VIROLOGY
(1976)
69, 332-335
Host Membrane
Lipid Synthesis Is Not Required T4 Infection
WILLIAM Department
of Molecular
Biophysics
D. NUNN’
AND
and Biochemistry, Connecticut Accepted
July
JOHN
for Successful
E. CRONAN,
Yale University 06510
School
Phage
JR. of Medicine,
New
Haven,
25,1975
Phage T4 infection of Escherichiu coli during the absence of membrane lipid synthesis was examined. Under these conditions the phage completed a productive lytic cycle. These results indicate that host de nova lipid synthesis is not a co-requisite for successful phage infection.
In 1971, Cronan and Vagelos (1) reported that infection by bacteriophage T4 of a mutant of Escherichia coli containing a temperature-sensitive defect in phospholipid synthesis was abortive at the restrictive temperature. These authors found that T4 infection of the mutant was normal at the permissive temperature but unsuccessful at the nonpermissive temperature due to premature lysis of the infected cell (I). These experiments were performed with a mutant of E. coli previously characterized as containing a single mutation resulting in a thermolabile glycerol-3phosphate acyltransferase, the first enzyme in the phospholipid biosynthetic pathway Cz). Since the mutant, designated as pZsA, is incapable of synthesizing phospholipid at the restrictive temperature, the authors interpreted their results as indicating that de nouo phospholipid synthesis is a necessary co-requisite for the successful infection of E. coli by phage T4. However, recent studies by Glaser and co-workers (3, 4) indicate that the phenotype of the plsA mutants at nonpermissive temperatures is more complex than originally perceived. When these mutants are shifted to temperatures between 35 and 37.5” they cease growth and phospholipid synthesis but continue to synthesize macromolecules and ATP at a normal rate Q-4). ’ Present address: Department of Molecular Biology and Biochemistry, University of California, Irvine, Calif. 92664. 332 Copy-right 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
However, upon shift to temperatures above 38” the plsA mutants abruptly and dramatically curtail macromolecular and ATP synthesis (3, 4). Since Glaser et al. (4) have recently shown that adenylate kinase as well as this acyltransferase is thermolabile in the pZsA mutants, the latter results can be explained as being due to the simultaneous inactivation of these enzymes at temperatures above 38”. Although this phenomenon is not yet understood, the present hypothesis (4) suggests that the adenylate kinase and sn-glycerol3-phosphate acyltransferase may have interacting and/or shared structural components. Since the experiments of Cronan and Vagelos (1) were performed at 42”, it seems likely that ATP and macromolecular synthesis as well as phospholipid synthesis were inhibited in their experiments. The complex behavior of the pZsA mutants makes these strains difficult to use in physiological experiments. Therefore, we have used two other mutants in phospholipid synthesis in order to reassess the dependence of T4 infection on host membrane lipid synthesis. Both classes of mutants require glycerol (or sn-glycerolS-phosphate) for growth. Strain BB26-36 has a defect in the plsB gene which results in a sn-glycerol-3-phosphate acyltransferase with a K, for snglycerolS-phosphate that is lo-fold higher than normal (5, 6). Since the endogenous supply of sn-glycerol-3-phosphate in this
333
SHORT COMMUNICATIONS
strain is insufficient to allow normal phospholipid synthesis and growth, it must be supplemented with exogenous glycerol and/or sn-glycerol-3-phosphate (6). Following glycerol deprival, strain BB26-36 continues to grow for about onehalf generation (Fig. la), although phospholipid synthesis is immediately reduced by more than 90% (Fig. lb). In addition, the incorporation of [3H]uracil into RNA and the incorporation of [3H]isoleucine into protein continued at about 50-70% of the normal rate for about one-half generation following glycerol starvation (Table 1). In view of the above observations, we initiated phage T4 infection of strain BB26-36 during a period following glycerol deprivation in which cellular