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Inhibition of protein synthesis in E. coli protoplasts by actinomycin-D
J. Mol. Biol. (1963) 6, 247-249
Inhibition of Protein Synthesis in E. coli Protoplasts by Actinomycin-D Actinomycin-D has been reported to inhibit DNA-dependent RNA synthesis (Goldberg & Rabinowitz, 1962 ; Hurwitz, Furth, Malamy & Alexander, 1962), but to allow RNA viruses to continue to replicate in animal cells (Reich, Franklin, Shatkin & Tatum, 1961, 1962.) In Gram-positive bacteria this inhibition of RNA synthesis has been followed by an inhibition of protein synthesis (Kirk, 1960; Levinthal, Keynan & Riga, 1962). Actinomycin-D has been reported not to affect E . coli (Hurwitz et al. 1962), most likely because the cell wall is impermeable to actinomycin. We have observed that actinomycin-D at 10 y/mI. will strongly inhibit protein synthesis in protoplasts of E. coli C-3000 without inhibiting the growth of an RNA bacteriophage, MS2 (Davis, Strauss & Sinsheimer, 1961). Protoplasts were made by a modified version of the method previously described (Guthrie & Sinsheimer, 1960; also manuscript in preparation). E. coli C-3000 cells were grown to a concentration of 5 x lOs/mI. in modified 3XD broth (Guthrie & Sinsheimer, 1960). The cells were centrifuged and washed with t volume of 0·05 M-tris, pH 7,8, and 0·1 M-NaCI. Per 20 mI. of the original culture were added 0·35 ml. of 1·5 M-sucrose, 0·17 ml. of 30% BSA, 20,\ of lysozyme (2 mg/mI. in 0·25 M-tris, pH 8,1), 40,\ of 4 % EDTA and 3·6 ml. of a synthetic nutrient medium consisting of 250 mg jml, of ea ch of the essential amino acids except leucine, TPG medium (Sinsheimer, Starman, Nagler & Guthrie, 1962) at half strength, 350 mg/I. of Na 2HP04 , and 100 gil. of su crose. After 10 minutes, 0·2 ml. of 10% MgS04 was added, and the protoplasts then diluted 1 : 10 into the synthetic nutrient plus 0'2 % MgS04. Actinomycin-D was added to a concentration of 10 y/ml. in the appropriate tubes. All tubes were wrapped with aluminum foil to prevent the entry of light. Either immediately after the addition of actinomycin or 25 minutes later, leucine and [14C]leucine (Nuclear-Chicago) were added to provide a final concentration of leucine of 20 y/mI. and 2·5 x 10 5 ctsjminjml. A 0·5 mI. sample was immediately removed and frozen, and the remaining protoplasts incubated at 39°C without aeration. If a volume of greater than 2 mI., or a cell concentration of greater than 5 x lOS/mI., was used, the protoplasts were gently ag itated in a volumetric flask . One-half mi. samples were taken at different times and frozen and thawed three times to lyse the protoplasts. After addition to each of 0·08 mi. of 1-5% BSA and 0·5 mi. of 10% TCA, the resultant precipitates were washed three times in the cold with 5% TCA, resuspended in 0·5 ml. of 2 M-NH40H, dried on planchets and counted. Viable counts obtained on protoplast preparations indicate a survival of about 1% of the cells. If phage-infected cells are made into protoplasts and osmotically shocked, the infective centers remaining are about 1 to 3 %. Protoplasts incorporate [14C]leucine into protein at about two-thirds the rate of whole cells. E. coli C-3000 cells do not alter their uptake of [14C]leucine when treated with 10 y/ml. of actinomycin-D. The time course of [14C]leucine uptake in actinomycintreated and untreated protoplasts is shown in Fig. 1. Protein synt hesis markedly slows after 9 minutes' exposure to actinomycin but is not completely blocked. The 247
248
A. M . HAYWOOD AN D R. L . SINSHEIMER
residual synthesis may represent synthesis in the whole cells left in the protoplast preparation. Dependent upon the protoplast preparation, the [14C]leucine upt ake from 9 minutes to 1 hour in actinomycin-treated protoplasts varies from about 4 to 12% of the uptake in the control protoplasts. If the cells are exposed to actinomycin in the cold for 25 minutes befor e incubation at 39°C, the residual uptake is lower, as shown in Fig. 1. 1440 1320 1200 1080 960 .!!1
840
0;
u
Q
720
x
~ci. li
600 480 360 240 120
.::-6~:.:.:.:.:.:.:::'.~':.::=":~.===~_=~ 8
16
24
32
40
48
56
64
Time(min aft er addition 14C -leucine )
FIG. 1. Incorporation of p'C)leucine into protein in E . coli C-3000 protoplasts. 0 - - 0 Control (no actinomycin) ; 6 - - - 6 , [lCC]leucine added immediately after actinomycin-D (lOy/mI.); O · . .. O . p'C]leucine added 25 min after actinomycin-D (lO y/mI.).
