Segregation Kinetics of Colicinogenic Factor Col E1 from a Bacterial Population Temperature Sensitive fbr DNA Polymerase I

Molec. gen. Genet. 121, 71--75 (1973) © by Springer-Verlag 1973

Segregation Kinetics of Colicinogenic Factor Col E1 from a Bacterial Population Temperature Sensitive fbr DNA Polymerase I B a r b a r a W . D u r k a c z a n d D a v i d J. S h e r r a t t School of Biological Sciences, University of Sussex, Falmer, Brighton, England Received September 30, 1972 Summary. We have studied the segregation kinetics of the bacterial plasmid ColE1 from a population of cells temperature sensitive for DNA polymerase L The results indicate that there are about twelve plasmid copies per cell of the strain used and that segregation is probably random.

Introduction T h e b a c t e r i a l n o n - s e x f a c t o r p l a s m i d , colicinogenic f a c t o r ColE1, is n o r m a l l y i s o l a t e d f r o m E. coli as a c o v a l e n t l y closed circle of d u p l e x D N A of m o l e c u l a r weight 4 . 2 x 1 0 s d a l t o n s ( B a z a r a l a n d Helinski, 1968). ColE1 r e q u i r e s D N A p o l y m e r a s e I for its r e p l i c a t i o n a n d hence for its m a i n t e n a n c e in a p o p u l a t i o n of b a c t e r i a l cells ( K i n g s b u r y a n d Helinski, 1970). I s o t o p i c labelling e x p e r i m e n t s , followed b y i s o l a t i o n of circular p l a s m i d D N A b y v e l o c i t y or e q u i l i b r i u m centrifugation, h a v e i n d i c a t e d t h a t t h e r e are a b o u t t w e n t y four circular copies of t h e p l a s m i d p e r cell of a n e x p o n e n t i a l l y growing pol A + c u l t u r e (Clewell a n d Helinski, 1972). B y t h e use of a m u t a n t t e m p e r a t u r e sensitive for D N A p o l y m e r a s e I , i t has been possible to s t u d y t h e segregation kinetics of ColE1 f r o m a b a c t e r i a l p o p u l a t i o n a n d also to m a k e a n e s t i m a t e of t h e n u m b e r of p l a s m i d copies p e r cell. T h e results i n d i c a t e t h a t u n d e r t h e conditions uesd, t h e r e are on a v e r a g e t w e l v e copies of t h e p l a s m i d p e r cell, a n d t h a t segregations is p r o b a b l y r a n d o m .

Materials and Methods Bacteria. The strains were a gift from D. R. Helinski. E. coli K12 DS708 is a ColEl + E-r V-r (resistant to colicins E and V) derivative of JG108 (met E lac- rha- thy- str-r). DS624 is a met E + TOl Ats21a (temperature sensitive for DNA polymerase I, Kingsbury, 1971) derivative of DS708. E. coli K12 strain ¥$40 V-r (his- pro- str-r, resistant to V colicin) was used as an E colicin-sensitive indicator strain. Media and Methods. Liquid medium was L broth (Bactopeptone 1%, yeast extract 0.5%, NaC1 0.5% and glucose 0.1%) supplemented with 20[~g/ml thymine. Solid medium was AB3 nutrient agar (Difco), supplemented with 20 ~tg/ml thymine. Bacteria were grown at 33°C unless otherwise stated. E1 colicin production was detected as follows: cells were plated (100-200 colony forming units per plate) and grown overnight at 33° C. The resultant colonies were killed by five minutes exposure to chloroform vapour and the plates were overlaid with l0 s cells of the indicator strain suspended in water/agar (0.6%) at 46 ° C. Production of E1 colicin was detected by the presence of clear "haloes" around E1 producing clones. Cell density was measured from the absorbance at 650 nm in a Bausch-Lomb Speetronic 20.

72

B.W. Durkacz and D. J. Sherratt: Mathematical Analysis

Let hro = number of cells at zero time (i.e. time at which cells shifted to 42° C) M 0 ~ number of plasmids at zero time hrm = number of cells after m generations (at 42° C) M m ~ number of plasmids after m generations Pm ~ average number of plasmids per cell after m generations = M,./Nm

F m = fraction of cells with plasmids after m generations 1 -- e-P,~. Then Nm = No eklm (]¢1= l°ge2 for exponential growth of cells) M m = Me ek.m (/¢~= 0 when the plasmid does not replicate) Pm = Me/No e(k'--kDm

Fm ~ 1 -- e-M~/N°e(km-kl)m 1/(1 --Fro)~--e~0/Noe(k'--kDm log~loge [1/(1 --Fro)] = --(k 1 --ks) m + loge(Me~No), which is of the form y = m x T C , therefore a plot of log~oge[1/(1--Em) ] versus m will give a line of slope -- (kl -- ks) and an intercept on the ordinate of loge(M0/N0).

