The Thermosensitive Lesion in the Replication of the Drug Resistance Factor, Rts1

The Thermosensitive Lesion in the Replication of the Drug Resistance Factor, Rts1Author(s): C. G. DiJoseph and Akira KajiSource: Proceedings of the National Academy of Sciences of the United States of America,Vol. 71, No. 6 (Jun., 1974), pp. 2515-2519Published by: National Academy of SciencesStable URL: http://www.jstor.org/stable/63460 .

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Proc. Nat. Acad. Sci. USA Vol. 71, No. 6, pp. 2515-2519, June 1974

The Thermosensitive Lesion in thle Replic of the Drug Resistance Factor, Rtsl

(plasmid/circularization of DNA/curing/alkaline sucrose)

C. G. DiJOSEPH AND AKIRA KAJI

Department of Microbiology, School of Medicine, University of Pennsyl

Communicated by W. D. McElroy, March 29, 1974

ABSTRACT The DNA of the thermosensitive R factor, Rtsl, has been examined by the technique of sedimentation in alkaline sucrose density gradients. Rtsl DNA was found as closed covalent circles in only a few copies per cell in an Escherichia coli host at the permissive temperature. Rtsl DNA appears to be synthesized at the nonpermissive temperature, but was not found as closed covalent circles. However, circular DNA could be recovered upon shift down to the permissive temperature. The large number of plasmid-negative cells which accumulate after prolonged culture at non-permissive temperature may be due to a strong selective pressure favoring the growth of rare R- segregants.

R factors are extrachromosomal genetic elements which confer on their host resistance to one or more antibiotics. Like other plasmids, they consist of double-stranded DNA, at least a portion of which is in the form of closed covalent circles (1, 2). An R factor Rtsl, has been described which has a lower frequency of transfer of kanamycin resistance at nonpermissive temperature, and therefore appeared to be temperature sensitive for replication (3). This hypothesis received further support from the observation that the ratio of R DNA to total DNA decreased during growth of Proteus mirabilis Rtsl cells at 42? (4). In addition, Terawaki et al. (5) have shown that host cell growth is altered at nonpermissive temperature. We have extended this observation and found that nonviable cells accumulated during growth at 42? (6). In this communi- cation we report that the R factor, Rtsl, has a molecular weight of approximately 120 million, and synthesis of Rtsl DNA takes place even at the nonpermissive temperature, but this newly synthesized Rtsl DNA could not be isolated as closed covalent circular DNA. However, this DNA. could be recovered as closed covalent circles after shift down to the permissive temperature.

MATERIALS AND METHODS

Bacterial Strains and R Factors. The properties of the R factors and host strains, and the conditions for culture have been described previously (6).

Preparation of Lysates and Analysis for Closed Covalent Circular DNA. Lysates were prepared and analyzed by a modification of the procedure of Freifelder et al. (7). Cultures were quickly chilled to 0? and centrifuged at 5000 rpm for 5 mrain in the Sorvall SS-34 rotor. The pellet was washed once with a buffer containing 0.01 M K-phosphate (pH 7), 1 mM MgSO4, 0.1 mM CaCl2, and 0.1 M NaCl, and gently suspended

Abbreviation: MW, molecular weight.

25]

ation

rania, Philadelphia, Pa. 19174

to 108 to 109 cells per ml in lysis buffer containing 0.05 M NaCl, 0.02 M ethylenediaminetetraacetic acid (EDTA), and 0.02 M tris(hydroxymethyl)aminomethane (Tris)-HCI buffer, pH 9.1 at 0?. For temperature shift experiments, all cell samples were suspended in lysis buffer containing 0.1% diethylpyro- carbonate to inhibit nuclease activity. All operations were carried out at 00-5?. A 0.1-ml portion was added to a 1 cm X 7.5 cm round-bottom test tube containing 5 ,l of Antifoam A (Dow-Corning). A motor-driven microsyringe was used to deliver alkaline SDS (0.8 N NaOH, 1% sodium dodecyl sulfate) at a rate of 12 pl/min. The tube was kept in an ice bath and rocked gently by hand during addition. The sample was sheared by vortexing for 30 sec, and 0.1 ml was analyzed on a 5-ml sucrose .density gradient (5%-20%), containing 0.5 M NaCl, 0.02 M ethylenediaminetetraacetic acid, and 0.3 N NaOH. The gradients were centrifuged at 25? for 20 min at 40,000 rpm in a Spinco SW-50L rotor and fractionated from the bottom onto Whatman filter discs. The radioactivity in each fraction insoluble in cold (4?) trichloroacetic acid was determined as described (6).

