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Effect of nalidixic acid on conjugational transfer and expression of episomal Lac genes in Escherichia coli K12
J. Mol. Biol. (1967) 28, 373-376
LETTERS TO THE EDITOR
Effect of Nalidixic Acid on Conjugational Transfer and Expression of Episomal Lac Genes in Escherichia coli K12 The relationship and contribution of DNA synthesis to the transfer of genes by bacterial conjugation are controversial (Jacob, Brenner & Cuzin, 1963; Bouck & Adetberg, 1963; Bonhoeffer, 1966) and have been reviewed (Freifelder, 1966a; Curtiss, 1966; Gross & Caro, 1966). Strong evidence has been presented that DNA synthesis occurs in the donor concomitant with transfer of a DNA duplex to the F cell (Gross & Caro, 1966). The transferred duplex consists of one strand synthesized before and the other strand synthesized after the conjugation is begun, and this duplex can be isolated after transfer to merozygotes that are unable to make DI~IA. This evidence is consistent with the model proposed by Jacob et ~. (1963) that DNA synthesis occurs in the donor during gene transfer. Other less convincing evidence consistent with the same conclusion is also available (Hollom & Pritehard, 1966; Ptashne, 1965; Freffelder, 19665). Recently, evidence suggesting a role of the recipient in the transfer of DNA has been presented (Bonhoeffer, 1966; Curtiss, 1966; Freifelder, 1967). Discussion and possible explanations of the conflicting experhnental results are presented in the references above and will not be discussed here. I f trivial explanations of a technical nature are not responsible for the conAiet, it would appear that the chromosomal transfer process is a complicated one. In the future it should become possible to organize the available information and to constr'uct a consistent model. I report here further evidence for genetic transfer by donor DNA replication during mating (Jacob et al., 1963). All bacterial strains used were derived from Escherichia r~li K12. Strain Ml~2 is F - ~ - ~ (×74) i - , Sm~, B T (obtained from Dr Austin Newton). A nalidi~e acid° resistant strain was selected from this F - after growth on nalidixic acid (20 ~g/ml.). The resistant isolate grows equally well on 200 ~g of the drug~ml. Smr derivatives of the nalidi~c acid-sensitive and resistant strains were isolated and are used as recipients in the experiments reported. F ' ~ + from R V ~ F ' ~ ÷ was transferred into nalidixic acid-sensitive and resistant, streptomycin-sensitive, strains of MR2 to make the donor strains used. Thus the strains used are isogenic with respect to genetic background and to the nalidixlc acid-resistance marker. Bacteria were grown either in i~63 (pH 6-3) medium (Pardee, Jacob & Monod, 1959) supplemented with 2 mg Casamlno acids~ml, and 2 mg glycerol]ml., or in L broth (Lennox, 1955) as indicated. Growth was carried out in a New Brunswick gyrotory shaker at 37°C to exponential phase. Generation times under these conditions were 60 minutes in synthetic medium and 25 minutes in rich medium. ~ indicator plates used were MacConkey (Difco) supplemented with 200 ~g streptomycin]mi. Na~di~c acid was obtained as a gh~ from Mr C. E. Carl of Winthrop Laboratories, to whom I am grateful. It was used as an inhibitor in both rich and synthetic media Abbreviations used: ~tc, lactose operon; × 74, deletion a~ec~ing the entire lactose operon; SmUttY,streptomycin-sensitive(resistant). 373
374
S. D. B A R B O U R
