PLASMID 1, 589-593 (1978)

Interactions Between Two Heterogenic R Plasmids: Cointegrative Suppression of the Thermosensitive Replication of Rtsl by a

Nonconjugative Derivative of NRl

NOBUICHI GOTO, YOSHIRO TERAWAKI,’ AND RINTARO NAKAYA

Department of Microbiology, Tokyo Medical and Dental University School of Medicine, l-5-45, Yushima, Bunkyo-ku, Tokyo 113, Japan

Accepted June 14, 1978

A recombinant plasmid, R428, between a temperature-sensitive R plasmid, Ws 1, and a transfer-deficient mutant of NRl, NRl-4, was isolated. Experiments on stability at higher temperature, transfer frequency, and density profiles of DNAs suggested that R428 is a cointegrate and that the thermosensitivity of Rts 1 replication was suppressed.

An R plasmid Rfsl confers resistance to kanamycin (Km) and is temperature sensi- tive in conjugational transfer as well as replication (24,15). It also inhibits the host cell growth at higher temperature (12). Ter- awaki et al. (23) and Yoshimoto and Yoshi- kawa (19) have independently isolated Hfr strains of Escherichia coli with Rtsl and found that the thermosensitive replication of Rts 1 and the detrimental effect on the host cell growth at higher temperature can be suppressed by its integration in the host chromosome.

This report describes the plasmid-plasmid interactions, using R plasmid transcon- jugants doubly infected with Rts 1 and a non- conjugative R plasmid NRl-4, which has been obtained by transduction of NRl by ultraviolet-irradiated bacteriophage Plkc (5,6). NRI-4 confers resistance to chlor- amphenicol (Cm), streptomycin (Sm), and sulfathiazole (Su). The plasmids generated during the course of this work are described in the text and illustrated in Fig. 1.

The parental plasmids, RZS 1 and NRl (Tc Cm Sm Su), have distinct properties in sev- eral aspects. First, NRl was stable in Es- cherichia co/i K12 CSH-2 (met) at either

1 Present address: Department of Bacteriology, Faculty of Medicine, Shinshu University, 3-l-l Asahi, Matsumoto 390, Japan.

25 or 42°C and slightly unstable in Proteus mirabilis Pm17 (nit gal trp thy ura) only at 25”C, whereas Rtsl was markedly un- stable at 42°C in both of the host strains (Table 1). NRl-4 was not tested for its stabil- ity in these strains because of its defective- ness for conjugational transfer. It was stable, however, in its original host K-12 W677 (lac thr leu thi) at 25 and 42°C (data not shown). Second, as seen in Table 2, NRI could be transferred more efficiently at 42°C than at 25X, while the frequency of trans- fer of Rts 1 was considerably reduced at the higher temperature, i.e., approximately 10e3 lower than at the lower temperature. NRl was transferred from Pml7(NRl) to CSH-2 at a very low frequency even at 25”C, but further study proved that the transfer was not temperature sensitive (data not shown). Third, Rrsl inhibits the growth of its host cell at 42”C, while NRl does not (II). Finally, the Rtsl DNA had a buoyant density of 1.705 g/ml [Fig. 2a; Ref. (4)], whereas that of NRI DNA was found to be 1.712 or 1.718 g/ml in the nontransitioned or transitioned state, respectively (7,9,10).

To isolate hybrid plasmids composed of NRI-4 and Rts 1, transconjugants exhibit- ing all of the four drug’resistance markers (Km Cm Sm Su) were constructed by mobili- zation as follows (see also Fig. 1). An over-

589 0147-619x178/0014-0589$02.00/O Copyright 0 1978 by Academic Press. Inc. All rights of reproduction in any form reserved.