physiology, excepting phospholipid synthesis, appears fairly normal. We find that lysis of T4infected, glycerol-starved cultures of BB26-36 occurs with kinetics identical to those of unstarved cultures and the phage progeny yielded by the glycerol-starved cultures was 50-60% of that of unstarved cultures (Fig. 2). These results show that although phospholipid synthesis is inhibited by greater than 90%, the infection of the host strain, BB26-36, by phage T4 is successful. The lower yield of phage progeny from the glycerol-starved host can probably be attributed to the reduced rate of protein synthesis (Table 1). We have also performed a similar experiment with another glycerol auxotroph, strain BB20-14, in order to substantiate further the above findings. Strain BB20-14 has a mutation in the gpsA gene which codes for the biosynthetic glycerol-3-phosphate dehydrogenase (6, 7), and therefore it is deficient in sn-glycerol-3-phosphate synthesis. Thus, this strain, like strain BB26-36, requires exogenously supplied glycerol (or sn-glycerol-3-phosphate) for growth. When glycerol was removed, growth of this strain continued for one generation (Fig. 3a) whereas phospholipid synthesis was immediately inhibited by greater than 90% (Fig. 3b). The incorporation of 13Hlisoleucine into protein and the incorporation of 13H]uracil into RNA continue at approximately 70% of the normal rate for one generation after glycerol star-
;
40 0
I
I
20
40
Minutes
: 2 2
I ., l ..,*-.-. 01 0
IS
I
I
60
afler
-0 30
90
glycerol
I
I20
-glycerol
-a-*-r 45
/
100
starvation
I 60
75
90
Minutes
1. Effect of glycerol starvation on growth and phospholipid synthesis in strain BB26-36. A culture of BB26-36 was grown at 37” in low-phosphate medium (6) supplemented with casamino acids (0.05%), glucose (0.4%) and glycerol (0.02%). Cell growth was determined at 540 nm in a Klett-Summerson calorimeter [one Klett Unit (KU) equals 5 x lo8 cells/ml]. Upon reaching a cell density of 2.5 x 1Oa cells/ml, the bacteria were harvested by tiltration, washed once, resuspended in the same medium minus glycerol, and divided into two equal portions. One portion was supplemented with glycerol (0.02%) and the other portion was left unsupplemented. The cultures were then further incubated at 37”. The rate of phospholipid synthesis was determined at the indicated times by briefly labeling 1 ml of cells from each culture with 40 &i of 32P, (40 &i/pmole) for 5 min as previously described (6, 8). The lipid was extracted into chloroform-methanol (1:2), and the incorporated label was extracted and quantitated as described by Nunn and Cronan (9). At the time indicated by the arrow in Fig. lb, a portion of the cells from each culture was infected with phage T4B (see Fig. 2). FIG.
vation (data not shown). Infection of the glycerol-starved culture of BB20-14 by phage T4 was successful, and the phage
334
SHORT COMMUNICATIONS TABLE RATES
OF ISOTOPE
RIBONUCLEIC BY E.
1
INCORPORATION ACID
PROTEIN
BB26-36”
coli
Growth conditions
AND
progeny yield was approximately 70% of that of the uninfected control culture (Fig. 4).
INTO
Time (min)
13H1uracil incorporation”
13Hlisoleucine incorg;.i
15 30 45 15 30 45
111 124 115 92 70 38
80 75 83 57 49 31
Plus glycerol Minus glycerol
a The cultures were treated as described in the legend to Fig. 1 except that, to assay for ribonucleic acid and protein synthesis, l-ml aliquots were removed from each culture at the indicated times and added to tubes containing 0.5 @i of [SH1uracil (50 Ci/mmole) and/or 1.0 @Zi of 13H]isoleucine (1.0 Ci/mole), respectively. After a 5-min incubation at 37”, 1.0 ml of 10% trichloroacetic acid was added. The samples were assayed as previously described (8). * Counts per minute per 5 X lo6 cells. IO9
I
a
1 so’
8820-14
401
I 0
20
1
so
40 Minutes
0
dtsr
so qtycerot
I 120
100 starvation
I
I
I
I
8826-36
FIG. 3. Effect of glycerol starvation on growth and phospholipid synthesis in strain BB20-14. The growth conditions and experimental protocol are identical to that described in Fig. 1 except that strain BB20-14 was used. The rate of phospholipid synthesis was determined as described in Fig. 1. I
T
1
I
8820-14
+ glycerol
l \.-,* /
‘.