a t a final concentration of 20 pg/ml. I t has been characterized as a potent and specific inhlbitor of D N A synthesis in E. co//(Goss, Deitz & Cook, 1965). I t s mode of action is not y e t known. Consistent with published results, control experiments under the conditions used in m y experiments have shown the following: D N A synthesis is immediately iuhlbited to less t h a n 5~/o of the control rate. Mass increase, fl-galaetosidase inducibility and R N A and protein synthesis continue for a b o u t a generation in the presence of the drug without significant effect. Nalidi~ic acid blocks the synthesis of fl-galactosidase in the e p i s o m a l / a c mating system (see Barbour & Pardee, 1966) ff the drug is present from the beglunlng of mating (Fig. 1). Addition of the drug after mating is complete (at 15 miuutes) has no
z%
;=
-8 .q ~o
L~ t
I0
20
30
40
SO 60
70
Time (mln)
FIG. 1. Effect of nalidi~i~ acid on the transfer and expression of genes by sensitive strains in the b~c mating system. Strain MR2 was the recipient and MR2/F'b~c+ the donor in this experiment. The strains were grown overnight in M63 (pH 6.3), 0-2% glycerol, 0.2% Casamino acids, th~mlne medium. Cultures were then diluted and grown to exponential phase (3 × l0 s bacteria/ml.) in the same media. 1~ cells were miTed with twice their volume of F - cells at the same density, and mating was carried out for 10 rnin with slow shaking at 37°C in a New Brunswick gyrotory shaker. Mating was stopped by the addition of 30 pg Duponol]ml., 200 pg streptomycin/ml., and rapid shaking. - - × - - × - - , Na]idixic acid (20 pg]ml.) at the beginning of mating; - - 0 - - 0 - - , nalidixic acid (20 pg/ml.) at 15 rui~; - - ~ - - A - - , no na~dixic acid. Samples were taken periodically for assay of fl-galactosidaas by a modification (Barbour & Pardce, 1966) of the method devised by Pardes e~ aL (1959).
effect on the synthesis of the enzyme. I t therefore appears t h a t inhibition of gene transfer is the reason for the absence of enzyme synthesis. To test this I measured the frequencies of F'b~c + transfer in couples of nalidi~e acid-sensitive a n d resistant strains m a t e d in the presence a n d absence of the drug (Table 1). ~'b~c +transfer was severely retarded b y the presence of the drug. Only the donor strain appeared to be the target. Normal episome transfer occurs only if the donor is resistant to the inhibi. tor. W h e n the recipient is resistant and the donor sensitive, gene transfer is severely iuhlbited. I t appears therefore t h a t some process, occurring in the donor and sensitive to nalidlxle acid, is necessary for the transfer of the F'/a~ + episome. No na]Jdi~ie acid-sensitive process occurring in the recipient is necessary for the transfer of the F'b~c + episome.
TABLE 1
Freque~wies of F ' l a o + transfer by nalidia:i~ twid.sen,gitive and resistant mating strains in the ~resence am~ abse~weof the drug F' X F-
F'/ac+/ml.
F-/ml.
% recombinants
R a t i o % ' s C/N
SXS C N
107 2.1 x 104
2.6 × l 0 T 2-8 × l 0 T
42.5 0.07
6O0
R×R C
2.3 × 107
5.2 × 107
1~
1.2 × 107
4.0 X 107
43.7 30
1.4
C N
2.4 × 107 4.7 x 104
5.7 × l 0 T 7 x l0 T
42 0.07
600
C N
9.8 x 10 e 8.9 x 10s
2.3 x l 0 T 2.4 X l 0 T
42 37.4
1-1
SxR
RxS
Nalidixic acid.sensitive (S) a n d resistant (R) strains of IvIR2 a n d iKR2fF'~c + were grown to exponential phase (3 x 10 s bacterial/ml.) in L broth. E q u a l volumes were m a t e d in t h e same m e d i a a t 37°C w i t h slow shaking in t h e presence (N) a n d absence (C) of nalidixio acid (20 pg/mL). Samples were t h e n plated on MacConkey streptomycin a n d i n c u b a t e d a t 37°C for 24 h r in order to count lac + a n d lac- streptomycin-resistant colonies.