RESULTS

Isolation and Characterization of Closed Covalent Circular Rtsl DNA. Host cells of Escherichia coli 20SO carrying either Rtsl alone or in combination with nonthermosensitive R factors were labeled with [3H]thymidine for several generations at 32? and lysates were analyzed by alkaline sucrose density gradient centrifugation. Fig. la shows that no fast- sedimenting material was present in lysates of plasmid- negative cells. Lysates of Rtsl cells, however, displayed a peak of radioactivity which sedimented much more rapidly than either R100 or R28K DNA (Fig. lb-e). This material is regarded as closed covalent circular Rtsl DNA (7).

The sedimentation rate of Rtsl DNA relative to either R100 or R28K DNA was used to estimate a molecular weight for Rtsl DNA of approximately 120 million (Table 1). This value was confirmed by electron microscopy of Rtsl DNA and by measurement of y-ray irradiation target size (8).

The number of copies of Rtsl DNA per chromosome can be estimated from R factors of known copy number. It is assumed that there was no major selective loss, since the copy numbers of either of the reference R factors (R28K, R100) cal- culated from these data corresponded with the values (9, 14) which were obtained under different experimental conditions, even though the molecular weight of R100 was approximately twice that of R28K. From these values we estimated that there were about 1.3 copies of Rtsl DNA per chromosome,

5



2516 Biochemistry: DiJoseph and Kaji

~~20~~1 2

I0 60

o 0

I 00

30

w' 0 25

0 0

(C)

20 5- 50

10 25

I 5 10 15 20 25 30 35 40 FRACTIOF

FIG. 1. Alkaline sucrose density gradient analysis of DNA fr< were inoculated into glucose minimal medium at 2 to 4 X 107 cells

(Schwarz/Mann, 6 Ci/mmole) and 250 ,g/ml of deoxyadenosine. A' described in Methods. Sedimentation is from right to left. The large of the host. The results are expressed as cpm per fraction versus fri Rtsl: R28K; (e) Rtsl. (0) and (0) refer to the scales on the left an

or about 2 copies per cell under these culture conditions (Table 1).

Replication of Rtsl DNA. Cells cultured in penicillin form long multinucleated filaments which form septa and divide within a short time after penicillin is removed (10). Division is not dependent on DNA synthesis after a round of replication has been completed (11). These observations permitted a test of the hypothesis that Rtsl DNA is thermosensitive for DNA replication. As shown in Table 2, Rtsl cells which had been precultured at 32? were shifted to 42? in the presence of penicillin. After 4 hr, the culture was diluted to reduce the penicillin concentration to a subinhibitory level, and shifted to 36? for an additional hour in the presence of nalidixic acid to prevent DNA synthesis (12). Samples were plated before and after the post-penicillin period and the colonies which developed were tested for the presence of Rtsl by replica plating to media with and without kanamycin, the antibiotic to which Rtsl confers resistance (3). A separate experiment had shown that no incorporation of [3H]thymnidine into R

DNA and only a small amount of incorporation into host DNA took place during the post-penicillin period. We would predict that if the replication and segregation of Rtsl DNA were temperature sensitive, some of the cells derived from a filament after septation would lack the R factor and would be detected as drug-sensitive clones. On the other hand, if Rtsl DNA was replicating and segregated along with the formation

Proc. Nat. Acad. Sci. USA 71 (1974)

(d)

8 - -200

6 10 -

4 100

2 - J 50

(e)