-glycerol
IO’ 0 Minutes
after
infection
FIG. 2. T4B growth in strain BB26-36 in the presence and absence of glycerol. Single-step growth experiments as described by Adams (10) were performed by adsorbing aliquots of 2.5-3.0 x lOa cells/ml from the glycerol-starved and unstarved cultures of strain BB26-36 (see Fig. 11 with T4B phage at a multiplicity of 0.05-0.10. Following adsorption for 5 min the mixtures (at the time indicated by the arrow in Fig. lb) were diluted by at least 10VJin the same medium (see Fig. 1) with and without glycerol, respectively. In order to determine the number of infective centers per milliliter, samples were removed at the indicated time intervals and plated on strain 8 (1, II) at 37”.
. :;
I
I07 0
10
20 Minutes
30 after
40 SO infection
60
FIG. 4. T4B growth in strain BB20-14 in the presence and absence of glycerol. Single-step growth experiments were performed on strain BB20-14 in the presence and absence of glycerol as described in Fig. 2.
335
SHORT COMMUNICATIONS
In order to preclude the possibility that phage T4 alters the phenotype of strains BB20-14 and BB26-36 such that phospholipid synthesis is not inhibited when they are starved for glycerol, we compared the rates of phospholipid synthesis in glycerolstarved infected and uninfected cultures. We found that the rate of lipid synthesis in a starved infected culture of strain BB2014 was inhibited to at least the same degree as lipid synthesis in the starved uninfected culture (Table 2). Comparable results were obtained when strain BB26-36 was used (data not shown). These results indicate that (as expected) T4 infection has no effect on the phenotype of either type of glycerol auxotroph. In conclusion, a drastic reduction in the TABLE
Plus glycerol Minus glycerolc
Time after infection (min)
3*P, incorporated (cpm/lOB cells) * InfecUnintedb fected
5 15
424
30 5
255
15
4.6 4.3
30
ACKNOWLEDGMENTS We would like to thank George F. Sprague and Tapas K. Ray for helpful discussions and comments on the manuscript. W. D. Nunn was supported by an N.I.H. Postdoctoral Fellowship (No. l-F02-AI-55,327). J. E. Cronan, Jr., is a United States Public Health Research Career Development Awardee (Na. GM 70,411). This investigation was supported by grants from the United States Public Health (No. AI-10186) and the National Science Foundation (No. GB-32063).
2
RATE OF PHOEPHOLIPID SYNTHESIS IN INFECTED AND UNINFECTED CULTURES OF E. coli BB20-14”
Growth conditions
rate of phospholipid synthesis in both these mutant strains does not prevent phage T4 from completing a productive cycle. Consequently, our results indicate that host de nouo membrane phospholipid synthesis is not a necessary co-requisite for successful infection by phage T4.
292 9.4
604 605 565 11.4 6.4 6.7
o The growth conditions and experimental protocol are identical to that described in Fig. 1 except that strain BB20-14 was used. The rate of phospholipid synthesis was determined as described in Fig. 1. b Cultures were infected with T4B phage at a multiplicity of 5. c Phage infection of strain BB20-14 was initiated 15 min after glycerol starvation.
REFERENCES 1. CRONAN, J. E., JR., and VAGELOS, P. R., Virology 43,412-421 (1971). 2. CRONAN, J. E., RAY, T. K. and VAGELOS, P. R., Proc. Nat. Acad. Sci. USA 65,737-744 (1970). 3. GLASER, M., BAUER, W. H., BELL, R. M., and VAGELOB, P. R., Proc. Nat. Acad. Sci. USA 70,385-389 (1973). 4. GLASER, M., NULTY, W., and VAGEL~S, P. R., J. Bacterial., 123, 128-136 (1975). 5. CRONAN, J. E., JR., and BELL, R. M., J. Bacteriol. 120, 227-233 (1974). 6. BELL, R. M., J. Bacterial. 117,1065-1076 (1973). 7. CRONAN, J. E., JR., and BELL, R. M., J. Bacteriol. 118, 598-605 (1974). 8. NIJNN, W. D., Biochim. Biophys. Acta 380,403413 (1975). 9. NUNN, W. D., and CRONAN, J. E., JR., J. Biol. Chem. 249, 3994-3996 (1974). 10. ADAMS, M. A., “The Bacteriophages.” WileyInterscience, New York (1959). 21. HAYASHI, S., KOCH, J. P., and LIN, E. C. C., J. Biol. Chem. 239,3098-3105 (1964).