_~ tO
0
30 40 50 60 70 Time (rain) FIa. 2. Effect of nalidixle acid on t h e transfer a n d expression of genes b y sensitive a n d resistant stralnR in t h e Zao m a t i n g system. The same strains were u s e d as those of t h e experiment of Table 1. The growth a n d m a t i n g were carried o u t as described in Fig. 1. Samples were t a k e n periodically a n d assayed for ~-galactosldase. Protocol was as follows:
26
10
20
Strains (P' x F - )
Nalidl,rlo acid (20 ~glmL)
Time added (rain)
R x S (@) RxS (x) S x R (O) S x R (A)
-+ -+
R x S (A) S x R (D)
-I+
-0 -0 T5
15
376
S. D . B A R B O U R
The effect of nalidi~c acid on the transfer and expression o f / a c genes b y sensitive and resistant strains was tested (Fig. 2). Here the only nalidixic acid-sensitive target in the process of episomal transfer and gene expression again appears to be in the donor. The sensitive process is involved only in the transfer of the genes. Genes transferred b y a resistant donor to a sensitive recipient can direct normal synthesis of fl-galactosidase, indicating t h a t there is no nalidixic acid-sensitive target in the synthesis of fl-galactosidase involved in the zygote. Also repression is established normally in the presence of nalidixic acid (Fig. 2). This is consistent with the results of Horiuchi & Ohsbima (1966), who have shown t h a t DNA synthesis is not necessary for the synthesis of repressor. As discussed above, it appears that the effect of nalidi~c acid in vivo is through its effect on DNA synthesis. I assume t h a t the effect of nalidixic acid on episome transfer results from this and not some lml~nown effect on conjugation. I t is important t h a t its mode of action in blocking DNA synthesis be ascertained. I conclude that: (1) DNA synthesis in the donor is required for the transfer of episomal genes b y conjugation. (2) DNA synthesis in the recipient is not required for episome transfer. (3) Once transferred, t h e / a c episome will direct the normal synthesis of fl-galactosidase and repressor in the presence of nalidixic acid, even though the episome m a y not be replicated after it enters the recipient cytoplasm. These conclusions are consistent with the model proposed b y Jacob et al., 1963. This work was supported by National Institutes of Health Grant AI 04409 to Dr A. B. Pardee. I am a National Science Foundation Graduate Follow (1964-1967). I thank Drs Pardee and R. Eisenberg for stimulating discussion. iVote added in proof: Experiments with nalldixic acid-sensitive strains have shown that pair formation occurs normally in the presence of the drug. Couples formed in the presence of nalicliYie acid show normal episome transfer upon subsequent removal of the drug.
Department of Biology Princeton University Princeton, New Jersey 08540, U.S.A.
STSPH~ D. BA~BOU~
Received 30 May 1967 REFERENCES Barbour, S. D. & Pardee, A. B. (1966). J. Mol. Biol. 20, 505. Bonhoeffer, F. (1966). Z. VererSungsl. 98, 141. Bouck, N. & Adelberg, E. A. (1963). Biochem. Biophys. Re~. Uomm. 11, 24. Curtiss, Roy, I I I (1966). Morgan Cent. Symp., in the press. Freffelder, D. L. R. (1966a). Thesis, University of California. lCreifelder, D. (1966b). Biochem. Biophys. Re~. Gomm. 23, 575. Freffelder, D. (1967). J. Bact., in the press. Goss, W., Deitz, W. & Cook, T. (1965). J. Bact. 89, 1068. Gross, J. D. & Caro, L. (1966). J. Mol. Biol. 16, 269. Hollom, S. & Pritehard, R. H. (1966). Genet. Re~. Camb. 6, 479. Horiuchi, T. & Ohshima, Y. (1966). J. Mol. Biol. 20, 517. Jacob, F., Brenner, S. & Cuzin, F. (1963). Co/d Spr. Harb. Syrup. Quant. Biol. 28, 329. Lennox, E. (1955). Virology, 1, 190. Pardee, A. B., Jacob, F. & Monod, J. (1959). J. MoZ. Biol. l, 165. Ptashne, M. (1965). J. Mol. Biol. l, 829.
