Vol. 19, No. 2 Printed in U.S.A.
JOURNAL OF VIROLOGY, Aug. 1976, p. 446-456 Copyright C) 1976 American Society for Microbiology
Phospholipid Metabolism in Pseudomonas BAL-31 Infected with Lipid-Containing Bacteriophage PM2 D. L. DIEDRICH'* AND E. H. COTA-ROBLES Thimann Laboratory, Biology Board of Studies, University of California, Santa Cruz, California 95064 Received for publication 16 April 1976
Infection of Pseudomonas BAL-31 with the lipid-containing bacteriophage PM2 resulted in no detectable change in the rate of phosphatidylglycerol (PG) or phosphatidylethanolamine (PE) biosynthesis. An increase in the PG content of infected cultures was not seen until the cultures began to lyse, and this increase was in fact only a relative increase resulting from the extensive turnover of PE at the onset of culture lysis. Turnover studies revealed that the glycerol, phosphorus, fatty acid, and ethanolamine moieties of PE turned over simultaneously at the time of lysis, and therefore made it unlikely that there was a PE to PG conversion during the latent period of the phage. The lipid found in the bacteriophage did not reflect a preferential selection for lipid synthesized before or after infection, but in fact reflected the composition of the host membrane at the time the phage were assembled. The use of a modified medium that allowed the cultivation of Pseudomonas BAL-31 as a prototroph and resulted in reliable lysis times of infected cultures led us to the conclusion that PM2 infection effects little change in host phospholipid metabolism, and that there is sufficient PG in the host cytoplasmic membrane to account for a full burst of phage. As a result of the reliable lysis times that we have achieved, we concluded that certain metabolic events, i.e., PE turnover, are lytic phenomena and must not be confused with events relevant to the biosynthesis and maturation of the phage.
Bacteriophage PM2 is a lipid-containing marine bacteriophage that was isolated and described by Espejo and Canelo (6). Braunstein and Franklin (3) subsequently showed that the phospholipid composition of the phage was approximately the inverse of the host, Pseudomonas BAL-31. The host contains approximately 70% phosphatidylethanolamine (PE) and 25% phosphatidylglycerol (PG), whereas the phage is 28% PE and 65 to 68% PG. Thus it appeared a priori that phage infection led to a control of host phospholipid metabolism, and resulted in elevated levels of PG in the host membrane. This was supported by the findings of Braunstein and Franklin (3), who showed that the phosphatide composition of infected cultures changed to give rise to relatively higher levels of PG late in infection. Tsukagoshi and Franklin (11) subsequently reported that phage infection led to an increase in the rate of PG biosynthesis and a decrease in the rate of PE biosynthesis, thus accounting for a net increase in PG in the membrane of infected cells. This observation was based on differences in the net incorporation of 32P into PE and PG of infected cultures. I Present address: Department of Microbiology, University of Virginia School of Medicine, Charlottesville, Va.
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In apparent contrast the the findings of Tsukagoshi and Franklin (11), Snipes et al. (10) interpreted their data from a double-isotopelabel experiment to indicate that the polar portion of PE was cleaved and the resulting phosphatidic acid (PA) received a glycerol moiety, thus leading to a conversion of PE to PG. These conclusions are not mutually exclusive, since Tsukagoshi and Franklin (11) measured the net biosynthesis of PG and PE rather than the instantaneous rate of phosphatide biosynthesis. Thus, it is possible that they were measuring the result of the mechanism proposed by Snipes et al. (10). Further disagreement concerns the origin of the phospholipid of bacteriophage PM2. Espejo and Canelo (7) reported that the lipids of the phage were synthesized before infection, whereas Tsukagoshi and Franklin (11) reported that approximately 66% of the phage lipid was synthesized after infection. Snipes et al. (10) calculated that approximately half of the phage lipid was synthesized after infection. To resolve the anomalies outlined above, we undertook a more detailed study of the influence of PM2 infection on the metabolism of the glycerol, fatty acid, phosphorus, and polar moieties of infected cultures of Pseudomonas BAL-31. Since it has been shown that the turn-
VOL. 19, 1976