15 300

10- 1 200

5 -1o00

o 0 I 510 15 20 25 30 35 40

I NUMBER

im E. coli with and without R factors. Exponential phase cultures

per ml. The medium also contained 25-40 ,Ci/ml of [3H] thymidine 'ter 2.5-3 generations at 32?, lysates were prepared and analyzed as

peak on the right (note different scale) is sheared endogenous DNA action number. (a) E. coli 20SO R-; (b) Rtsl: RIOO; (c) RIOO; (d) i right, respectively.

of multiple host nuclei, all clones of cells derived from a filament should be resistant to kanamycin. Table 2 shows that, on the average, a filament formed four cells during the post- penicillin period, and that 100% of the clones from these cells were resistant to kanamycin. Since the number of copies of Rtsl DNA at the time of the shift to 42? was less than 4 (Table 1), we conclude that this factor is not temperature sensitive for DNA synthesis.

As a more direct test of this question, we attempted to iso- late closed circular Rtsl DNA from cells which had been cultured at 42?. In the experiment shown in Fig. 2, cultures of E. coli Rtsl grown at 32? were shifted to 42?. After 30 nmin, [3H]thymidine was added for an additional 30 min at 42? before chasing with a large excess of cold thymidine. Half of this culture was immediately analyzed on an alkaline sucrose gradient. Only a small peak of radioactivity was detected at the position of Rtsl closed circular DNA, which corresponded to only 0.09% of the radioactivity in the host peak.

When the cells in the remaining half of the culture labeled at 42? were pelleted to remove label and incubated at 36? for 90 mrain in a medium containing unlabeled thymidine, a peak of radioactivity was found at the position of closed circular Rtsl DNA (Fig. 2). The ratio of Rtsl DNA to host DNA in this experiment was 0.015. Separate experiments have shown that the ratio of closed circular R DNA to host DNA was always less at 36? or 42? than at 32?. Thus, most, if not all, of the Rtsl which could be isolated as closed covalent




TABLE 1. Quantitative analysis of labeled closed covalent circular R factor DNA after alkaline sucrose density

gradient centrifugation

Radioactivity Radioactivity ratio R copies copies

R (cpm*) DNA/ MW per per factor R host host DNA (million) genome cell

R100 7,135 293,704 0.026 66t 1.8? 2.9 9,094 262,055

R28K 11,899 572,380 0.020 44t 2.2? 3.6 Rtsl 9,865 293,704 0.033 120T 1.3? 2.1

18,852 572,308 34,885 1,044,585

* Total cpm in fast-sedimenting peaks (R DNA) or in slow- sedimenting major peak (host DNA).

t Average molecular weight cited for RiOO (14, 22) or R28K (9). : From the relationship, s20,w = 7.44 + 0.00243 MW?.5s, where

MW is molecular weight, (19) using the distance migrated relative to either RIOO or R28K. While the s values cited are for neutral sucrose, it is assumed that s values under alkaline conditions are increased but remain proportional to molecular weight.

? RIOO (14), R28K (9). ? Copies of Rtsl DNA/genome = (ratio Rtsl DNA to host

DNA/ratio reference DNA to host DNA) X (MW of reference DNA/MW of Rtsl DNA) X (copies per genome of reference DNA).

1 Copies per cell'= (copies per genome) X (genome equiv- alents, G, per cell), where G = 1.6 under these culture conditions (20, 21). The validity of this relationship was checked by predicting the copy number/genome of R100 based on the data of R28K and vice versa. The predicted copy number/genome was found to be 1.85 for R100 and 2.19 for R28K, in good agreement with the values reported (9, 14).

circles at 36? was obtained in this experiment. Since labeling was done at 42?, these results suggest that this material represent Rtsl DNA which had been synthesized at 42?.

Labeled closed circular Rtsl DNA could be isolated from cells cultured at 42? if these cells were labeled at 32? before shifting to 42? (Fig. 2). Thus the absence of closed circular Rtsl DNA at 42? was not an artifact of isolation peculiar to cells cultured at 42?, or due to an activation of endonuclease

TABLE 2. Kanamycin resistance of clones from p

0 min post-penicillin at 36?

