Effects of recombinant plasmid size on cellular processes in Escherichia coli

PLASMID

18. 127-134 (1987)

Effects of Recombinant Plasmid Size on Cellular Processes in Escherichia co/i U. EONG CHEAH,* *Department

of Biology

WILLIAM and fChemica1

of Technology,

A. WEIGAND,~ Engineering IIT Center,

AND BENJAMIN

and Biotechnology Program. Chicago, Illinois 6Ob16

C. STARK*” Illinois

Institute

Received May 29, 1987; revised September 3, 1987 The effects of recombinant plasmid size on cell growth and viability, plasmid copy number, and synthesis of plasmidencoded protein were investigated in Escherichia co/i using plasmid pUC8 and four recombinant derivatives containing inserts of Drosophila melanogaster DNA of 1.7-6.0 kb. Growth in log phase was unaffected by plasmid size, but as plasmid size increased, maximum cell density decreased and, with the largest plasmid, cell death was accelerated after the stationary phase was reached. There was also a correlation between increasing plasmid size and decreased viability at high ampicillin concentrations, resistance to which is conferred by the plasmids. These effects were shown not to be due to transcription or translation of Drosophila sequences carried on the recombinant plasmids. Cells harboring the largest plasmid, pBS5 (8.7 kb), fared poorly in competition with plasmid-free cells in mixed cultures, compared with cells harboring pUC8 (2.7 kb). In addition, pBS5 was harbored at significantly fewer copies per cell than pUC8 at ah phases of growth and supported much less production of the plasmid-encoded protein, &lactamase, than did pUC8. The results suggest that recombinant plasmid size may be an important parameter in the optimization of large-scale production of plasmid-encoded proteins. 8 1987 Academic Press. Inc.

Plasmid vectors are indispensable in modem molecular genetic research, particularly for the large-scale production of DNA probes and relevant proteins in bacteria. This is refleeted in the many studies concerning the molecular biology of plasmid replication and stability, the effects of physiological conditions on plasmid copy number and stability, and mathematical models of the behavior of cells containing genes cloned into plasmid vectors (Scott, 1984; Imanaka and Aiba, 198 1; Ollis and Chang, 1982; Lee and Bailey, 1984; Ryder and DiBiasio, 1984). Among the physiological conditions which have been examined with regard to plasmid replication are composition of the growth medium (Engberg and Nordstrom, 1980; Jones et al., 1980; Wouters et al., 1980; Adams and Hatfield, 1984), phase in the growth cycle (Steuber and Bujard, 1982; Frey and Timmis, 198% dilution rate in a chemostat (Jones et al., 1980; ’ To whom correspondence should be addressed.

Wouters ef ul., 1980), and inhibition of protein synthesis (Clewell, 1972). The effects of plasmid size on cell growth and plasmid replication and stability have been examined, but systematic studies of this parameter are rare. The available evidence indicates that an increase in plasmid size may decrease the growth rate of host cells (Zund and Lebek, 1980) and lower the plasmid copy number (Hershfield el al., 1976; Gelfand ef al., 1978; Summers and Sherratt, 1984; Bron and Luxen, 1985); the most extensive study of the latter phenomenon is complicated by the presence of two replicons in and inherent instability of some of the plasmids used (Bron and Luxen, 1985). Because of this we chose to investigate the effects of increasing size of recombinant plasmids derived from the E. coli vector pUC8 on plasmid copy number and stability, cell growth and viability, and levels of production of plasmid-encoded protein (/3lactamase). Our work is aimed at providing both a better understanding of the physiology 127

0147-619X/87

$3.00

Copyright 0 1987 by Academic Press. Inc. All rights ~Creprcduaion in any form mrved.

128

CHEAH, WEIGAND.