over of these moieties in phospholipids can occur at different rates (12), we were concerned about the validity of data derived from ratios of radioactivity in double-label experiments popularly employed in the above studies. Furthermore, considerable variations in the lysis times of infected cultures were reported within the individual studies. This made it difficult to distinguish data describing postlysis events from events relevant to the maturation of the phage. MATERIALS AND METHODS Cultivation of Pseudomonas BAL-31. Cultures of Pseudomonas BAL-31 were grown on TES-minimal medium. This medium contained, per liter of doubledistilled water: 0.7 g of KCl, 1.1 g of NH4Cl, 1.5 g of CaCl2 2H2O, 12.0 g of MgSO4 * 7H2O, 26.0 g of NaCl, 25mg of KH2PO4, 2.5 g of glucose, and 11.45 g of TES [N - tris(hydroxymethyl)methyl - 2 - aminoethanesulfonic acid] buffer. The medium was adjusted to pH 7.2 with 1 N NaOH and filter-sterilized through 0.22-pm membrane filters (Millipore Corp.). This medium is a TES buffer modification of the medium of Franklin et al. (9). Growth and preparation of bacteriophage PM2. Exponentially growing cultures of Pseudomonas BAL-31 at 2 x 108 to 4 x 108 viable cells per ml were infected with bacteriophage PM2 at multiplicities carefully controlled not to exceed 10 PFU per viable cell. Lysates were processed promptly by two centrifugations at 10,400 x g for 20 min to remove cell debris. The low-speed lysate supernatants were centrifuged at 50,000 x g for 1.5 h at 5°C. The pelleted phage were allowed to disaggregate for several hours in NTC buffer at 5°C. NTC buffer (9) was modified to contain 0.05 M TES buffer instead of Tris. The resuspended pellets were subjected to two more low-speed centrifugations to remove cell debris, and the phage-containing supernatants were filtered through 0.22-ttm filters into sterile containers for storage at 5°C. Bacteriophage prepared in this manner were used for most experiments. Some experiments required more extensive purification of the phage. This was accomplished by employing sucrose density gradients. Linear sucrose gradients were prepared from 20 to 35% sucrose (wt/ vol) in NTC buffer. The gradients were run at 20°C in a Beckman SW27 rotor at 25,000 rpm for 3 h. The phage band was in the bottom one-third of the tube. The phage in sucrose were dialyzed for 24 h against several changes of NTC buffer at 5°C. Bacteriophage PM2 was assayed by the doublelayer plating technique employing AMS-nutrient agar (6). Care was taken to ensure that the host culture employed for plating in this medium had a density of less than 4 x 108 viable cells per ml. The plating efficiency was observed to drop considerably at higher culture densities. Lipid techniques. Phospholipids were extracted, fractionated, and quantitated as previously described (5). Lipid phosphorus was determined by the Bartlett procedure (2). Isotope labeling of lipids. Cultures were labeled with H332PO4 (carrier free) at 2 ,uCi per ml, [23H]glycerol (specific activity of 8.81 Ci/mmol) at 4 -
BACTERIOPHAGE PM2 LIPIDS
447
ACi per ml, [1-_4C]sodium acetate (specific activity of 59 mCi/mmol) at 1 ,uCi per ml, or DL-[3-'4C]serine (specific activity of 12.5 mCi/mmol) at 0.5 ZCi per ml. The details of labeling are given for each experiment. The suitability of these radioactive isotopes for labeling the lipids of Pseudomonas BAL-31 was determined by subjecting labeled lipids to mild alkaline methanolysis (5) and determining the distribution of the label in the resultant glycerol phosphate esters and in the fatty acid moieties. Labeling cultures with acetate resulted in more than 95% of the lipid label appearing in the fatty acids. Labeling the cultures with serine or glycerol resulted in more than 95% of the lipid label appearing in the watersoluble glycerolphosphate ester. Rate of PE and PG biosynthesis. The rate of PE and PG biosynthesis was determined by removing samples from infected and uninfected cultures and pulsing the samples with 5 ,uCi of 32P per ml. Samples were removed in 60-s intervals (six samples) from the pulsed samples, and the lipids were extracted. After separation of the lipids by thin-layer chromatography (TLC), it was possible to examine the net incorporation of isotope into PE and PG over a 5-min interval. From these plots a rate was calculated for each 5-min pulse period, and these rates were plotted against time. Each point in Fig. 2 is plotted at the time corresponding to 2.5 min into the 5-min pulse. Phospholipid turnover studies. Exponentially growing cultures of Pseudomonas