Results The ColE1 plasmid is maintained stably in strain DS624 grown in liquid culture for m a n y generations at 33 ° C: ~ 1/1000 of the cells in a given population has lost the plasmid. I n contrast, growth at 42 ° C results in a rapid loss of the plasmid from a population (Fig. 1). No segregants were observed until the third or fourth generation at 42 ° C, after which the proportion of plasmid free cells increased with time. To analyse the d a t a further t h e y were plotted as shown in Fig. 2. The slopes were drawn from least square fit values. The intercepts on the ordinate give an average value of plasmid copies per cell (Mo/21o) o f 12. The three values of k s were determined to be 0.20, 0.29 and 0.37; as these were significantly greater t h a n 0, then either there is residual synthesis of ColE1 at the restrictive temperature or some other p h e n o m e n o n is affecting the appearance of segregants. Isotopic labelling experiments (Kingsbury, 1971 and Sherratt, unpublished data) have shown t h a t residual ColE1 synthesis is less t h a n 5% of wild-type level after a shift to 42 ° C. W e therefore eonlude t h a t residual ColE1 synthesis is unlikely to be responsible for the observed k~ values. I t was observed t h a t within one generation after shifting to 42 ° C, DS624 had begun to filament extensively (both a non-eolieinogenic derivative of the same strain, and DS708 showed considerably less filamentation under the same conditions), though the generation time, measured b y absorbance increase, remained constant, indicating t h a t the filaments were remaining viable. W e were unable to make a quantitative estimate of filament size and frequency in the microscope, t h o u g h the n u m b e r of viable colony forming units per unit of absorbance dropped 2-3 fold during each of the experiments. The effect of filamentation would be to reduce the observed rate of segregation due to the colony forming unit changing from a single cell to a n u m b e r of cells; in fact k 2 m a y be considered as a measure of filamentation, the extreme cases being when there is no filamentation (]cu~- 0) and when there is no cell separation (/~ ----]cl----log~2). I t should be emphasized t h a t the value of M o / N o determined from Fig. 2 is independent of the value o f / ~ . I n isotopic labelling

Segregation Kinetics of Colicinogenie Factor ColE1

73

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Fig. 1. Kinetics of loss of the ColE1 plasmid from colicinogenic cells temperature sensitive for DNA polymerase I grown at 42 ° C. Bacteria were grown at 33 ° C to an absorbanee at 650 nm of 0.1 (equivalent to about 5 × 107 colony forming units/ml). They were then shifted t~ 42 ° C and grown for one generation, measured by absorbance increase. Samples were removed, plated and grown at 33 ° C and the rest of the culture diluted back down to the initial absorbance in pre-warmed medium and the culture grown for one more generation at 42 ° C. This protocol was followed for ten generations at 42 ° C. The bacteria were also examined microscopically in a counting chamber. At least 500 colonies were screened for coliein E1 production for each generation. (The results from three separate experiments are shown). ~ - - o DS708, o - - o DS624

1

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6

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m Fig. 2. Data in :Fig. 1 replotted to determine the number of plasmid copies per cell and the values for ks. Y =logeloge[1/1 --Fro) ] m ~ number of generations at 42 ° C . . . . Expt. 1, o - - o Expt. 2, , - - , Expt. 3, - . . . . . Theoretical slope when k 2 = 0

e x p e r i m e n t s (e.g. see Clewell a n d H e l i n s k i , 1972), w e h a v e s h o w n t h a t t h e cells of D S 6 2 4 g r o w n a t 3 3 ° C in L - b r o t h , c o n t a i n o n a v e r a g e b e t w e e n 10 a n d 17 copies of c i r c u l a r C o l E 1 p l a s m i d ( D u r k a c z a n d S h e r r a t t , u n p u b l i s h e d d a t a ) , in g o o d a g r e e m e n t w i t h t h e g e n e t i c d a t a p r e s e n t e d here.

