JouRNAL oF BACTERIOLOGY, Mar. 1976, p. 1229-1231 Copyright 0 1976 American Society for Microbiology
Vol. 125, No. 3 Printed in U.SA.
Translocation in Bacillus subtilis: Irreversible Inactivation of Elongation Factor G and Viability of a TemperatureSensitive Mutant YAIR AHARONOWITZI* AiN ELIORA Z. RON Department of Microbiology, The (jeorge S. Wise Center for Life Sciences, Tel Aviv University, Ramat Aviv, Israel
Received for publication 10 October 1975
A temperature-sensitive mutant of Bacillus subtilis 168 lost its viability irreversibly when grown at temperatures higher than 50 C. It is suggested that this loss of viability is due to irreversible alteration of elongation factor G activity, which was shown in vitro by two different assay methods.
In a previous communication (1) we described the isolation, partial characterization, and genetic mapping of a mutant of Bacillus subtilis blocked in its ability to synthesize protein at high temperatures. The mutation responsible for the temperature sensitivity is closely linked to the str and fus markers and is probably in elongation factor G (EF-G). A similar mutant was isolated independently by Kimura et al. (3). The studies presented in this communication provide evidence that the purified EF-G of the temperature-sensitive mutant is, indeed, temperature sensitive in vitro and deal with the effect of this mutation on the growth and viability of the mutant. Mutant 168 ts-3 ofB. subtilis does not grow at temperatures higher than 50 C whereas the wild type grows up to 55 C. Moreover, exposure to temperatures above 50 C results in immediate cell death (Fig. 1). Cell death is not due to a deficiency of a growth requirement caused by the high temperature since it occurs in rich growth media as well as in minimal ones (Fig. 2). Furthermore, killing does not depend on activity or cycling of ribosomes since it is not prevented by the addition of chloramphenicol at concentrations that inhibit protein synthesis by 98% (Fig. 2). The following experiments suggest that killing is due to the irreversible thermal inactivation of EF-G. EF-G was purified as previously described (2), and its activity in the mutant and in the wild type was assayed by the use of two methods. In the first method, EF-G activity was measured by assaying ribosome-dependent hydrolysis of guanosine 5'-triphosphate (GTP) (guanosine triphosphatase [GTPase] activity). This reaction is uncoupled to peptide chain I Present address: Department of Nutritional and Food
elongation or to translocation but depends on EF-G activity (4). GTPase activity was measured in mutant and wild type after preincubation at 55 C. The results presented in Fig. 3 indicate that, after 20 min of preincubation at 55 C, there was total loss of GTPase activity of EF-G from the mutant, whereas the wild-type factor still retained 50% of its activity.
n IL
Q
TIME (men)
FIG. 1. Viability of mutant and wild-type strains at 50 C. Cells were grown at 37 C with vigorous aeration in supplemented salt solution (5) containing tryptophan. When the culture contained about 108 colony-forming units (CFU) per ml, it was transferred to 50 C and samples were removed at interScience, Massachusetts Institute of Technology, Cam- vals, diluted, and plated on nutrient agar (Difco) bridge, Mass. 02139. plates. 1229
1230
NOTES
J. BACTERIOL.
500
F ~168 50 \
100
I-50 IL
w
U. l0
10
20
TIME (min) AT 55-C
20
40
60
80
TIME (min)
FIG. 2. Effect of chloramphenicol on viability of the mutant strain at 37 and 52 C. A culture of 168 ts3, growing exponentially in nutrient broth containing tryptophan (50 pg/ml), was divided into four parts. Two parts were grown at 37 C with (A) and without (A) chloramphenicol (50 pglml). The other two parts were transferred to 52 C and were grown with (a) and without (0) chloramphenicol. CFU, Colony-forming units.