590 SHORT COMMUNICATIONS

NR 1 (Tc Cm Sm Su Tra+) Rlr 1 [Km Rep(Ts) Tra(Ts)]

R428C (Km Rep(Ts) Tra(Ts)l 42%

R428A (Cm Sm Su Tra(Ts)] R4288 [Km Tra(Ts))

FIG. 1. Phylogeny of the plasmids. Plasmids are un- derlined. Tc, Cm, Sm, Su, and Km: resistance to tetra- cycline, chloramphenicol, streptomycin, sulfathiazole, and kanamycin, respectively. Tra- and Tra(Ts): defec- tive and thermosensitive, respectively, in mediating conjugation. Rep(Ts): temperature sensitive in rephca- tion. Refer to the text for bacterial strains (en- circled).

night culture of W677 (NRl-4) was mixed with equal volumes of the overnight cul- tures of Pm17 (Rtsl) and CSH-2. The mix- ture was diluted twofold in fresh broth and in- cubated at 25°C overnight. For selection of R plasmid transconjugants of CSH-2 cells, the mating mixture was then streaked onto the minimal agar plates supplemented with lactose, methionine, Cm, and Km, and in- cubated at 30°C for 3 days. A number of colonies thus formed were purified and tested for the growth rate turbidimetrically in Penassay broth at 42°C. While most strains grew more slowly than R- strain, some of them grew normally as well as the control. One such strain was operationally designated R428 and used in the following experiment.

R428 looked like a cointegrate of Rfs 1 and NRl-4 based on the following observations. First, as shown in Table 2, Pm17(R428) transferred the Cm Sm Su markers as ef- ficiently as the Km marker, i.e., the four determinants were transferred en bloc. Sec- ond, the Km resistance was eliminated at 42°C at a much reduced frequency, and elimination of the four determinants en bloc was observed at a low frequency only when

they were harbored in CSH-2 (Table 1). Third, CsCl density gradient analysis showed that a satellite DNA band corresponding to NRl-4 (density of 1.712 g/ml) was not ob- served in Pm17(R428) DNA (Fig. 2b).

Although these results seem to indicate that R428 is a cointegrate R plasmid, the observation that the Km marker or the Cm Sm Su markers were independently sep- arated from R428 in a few cases and that the 1.718 g/ml DNA was demonstrated in the DNA preparation from Pm17(R428) cells cultured in the presence of Cm (Fig. 2c) suggest that the Km and the Cm Sm Su determinants may occasionally dissociate from the cointegrate plasmid. Since the difference in buoyant density between Rts 1 and R428 DNA was negligible, the size of NRl-4 fragment integrated in R428 may be very small.

A Km-sensitive clone and Cm, Sm, and Su-sensitive clones were isolated in the experiment on the stability in maintenance of R428 in CSH-2 at 42°C (Table l), and the plasmids were designated R428A and R428B, respectively (Fig. 1). After being transferred to Pm17, they were tested for stability in maintenance (Table l), frequency of transfer (Table 2), and CsCl density gra- dient profiles of DNA (Fig. 2). R428A still retained the stability and the transferability of R428 at 42°C. The CsCl density profile of R428A (Fig. 2d) was also essentially the same as that of R428. When Pm17(R428A) was grown in the presence of Cm, the DNA showed a new satellite band of a buoyant density of 1.718 g/ml in addition to a 1.705- g/ml band (Fig. 2e). This transition of the plasmid DNA was identical to that of R428 DNA. These findings suggest that the loss of Km resistance determinant from R428 did not affect the DNA profile.

R428B conferred resistance only to Km as Rts 1, but was stable at 42°C in contrast to Rfsl (Table 1). The frequency of transfer and the density profile of DNA of R428B were similar to those of Rtsl (Table 2 and Fig. 2f).

The stable maintenance of R428 and its

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TABLE I

EFFECTS OF INCUBATION TEMPERATURE ON THE STABILITY IN MAINTENANCE OF R PLASMIDS”

Percentage of Tempera- Resistance sensitive colonies

R plasmid and resistance ture’ markers complex (phenotype)” (“Cl lost Pm17d CSH-2d

Rfs 1 [Km Rep(Ts) Tra(Ts)] 25 Km 0 0 42 Km 58 92

NRl [Tc Cm Sm Su Tra] 2.5 Tc Cm Sm Su 4 0 42 Tc Cm Sm Su 0 0

R428 [Km Cm Sm Tu Tra(Ts)] 25 Km 1 0 Cm Sm Su 1 0

42 Km 0 1 Cm Sm Su I 1 Km Cm Sm Su 0 5

R428A [Cm Sm Su Tra(Ts)] 25 Cm Sm Su 0 NTC 42 Cm Sm Su 1 NT

R428B [Km Tra(Ts)] 25 Km 0 NT 42 Km 2 NT

R428C [Km Rep(Ts) Tra(Ts)] 25 Km 0 NT 42 Km 29 NT

a A colony on a selective plate was inoculated into Penassay broth and cultured overnight at 25°C. The culture was diluted to 10e4 in fresh broth and incubated for 24 h at the temperatures indicated. An aliquot (0.1 ml) of a lO-5 dilution of the culture was spread on a nutrient agar plate and incubated overnight at 30°C. At least 100 colonies were tested for their drug resistance by the replica plating method.