LETTERS TO THE EDITOR
Effect of Nalidixic Acid on Conjugational Transfer and Expression of Episomal Lac Genes in Escherichia coli K12 The relationship and contribution of DNA synthesis to the transfer of genes by bacterial conjugation are controversial (Jacob, Brenner & Cuzin, 1963; Bouck & Adetberg, 1963; Bonhoeffer, 1966) and have been reviewed (Freifelder, 1966a; Curtiss, 1966; Gross & Caro, 1966). Strong evidence has been presented that DNA synthesis occurs in the donor concomitant with transfer of a DNA duplex to the F cell (Gross & Caro, 1966). The transferred duplex consists of one strand synthesized before and the other strand synthesized after the conjugation is begun, and this duplex can be isolated after transfer to merozygotes that are unable to make DI~IA. This evidence is consistent with the model proposed by Jacob et ~. (1963) that DNA synthesis occurs in the donor during gene transfer. Other less convincing evidence consistent with the same conclusion is also available (Hollom & Pritehard, 1966; Ptashne, 1965; Freffelder, 19665). Recently, evidence suggesting a role of the recipient in the transfer of DNA has been presented (Bonhoeffer, 1966; Curtiss, 1966; Freifelder, 1967). Discussion and possible explanations of the conflicting experhnental results are presented in the references above and will not be discussed here. I f trivial explanations of a technical nature are not responsible for the conAiet, it would appear that the chromosomal transfer process is a complicated one. In the future it should become possible to organize the available information and to constr'uct a consistent model. I report here further evidence for genetic transfer by donor DNA replication during mating (Jacob et al., 1963). All bacterial strains used were derived from Escherichia r~li K12. Strain Ml~2 is F - ~ - ~ (×74) i - , Sm~, B T (obtained from Dr Austin Newton). A nalidi~e acid° resistant strain was selected from this F - after growth on nalidixic acid (20 ~g/ml.). The resistant isolate grows equally well on 200 ~g of the drug~ml. Smr derivatives of the nalidi~c acid-sensitive and resistant strains were isolated and are used as recipients in the experiments reported. F ' ~ + from R V ~ F ' ~ ÷ was transferred into nalidixic acid-sensitive and resistant, streptomycin-sensitive, strains of MR2 to make the donor strains used. Thus the strains used are isogenic with respect to genetic background and to the nalidixlc acid-resistance marker. Bacteria were grown either in i~63 (pH 6-3) medium (Pardee, Jacob & Monod, 1959) supplemented with 2 mg Casamlno acids~ml, and 2 mg glycerol]ml., or in L broth (Lennox, 1955) as indicated. Growth was carried out in a New Brunswick gyrotory shaker at 37°C to exponential phase. Generation times under these conditions were 60 minutes in synthetic medium and 25 minutes in rich medium. ~ indicator plates used were MacConkey (Difco) supplemented with 200 ~g streptomycin]mi. Na~di~c acid was obtained as a gh~ from Mr C. E. Carl of Winthrop Laboratories, to whom I am grateful. It was used as an inhibitor in both rich and synthetic media Abbreviations used: ~tc, lactose operon; × 74, deletion a~ec~ing the entire lactose operon; SmUttY,streptomycin-sensitive(resistant). 373
374
S. D. B A R B O U R