In the second method, purified preparations preincubated at 55 C and assayed for EFG activity by the peptidyl-puromycin reaction (2). In this reaction, translocation was followed by the EF-G-dependent increase in the synthesis of peptidyl-[3H]puromycin, using wild-type ribosomes carrying in vivo-bound peptidyltransfer ribonucleic acid ("native" peptidetransfer ribonucleic acid) as peptide donors. After 10 min of preincubation at 55 C, there was 80% loss of the EF-G activity of the mutant, whereas wild-type extracts retained their original activity (Fig. 4). The difference in the effect of high temperatures on the wild-type extract, shown by the two reactions, might suggest that different active sites are involved. These two sites may well have a different response to high temperatures. were
FIG. 3. Effect of temperature on EF-G from mutant and wild-type strains. EF-G was purified from cell extracts by Sepharose 6B and diethylaminoethylSephadex chromatography as previously described (2). EF-G preparations were preincubated at 55 C and transferred to ice at intervals. EF-G was determined by assaying for ribosome-dependent GTPase (2). Reaction mixtures were in a total volume of 0.13 ml and contained tris(hydroxymethyl)aminomethane-hydrochloride, pH 7.8, 10 mM; NH4C1, 100 mM; magnesium acetate, 10 mM; 2-mercaptoethanol, 6 mM; y[32P]GTP, 945 pmol (321 counts/min per pmol); and 1.38 absorbance at 260 nm units of wildtype ribosomes, previously washed three times in buffer containing 1 N NH4Cl. EF-G of wild-type strain and mutant 168 ts-3 were 1.15 and 1.2 pglml, respectively. After preincubation of EF-G extracts for the indicated times, the reaction mixtures were incubated 10 min at 37 C. All readings were corrected by subtracting control activities of reaction mixtures in which EF-G was eliminated.
The results of Fig. 3 and Fig. 4 indicate that mutant 168-ts-3 has an altered, temperaturesensitive EF-G activity. Moreover, since in both assays EF-G activity was measured after incubation at high temperatures and not during such incubation, the results indicate that the thermal inactivation of EF-G is irreversible. The irreversibility of the thermal inactivation of EF-G provides an explanation for the loss of viability that occurs at the higher temperature. This explanation is supported by the findings that the rate of EF-G inactivation at 55 C is very similar to the rate at which viability is lost. In addition, this rate might be ex-
NOTES
VOL. 125, 1976
1231
pected to be independent of growth conditions or cellular protein synthesis, as was, indeed, the case (Fig. 2). Apparently, after exposure to high temperatures, the thermally inactivated EF-G cannot be replaced by functional EF-G since for such replacement an active proteinsynthesizing machinery would be required.
5; 4% 0
5
10
TIME (min) AT 55-C
FIG. 4. Effect of temperature on the formation of peptidyl-[3H]puromycin by mutant and wild-type extracts. EF-G preparations were obtained and treated as described in the legend to Fig. 3, except their activity was assayed by determining the EF-G-dependent formation of peptidyl-[3H)puromycin (2): EF-G (1.2 pg/ml) was incubated with 6.9 absorbance at 260 nm units of wild-type ribosomes washed three times in buffer containing 1 N CH4Cl, 10 mM tris(hydroxymethyl)aminomethane-hydrochloride, pH
LITERATURE CITED 1. Aharonowitz, Y., and E. Z. Ron. 1972. A temperature sensitive mutant in Bacillus subtilis with an altered elongation factor G. Mol. Gen. Genet. 119:131-138. 2. Aharonowitz, Y., and E. Z. Ron. 1975. Translocation in Bacillus subtilis: characterization of EF-G by peptidyl-[3H]puromycin synthesis. J. Bacteriol. 125:10741079. 3. Kimura, A., A. Muto, and S. Osawa. 1974. Control of stable RNA synthesis in a temperature-sensitive mutant of EF-G of Bacillus subtilis. Mol. Gen. Genet. 130:203-214. 4. Nishizuka, Y., and F. Lipmann. 1966. Comparison of guanosine triphosphate split and polypeptide synthesis with a purified E. coli system. Proc. Natl. Acad. Sci. U.S.A. 55:212-219. 5. Spizizen, J. 1958. Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proc. Natl. Acad. Sci. U.S.A. 44:1072-1078.
7.8, 100 mM NH4Cl, 10 mM magnesium acetate, 1 mM dithiothreitol, 0.6 mM GTP, and 238 pmol of [3H]puromycin (Radiochemical Center, Amersham, England). Total volume was 0.05 ml. The reaction was carried out at 37 C for 5 min. One hundred percent represents 1,800 counts/min (net EF-Gderived counts after subtraction of a control value of reaction carried by ribosomes + GTP).