* Cm, Sm, Su, Tc, and Km; resistance to chloramphenicol, streptomycin, sulfathiazole, tetracycline, and kanamycin, respectively. Tra; mediating conjugation. Rep; replication. Ts; thennosensitive.

c Temperature at which the incubation was carried out. d Host strains. e NT. not tested.

derivatives at 42°C seemed to imply that the temperature-sensitive replication of Rts 1 was suppressed by NRl-4 replication genes. To prove this assumption, isolation of a temperature-sensitive revertant from R428 was attempted. Considering the slight in- stability of NRI in Pm17 at a lower tem- perature, such a revertant that has lost the NRI-4 genome was expected to be found in Pm17(R428) stock culture. Twelve Km- resistant but Cm-sensitive colonies were isolated from a 3-year-old stock culture of Pm17(R428) in cooked meat medium and tested for stability in maintenance of the plasmid at 42°C. At least one of the Km- resistant clones was found to be thermosen-

sitive with respect to the plasmid main- tenance and the plasmid was named R428C. Further experimentation revealed that R428C was as unstable at 42°C as Rts 1 within the limits of experimental variation (Table 1). The frequency of transfer and the density profile of DNA both indicated that R428C was indistinguishable from Rts 1 (Table 2 and Fig. 2g). These results indicate that the intact Rrs 1 genome exists in R428 as a part of a cointegrate plasmid and is suppressed in its thermosensitive replication by NRI-4. In contrast to this, the conjugational transfer of R428 at 42°C was not changed in its thermosensitivity.

Although there is no direct evidence that

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4/ I ’ (a) I

1 I I I I I I (b) / f R428

R428 CMx.5

(9) ;

1.718 1.705 l.iOO 1.718 1.705 1.700

+ DENSITY

FIG. 2. CsCl density gradient profiles of the R plasmid DNA in Proteus mirubilis Pm17. The plasmids are listed to the right of the density profiles. CM x 5 in (c) and (e): cells were subcultured in Penassay broth containing chloramphenicol (100 j&ml) five times consecutively before DNA extraction.

the entire NRI-4 genome was incorporated into R428, R428 must bear at least the r- determinants and some replication genes of NRl-4. It was reported by different authors [Yoshikawa, personal communication; Refs. (2,17)] that NRl (alternatively designated RlOO) has two replication genes. both on the conjugative component. R428B, which is a mutant of R428 that lost the r-determin- ants originating from NRl-4, still showed temperature-insensitive replication similar to that shown by R428. It is, therefore, rea- sonable to assume that the defective con- jugative component of NRl-4 still retained at least one of the replication genes and that it was sufficient for the cointegrative sup- pression of the thermosensitivity in re- plication. Since only repA was reported to be responsible for integrative suppression of dnuA (17), Rrsl is more likely to be sup- pressed by repA. However, the possibility may not be ruled out that repB could

mediate the cointegrative suppression of RtSl.

Yokota et al. (16) and Yoshikawa and Sakai (18) have isolated a number of re- combinants between Rrsl and RlOO (same as NRl) and determined their characteris- tics. All of the recombinants were found to be temperature sensitive in maintenance. The reason these authors have failed to ob- tain a temperature-insensitive recombinant might be explained as follows. They have used intact NRl plasmid while a transfer- deficient deletion mutant of NRl was em- ployed in this study. The former might have molecular decomposition upon recombina- tion probably due to the unusually large molecular weight of the recombinant [esti- mated as 186 million; Ref. (3)] and resulted in the formation of smaller recombinants consisting of the Rrsl genome and a small fragment of NRl containing no replication genes. Purification of DNA of Rrs 1 and of

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TABLE 2 248160 from the Ministry of Education, Japan, a Naito

FREQUENCY OF CONJUGATIONAL TRANSFER Foundation grant, and a Tokyo Medical and Dental

OF R PLASMIDS” University special grant.