AND STARK

TABLE I BACTERIAL~RAINSAND Strain

Plasmid

JMl03 Xf3 DBI BS4 DB2 BS5

None pUC8 pDB 1 pBS4 pDB2 pBS5

Plasmid size (kb)

PLASMIDS Restriction sites for insert

Insert transcribed in Drosophila”

2.7 4.4

Hind111

Yes

4.4 5.3 8.7

EcoRl, Hind111 BarnHI, Hind111 EcoRl

No Yes Yes

’ As determined by Bums et al. ( 1984).

of bacteria harboring recombinant plasmids and data required for the modeling of recombinant cell cultures for purposes of optimizing production of recombinant plasmid-encoded proteins under scale-up conditions. MATERIALS

AND METHODS

Bacterial strains and plasmids. The bacterial host strain used was Escherichia coli JM103 (Messing, 1983). The recombinant plasmids were derivatives of pUC8 (Messing, 1983), constructed for a previous study of Drosophila melanogaster ribosomal protein genes (Bums et al., 1984). The sizes of these plasmids varied as they contained differentsized Drosophila DNA inserts, but each contained one complete pUC8 sequence and thus conferred resistance to ampicillin. Details on the sizes and properties of the plasmids are presented in Table 1. Chemicals and enzymes. Ethidium bromide, sodium ampicillin, isopropyl thiogalactoside (IPTG),* penicillinase (Type 1) (EC 3.5.2.6), chicken egg-white lysozyme (EC 3.2.1.17), and pancreatic RNase (EC 3.1.27.5) were obtained from Sigma Chemical Co. (St. Louis, MO). Restriction enyzmes (EcoRI, HindIII, and BamHI) were purchased from Bethesda Research Laboratories (Gaithersburg, MD); tryptone and yeast ex* Abbreviations used: IPTG, isopropyl thiogalactoside; Ap, ampicillin.

tract were from Difco (Detroit, MI); agarose was from Bio-Rad (Richmond, CA); and sodium neutral cephalothin was from Eli Lilly (Indianapolis, IN). Media. Unless otherwise stated, cells were cultured in LB (Miller, 1972) containing 0.1 mg/ml sodium ampicillin (Ap-LB) for the inocula and in LB without ampicillin for the growth curves. IPTG was used at a final concentration of 1 mM. Plates were LB with or without 0.1 mg/ml sodium ampicillin as indicated. Growth curves. For each inoculum, an overnight Ap-LB culture was diluted 1:20 with fresh Ap-LB and grown for 1 h at 37°C. The cells were harvested by a 5-min centrifugation at 13,500g in a Fisher 235B microcentrifuge, washed twice in 37°C LB (followed each time by a 5-min centrifugation at 13,500g in a Fisher 235B microcentrifuge), and inoculated into fresh LB (100 ml for copy number experiments and 25 ml for all others) to a final density of 2 X 10’ cells per milliliter. In mixed culture experiments, the two strains were not mixed until the time of inoculation. Cultures were grown in a shaking water bath set at 37°C and 120 rpm. The numbers of viable cells and plasmid-bearing cells were determined by plating on LB and Ap-LB, respectively; the difference between them represented the number of plasmidfree cells (this was also used to differentiate between plasmid-bearing and plasmid-free cells in mixed culture experiments). All sam-

PLASMID

129

SIZE EFFECTS IN E. coli

ples were serially diluted with 0.85% NaCl and appropriate dilutions were plated in duplicate or triplicate. Mini-preparations of plasmids and plasmid copy-number determinations. For each preparation, about lo8 cells were harvested and mini-preparations of plasmids were performed as previously described (Holmes and Quigley, 198 1). Each plasmid preparation was cleaved with the appropriate restriction enzyme(s) to yield one vector and one insert fragment using conditions recommended by the supplier. All preparations were done in triplicate for each time point. The resulting samples were electrophoresed in 0.8 1.O% agarose gels along with standards of 10, 20, 50,100, and 200 ng of linearized pUC8. All gels were run at 20 V/cm using as buffer 100 mM Tris-base, 2 mM EDTA, brought to pH 8.0 with 85% phosphoric acid (Maniatis et al., 1982). They were stained with 250 rig/ml ethidium bromide for 30 min and destained in water for 30-60 min. The DNA bands were visualized under uv light (300 nm) and photographed using an orange filter and Type 55 Polaroid film. The negative was scanned on an EC9 10 densitometer (EC Apparatus, St. Petersburg, FL) at 540 nm to quantitate the amount of pUC8 in each standard lane as well as the amount of DNA in the pUC8 fragment of each sample lane. The area under the densitometric trace of each pUC8 standard was plotted against the amount of pUC8 loaded. The linear portion of this standard curve was used to determine the amount of pUC8 DNA present in each sample lane (D). The plasmid copy number (C) was calculated by the formula C = D/KV, where V is the number of viable cells in the sample from which the plasmid was isolated (determined by titering as described above) and K is 3.0 X IO-l2 pg, the mass of one copy of pUC8. This method is accurate within 68% down to 67 ng of DNA (Projan et al., 1983; Spangler et al., 1985). Large-scale preparation ofpUC8. pUC8 in JM 103 (our isolate denoted Xt3) was amplified by the use of chloramphenicol and isolated as previously described (Maniatis et al.,