BAL-31 were diluted to an absorbance at 600 nm (A,,,,,) of 0.02 in fresh medium and allowed to grow in the abovementioned amount of isotope for six generations. These labeled, exponentially growing cultures were pelleted at 10,000 x g for 10 min at 5°C, washed twice in fresh medium, and resuspended to an A,,, of 0.2 in fresh prewarmed medium. The cultures were divided into two fractions, and one was infected with PM2 at a multiplicity of 10. The cultures were incubated at 28°C on a gyratory shaker at 250 rpm. Samples were periodically taken from the infected and uninfected cultures, and the lipids were extracted and fractionated. After TLC, the amount of radioactivity in each of the phosphatides was determined. For 32P-labeled lipids, the spots on the TLC plates were scraped directly into vials and counted with a liquid scintillation spectrometer. For 3H- and "4C-labeled lipids, the spots were collected into small columns, and the lipids were eluted with methanol into counting vials. Counting fluid was added after drying the lipids in the vials with dry nitrogen. The elution of the lipids from the columns was found to be complete. This was determined with 32P-labeled lipids by counting the eluted silica gel. Silica gel does not significantly quench 32p radioactivity. To examine the turnover of the diacyl and nonacylated glycerols of PG in an experiment in which the PG was labeled with [2-3H]glycerol, the PG was isolated as above. After drying under nitrogen, the PG was suspended in 1.0 ml of diethyl ether in screw-cap tubes. To the tubes was added 0.42 ml of water that contained 20 ,uM acetate buffer (pH 5.6), 40 ,uM CaCl2, and 1.6 mg of cabbage phospholipase D (Calbiochem). The reaction mixture was vigorously agitated and incubated at 25°C for 3 h. The ether
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DIEDRICH AND COTA-ROBLES
was removed with a stream of nitrogen, and 1.85 ml of water, 2.5 ml of methanol, and 2.5 ml of chloroform were added. The water-soluble unacylated glycerol from PG was recovered in the aqueous phase, and the resulting PA and unreacted PG were found in the organic phase. These were separated by TLC and quantitated. This procedure allowed for 95 to 98% hydrolysis of the PG. Lipid translocation. A culture of Pseudomonas BAL-31 was grown to an A,;. of 0.2 and divided into two cultures. One was infected with PM2 at a multiplicity of 10. After 30 min, both cultures received 2.5 gCi of [2-3H]gilycerol. At the times indicated, samples were removed and diluted into ice-cold 0.5 M NaCl. The cells were washed once in NaCl, and half of the sample was extracted of its lipids. The other half of the sample was processed by the procedure of Forsberg et al. (8) to separate the outer membrane from the cytoplasmic membrane. The resulting membrane fractions were then extracted of their lipids, and the net incorporation of radioactivity into each membrane could be determined.
RESULTS Cultivation of phage and host. Pseudomonas BAL-31 was originally described as a prototroph, but Franklin et al. (9) reported that growth was poor in a glucose and salts medium unless arginine and proline were added. These supplements in turn proved to be inadequate in our laboratory and others, to the extent that recent studies describe the routine inclusion of most amino acids into their defined medium (4, 10). We have resolved the apparent complex nutritional requirements ofPseudomonas BAL-31 by substituting TES buffer for Tris buffer in the medium described by Franklin et al. (9). Pseudomonas BAL-31 grows as a prototroph in this medium with a mean generation time of 60 min. Experiments in which the pH of the medium was controlled during growth in Trisbuffered medium revealed that the poor buffering capacity of Tris at pH 7.2 was not responsible for the prolonged generation time and low yields (data not shown). Since Tris from several commercial suppliers and of different degrees of purity had little effect on growth, it was concluded that the difficulty in culturing Pseudomonas BAL-31 in a Tris-buffered medium was a direct effect of the Tris on cellular function, possibly the result of the potent chelating ability of Tris. The ability of a TES-buffered medium to support prototrophic growth of PseudomonasBAL-31 was not restricted to our strain, since a strain obtained from R. L. Sinsheimer's laboratory grew equally well on a TESbuffered minimal medium. The success of the TES-buffered medium is dependent on filter sterilization. When exponentially growing cultures of