Vol. 125, No. 3 Printed in U.SA.
Translocation in Bacillus subtilis: Irreversible Inactivation of Elongation Factor G and Viability of a TemperatureSensitive Mutant YAIR AHARONOWITZI* AiN ELIORA Z. RON Department of Microbiology, The (jeorge S. Wise Center for Life Sciences, Tel Aviv University, Ramat Aviv, Israel
Received for publication 10 October 1975
A temperature-sensitive mutant of Bacillus subtilis 168 lost its viability irreversibly when grown at temperatures higher than 50 C. It is suggested that this loss of viability is due to irreversible alteration of elongation factor G activity, which was shown in vitro by two different assay methods.
In a previous communication (1) we described the isolation, partial characterization, and genetic mapping of a mutant of Bacillus subtilis blocked in its ability to synthesize protein at high temperatures. The mutation responsible for the temperature sensitivity is closely linked to the str and fus markers and is probably in elongation factor G (EF-G). A similar mutant was isolated independently by Kimura et al. (3). The studies presented in this communication provide evidence that the purified EF-G of the temperature-sensitive mutant is, indeed, temperature sensitive in vitro and deal with the effect of this mutation on the growth and viability of the mutant. Mutant 168 ts-3 ofB. subtilis does not grow at temperatures higher than 50 C whereas the wild type grows up to 55 C. Moreover, exposure to temperatures above 50 C results in immediate cell death (Fig. 1). Cell death is not due to a deficiency of a growth requirement caused by the high temperature since it occurs in rich growth media as well as in minimal ones (Fig. 2). Furthermore, killing does not depend on activity or cycling of ribosomes since it is not prevented by the addition of chloramphenicol at concentrations that inhibit protein synthesis by 98% (Fig. 2). The following experiments suggest that killing is due to the irreversible thermal inactivation of EF-G. EF-G was purified as previously described (2), and its activity in the mutant and in the wild type was assayed by the use of two methods. In the first method, EF-G activity was measured by assaying ribosome-dependent hydrolysis of guanosine 5'-triphosphate (GTP) (guanosine triphosphatase [GTPase] activity). This reaction is uncoupled to peptide chain I Present address: Department of Nutritional and Food
elongation or to translocation but depends on EF-G activity (4). GTPase activity was measured in mutant and wild type after preincubation at 55 C. The results presented in Fig. 3 indicate that, after 20 min of preincubation at 55 C, there was total loss of GTPase activity of EF-G from the mutant, whereas the wild-type factor still retained 50% of its activity.
n IL
Q
TIME (men)
FIG. 1. Viability of mutant and wild-type strains at 50 C. Cells were grown at 37 C with vigorous aeration in supplemented salt solution (5) containing tryptophan. When the culture contained about 108 colony-forming units (CFU) per ml, it was transferred to 50 C and samples were removed at interScience, Massachusetts Institute of Technology, Cam- vals, diluted, and plated on nutrient agar (Difco) bridge, Mass. 02139. plates. 1229
1230
NOTES
J. BACTERIOL.
500
F ~168 50 \
100
I-50 IL
w
U. l0
10
20
TIME (min) AT 55-C
20
40
60
80
TIME (min)
FIG. 2. Effect of chloramphenicol on viability of the mutant strain at 37 and 52 C. A culture of 168 ts3, growing exponentially in nutrient broth containing tryptophan (50 pg/ml), was divided into four parts. Two parts were grown at 37 C with (A) and without (A) chloramphenicol (50 pglml). The other two parts were transferred to 52 C and were grown with (a) and without (0) chloramphenicol. CFU, Colony-forming units.