The conditions for this experiment permit synthesis of a normal yield of the RNAcontaining bacteriophage. MS2, in protoplasts made from pre -infected cells at 5 minutes after infection. Similar conditions cause a 97% reduction of the yield of the DNA-containing bacteriophage c/JX174o in protoplasts made at 10 minutes after infection of cells, infected and maintained in cyanide. It seems reasonable to assume that the mechanism of inhibition of protein synthesis is analogous to that already demonstrated in other systems, i.e. suppression of the synthesis of DNA-dependent RNA. It thus appears that DNA-dependent RNA synthesis is not necessary for MS2 replication in agreement with the results obtained with RNA-containing viruses in animal cells. This research was supported in part by grant RG 6965 from the National Institutes of H eal th. Di v ision of Biology California Institute of Technology P asadena. Ca liforn ia , U.S.A. R eceived 14 January 1963
ANNE
M.
R OBERT
HAYWOOD
L.
SINSHETh1ER
LETTERS TO THE EDITOR
249
REFERENCES Davis, J. E., Strauss, J. H., Jr., & Sinsheimer, R. L . (1961). S cience, 134, 1427. Goldberg, 1. H. & Rabinowitz, M. (1962). Science, 136, 315. Guthrie, G. D. & Sinsheimer, R. L. (1960) . J. il'I ol. R iolo 2, 297. Hurwitz, J., Furth, J. J., Malamy, M. & Alexander, 1\'1. (1962) . Proc. Nat. Acad. sa; Wash. 48, 1222. Kirk, J. M. (1960). Biochim , biophys. Acta, 42, 167. Levinthal, C., Keynan, A. & Higa, A. (1962). Proc . Nat. Acad. s«, Wash. 48, 1631. R eich, E., Franklin, R. M., Shatkin, A. J. & Tatum, E. L. (1961). S cience, 134, 556. Reich, E ., Franklin, R. M., Shatkin, A. J . & Tatum, E. L. (1962) . Proc . Nat. Acad. sa; Wash. 48, 1238. Sinsheimer, R. L., Starman, B., Nagler, C. & Guthrie, S. (1962). J. Mol. Riol. 4, 142.
Inhibition of Protein Synthesis in E. coli Protoplasts by Actinomycin-D Actinomycin-D has been reported to inhibit DNA-dependent RNA synthesis (Goldberg & Rabinowitz, 1962 ; Hurwitz, Furth, Malamy & Alexander, 1962), but to allow RNA viruses to continue to replicate in animal cells (Reich, Franklin, Shatkin & Tatum, 1961, 1962.) In Gram-positive bacteria this inhibition of RNA synthesis has been followed by an inhibition of protein synthesis (Kirk, 1960; Levinthal, Keynan & Riga, 1962). Actinomycin-D has been reported not to affect E . coli (Hurwitz et al. 1962), most likely because the cell wall is impermeable to actinomycin. We have observed that actinomycin-D at 10 y/mI. will strongly inhibit protein synthesis in protoplasts of E. coli C-3000 without inhibiting the growth of an RNA bacteriophage, MS2 (Davis, Strauss & Sinsheimer, 1961). Protoplasts were made by a modified version of the method previously described (Guthrie & Sinsheimer, 1960; also manuscript in preparation). E. coli C-3000 cells were grown to a concentration of 5 x lOs/mI. in modified 3XD broth (Guthrie & Sinsheimer, 1960). The cells were centrifuged and washed with t volume of 0·05 M-tris, pH 7,8, and 0·1 M-NaCI. Per 20 mI. of the original culture were added 0·35 ml. of 1·5 M-sucrose, 0·17 ml. of 30% BSA, 20,\ of lysozyme (2 mg/mI. in 0·25 M-tris, pH 8,1), 40,\ of 4 % EDTA and 3·6 ml. of a synthetic nutrient medium consisting of 250 mg jml, of ea ch of the essential amino acids except leucine, TPG medium (Sinsheimer, Starman, Nagler & Guthrie, 1962) at half strength, 350 mg/I. of Na 2HP04 , and 100 gil. of su crose. After 10 minutes, 0·2 ml. of 10% MgS04 was added, and the protoplasts then diluted 1 : 10 into the synthetic nutrient plus 0'2 % MgS04. Actinomycin-D was added to a concentration of 10 y/ml. in the appropriate tubes. All tubes were wrapped with aluminum foil to prevent the entry of light. Either immediately after the addition of actinomycin or 25 minutes later, leucine and [14C]leucine (Nuclear-Chicago) were added to provide a final concentration of leucine of 20 y/mI. and 2·5 x 10 5 ctsjminjml. A 0·5 mI. sample was immediately