1982). The concentration of the purified pUC8 was determined from its absorbance at 260 nm; a conversion factor of one absorbance unit to 50 &ml DNA was used. /3-Lactamase assay. @-Lactamase was released from cells by osmotic shock as previously described (Anraku and Hepple, 1967) and was assayed spectrophotometrically by measuring the rate of hydrolysis of the penicillin analog cephalothin, according to published procedures (CYCallaghan et al., 1968). RESULTS

Growth Curves During exponential growth in LB, plasmid-free JM 103, as well as the five plasmidbearing strains listed in Table 1, had a doubling time of about 19 min (Figs. la and 1b). Five hours after inoculation, however, cells bearing the larger plasmids (pDB2 (5.3 kb) and pBS5 (8.7 kb)) entered the stationary phase while the other strains remained in the late exponential phase (Fig. la). After 6 h growth, the six strains showed distinct differences in their cell densities, which persisted throughout the stationary phase. Overall, an increase in a plasmid’s size lowered the maximum cell density attained by its host (Fig. 1c); only strain BS4 deviated from this trend, growing more poorly than strain DB2, which contains a larger plasmid. At 18 h, all strains except BS5 had a viable cell count of 69-78% of the maximum viable cell count; BS5 (which contains the largest plasmid (8.7 kb) used in our study) had a viable cell count of 25% of maximum (Fig. la). In addition to titering by plating, the cell densities of 18-h cultures of Xt3 and BS5 were determined using a hemocytometer; these hemocytometer counts were approximately the same as the maximum viable cell counts achieved by these cultures but much higher than the 18-h viable cell counts. Thus, cell death in the post-growth period appears to occur without cell lysis. Despite the loss of cell viability, all viable cells of each of the five plasmid-bearing strains remained ampicillin resistant throughout the 18 h of growth. The

130

CHEAH, WEIGAND,

AND STARK

IO’O a

a--c lo'-

L. .Z ; "

;

-O...

--..

-*..

4-

ZZ $2

--..

_

.

t -

E

i .

.$

0 z

;: E I

IO6 0

2

I 4

I

I

I

I

1

I

6

a

10

12

1.4

16

1

D E

I’

-...-... *...-...

-...

2-

$

P 0

”

2

”

4

”

6

”

a

’

1 IO

18

hme ‘h

p&mid

size (kb)

FIG. 1. Effects of plasmid size on various growth parameters. (a) Growth of cells. Due to the similarities in doubling times, for clarity a single dotted line was used to represent all six strains between 1 and 5 h. All points are the means of two to eight independent trials, each done in duplicate or triplicate. (b) Exponential phase doubling time. (c) Maximum cell density. (Cl) JM103; (A) Xf3; (A) BS4; (0) DBl; (m) DB2; (0) BS5; maximum values were reached at 8, 10,6, 12,12, and 6 h of growth, respectively. Error bars in (b) and (c) denote range of values.

percentage of total cells that were ampicillin resistant as calculated from a total of 249 individual measurements (from 44 independent trials including all plasmid-bearing strains) had a mean of 99.4% and a standard deviation of 10.2%. Only once was plasmid loss observed; when a colony from a BS5 plate stored for four months at 4°C was inoculated into LB, the overnight culture was 40% plasmid free. IPTG Control Each recombinant plasmid used in our study contains a segment of Drosophila DNA

cloned into pUC8 downstream of 1acZp; this location provided the possibility that portions of the Drosophila DNA inserts could be transcribed and translated. Production of such Drosophila products could have impeded cell growth by diverting essential resources or decreased cell viability if Drosophila-encoded proteins were toxic to the host cells. This may have been of particular importance after the exponential phase (see below); at these times plasmid copy numbers (and thus 1acZo sequences) may have exceeded the number of lac repressors (even in the I4 background of JM103) and thus sequences downstream of 1acZp would have been induced.

PLASMID

131

SIZE EFFECTS IN E. coli

I b

012345

c

d

/, 12345

FIG. 2. Growth of plasmid-bearing strains in medium containing IPTG. (a) XD; (b) BS4; (c) DBl; (d) BS5. IPTG also had no effect on the growth of DB2 (not shown). (0) No IPTG, (0) with IPTG.