Viable (VC) or Penicillin total (TC) count Clones resistanrt Nalii treatment (millions) to kanamycin ac

36? 4.37 (TC) 2.1 (VC) (41/41) 100% -

4 42? 4.5 (TC)

1.15 (VC) (50/50) 100% -

Cultures of E. coli 20S0 Rtsl were inoculated at 4.6 X 106 cells per lin G (E. Lilly). During 4 hr at 36? or 42? all cells were converted to fi the concentration of penicillin, and portions were incubated at 36? fom Winthrop). Microscopic observation showed that septation was corn before and after penicillin treatment by plating onto trypticase soy were tested for resistance to kanamycin by replica plating to MacCor

Rtsl DNA 2517

which may nick the closed covalent circular Rtsl DNA synthesized at 42?. This point was further strengthened by an experiment in which R DNA was examined in cells carrying both R100 and Rtsl. As shown in Fig. 3, R100 DNA was synthesized and recovered as closed circular DNA at 42? even in the simultaneous presence of Rtsl DNA, whereas Rtsl DNA labeled at 42? could not be isolated as covalent circular DNA even in the presence of the normally replicating plasmid. The temperature sensitivity of Rtsl may, therefore, be as- sociated with some step in the formation of closed circular DNA, and not in replication, per se.

Loss of Rtsl from Host Cells at 42?. During prolonged incubation of low density cultures of Rtsl cells at 42?, large numbers of R- segregants accumulated (3). It appeared that Rtsl was lost from its host at high frequency, and this supported a hypothesis that this R factor was temperature sensitive for replication (3, 4). In confirmation of Terawaki et al. (3), Table 3 shows that 77% of the viable cells cultured at 42? for 24 hr were plasmid-negative. However, under these culture conditions, the number of viable Rtsl cells remained constant after an initial 6-fold increase (6). In addition, R- cells grow normally at 42? even in the presence of abnormally growing Rtsl cells (unpublished observation). A corrected frequency of curing of Rtsl from its host can be derived from the observed percentage of drug sensitive segregants after correction for the differential rates of growth at 42? of Rtsl cells compared to R- segregants (Table 3). Thus we calculate the loss of Rtsl from its host to occur at a rate of approximately 10-8/cell per generation. The appearance of large numbers of R- cells, then, is due to a strong selective pressure favoring the overgrowth of rare, spontaneous R- segregants.

DISCUSSION

When Rtsl cells were treated with penicillin at the nonpermissive temperature, elongated cells with multiple host nuclei were produced. Upon removal of penicillin, multinucleated Rtsl cells formed at 42? in the presence of penicillin divided in the absence of DNA synthesis into approximately four daughter cells, and each contained Rtsl. Since the original cells had approximately 2.1 copies of the plasmid, these results suggest that Rtsl DNA replicated at the nonpermissive temperature.

enicillin-induced filaments of E. coli 20SO Rtsl

60 min post-penicillin at 36?

Number of cells iixic Viable count Clones resistant produced from id (millions) to kanamycin each filament

10.7 (100/100) 100% 5 - 10.0 (100/100) 100% 4.8

- 4.55 (100/100) 100% 4 - 4.45 (100/100) 100% 3.9

ml into glucose minimal medium containing 20 units per ml penicil- aments. Each culture was diluted 100-fold in fresh medium to lower 1 hr in the presence or absence of 5 pg/ml of nalidixic acid (Sterling-

pleted within 60-75 min. Viable and/or total counts were measured agar plates. After 2 days at 27?, clones from each series of plates

key's agar plates with and without 25 ,g/ml of kanamycin (Sigma).



2518 Biochemistry: DiJoseph and Kaji

(x) (o, A) 35 --140

15 - i 30 - -120

25--1 0 I

r~~~~~Io 1

10 20 3o 0 x x

0- 115 60

5- j E cl Rl 1 10 --40

5 20

0 !