J. VIROL.
Pseudomonas BAL-31 were infected with bacteriophage PM2 at input multiplicities carefully controlled not to exceed 10 PFU per viable cell, the cultures lysed 55 to 60 min postinfection. Lysis was complete by 100 min postinfection. Intracellular phage could be released at full titer at 50 to 55 min postinfection by sparging the culture vigorously for 30 s with nitrogen (data not shown). If input multiplicities exceeded 15, lysis was delayed often by an additional 60 min. Lipid metabolism. Tsukagoshi and Franklin (11) reported that infection of Pseudomonas BAL-31 with bacteriophage PM2 led to an increase in the rate of PG biosynthesis and a decrease in the rate of biosynthesis of PE. This was determined by examining the net uptake of 32p into PE and PG in infected and uninfected cultures. Examination of the data of Tsukagoshi and Franklin (11), however, showed that the increase in the rate of PG biosynthesis occurred early in infection, whereas the decrease in the rate of PE biosynthesis occurred late in infection. This would be expected to lead to an increase in the total lipid phosphorus of an infected culture when compared with an uninfected culture during the latent period of the phage. We examined the increase in total lipid phosphorus by extracting the lipids from an infected and uninfected culture at intervals and determining the amount of inorganic phosphorus by the Bartlett procedure (2). The results of this experiment, in Fig. 1, show that phage infection did not alter the net biosynthesis or amount of lipid until the onset of culture lysis. At this time, there was a net loss of lipid phosphorus. Therefore, if PG is synthesized at a faster rate in infected cultures, the net decrease in the rate of PE biosynthesis must occur early in infection and must be of the same magnitude. Net biosynthesis of PE could be decreased by a slower rate of synthesis or by an enhanced rate of turnover. To examine the rates of synthesis of PE and PG and the relationship of these rates to phage assembly and culture lysis, an experiment was performed that examined the instantaneous rate of PE and PG synthesis. This experiment was performed according to Materials and Methods by removing samples from infected and uninfected control cultures and pulsing the samples for 5 min with 32P. At 1-min intervals samples were removed from the pulsed samples, and the lipids were extracted and fractionated. The rate of PE and PG biosynthesis was calculated over these 5-min intervals, and the rates were plotted versus time. It can be seen in Fig. 2 that the rates of PE
VOL. 19, 1976
BACTERIOPHAGE PM2 LIPIDS 0
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105 40 60 80 TI M E ( M N ) FIG. 2. Rateoj 32P incorporation into the phospholipids PE and PGrof bacteriophage PM2-infected and uninfected cultur res of Pseudomonas BAL-31. Each point represents t he rate of isotope incorporation calculated from plot 'sof the net incorporation of32P over a5-min interval. Symbols: Incorporation oflabel into PG in uninfectedctculture (@); incorporation of label culture (-); incorporationof into PE of uninf label into PG ofi nfected culture (0); and incorporacul ture (O). tion of label into PE of infected 20
Iected
and PG biosyntthesis in the infected culture are the same as the ir respective rates in uninfected cultures for the period of time corresponding to the latent period of the phage. At 60 min postinfection there is a dramatic increase in the rates of synthesis of PE and PG. This corresponds to
449
the lysis time of the culture. The increase in the rates of synthesis of PE and PG in both the infected and uninfected cultures for the first 60 min is a reflection of growth, since the data were not normalized for growth. These data are in contrast to the findings of Tsukagoshi and Franklin (11) and suggest that PM2 infection does not lead to a change in the rate of PE and PG biosynthesis until the onset of culture lysis, a time when lipid synthesis should be incidental to the maturation of the phage. We have observed that the intracellular phage are at full titer at 50 to 55 min infection. In the event that the increase in the rate of phospholipid biosynthesis at the onset of lysis may play some role in the late maturation stages of the phage, we examined the significance of these increased rates by pulsing an infected culture at 60 min postinfection with 32p (5 gCi per ml). At 90 min postinfection, when there is a decline in the rate of phospholipid biosynthesis, half of the culture was extracted and the specific activity of the lipid phosphorus was determined. The other half of the culture was allowed to lyse completely, and the phage were isolated and purified on sucrose gradients. The lipids were extracted from the phage, and the specific activity of the lipid phosphorus was determined. The first sample would show the specific activity of the phage and the host membrane. The second sample would show the specific activity of only the phage lipids. It was found that the phage isolated possessed lipid phosphorus at a specific activity of 6.8 ,uCi per,umol of lipid phosphorus, whereas the lysate lipids had a specific activity of 12.0 ,uCi per,Lmol of lipid phosphorus. The specific activity of the lysate would represent a minimum specific activity of the host membrane since the presence of phage lipid would dilute the radioactivity of the membrane. The dilution factor would depend on the amount of the phage lipid in the lysate lipid. The conclusion deduced from this experiment is that the increase in the rate of phospholipid biosynthesis at 50 min postinfection is of no significance to the assembly of the phage, and this lipid consists primarily of host membrane lipid.
