA fragments inserted into an E. coli/ plex binds in its turn .to a membrane site on the B. subtilis shuttle vector caused a dramatic reduction plane of cell division. Septum formation separates in both copy number and stability in the Gram- the plasmid pair and subsequent DNA replication positive host. In Staphylococcus aureus, however, releases the complex from the ~cell membrane. there is evidence that during the regeneration of Stable maintenance of Pl requires, in addition to TIBTECHAUGUST1991(VOL9)

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reviews the components of the partition apparatus, a site- cell. It is probably more practical to address the specific recombination system 21 which inter- problem at source by ensuring effective copyconverts plasmid monomers and dimers. In the number control and multimer resolution. absence of this function, plasmid dimers interfere with effective partitioning in about one percent of References cell divisions. 1 Boe, L., Gerdes, K. and Molin, S. (1987)J. Bacteriol. 169, 4646--4650 In contrast to the partition systems of most lowcopy-number plasmids, the 375 bp par region of 2 Chiang, C. S. and Bremer, H. (1988) Ptasmid 20, 207-220 E. M., Humphreys, G. O. and Yarranton, G. T. (1986) pSC10122 encodes no protein but does contain a 3 Wright, Gene 49, 311-321 binding site for DNA gyrase23. pSC101 derivatives 4 Thompson, R. (1988) in Methodsin Microbiology (Grinstead, J. and lacking par are unstable and have decreased negative Bennett, P. M., eds), pp. 179-204, Academic Press supercoiling. One possibility is that supercoiling is 5 Steuber, D. and Bujard, H. (1982) EMBOJ. 1, 1399-1404 necessary for the interaction of the plasmid with 6 Summers, D. K. and Sherratt, D.J. (1984) Cell 36, 1097-1103 host-encoded proteins involved in partitioning 24. 7 Biek, D. P. and Cohen, S. N. (1986)J. Bacteriol. 167, 594-603 Alternatively, it has been suggested2° that par may 8 James, A. A., Morrison, P. T. and Kolodner, R. (1983) Naalre 303, 256-259 be analogous to the crop locus of S. aureus plasmid 9 Berg, C. M., Liu, L., Coon, M., Strausbaugh, L. D., Gray, P., pT1812s which stimulates binding of the replication Vartak, N. B., Brown, M., Talbot, D. and Berg, D. E. (1989) initiator protein to the origin. Deletion of par might Piasmid 21, 138-141 then cause instability through loss of efficient copy- 10 Hakkaart, M. J. J., van den E!zen, P. J. M., Vdtkamp, E. and number control. It is known, however, that par Nijkamp, H. J. J. (1984) Cell ~ , 203-209 stabilizes wild-type plasmids at a copy number 11 Stirling, C. J., Szatmari, G., Stewart, G., Smith, M. C. M. and Sherratt, D.J. (1988) EMBOJ. 7, 4389-4395 which is insufficient to explain stability by random C. J., Colloms, S. D., Collins, J. F., Szatmari, G. and distribution, implying that some active partition 12 Stifling, Sherratt, D. J. (1989) EMBOJ. 8, 1623-1627 process must be involved. 13 CoUoms, S. D., Sykora, P., Szatmari, G. and Sherratt, D. J.

Host-killing systems Many low-copy-number plasmids encode functions which linfit the proliferation of plasmid-free cells which arise when dimer formation or abnormally low copy number prevent effective partitioning. The parB locus of plasmid R1 is an example of such a host-killing system. Cells which loose the plasmid are inviable, appearing as transparent 'ghosts' by phase-contrast microscopy 26. Genetic analysis has identified two genes (hok and sok) within parB. These genes are transcribed in opposite directions with a 128 bp overlap at their 5' ends. hok is responsible for host cell killing while sok encodes a trans-acting antagonist of hok gene activity. In plasmid-containing cells, translation of the Hok protein is repressed by the binding of the sok transcript to the 5' end ofhok mRNA, making the hok Shine-Dalgarno sequence inaccessible to ribosomes. In plasmid-free segregants, there is no further transcription of either hok or sok. hok mRNA is extremely stable (it has a half-life of--.20 min) and, as the concentration of the unstable sok transcript falls, Hok protein is translated and the cell dies 27. Host-killing functions are widespread among low-copy-number plasmids. RI carries a second host-killing region2a (parD), apparently unrelated toparB. Plasmid F also carries at least two host-killing systems; ccd29 and stm/flm 3°. What are the prospects of using stability functions from low-copy-number plasmids to stabilize cloning vectors? Active partition is essential for low-copy-number vectors but although it has been shown that an active partition cassette cloned into a high-copy-number vector does improve stability 3t, the size of the cassette means that a substantial extra replication load is imposed upon the host TIBTECHAUGUST1991(VOL9)

(1990)J. Bacteriol. 172, 69736980 14 Luria, S. E. and Suit, J. L. (1987) in Escherichia coli and Salmonella typhimurium Celbdar and Moleadar Biology (Neidhardt, F. C., ed,), pp. 1615-1624, American Society for

Microbiology 15 Summers, D. K. (1989) EMBOJ. 8, 309-315 16 Chang, S., Chang, S-¥. and Gray, O. (1987)J. Baaeriol. 169, 3952-3962 17 Bron, S. and Luxen, E. (1985) Plas,,id 14, 235-244 18 Gruss, A. and Novick, R. (1986)J. Baaeriol. 165, 878-883 19 Austin, S. J, (1988) Plasmid20, 1-9 20 Nordstrom, K. and Austin, S. J. (1989) Atom. Re,,. Genet. 23, 37-69 21 Austin, S., Ziese, M. and Steinberg, N. (1981) Cell 25, 729-736 22 Meacock, P. A, and Cohen, S. N, (1980) Cell 20, 529--542 23 Wahle, E. and Komberg, A. (1988) EMBOJ. 7, 1889-1895 24 Miller, C. A., Beaucage, S. L. and Cohen, S. N. (1990) Cell 62, 127-133 25 Gennaro, M. L. and Novick, R. P. (1988)J. Baaeriol. 170, 5709.-5717 26 Gerdes, K., Rasmussen, P. B. and Molin, S. (1986) Doc. Nail Acad. Sci. USA 83, 3116-3120 27 Gerdes, K., Helin, K., Christeusen, O. W. and Lobner-Olesen, A. (1988)J. Mol. Biol. 203, 119-129 211 Bravo, A., Ortega, S., de Torrontegui, G. and Diaz, R. (1988) Mol. Gen. Genet. 215, 146-151 29 Jaffe, A., Ogura, T. and Hiraga, S. (1985)J. Bacteriol. 163, 841-849 30 Golub, E. !. and Panzer, H. A. (1988) Mol. Gen. Genet. 214, 353-357 ;31 Austin, S., Friedman, S. and Ludtke, D. (1986)J. Bacteriol. 168, 1010--1013

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