1 10 20 3032 Fraction NUMBER

FIG. 2. Alkaline sucrose density gradient analysis of DNA from E. coli Rtsl before and after temperature shiftdown. A culture of E. coli 20SO Rtsl at 2 X 107 cells per ml in glucose minimal medium was divided into two. One portion was labeled by incubation at 32? with 50 /Ci/ml (1.8 pg/ml) [3H]thymidine and 250 pg/ml of deoxyadenosine for 30 min and then chased with a 200-fold excess of unlabeled thymidine for 10 min. The culture was chilled, pelleted, and resuspended in a double volume of fresh medium containing 250 Ag/ml of deoxyadenosine and a 50- fold excess of unlabeled thynoidine over the original [3H] thymidine concentration. After 70 min at 42?, the culture was harvested and analyzed as in Fig. 1 (X--X). Another portion of the original culture was incubated at 32? without radioactive thymidine for 40 min., diluted 2-fold, and shifted to 42?. After 30 min, [3H]thymidine at 25 pCi/ml and deoxyadenosine at 250 ug/ml were added. After labeling for 30 min, a 200-fold excess of unlabeled thymidine was added for 10 min. The culture was then quickly chilled, divided into two equal parts, and centrifuged. One half of the 42?-labeled culture was processed and analyzed (A,A). The remaining half of the cells was resuspended in a double volume of fresh medium containing 250 pg/ml of deoxyadenosine and a 50-fold excess of unlabeled thymidine. After 90 min at 36?, this culture was harvested for density gradient analysis (,0O). Note different scale for host DNA radioactivity (open symbols, dashed lines).

Analysis of cell extracts on alkaline sucrose density gradients indicated that Rtsl DNA, like other R factors, exists as closed covalent circles. Most, if not all of the R DNA in the cell is presumed to be in the form of closed covalenit circles (17, 23). Rtsl DNA was estimated to be around 120 X 106 daltons, approximately twice the mass suggested for Rtsl in P. mirabilis (4). However, a separate experiment indicated that the molecular weight of Rtsl DNA in P. mirabilis was also approximately 120 million. Rtsl DNA may dimerize as reported for F'451 DNA (13). The copy number of Rtsl was estimated to be 1.3 copies per genome, which is similar to the reported values for R100 and R28K (14, 9). Thus, Rtsl, like other plasmids of this size class (15), is stringently replicated in E. coli.

Further evidence that Rtsl DNA can be synthesized in E. coli at nonpermissive temperature was obtained from an


12 - 60

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FRACTION NUMBER

FIG. 3. Formation of covalently circular R100 I)OO DNA at 42? in the presence of IRttsl. E. coli 20SO Rtsl:R1i00 cells at 4 X 100 cells per ml were grown for 3 hr at 36? or 42? with 20 units/ml of penicillin G in glucose minimal medium. I)eoxyadenosine (250 pg/ml) and [RH] thymidine (50 pCi/ml) were added to each culture and incubation was continued for an additional hour and lysates were analyzed on alkaline sucrose density gradients. (a) E. coli RItsl: RI10 at 36?; (b) Rtsl: ROO at 42?. The ratios of closed covalent circular R DNA to host DNA were 0.014 for R,100 at 36? or 42?, 0.016 for Rtsl at 36?, and 0.0008 for R.tsl at 42?. The difference between the total counts on the two gradients is not significant, since the two cultures were suspended in different volumes of lysis buffer. (*) and (0) refer to the scales on the left and right side, respectively. A separate experiment revealed that recovery of prelabeled Rtsl DNA was not influenced by the presence of penicillin at 42?.

experiment in which [3H]thymidine was incorporated into DNA at 42?. It should be emphasized that closed circular DNA was not observed if cells were labeled at 42?. It subse- quently appeared after further incubation at 36? in the presence of excess nonlabeled thymidine. However, closed circular Rtsl DNA was found in cells labeled at 42? if [3H]thymidine was incorporated at 32? before the shift to 42?. This prelabeled Rtsl DNA may represent a portion of the plasmid DNA pool which had not yet replicated at 42?. These data suggest that some step in the processing of Rtsl DNA into the closed covalent circular form may be temperature sensitive. The Rtsl DNA which is replicating at 42? may be in the form of a relax- ation complex or a complex with RNA (16).