a t a final concentration of 20 pg/ml. I t has been characterized as a potent and specific inhlbitor of D N A synthesis in E. co//(Goss, Deitz & Cook, 1965). I t s mode of action is not y e t known. Consistent with published results, control experiments under the conditions used in m y experiments have shown the following: D N A synthesis is immediately iuhlbited to less t h a n 5~/o of the control rate. Mass increase, fl-galaetosidase inducibility and R N A and protein synthesis continue for a b o u t a generation in the presence of the drug without significant effect. Nalidi~ic acid blocks the synthesis of fl-galactosidase in the e p i s o m a l / a c mating system (see Barbour & Pardee, 1966) ff the drug is present from the beglunlng of mating (Fig. 1). Addition of the drug after mating is complete (at 15 miuutes) has no
z%
;=
-8 .q ~o
L~ t
I0
20
30
40
SO 60
70
Time (mln)
FIG. 1. Effect of nalidi~i~ acid on the transfer and expression of genes by sensitive strains in the b~c mating system. Strain MR2 was the recipient and MR2/F'b~c+ the donor in this experiment. The strains were grown overnight in M63 (pH 6.3), 0-2% glycerol, 0.2% Casamino acids, th~mlne medium. Cultures were then diluted and grown to exponential phase (3 × l0 s bacteria/ml.) in the same media. 1~ cells were miTed with twice their volume of F - cells at the same density, and mating was carried out for 10 rnin with slow shaking at 37°C in a New Brunswick gyrotory shaker. Mating was stopped by the addition of 30 pg Duponol]ml., 200 pg streptomycin/ml., and rapid shaking. - - × - - × - - , Na]idixic acid (20 pg]ml.) at the beginning of mating; - - 0 - - 0 - - , nalidixic acid (20 pg/ml.) at 15 rui~; - - ~ - - A - - , no na~dixic acid. Samples were taken periodically for assay of fl-galactosidaas by a modification (Barbour & Pardce, 1966) of the method devised by Pardes e~ aL (1959).
effect on the synthesis of the enzyme. I t therefore appears t h a t inhibition of gene transfer is the reason for the absence of enzyme synthesis. To test this I measured the frequencies of F'b~c + transfer in couples of nalidi~e acid-sensitive a n d resistant strains m a t e d in the presence a n d absence of the drug (Table 1). ~'b~c +transfer was severely retarded b y the presence of the drug. Only the donor strain appeared to be the target. Normal episome transfer occurs only if the donor is resistant to the inhibi. tor. W h e n the recipient is resistant and the donor sensitive, gene transfer is severely iuhlbited. I t appears therefore t h a t some process, occurring in the donor and sensitive to nalidlxle acid, is necessary for the transfer of the F'/a~ + episome. No na]Jdi~ie acid-sensitive process occurring in the recipient is necessary for the transfer of the F'b~c + episome.
TABLE 1
Freque~wies of F ' l a o + transfer by nalidia:i~ twid.sen,gitive and resistant mating strains in the ~resence am~ abse~weof the drug F' X F-
F'/ac+/ml.
F-/ml.
% recombinants
R a t i o % ' s C/N
SXS C N
107 2.1 x 104
2.6 × l 0 T 2-8 × l 0 T
42.5 0.07
6O0
R×R C
2.3 × 107
5.2 × 107
1~
1.2 × 107
4.0 X 107
43.7 30
1.4
C N
2.4 × 107 4.7 x 104
5.7 × l 0 T 7 x l0 T
42 0.07
600
C N
9.8 x 10 e 8.9 x 10s
2.3 x l 0 T 2.4 X l 0 T
42 37.4
1-1
SxR
RxS
Nalidixic acid.sensitive (S) a n d resistant (R) strains of IvIR2 a n d iKR2fF'~c + were grown to exponential phase (3 x 10 s bacterial/ml.) in L broth. E q u a l volumes were m a t e d in t h e same m e d i a a t 37°C w i t h slow shaking in t h e presence (N) a n d absence (C) of nalidixio acid (20 pg/mL). Samples were t h e n plated on MacConkey streptomycin a n d i n c u b a t e d a t 37°C for 24 h r in order to count lac + a n d lac- streptomycin-resistant colonies.