To test whether this may have been the reason for the deleterious effects of our recombinant plasmids, we compared growth of strains containing pUC8 and pDB1, pDB2, pBS4, and pBS5 in the presence and absence of IPTG (Fig. 2). IPTG, although inducing expression from 1acZp continuously after inoculation, had no effect on the growth of any of the strains containing a recombinant plasmid, but did inhibit the growth of JM103 containing pUC8, presumably due to overproduction of the portion of ,&galactosidase carried by this plasmid (the ,&galactosidase sequence is under the control of lacZp, but is disrupted by the Drosophila sequences in our recombinant plasmids). These results are exactly contrary to those expected if transcription or translation of Drosophila sequences impeded growth and/or viability.

being outgrown by JM103. In contrast, XD, which harbors the smaller pUC8, was not outgrown by JM103 unless it comprised a

Mixed Culture Experiments When an equal number of plasmid-bearing BS5 and plasmid-free JM 103 were inoculated together into growth medium (Fig. 3a), the shape of the growth curve for BS5 resembled the one obtained for a pure culture (Fig. la) but with accelerated stationary and death phases. A higher initial fraction of BS5 allowed it to attain a greater cell density (Fig. 3b) which delayed, but did not prevent, its

mm

Ihl

FIG. 3. Growth curves of mixed cultures. (a) BS5 and JM103; (b) BS5* and JM103; (c) XD and JM103*; (d) Xf3* and JM 103. * denotes strain with higher inoculum. (0) JM103, plasmid-free; (A) XD; (0) BSS.

132

CHEAH.

lesser fraction of the inoculum (Figs. 3c and 3d). P-Lactamase Production Copy Number

WEIGAND.

than JM103

and Plasmid

When strains Xf3, BS4. DB2, and BS5 were grown at 5 mg/ml ampicillin, there was an initial loss of viable cells followed by a recovery period until growth resumed at an ampicillin-free rate. There was a trend (although not as strong as the variation of maximum cell density with plasmid size) for both the loss of viability and the time until recovery to increase as plasmid size increased; this indicated that there may be an inverse relationship between plasmid size and (plasmidencoded) /I-lactamase production. To investigate this further plasmid copy number and /3-lactamase production were measured in strains Xf3 and BS5 under a variety of growth conditions. For both BS5 and Xf3, /?-lactamase levels were 20-30s higher in log-phase cells grown in 0.1 and 1 mg/ml ampicillin than in log-phase cells grown in the absence of ampicillin. In addition, Xf3 produced considerably more P-lactamase per cell than did BS5 at all three concentrations (3.3-4.4 units/IO” cells vs 0.76-0.92 units/ 10” cells). For growth in the absence of ampicillin, the disparity in P-lactamase production between Xf3 and BS5 occurred during both log and stationary phases and was paralleled by a similar disparity in plasmid copy numbers between the two strains (e.g., 420/ cell for Xf3 vs 250/&l for BS5 in log phase). Both strains, however, underwent dramatic increases in plasmid copy number beginning in late log phase, reaching at least 1400/tell for Xf3 and 600/tell for BS5 in stationary phase; in both cases this was accompanied by a gradual decrease (of about 50%) rather than an increase in @-lactamase production. DISCUSSION

The copy numbers we have measured for pUC8 and pBS5 are somewhat higher than those reported for at least one similar plasmid. pCAP 1, a pBR322 derivative, has been