Turnover studies. Tsukagoshi and Franklin previously showed that PG which was synthesized before infection was stable and did not turn over after infection. On the other hand, they showed that PE synthesized before infection was degraded at the onset of culture lysis, although they claimed that the rate of PE biosynthesis had declined before detectable turnover of PE. They employed [ 3P]serine and [14C]serine to label PE in two separate experiments, and these two experiments showed a
450
DIEDRICH AND COTA-ROBLES
difference of 30 min in the onset of culture lysis and PE turnover. We reexamined the turnover of the phosphatides in infected and uninfected cultures of Pseudomonas BAL-31 to determine if the phosphorus, glycerol, fatty acid, and polar moieties turned over at the same rates and if the onset of tumover could be closely related to culture lysis under conditions in which the lysis times of the cultures were reliable. Furthermore, these experiments would be used to evaluate the data of Snipes et al. (10), who interpreted their data to mean that PM2 infection leads to elevated levels of PG by effecting a PE to PG interconversion. If their hypothesis was correct, we predicted that radioactivity lost from PE labeled in the glycerol and perhaps fatty acid and phosphorus moieties would be recovered in PG. The data in Fig. 3 show that PE is not turned over in the uninfected culture during the time period examined. PE turnover in infected cultures commences at the onset of culture lysis, and the turnover of the glycerol, fatty acids, phosphorus, and ethanolamine (Fig. 4) is coincident. This suggests that the entire molecule is degraded simultaneously. The suitability of employing these radioactive isotopes to specifically label the different moieties in Pseudomonas BAL-31 has been discussed in Materials and Methods. Tsukagoshi and Franklin (11) previously observed that PG did not turn over in infected cultures. Our examination of the turnover of PG is given in Fig. 5. It can be seen that PG does not turn over during the period of time of phage eclipse; however, at the time of culture lysis, there is a slow turnover of the PG phosphorus and glycerol, along with an increase in the radioactivity associated with the PG fatty acids. Although the turnover of PG is more complex than that of PE, the events observed are postlytic phenomena. The turnover of the PG glycerol observed in the center panel of Fig. 5 represents the net turnover of the diacyl and nonacylated moieties. For this reason, we attempted to examine the turnover of each glycerol moiety by isolating the PG from turnover experiments and cleaving it with cabbage phospholipase D as described in Materials and Methods. Figure 6 shows that infection of Pseudomonas BAL-31 with bacteriophage PM2 results in turnover of diacyl-glycerol with an opposite and equal turnover of the unacylated glycerol. Radioactivity lost from the diacyl-glycerol is completely regained in the nonacylated glycerol, thus accounting for no net change in total radioactivity of the PG molecule seen in Fig. 5. In addition to a change in the distribution of radio-
J. VIROL.
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activity in PG, it can also be seen from Fig. 6
that at zero time there is approximately fourfold more radioactivity in the nonacylated glycerol than in the diacyl-glycerol. This is observed in both infected and uninfected cultures (see Discussion). Furthermore, the turnover of the two PG glycerol moieties occurs during the latent period of the phage, and represents to this point the only detectable influence of bacteriophage PM2 infection on host lipid metabolism before culture lysis.