The notion that a step in the processing of Rtsl D)NA into closed circular form is temperature sensitive is consistent with the observation that, during conjugation, newly ac- quired linear single-stranded R DNA is converted to closed covalent circles through linear duplex and open circular membrane-bound intermediates (17). In this connection, we have previously shown that one of the effects on Rtsl on its




TABLE 3. Loss of R factors from host cells

Incu- Incu- Percent colonies bation bation Viable sensitive to

temper- period count kanamnycin or Culture ature (?) (hr) (cells/ml) ampicillin (%)

R28K 0 0.42 X 103 0 27 24 2.2 X 109 0 42 24 5.8 X 1CG 0

Rtsl 0 0.98 X 103 0 27 24 1.3 X 109 0 42 24 2.5 X 104 77

Cultures of E. coli 20SO R.tsl and R,28K were diluted to 0.5 to 1 X 103 cells per ml and incubated in trypticase soy broth at 27? or 42? for 24. hr without shaking. Before and after incubation samples were plated and the resulting clones were tested for resistance to 25 pg/ml kanamycin or 20 ,g/ml of ampicillin (Bristol Labs) as in Table 2. The percentage of cells sensitive to kanamycin after incubation at 42? was used to calculate a corrected frequency of curing, f, as follows. In a growing culture without selective loss in viability f can be derived from the

IN relationship; NR- = E (f NRn+) 2N, where NRO+ is the ihitial

n=--0

R,tsl cell density and NR- is the number of R- segregants which accumulate after N generations. Since Rtsl does not reach saturation cell densities within 24 hr, we assume n = 32, or a generation time of about 45 main. Since NR0+ is constant after an initial 6-fold increase, the expression of f then becomes NR- = (f NRo+) (232 + 232 + 232 + 232 + (3/4)232) + (fNRt+) (6) (228 + 227 + ... + 20), assuming that the nonviable cells receive a copy of Rtsl DNA as suggested by Table 2 and Fig. 2. The expression reduces to f = (NR-/NRo+)(1/t.9 X 109). The final culture was composed of 0.6 X 104 kanamycin-resistant cells and 1.9 X 104 cells per ml of kanamycin-sensitive cells. Therefore, f is approximately 10-8/cell per generation.

host is to alter the cell envelope (6). Our conclusion that Rtsl DNA can replicate at the nonpermissive temperature does not contradict the observation that R- segregants accumulate at 42? (3). Since Rtsl viable cell numbers do not increase at the nonpermissive temperature, rare spontaneous R- segregants overgrow the Rtsl cells. Loss of a nonthermosensitive F' at low cell density, which reduces the chance of reinfection (18), has been reported. The present observation that Rtsl replicates at 42? does not necessarily imply that

Rtsl DNA 2519

replication continues indefinitely at 42?. It is possible that after prolonged exposure to 42? the replication of Rtsl may stop. Data in Table 2, however, indicate that curing can take place simply from mutational events. The elucidation of the exact biochemical step influenced by Rtsl is possible with an in vitro system.

We thank Dr. H. Blum for help in deriving the expression for curing frequency. This work was supported by Public Health Service Grant PHS GM 12053 and by NIH Predoctoral Training Grant 5 TO1 GM 00849-10.

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M. (1968) Nature 219, 284-285. 6. DiJoseph, C. G., Bayer, M. E. & Kaji, A. (1973) J. Bacteriol.

115, 399-410. 7. Freifelder, D., Folkmanis, A. & Kirshner, I. (1971) J.

Bacteriol. 105, 722-727. 8. DiJoseph, C. G. & Kaji, A., manuscript in preparation. 9. Kontomichalou, P., Mitani, M. & Clowes, R. C. (1970) J.

Bacteriol. 104, 34-44. 10. Starka, J. & Moravava, J. (1967) Folia Microbiol. i2, 240-

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103, 166-177. 15. Clowes, R.. C. (1972) Bacteriol. Rev. 36, 361-405. 16. Helinski, 1). R. (1973) Annu. Rev. Microbiol. 27, 437-470. 17. Falkow, S., Tompkins, L. S., Silver, R. P., Guerry, P. &

LeBlanc, D. J. (1971) in The Problems of Drug Resistant Pathogenic Bacteria, eds. Dulaney, E. L. & Laskin, A. I. Ann. N.Y. Acad. Sci. 182, 153-171.

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The Thermosensitive Lesion in the Replication of the Drug Resistance Factor, Rts1