_~ tO
0
30 40 50 60 70 Time (rain) FIa. 2. Effect of nalidixle acid on t h e transfer a n d expression of genes b y sensitive a n d resistant stralnR in t h e Zao m a t i n g system. The same strains were u s e d as those of t h e experiment of Table 1. The growth a n d m a t i n g were carried o u t as described in Fig. 1. Samples were t a k e n periodically a n d assayed for ~-galactosldase. Protocol was as follows:
26
10
20
Strains (P' x F - )
Nalidl,rlo acid (20 ~glmL)
Time added (rain)
R x S (@) RxS (x) S x R (O) S x R (A)
-+ -+
R x S (A) S x R (D)
-I+
-0 -0 T5
15
376
S. D . B A R B O U R
The effect of nalidi~c acid on the transfer and expression o f / a c genes b y sensitive and resistant strains was tested (Fig. 2). Here the only nalidixic acid-sensitive target in the process of episomal transfer and gene expression again appears to be in the donor. The sensitive process is involved only in the transfer of the genes. Genes transferred b y a resistant donor to a sensitive recipient can direct normal synthesis of fl-galactosidase, indicating t h a t there is no nalidixic acid-sensitive target in the synthesis of fl-galactosidase involved in the zygote. Also repression is established normally in the presence of nalidixic acid (Fig. 2). This is consistent with the results of Horiuchi & Ohsbima (1966), who have shown t h a t DNA synthesis is not necessary for the synthesis of repressor. As discussed above, it appears that the effect of nalidi~c acid in vivo is through its effect on DNA synthesis. I assume t h a t the effect of nalidixic acid on episome transfer results from this and not some lml~nown effect on conjugation. I t is important t h a t its mode of action in blocking DNA synthesis be ascertained. I conclude that: (1) DNA synthesis in the donor is required for the transfer of episomal genes b y conjugation. (2) DNA synthesis in the recipient is not required for episome transfer. (3) Once transferred, t h e / a c episome will direct the normal synthesis of fl-galactosidase and repressor in the presence of nalidixic acid, even though the episome m a y not be replicated after it enters the recipient cytoplasm. These conclusions are consistent with the model proposed b y Jacob et al., 1963. This work was supported by National Institutes of Health Grant AI 04409 to Dr A. B. Pardee. I am a National Science Foundation Graduate Follow (1964-1967). I thank Drs Pardee and R. Eisenberg for stimulating discussion. iVote added in proof: Experiments with nalldixic acid-sensitive strains have shown that pair formation occurs normally in the presence of the drug. Couples formed in the presence of nalicliYie acid show normal episome transfer upon subsequent removal of the drug.
Department of Biology Princeton University Princeton, New Jersey 08540, U.S.A.
STSPH~ D. BA~BOU~
Received 30 May 1967 REFERENCES Barbour, S. D. & Pardee, A. B. (1966). J. Mol. Biol. 20, 505. Bonhoeffer, F. (1966). Z. VererSungsl. 98, 141. Bouck, N. & Adelberg, E. A. (1963). Biochem. Biophys. Re~. Uomm. 11, 24. Curtiss, Roy, I I I (1966). Morgan Cent. Symp., in the press. Freffelder, D. L. R. (1966a). Thesis, University of California. lCreifelder, D. (1966b). Biochem. Biophys. Re~. Gomm. 23, 575. Freffelder, D. (1967). J. Bact., in the press. Goss, W., Deitz, W. & Cook, T. (1965). J. Bact. 89, 1068. Gross, J. D. & Caro, L. (1966). J. Mol. Biol. 16, 269. Hollom, S. & Pritehard, R. H. (1966). Genet. Re~. Camb. 6, 479. Horiuchi, T. & Ohshima, Y. (1966). J. Mol. Biol. 20, 517. Jacob, F., Brenner, S. & Cuzin, F. (1963). Co/d Spr. Harb. Syrup. Quant. Biol. 28, 329. Lennox, E. (1955). Virology, 1, 190. Pardee, A. B., Jacob, F. & Monod, J. (1959). J. MoZ. Biol. l, 165. Ptashne, M. (1965). J. Mol. Biol. l, 829.