(1976)
69, 332-335
Host Membrane
Lipid Synthesis Is Not Required T4 Infection
WILLIAM Department
of Molecular
Biophysics
D. NUNN’
AND
and Biochemistry, Connecticut Accepted
July
JOHN
for Successful
E. CRONAN,
Yale University 06510
School
Phage
JR. of Medicine,
New
Haven,
25,1975
Phage T4 infection of Escherichiu coli during the absence of membrane lipid synthesis was examined. Under these conditions the phage completed a productive lytic cycle. These results indicate that host de nova lipid synthesis is not a co-requisite for successful phage infection.
In 1971, Cronan and Vagelos (1) reported that infection by bacteriophage T4 of a mutant of Escherichia coli containing a temperature-sensitive defect in phospholipid synthesis was abortive at the restrictive temperature. These authors found that T4 infection of the mutant was normal at the permissive temperature but unsuccessful at the nonpermissive temperature due to premature lysis of the infected cell (I). These experiments were performed with a mutant of E. coli previously characterized as containing a single mutation resulting in a thermolabile glycerol-3phosphate acyltransferase, the first enzyme in the phospholipid biosynthetic pathway Cz). Since the mutant, designated as pZsA, is incapable of synthesizing phospholipid at the restrictive temperature, the authors interpreted their results as indicating that de nouo phospholipid synthesis is a necessary co-requisite for the successful infection of E. coli by phage T4. However, recent studies by Glaser and co-workers (3, 4) indicate that the phenotype of the plsA mutants at nonpermissive temperatures is more complex than originally perceived. When these mutants are shifted to temperatures between 35 and 37.5” they cease growth and phospholipid synthesis but continue to synthesize macromolecules and ATP at a normal rate Q-4). ’ Present address: Department of Molecular Biology and Biochemistry, University of California, Irvine, Calif. 92664. 332 Copy-right 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
However, upon shift to temperatures above 38” the plsA mutants abruptly and dramatically curtail macromolecular and ATP synthesis (3, 4). Since Glaser et al. (4) have recently shown that adenylate kinase as well as this acyltransferase is thermolabile in the pZsA mutants, the latter results can be explained as being due to the simultaneous inactivation of these enzymes at temperatures above 38”. Although this phenomenon is not yet understood, the present hypothesis (4) suggests that the adenylate kinase and sn-glycerol3-phosphate acyltransferase may have interacting and/or shared structural components. Since the experiments of Cronan and Vagelos (1) were performed at 42”, it seems likely that ATP and macromolecular synthesis as well as phospholipid synthesis were inhibited in their experiments. The complex behavior of the pZsA mutants makes these strains difficult to use in physiological experiments. Therefore, we have used two other mutants in phospholipid synthesis in order to reassess the dependence of T4 infection on host membrane lipid synthesis. Both classes of mutants require glycerol (or sn-glycerolS-phosphate) for growth. Strain BB26-36 has a defect in the plsB gene which results in a sn-glycerol-3-phosphate acyltransferase with a K, for snglycerolS-phosphate that is lo-fold higher than normal (5, 6). Since the endogenous supply of sn-glycerol-3-phosphate in this
333
SHORT COMMUNICATIONS
strain is insufficient to allow normal phospholipid synthesis and growth, it must be supplemented with exogenous glycerol and/or sn-glycerol-3-phosphate (6). Following glycerol deprival, strain BB26-36 continues to grow for about onehalf generation (Fig. la), although phospholipid synthesis is immediately reduced by more than 90% (Fig. lb). In addition, the incorporation of [3H]uracil into RNA and the incorporation of [3H]isoleucine into protein continued at about 50-70% of the normal rate for about one-half generation following glycerol starvation (Table 1). In view of the above observations, we initiated phage T4 infection of strain BB26-36 during a period following glycerol deprivation in which cellular