VOL. 19, 1976
BACTERIOPHAGE PM2 LIPIDS
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JOURNAL OF VIROLOGY, Aug. 1976, p. 446-456 Copyright C) 1976 American Society for Microbiology
Phospholipid Metabolism in Pseudomonas BAL-31 Infected with Lipid-Containing Bacteriophage PM2 D. L. DIEDRICH'* AND E. H. COTA-ROBLES Thimann Laboratory, Biology Board of Studies, University of California, Santa Cruz, California 95064 Received for publication 16 April 1976
Infection of Pseudomonas BAL-31 with the lipid-containing bacteriophage PM2 resulted in no detectable change in the rate of phosphatidylglycerol (PG) or phosphatidylethanolamine (PE) biosynthesis. An increase in the PG content of infected cultures was not seen until the cultures began to lyse, and this increase was in fact only a relative increase resulting from the extensive turnover of PE at the onset of culture lysis. Turnover studies revealed that the glycerol, phosphorus, fatty acid, and ethanolamine moieties of PE turned over simultaneously at the time of lysis, and therefore made it unlikely that there was a PE to PG conversion during the latent period of the phage. The lipid found in the bacteriophage did not reflect a preferential selection for lipid synthesized before or after infection, but in fact reflected the composition of the host membrane at the time the phage were assembled. The use of a modified medium that allowed the cultivation of Pseudomonas BAL-31 as a prototroph and resulted in reliable lysis times of infected cultures led us to the conclusion that PM2 infection effects little change in host phospholipid metabolism, and that there is sufficient PG in the host cytoplasmic membrane to account for a full burst of phage. As a result of the reliable lysis times that we have achieved, we concluded that certain metabolic events, i.e., PE turnover, are lytic phenomena and must not be confused with events relevant to the biosynthesis and maturation of the phage.
Bacteriophage PM2 is a lipid-containing marine bacteriophage that was isolated and described by Espejo and Canelo (6). Braunstein and Franklin (3) subsequently showed that the phospholipid composition of the phage was approximately the inverse of the host, Pseudomonas BAL-31. The host contains approximately 70% phosphatidylethanolamine (PE) and 25% phosphatidylglycerol (PG), whereas the phage is 28% PE and 65 to 68% PG. Thus it appeared a priori that phage infection led to a control of host phospholipid metabolism, and resulted in elevated levels of PG in the host membrane. This was supported by the findings of Braunstein and Franklin (3), who showed that the phosphatide composition of infected cultures changed to give rise to relatively higher levels of PG late in infection. Tsukagoshi and Franklin (11) subsequently reported that phage infection led to an increase in the rate of PG biosynthesis and a decrease in the rate of PE biosynthesis, thus accounting for a net increase in PG in the membrane of infected cells. This observation was based on differences in the net incorporation of 32P into PE and PG of infected cultures. I Present address: Department of Microbiology, University of Virginia School of Medicine, Charlottesville, Va.
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In apparent contrast the the findings of Tsukagoshi and Franklin (11), Snipes et al. (10) interpreted their data from a double-isotopelabel experiment to indicate that the polar portion of PE was cleaved and the resulting phosphatidic acid (PA) received a glycerol moiety, thus leading to a conversion of PE to PG. These conclusions are not mutually exclusive, since Tsukagoshi and Franklin (11) measured the net biosynthesis of PG and PE rather than the instantaneous rate of phosphatide biosynthesis. Thus, it is possible that they were measuring the result of the mechanism proposed by Snipes et al. (10). Further disagreement concerns the origin of the phospholipid of bacteriophage PM2. Espejo and Canelo (7) reported that the lipids of the phage were synthesized before infection, whereas Tsukagoshi and Franklin (11) reported that approximately 66% of the phage lipid was synthesized after infection. Snipes et al. (10) calculated that approximately half of the phage lipid was synthesized after infection. To resolve the anomalies outlined above, we undertook a more detailed study of the influence of PM2 infection on the metabolism of the glycerol, fatty acid, phosphorus, and polar moieties of infected cultures of Pseudomonas BAL-31. Since it has been shown that the turn-
VOL. 19, 1976