In the second method, purified preparations preincubated at 55 C and assayed for EFG activity by the peptidyl-puromycin reaction (2). In this reaction, translocation was followed by the EF-G-dependent increase in the synthesis of peptidyl-[3H]puromycin, using wild-type ribosomes carrying in vivo-bound peptidyltransfer ribonucleic acid ("native" peptidetransfer ribonucleic acid) as peptide donors. After 10 min of preincubation at 55 C, there was 80% loss of the EF-G activity of the mutant, whereas wild-type extracts retained their original activity (Fig. 4). The difference in the effect of high temperatures on the wild-type extract, shown by the two reactions, might suggest that different active sites are involved. These two sites may well have a different response to high temperatures. were
FIG. 3. Effect of temperature on EF-G from mutant and wild-type strains. EF-G was purified from cell extracts by Sepharose 6B and diethylaminoethylSephadex chromatography as previously described (2). EF-G preparations were preincubated at 55 C and transferred to ice at intervals. EF-G was determined by assaying for ribosome-dependent GTPase (2). Reaction mixtures were in a total volume of 0.13 ml and contained tris(hydroxymethyl)aminomethane-hydrochloride, pH 7.8, 10 mM; NH4C1, 100 mM; magnesium acetate, 10 mM; 2-mercaptoethanol, 6 mM; y[32P]GTP, 945 pmol (321 counts/min per pmol); and 1.38 absorbance at 260 nm units of wildtype ribosomes, previously washed three times in buffer containing 1 N NH4Cl. EF-G of wild-type strain and mutant 168 ts-3 were 1.15 and 1.2 pglml, respectively. After preincubation of EF-G extracts for the indicated times, the reaction mixtures were incubated 10 min at 37 C. All readings were corrected by subtracting control activities of reaction mixtures in which EF-G was eliminated.
The results of Fig. 3 and Fig. 4 indicate that mutant 168-ts-3 has an altered, temperaturesensitive EF-G activity. Moreover, since in both assays EF-G activity was measured after incubation at high temperatures and not during such incubation, the results indicate that the thermal inactivation of EF-G is irreversible. The irreversibility of the thermal inactivation of EF-G provides an explanation for the loss of viability that occurs at the higher temperature. This explanation is supported by the findings that the rate of EF-G inactivation at 55 C is very similar to the rate at which viability is lost. In addition, this rate might be ex-
NOTES
VOL. 125, 1976
1231
pected to be independent of growth conditions or cellular protein synthesis, as was, indeed, the case (Fig. 2). Apparently, after exposure to high temperatures, the thermally inactivated EF-G cannot be replaced by functional EF-G since for such replacement an active proteinsynthesizing machinery would be required.
5; 4% 0
5
10
TIME (min) AT 55-C
FIG. 4. Effect of temperature on the formation of peptidyl-[3H]puromycin by mutant and wild-type extracts. EF-G preparations were obtained and treated as described in the legend to Fig. 3, except their activity was assayed by determining the EF-G-dependent formation of peptidyl-[3H)puromycin (2): EF-G (1.2 pg/ml) was incubated with 6.9 absorbance at 260 nm units of wild-type ribosomes washed three times in buffer containing 1 N CH4Cl, 10 mM tris(hydroxymethyl)aminomethane-hydrochloride, pH
LITERATURE CITED 1. Aharonowitz, Y., and E. Z. Ron. 1972. A temperature sensitive mutant in Bacillus subtilis with an altered elongation factor G. Mol. Gen. Genet. 119:131-138. 2. Aharonowitz, Y., and E. Z. Ron. 1975. Translocation in Bacillus subtilis: characterization of EF-G by peptidyl-[3H]puromycin synthesis. J. Bacteriol. 125:10741079. 3. Kimura, A., A. Muto, and S. Osawa. 1974. Control of stable RNA synthesis in a temperature-sensitive mutant of EF-G of Bacillus subtilis. Mol. Gen. Genet. 130:203-214. 4. Nishizuka, Y., and F. Lipmann. 1966. Comparison of guanosine triphosphate split and polypeptide synthesis with a purified E. coli system. Proc. Natl. Acad. Sci. U.S.A. 55:212-219. 5. Spizizen, J. 1958. Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proc. Natl. Acad. Sci. U.S.A. 44:1072-1078.
7.8, 100 mM NH4Cl, 10 mM magnesium acetate, 1 mM dithiothreitol, 0.6 mM GTP, and 238 pmol of [3H]puromycin (Radiochemical Center, Amersham, England). Total volume was 0.05 ml. The reaction was carried out at 37 C for 5 min. One hundred percent represents 1,800 counts/min (net EF-Gderived counts after subtraction of a control value of reaction carried by ribosomes + GTP).