removed and frozen, and the remaining protoplasts incubated at 39°C without aeration. If a volume of greater than 2 mI., or a cell concentration of greater than 5 x lOS/mI., was used, the protoplasts were gently ag itated in a volumetric flask . One-half mi. samples were taken at different times and frozen and thawed three times to lyse the protoplasts. After addition to each of 0·08 mi. of 1-5% BSA and 0·5 mi. of 10% TCA, the resultant precipitates were washed three times in the cold with 5% TCA, resuspended in 0·5 ml. of 2 M-NH40H, dried on planchets and counted. Viable counts obtained on protoplast preparations indicate a survival of about 1% of the cells. If phage-infected cells are made into protoplasts and osmotically shocked, the infective centers remaining are about 1 to 3 %. Protoplasts incorporate [14C]leucine into protein at about two-thirds the rate of whole cells. E. coli C-3000 cells do not alter their uptake of [14C]leucine when treated with 10 y/ml. of actinomycin-D. The time course of [14C]leucine uptake in actinomycintreated and untreated protoplasts is shown in Fig. 1. Protein synt hesis markedly slows after 9 minutes' exposure to actinomycin but is not completely blocked. The 247
248
A. M . HAYWOOD AN D R. L . SINSHEIMER
residual synthesis may represent synthesis in the whole cells left in the protoplast preparation. Dependent upon the protoplast preparation, the [14C]leucine upt ake from 9 minutes to 1 hour in actinomycin-treated protoplasts varies from about 4 to 12% of the uptake in the control protoplasts. If the cells are exposed to actinomycin in the cold for 25 minutes befor e incubation at 39°C, the residual uptake is lower, as shown in Fig. 1. 1440 1320 1200 1080 960 .!!1
840
0;
u
Q
720
x
~ci. li
600 480 360 240 120
.::-6~:.:.:.:.:.:.:::'.~':.::=":~.===~_=~ 8
16
24
32
40
48
56
64
Time(min aft er addition 14C -leucine )
FIG. 1. Incorporation of p'C)leucine into protein in E . coli C-3000 protoplasts. 0 - - 0 Control (no actinomycin) ; 6 - - - 6 , [lCC]leucine added immediately after actinomycin-D (lOy/mI.); O · . .. O . p'C]leucine added 25 min after actinomycin-D (lO y/mI.).
The conditions for this experiment permit synthesis of a normal yield of the RNAcontaining bacteriophage. MS2, in protoplasts made from pre -infected cells at 5 minutes after infection. Similar conditions cause a 97% reduction of the yield of the DNA-containing bacteriophage c/JX174o in protoplasts made at 10 minutes after infection of cells, infected and maintained in cyanide. It seems reasonable to assume that the mechanism of inhibition of protein synthesis is analogous to that already demonstrated in other systems, i.e. suppression of the synthesis of DNA-dependent RNA. It thus appears that DNA-dependent RNA synthesis is not necessary for MS2 replication in agreement with the results obtained with RNA-containing viruses in animal cells. This research was supported in part by grant RG 6965 from the National Institutes of H eal th. Di v ision of Biology California Institute of Technology P asadena. Ca liforn ia , U.S.A. R eceived 14 January 1963
ANNE
M.
R OBERT
HAYWOOD
L.
SINSHETh1ER
LETTERS TO THE EDITOR
249
REFERENCES Davis, J. E., Strauss, J. H., Jr., & Sinsheimer, R. L . (1961). S cience, 134, 1427. Goldberg, 1. H. & Rabinowitz, M. (1962). Science, 136, 315. Guthrie, G. D. & Sinsheimer, R. L. (1960) . J. il'I ol. R iolo 2, 297. Hurwitz, J., Furth, J. J., Malamy, M. & Alexander, 1\'1. (1962) . Proc. Nat. Acad. sa; Wash. 48, 1222. Kirk, J. M. (1960). Biochim , biophys. Acta, 42, 167. Levinthal, C., Keynan, A. & Higa, A. (1962). Proc . Nat. Acad. s«, Wash. 48, 1631. R eich, E., Franklin, R. M., Shatkin, A. J. & Tatum, E. L. (1961). S cience, 134, 556. Reich, E ., Franklin, R. M., Shatkin, A. J . & Tatum, E. L. (1962) . Proc . Nat. Acad. sa; Wash. 48, 1238. Sinsheimer, R. L., Starman, B., Nagler, C. & Guthrie, S. (1962). J. Mol. Riol. 4, 142.