over of these moieties in phospholipids can occur at different rates (12), we were concerned about the validity of data derived from ratios of radioactivity in double-label experiments popularly employed in the above studies. Furthermore, considerable variations in the lysis times of infected cultures were reported within the individual studies. This made it difficult to distinguish data describing postlysis events from events relevant to the maturation of the phage. MATERIALS AND METHODS Cultivation of Pseudomonas BAL-31. Cultures of Pseudomonas BAL-31 were grown on TES-minimal medium. This medium contained, per liter of doubledistilled water: 0.7 g of KCl, 1.1 g of NH4Cl, 1.5 g of CaCl2 2H2O, 12.0 g of MgSO4 * 7H2O, 26.0 g of NaCl, 25mg of KH2PO4, 2.5 g of glucose, and 11.45 g of TES [N - tris(hydroxymethyl)methyl - 2 - aminoethanesulfonic acid] buffer. The medium was adjusted to pH 7.2 with 1 N NaOH and filter-sterilized through 0.22-pm membrane filters (Millipore Corp.). This medium is a TES buffer modification of the medium of Franklin et al. (9). Growth and preparation of bacteriophage PM2. Exponentially growing cultures of Pseudomonas BAL-31 at 2 x 108 to 4 x 108 viable cells per ml were infected with bacteriophage PM2 at multiplicities carefully controlled not to exceed 10 PFU per viable cell. Lysates were processed promptly by two centrifugations at 10,400 x g for 20 min to remove cell debris. The low-speed lysate supernatants were centrifuged at 50,000 x g for 1.5 h at 5°C. The pelleted phage were allowed to disaggregate for several hours in NTC buffer at 5°C. NTC buffer (9) was modified to contain 0.05 M TES buffer instead of Tris. The resuspended pellets were subjected to two more low-speed centrifugations to remove cell debris, and the phage-containing supernatants were filtered through 0.22-ttm filters into sterile containers for storage at 5°C. Bacteriophage prepared in this manner were used for most experiments. Some experiments required more extensive purification of the phage. This was accomplished by employing sucrose density gradients. Linear sucrose gradients were prepared from 20 to 35% sucrose (wt/ vol) in NTC buffer. The gradients were run at 20°C in a Beckman SW27 rotor at 25,000 rpm for 3 h. The phage band was in the bottom one-third of the tube. The phage in sucrose were dialyzed for 24 h against several changes of NTC buffer at 5°C. Bacteriophage PM2 was assayed by the doublelayer plating technique employing AMS-nutrient agar (6). Care was taken to ensure that the host culture employed for plating in this medium had a density of less than 4 x 108 viable cells per ml. The plating efficiency was observed to drop considerably at higher culture densities. Lipid techniques. Phospholipids were extracted, fractionated, and quantitated as previously described (5). Lipid phosphorus was determined by the Bartlett procedure (2). Isotope labeling of lipids. Cultures were labeled with H332PO4 (carrier free) at 2 ,uCi per ml, [23H]glycerol (specific activity of 8.81 Ci/mmol) at 4 -
BACTERIOPHAGE PM2 LIPIDS
447
ACi per ml, [1-_4C]sodium acetate (specific activity of 59 mCi/mmol) at 1 ,uCi per ml, or DL-[3-'4C]serine (specific activity of 12.5 mCi/mmol) at 0.5 ZCi per ml. The details of labeling are given for each experiment. The suitability of these radioactive isotopes for labeling the lipids of Pseudomonas BAL-31 was determined by subjecting labeled lipids to mild alkaline methanolysis (5) and determining the distribution of the label in the resultant glycerol phosphate esters and in the fatty acid moieties. Labeling cultures with acetate resulted in more than 95% of the lipid label appearing in the fatty acids. Labeling the cultures with serine or glycerol resulted in more than 95% of the lipid label appearing in the watersoluble glycerolphosphate ester. Rate of PE and PG biosynthesis. The rate of PE and PG biosynthesis was determined by removing samples from infected and uninfected cultures and pulsing the samples with 5 ,uCi of 32P per ml. Samples were removed in 60-s intervals (six samples) from the pulsed samples, and the lipids were extracted. After separation of the lipids by thin-layer chromatography (TLC), it was possible to examine the net incorporation of isotope into PE and PG over a 5-min interval. From these plots a rate was calculated for each 5-min pulse period, and these rates were plotted against time. Each point in Fig. 2 is plotted at the time corresponding to 2.5 min into the 5-min pulse. Phospholipid turnover studies. Exponentially growing cultures of Pseudomonas BAL-31 were diluted to an absorbance at 600 nm (A,,,,,) of 0.02 in fresh medium and allowed to grow in the abovementioned amount of isotope for six generations. These labeled, exponentially growing cultures were pelleted at 10,000 x g for 10 min at 5°C, washed twice in fresh medium, and resuspended to an A,,, of 0.2 