physiology, excepting phospholipid synthesis, appears fairly normal. We find that lysis of T4infected, glycerol-starved cultures of BB26-36 occurs with kinetics identical to those of unstarved cultures and the phage progeny yielded by the glycerol-starved cultures was 50-60% of that of unstarved cultures (Fig. 2). These results show that although phospholipid synthesis is inhibited by greater than 90%, the infection of the host strain, BB26-36, by phage T4 is successful. The lower yield of phage progeny from the glycerol-starved host can probably be attributed to the reduced rate of protein synthesis (Table 1). We have also performed a similar experiment with another glycerol auxotroph, strain BB20-14, in order to substantiate further the above findings. Strain BB20-14 has a mutation in the gpsA gene which codes for the biosynthetic glycerol-3-phosphate dehydrogenase (6, 7), and therefore it is deficient in sn-glycerol-3-phosphate synthesis. Thus, this strain, like strain BB26-36, requires exogenously supplied glycerol (or sn-glycerol-3-phosphate) for growth. When glycerol was removed, growth of this strain continued for one generation (Fig. 3a) whereas phospholipid synthesis was immediately inhibited by greater than 90% (Fig. 3b). The incorporation of 13Hlisoleucine into protein and the incorporation of 13H]uracil into RNA continue at approximately 70% of the normal rate for one generation after glycerol star-
;
40 0
I
I
20
40
Minutes
: 2 2
I ., l ..,*-.-. 01 0
IS
I
I
60
afler
-0 30
90
glycerol
I
I20
-glycerol
-a-*-r 45
/
100
starvation
I 60
75
90
Minutes
1. Effect of glycerol starvation on growth and phospholipid synthesis in strain BB26-36. A culture of BB26-36 was grown at 37” in low-phosphate medium (6) supplemented with casamino acids (0.05%), glucose (0.4%) and glycerol (0.02%). Cell growth was determined at 540 nm in a Klett-Summerson calorimeter [one Klett Unit (KU) equals 5 x lo8 cells/ml]. Upon reaching a cell density of 2.5 x 1Oa cells/ml, the bacteria were harvested by tiltration, washed once, resuspended in the same medium minus glycerol, and divided into two equal portions. One portion was supplemented with glycerol (0.02%) and the other portion was left unsupplemented. The cultures were then further incubated at 37”. The rate of phospholipid synthesis was determined at the indicated times by briefly labeling 1 ml of cells from each culture with 40 &i of 32P, (40 &i/pmole) for 5 min as previously described (6, 8). The lipid was extracted into chloroform-methanol (1:2), and the incorporated label was extracted and quantitated as described by Nunn and Cronan (9). At the time indicated by the arrow in Fig. lb, a portion of the cells from each culture was infected with phage T4B (see Fig. 2). FIG.
vation (data not shown). Infection of the glycerol-starved culture of BB20-14 by phage T4 was successful, and the phage
334
SHORT COMMUNICATIONS TABLE RATES
OF ISOTOPE
RIBONUCLEIC BY E.
1
INCORPORATION ACID
PROTEIN
BB26-36”
coli
Growth conditions
AND
progeny yield was approximately 70% of that of the uninfected control culture (Fig. 4).
INTO
Time (min)
13H1uracil incorporation”
13Hlisoleucine incorg;.i
15 30 45 15 30 45
111 124 115 92 70 38
80 75 83 57 49 31
Plus glycerol Minus glycerol
a The cultures were treated as described in the legend to Fig. 1 except that, to assay for ribonucleic acid and protein synthesis, l-ml aliquots were removed from each culture at the indicated times and added to tubes containing 0.5 @i of [SH1uracil (50 Ci/mmole) and/or 1.0 @Zi of 13H]isoleucine (1.0 Ci/mole), respectively. After a 5-min incubation at 37”, 1.0 ml of 10% trichloroacetic acid was added. The samples were assayed as previously described (8). * Counts per minute per 5 X lo6 cells. IO9
I
a
1 so’
8820-14
401
I 0
20
1
so
40 Minutes
0
dtsr
so qtycerot
I 120
100 starvation
I
I
I
I
8826-36
FIG. 3. Effect of glycerol starvation on growth and phospholipid synthesis in strain BB20-14. The growth conditions and experimental protocol are identical to that described in Fig. 1 except that strain BB20-14 was used. The rate of phospholipid synthesis was determined as described in Fig. 1. I
T
1
I
8820-14
+ glycerol
l \.-,* /
‘.