74

B.W. I)urkacz and D. J. Sherratt: Discussion

Bacterial plasmids may be divided into two classes, depending on the average number of plasmid copies per cell. One class, represented by the sex factors, consists of plasmids which are present in small numbers per cell (Jacob, Brenner and Cuzin, 1963); to ensure inheritance of these plasmids to daughter cells some active segregation mechanism must exist. The other class, represented by the Cole plasmids, plasmid 15 (Lee and Davidson, 1970), /t dv (Matsubara and Kaiser, 1968) and the Salmonella pullorum plasmid 35 (Kline, 1972), consists of plasmids which exist in multiple copies (~10) per cell. Consequently their inheritance at high frequency by daughter cells may result from a random (stochastic) process. Our results indicate that the ColE1 plasmid may well be inherited b y such a process and moreover our estimate of the number of plasmid copies per cell indicates that random segregation would result in the generation of plasmid free cells at a low frequency (~e-12----6 × 10-e), which unfortunately is difficult to measure experimentally. The agreement between the genetic and biochemical estimates for the number of ColE1 molecules per cell indicates the validity of both of these methods for determining the ColE1 content of cells. In addition, if our interpretation of the segregation kinetics is correct, then we can also conclude that the population of colieinogenic cells is relatively homogenous with regard to plasmid content; if a significant fraction of the population contained a small number of plasmids, it would have been reflected by the appearance of segregants during the first two generations at 42 ° C. There are a number of obvious uses of this system. Firstly, it should facilitate the isolation of ColE1 mutants; because of the relatively large number of plasmids normally in a cell, mutation of a single plasmid will only be expressed when it is in a cell containing a homogeneous mutant population (unless the mutation is dominant). If segregation is random, the generation of such a cell from a cell containing a single mutant plasmid would not be expected to occur till between the seventh and twelfth generations when there are twelve plasmids in a cell (Haigh, personal communication), consequently reducing the mutant frequency by a factor of at least 8 X 10-3. Mutagenesis of a colicinogenic population temperature sensitive for DNA polymerase I, should allow one to easily achieve the situation in which there is a single plasmid per cell on average and consequently the m u t a n t frequency should be restored. Secondly, b y growing colicinogenie cells temperature sensitive for DNA polymerase I at temperatures intermediate between 33°C and 42°C it might be possible to obtain situations where there is a constant low number of plasmids per cell (e.g. 5). A study of the generation of plasmid free cells under these conditions might help test the hypothesis of random segregation of ColE1. The observation that colicinogenic cells filament extensively when ColE1 replication is inhibited agrees with other reports that inhibition of plasmid replication interferes with cell division (Tcrawaki, Kakizawa and Takayasu, 1968; Monk, 1967) and supports the idea that rephcation of all replicons in a cell may be coupled to cell division.

Segregation Kinetics of Colieinogenie Factor ColE1

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Acknowledgements. We wish to thank members of the microbial genetics unit for their valuable discussions during this work. B. W. Durkacz is supported by an M. R. C. studentship for training in research methods. Reterences Bazaral, M., Helinski, D. R.: Circular forms of colicinogenic factors El, E2 and E3 from Escherichia coll. J. molec. Biol. 36, 185-194 (1968). Clewell, D. B., Helinski, D. R. : Effect of growth conditions on the formation of the relaxation complex of supercoiled ColE1 deoxyribonucleic acid and protein in Escherichia coll. J. Bact. 110, 1135-1146 (1972). Jacob, F., Brenner, S., Cuzin, F. : On the regulation of DNA synthesis in bacteria. Cold Spr. Harb. Syrup. quant. Biol. 28, 329428 (1963). Kingsbury, D. T. : Isolation and characterization of mutants defective in plasmid replication. Ph.D. thesis, University of California, San Diego (1971). Kingsbury, D. T., Helinski, D. R.: DNA polymerase as a requirement for the maintenance of the bacterial plasmid colieinogenic factor El. Biochem. biophys. Res. Commun. 41, 1538-1544 (1970). Kline, B. C. : Inhibition of plasmid DNA replication by rifampicin in Salmonella pullorum. Biochem. biophys. Res. Commun. 46, 2019-2025 (1972). Lee, C. S., Davidson, N.: Physiochemieal studies on the minieircular DNA in Escherichia cvli 15. Bioehim. biophys. Aeta (Amst.) 204, 284-285 (1970). Matsubara, K., Kaiser, A. D. : ~ de: an autonomously replicating DNA fragment. Cold Spr. Harb. Syrup. quant. Biol. 33, 769-775 (1968). Monk, M. : Observations on the mechanism of indirect induction by mating with ultraviolet irradiated coll doners. Molec. gen. Genet. 100, 264-274 (1967). Terawaki, Y., Kakizawa, Y., Takayasu, H.: Temperature sensitivity of cell growth in F~scherichia coli associated with the temperature sensitive R(KM) factor. Nature (Lond.) 219, 284-285 (1968}. C o m m u n i c a t e d b y E. W i t k i n Barbara W. Durkacz Dr. David J. Sherratt School of Biological Sciences University of Sussex Falmer, Brighton BN1 9 QC England