AND

STARK

measured at 54 copies per cell when grown in broth and 330 copies per cell when grown under conditions of isoleucine starvation (Adams and Hatfield, 1984). Both pCAPl (Adams and Hatfield, 1984) and pUC8 lack the rop (Cesarini et al., 1982) (or rom (Tomizawa and Som, 1984)) function, which normally decreases copy number by 1.5-7~ (Twig and Sherratt, 1980). pBR322 has been measured for growth in broth at about 50 copies per cell in log phase and 14 I copies per cell in stationary phase with the rop function and about 85 copies per cell in log phase and 223 copies per cell in stationary phase without the rop function (Steuber and Bujard, 1982). These high copy numbers probably explain the high plasmid stability we see as it renders nearly impossible the generation of a plasmid-free segregant, even in the absence of an active partitioning mechanism. The increase in copy number of pUC8 and its derivative, pBS5, in late log and stationary phases is also consistent with results obtained with other plasmids (Frey and Timmis, 1985; Steuber and Bujard, 1982). It is likely, as has been suggested by others (Adams and Hatfield, 1984) that this increase is the result of lowered protein synthesis due to exhaustion of nutrients and thus mimics the plasmid amplification seen upon chloramphenicol addition. The inverse relationship between plasmid size and plasmid copy number is consistent with the results of others (Hershfield et al., 1976; Gelfand et al., 1978; Bron and Luxen, 1985). The decrease of maximum cell density with increasing plasmid size holds for all strains tested except BS4, which has a maximum cell density lower than would be expected for its size. The reason for this is as yet undetermined but it is not due to expression of Drosophila-encoded sequences by this strain (Fig. 2). These results are paralleled by the generally lower resistance to high ampicillin concentrations as plasmid size increases and the lowered plasmid copy number and /3-lactamase production of BS5 vs Xf3. In sum, they indicate that the metabolic cost of maintaining a large, high-copy-num-

PLASMID

133

SIZE EFFECTS IN E. coli

ber plasmid (BS5, for example, has approximately 2200 kb of plasmid DNA during log phase as opposed to about 1100 kb for Xf3) may place a generalized stress on the cell. This idea is supported by the mixed culture experiments depicted in Figs. 3a and 3b. At the time when BS5 cells can no longer multiply there are still enough nutrients in the culture medium to support significant growth of plasmid-free cells. The lowered /3lactamase production by BS5 compared with Xf3 may be due more to this generalized stress than the lowered copy number of pBS5 vs pUC8; the copy numbers of both plasmids may be above the point at which gene copy is linearly related to protein production (Dennis et al., 1985). Our results indicate that recombinant plasmid size may be of significant importance in the overall viability and production of plasmid-encoded protein by recombinant microorganisms. In addition, the results of the competition experiments indicate that plasmid-free segregants may have a large advantage over and may easily outgrow cells containing recombinant plasmids, particularly if these recombinant plasmids are large. Since continuous enzyme and antibiotic fermentations are operated under slow-growth conditions (Parulekar and Lim, 1985) (the point at which the plasmid size effect becomes most severe) this problem may be of particular importance in certain industrial applications. ACKNOWLEDGMENTS We thank Drs. D. Bums, S. Feinstein, and S. Parulekar, and C. Divencenzo for many helpful discussions. This work was supported by Public Health Service Grant 2 SO7 RR07027 from the National Institutes of Health and the Paul V. Galvin Venture Fund.

REFERENCES ADAMS, C. W., AND HATRELD, G. W. (1984). Effects of promoter strengths and growth conditions on copy number of transcription-fusion vecton. J. Eiol. Chem. 259.7398-7408.

ANRAKU, Y., AND HEPPLE, L. A. (1967). On the nature of the changes induced in Escherichia coli by osmotic shock. J. Biol. Chem. 242,2561-2569.

BRON, S., AND LUXEN, E. (1985). Segregational instability of pUBI derived recombinant plasmids in Bacillus subtilis.

Plasmid

14, 235-244.

BURNS, D. K., STARK, B. C., MACKLIN, M. D., AND CHOOI, W. Y. (1984). Isolation and characterization of cloned DNA sequences containing ribosomal protein genes of Drosophila melanogaster. Mol. Cell. Biol.

12, 2643-2652.

CESARINI, G., MUESING, M. A., AND POLISKY, B. (I 982). Control of ColEI DNA replication: The rop gene product negatively affects transcription from the replication primer promoter. Proc. Nafl. Acad. Sci. USA 79.63

13-63

17.

CLEWELL, D. B. (1972). Nature of ColE I plasmid replication in Escherichia coli. J. Bacreriol. 110,667-676. DENNIS, K., SRIENC, F., AND BAILEY, J. E. (I 985). Ampicillin effects on five recombinant Escherichia coli strains: Implications for selection pressure design. Biotechnol.

Bioeng.

27, 1490- 1494.