-glycerol
IO’ 0 Minutes
after
infection
FIG. 2. T4B growth in strain BB26-36 in the presence and absence of glycerol. Single-step growth experiments as described by Adams (10) were performed by adsorbing aliquots of 2.5-3.0 x lOa cells/ml from the glycerol-starved and unstarved cultures of strain BB26-36 (see Fig. 11 with T4B phage at a multiplicity of 0.05-0.10. Following adsorption for 5 min the mixtures (at the time indicated by the arrow in Fig. lb) were diluted by at least 10VJin the same medium (see Fig. 1) with and without glycerol, respectively. In order to determine the number of infective centers per milliliter, samples were removed at the indicated time intervals and plated on strain 8 (1, II) at 37”.
. :;
I
I07 0
10
20 Minutes
30 after
40 SO infection
60
FIG. 4. T4B growth in strain BB20-14 in the presence and absence of glycerol. Single-step growth experiments were performed on strain BB20-14 in the presence and absence of glycerol as described in Fig. 2.
335
SHORT COMMUNICATIONS
In order to preclude the possibility that phage T4 alters the phenotype of strains BB20-14 and BB26-36 such that phospholipid synthesis is not inhibited when they are starved for glycerol, we compared the rates of phospholipid synthesis in glycerolstarved infected and uninfected cultures. We found that the rate of lipid synthesis in a starved infected culture of strain BB2014 was inhibited to at least the same degree as lipid synthesis in the starved uninfected culture (Table 2). Comparable results were obtained when strain BB26-36 was used (data not shown). These results indicate that (as expected) T4 infection has no effect on the phenotype of either type of glycerol auxotroph. In conclusion, a drastic reduction in the TABLE
Plus glycerol Minus glycerolc
Time after infection (min)
3*P, incorporated (cpm/lOB cells) * InfecUnintedb fected
5 15
424
30 5
255
15
4.6 4.3
30
ACKNOWLEDGMENTS We would like to thank George F. Sprague and Tapas K. Ray for helpful discussions and comments on the manuscript. W. D. Nunn was supported by an N.I.H. Postdoctoral Fellowship (No. l-F02-AI-55,327). J. E. Cronan, Jr., is a United States Public Health Research Career Development Awardee (Na. GM 70,411). This investigation was supported by grants from the United States Public Health (No. AI-10186) and the National Science Foundation (No. GB-32063).
2
RATE OF PHOEPHOLIPID SYNTHESIS IN INFECTED AND UNINFECTED CULTURES OF E. coli BB20-14”
Growth conditions
rate of phospholipid synthesis in both these mutant strains does not prevent phage T4 from completing a productive cycle. Consequently, our results indicate that host de nouo membrane phospholipid synthesis is not a necessary co-requisite for successful infection by phage T4.
292 9.4
604 605 565 11.4 6.4 6.7
o The growth conditions and experimental protocol are identical to that described in Fig. 1 except that strain BB20-14 was used. The rate of phospholipid synthesis was determined as described in Fig. 1. b Cultures were infected with T4B phage at a multiplicity of 5. c Phage infection of strain BB20-14 was initiated 15 min after glycerol starvation.
REFERENCES 1. CRONAN, J. E., JR., and VAGELOS, P. R., Virology 43,412-421 (1971). 2. CRONAN, J. E., RAY, T. K. and VAGELOS, P. R., Proc. Nat. Acad. Sci. USA 65,737-744 (1970). 3. GLASER, M., BAUER, W. H., BELL, R. M., and VAGELOB, P. R., Proc. Nat. Acad. Sci. USA 70,385-389 (1973). 4. GLASER, M., NULTY, W., and VAGEL~S, P. R., J. Bacterial., 123, 128-136 (1975). 5. CRONAN, J. E., JR., and BELL, R. M., J. Bacteriol. 120, 227-233 (1974). 6. BELL, R. M., J. Bacterial. 117,1065-1076 (1973). 7. CRONAN, J. E., JR., and BELL, R. M., J. Bacteriol. 118, 598-605 (1974). 8. NIJNN, W. D., Biochim. Biophys. Acta 380,403413 (1975). 9. NUNN, W. D., and CRONAN, J. E., JR., J. Biol. Chem. 249, 3994-3996 (1974). 10. ADAMS, M. A., “The Bacteriophages.” WileyInterscience, New York (1959). 21. HAYASHI, S., KOCH, J. P., and LIN, E. C. C., J. Biol. Chem. 239,3098-3105 (1964).