R plasmid REFERENCES and Transfer frequency’

resistance Selective 1. ANDERSON, E. S., Nature (London) 208, 1016-

complex markers* 25°C 42°C 1017 (1965). 2. DEMPSEY, W. B., AND WILLETTS, N. S. J.

RtSl Km 1.1 x 10-I 1.5 x 10-4 Bacterial. 126, 166- 176 (1976).

NRl Cm, Sm, Su 8.0 x lO-9 1.5 x 1O-7 3. DIJOSEPH, C. G., AND KAJI, A., Proc. Nat. Acad.

R428 Km 1.6 x 10-i 2.9 x lO-4 Sci. USA 71, 2515-2519 (1974).

Cm, Sm, Su 2.7 x lo-* 8.6 x 1O-5 4. GOTO, N., YOSHIDA, Y.,TERAWAKI, Y., NAKAYA,

R428A Cm, Sm, Su 3.8 x 1O-2 1.2 x lO-6 R., AND SUZUKI, K., J. Bacterial. 102, 856-

R428B Km 5.2 x 10-Z 1.1 x 10-S 859 (1970).

R428C Km 2.7 x 1O-2 2.9 x lO-6 5. NAKAYA, R., NAKAMURA, A., AND MURATA, Y., Biochem. Biophys. Res. Commun. 3, 654-659

a R donor, Proteus mirabilis Pm17; Recipient, (1960).

Escherichia coli K-12 CSH-2. Conjugation was carried 6. NAKAYA, R., NAKAMURA, A., AND YAMADA, C.,

out for 3 h at the temperatures indicated. Pm17 was Igaku-no Ayumi 56, 363-373 (1966).

employed as a donor because DNA analysis was carried 7. PERLMAN, D., AND ROWND, R. H., J. Bacteriof.

out using this strain as a host. The transfer frequencies 123, 1013-1034 (1975).

of above plasmids except NRl were essentially the 8. ROWND, R. H., .I. Mol. Biol. 44, 387-402 (1969).

same when CSH-2 was used as a donor. NRl was 9. ROWND, R., KASAMATSU, H., AND MICKEL, S.,

transferred from CSH-2 very efficiently (on the order of Ann. N. Y. Acad. Sci. 182, 188-206 (1971).

lo-*). The reason NRI was transferred so inefficiently IO. ROWND, R., AND MICKEL, S., Nature New Biol.

from Pm17 is unknown. 234, 40-43 (1971).

b See Footnote b to Table 1. II. ROWND, R., NAKAYA, R., AND NAKAMURA, A.,

c Number of R plasmid transconjugants per donor J. Mol. Biol. 17, 376-393 (1966).

cell. 12. TERAWAKI, Y., KAKIZAWA, Y., TAKAYASU, H.,

AND YOSHIKAWA, M., Nature (London) 219,

R428 has been attempted many times using 284-285 (1968).

cleared lysate, ethidium bromide and neutral 13. TERAWAKI, Y., KISHI, H., AND NAKAYA, R., J.

Bncteriol. 121, 857-862 (1975).

CsCl density gradient centrifugation, and 14. TERAWAKI, Y., AND ROWND, R. H. .I. Bacterial.

nitrocellulose column chromatography. None 109, 492-498 (1972).

of these techniques was successful for iso- 15. TERAWAKI, Y., TAKAYASU, H., AND AKIBA, T.,

lating a sufficient amount of the plasmid J. Bacterial. 94, 687-690 (1967).

DNA for molecular analysis. 16. YOKOTA, T., KANAMARU, Y., MORI, R., AND

AKIBA, T., J. Bacterial. 98, 863-873 (1969). 17. YOSHIKAWA, M., J. Bacterial. 19, 1123-1131

ACKNOWLEDGMENTS (1974). 18. YOSHIKAWA, M., AND SAKAI, K., Japan. J. Micro-

The authors thank Masanosuke Yoshikawa for his biol. 16, 7-14 (1972). valuable suggestion and informative discussion. 19. YOSHIMOTO, H., AND YOSHIKAWA, M., J. Bac-

This study was supported by grants 844026 and teriol. 124, 661-667 (1975).