ENGBERG, B., AND NORDSTROM, K. (1980). Replication of R-factor RI in Escherichia coli K12 at different growth rates. J. Bacreriol. 123, I79- 186. FREY, J., AND TIMMIS, K. N.( 1985). ColDderived cloning vectors that autoamplify in the stationary phase of bacterial growth. Gene 35, 103-l 1 I. GELFAND, D. H., SHEPARD, H. M., O’FARRELL, P. H., AND POLISKY, B. (I 978). Isolation and characterization of a ColElderived plasmid copy-number mutant. Proc. Natl. Acad. Sci. USA 75, 5869-5873. HERSHRELD, V., BOYER, H. W., CHOW, L., AND HELINSKI, D. R. (1976). Characterization of a miniColE I plasmid. J. Bacferiol. 126, 447-453. HOLMES, D. S., AND QUIGLEY, M. ( I98 I). A rapid boiling method for the preparation of bacterial plasmids. Anal.

B&hem.

114, 193-197.

IMANAKA, T., AND AIBA, S. (I 98 1). A perspective on the application of genetic engineering: Stability of recombinant plasmids. Ann. N. Y. Acad. Sci. 369, I-14. JONES, I. M., PRIMROSE, S. B., ROBINSON, A., AND ELLWOOD, D. C. (1980). Maintenance of some ColEI-type plasmids in chemostat culture. Mol. Cm. Genet.

180, 579-584.

LEE, S. B., AND BAILEY, J. E. (1984). Analysis of growth rate effects on productivity of recombinant Escherichia coli populations using molecular mechanism models. Biotechnol. Bioeng. 26, 66-73. MANIATIS, T., FRITSCH, E. F., AND SAMBROOK, J. (1982). “Molecular Cloning: A Laboratory Manual,” p. 454. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. MESSING, J. (1983). New Ml3 vectors for cloning. In “Methods in Enzymology” (R. Wu, L. Grossman, and K. Moldave, Eds.), Vol. 101, pp. 20-78. Academic Press, New York. MILLER, J. H. (1972). “Experiments in Molecular Genetics,” p. 433. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Q’CALLAGHAN, C. H., MUGGLETON, P., AND Ross, G.

134

CHEAH, WEIGAND,

(1968). Effects of /3-lactamase from gram-negative organisms on cephalothins and penicillins. Antimicrob. Agents Chemother., 337-343. OLLIS, D. F., AND CHANG, H. (1982). Batch fermentation kinetics with (unstable) recombinant cultures. Biotechnol. Bioeng. 24,2583-2586. PARULEKAR, S. J., AND LIM, H. C. (1985). Modeling, optimization and control of semi-batch reactors. Adv. Biochem. EnginJBiotechnol. 32,207-258. PROJAN, S. J., CARLETON, S., AND NOVICK, R. P. (1983). Determination of plasmid copy number by fluorescence densitometry. PZasmid 9, 182-190. RYDER, D. F., AND DIBIASIO, D. (1984). An operational strategy for unstable recombinant DNA cultures. Biotechnol. Bioeng. 26, 942-947. SCOTT, J. R. (1984). Regulation of plasmid replication. Microbial. Rev. 48, l-23. SPANGLER, R., ZHANG, S., KRUEGER, J., AND ZVBAY, G. (1985). Colicin synthesis and cell death. J. Bacteriol. 163, 167-173. STEUBER, D., AND BUJARD, H. (1982). Transcription from efficient promoters can interfere with plasmid replication and diminish expression of plasmid specified genes. EMBO J. 11, 1399- 1404.

AND STARK SUMMERS, D. K., AND SHERRATT, D. J. (1984). Multimerization of high copy number plasmids causes instability: ColEl encodes a determinant essential for plasmid monomerization and stability. Cell 36, 1097-I 103. TOMIZAWA, J., AND SOM, T. (1984). Control of ColEl plasmid replication: Enhancement of binding of RNA I to the primer transcript by the rom protein. Cell 38, 871-878. TWIG, A. J., AND SHERRATT, D. (1980). Trans-complementable copy-number mutants of plasmid ColE 1. Nature (London) 283,2 16-2 18. WOUTERS, J. T. M., DRIEHUIS, F. L., POLACZEK, P. J., VAN OPPENRAAY, M. H. A., AND VAN ANDEL, J. G. (1980). Persistance of the pBR322 plasmid in Escherichia coli grown in chemostat cultures. Antonie van Leeuwenhoek 46,353-362. ZUND, P., AND LEBEK, G. ( 1980). Generation time prolonging R plasmids: Correlation between increases in the generation time of Escherichia coli caused by R plasmids and their molecular size. Plasmid 3,65-69. Communicated by Richard D. Kolodner