in fresh prewarmed medium. The cultures were divided into two fractions, and one was infected with PM2 at a multiplicity of 10. The cultures were incubated at 28°C on a gyratory shaker at 250 rpm. Samples were periodically taken from the infected and uninfected cultures, and the lipids were extracted and fractionated. After TLC, the amount of radioactivity in each of the phosphatides was determined. For 32P-labeled lipids, the spots on the TLC plates were scraped directly into vials and counted with a liquid scintillation spectrometer. For 3H- and "4C-labeled lipids, the spots were collected into small columns, and the lipids were eluted with methanol into counting vials. Counting fluid was added after drying the lipids in the vials with dry nitrogen. The elution of the lipids from the columns was found to be complete. This was determined with 32P-labeled lipids by counting the eluted silica gel. Silica gel does not significantly quench 32p radioactivity. To examine the turnover of the diacyl and nonacylated glycerols of PG in an experiment in which the PG was labeled with [2-3H]glycerol, the PG was isolated as above. After drying under nitrogen, the PG was suspended in 1.0 ml of diethyl ether in screw-cap tubes. To the tubes was added 0.42 ml of water that contained 20 ,uM acetate buffer (pH 5.6), 40 ,uM CaCl2, and 1.6 mg of cabbage phospholipase D (Calbiochem). The reaction mixture was vigorously agitated and incubated at 25°C for 3 h. The ether
448
DIEDRICH AND COTA-ROBLES
was removed with a stream of nitrogen, and 1.85 ml of water, 2.5 ml of methanol, and 2.5 ml of chloroform were added. The water-soluble unacylated glycerol from PG was recovered in the aqueous phase, and the resulting PA and unreacted PG were found in the organic phase. These were separated by TLC and quantitated. This procedure allowed for 95 to 98% hydrolysis of the PG. Lipid translocation. A culture of Pseudomonas BAL-31 was grown to an A,;. of 0.2 and divided into two cultures. One was infected with PM2 at a multiplicity of 10. After 30 min, both cultures received 2.5 gCi of [2-3H]gilycerol. At the times indicated, samples were removed and diluted into ice-cold 0.5 M NaCl. The cells were washed once in NaCl, and half of the sample was extracted of its lipids. The other half of the sample was processed by the procedure of Forsberg et al. (8) to separate the outer membrane from the cytoplasmic membrane. The resulting membrane fractions were then extracted of their lipids, and the net incorporation of radioactivity into each membrane could be determined.
RESULTS Cultivation of phage and host. Pseudomonas BAL-31 was originally described as a prototroph, but Franklin et al. (9) reported that growth was poor in a glucose and salts medium unless arginine and proline were added. These supplements in turn proved to be inadequate in our laboratory and others, to the extent that recent studies describe the routine inclusion of most amino acids into their defined medium (4, 10). We have resolved the apparent complex nutritional requirements ofPseudomonas BAL-31 by substituting TES buffer for Tris buffer in the medium described by Franklin et al. (9). Pseudomonas BAL-31 grows as a prototroph in this medium with a mean generation time of 60 min. Experiments in which the pH of the medium was controlled during growth in Trisbuffered medium revealed that the poor buffering capacity of Tris at pH 7.2 was not responsible for the prolonged generation time and low yields (data not shown). Since Tris from several commercial suppliers and of different degrees of purity had little effect on growth, it was concluded that the difficulty in culturing Pseudomonas BAL-31 in a Tris-buffered medium was a direct effect of the Tris on cellular function, possibly the result of the potent chelating ability of Tris. The ability of a TES-buffered medium to support prototrophic growth of PseudomonasBAL-31 was not restricted to our strain, since a strain obtained from R. L. Sinsheimer's laboratory grew equally well on a TESbuffered minimal medium. The success of the TES-buffered medium is dependent on filter sterilization. When exponentially growing cultures of
J. VIROL.
Pseudomonas BAL-31 were infected with bacteriophage PM2 at input multiplicities carefully controlled not to exceed 10 PFU per viable cell, the cultures lysed 55 to 60 min postinfection. Lysis was complete by 100 min postinfection. Intracellular phage could be released at full titer at 50 to 55 min postinfection by sparging the culture vigorously for 30 s with nitrogen (data not shown). If input multiplicities exceeded 15, lysis was delayed often by an additional 60 min. Lipid metabolism. Tsukagoshi and Franklin (11) reported that infection of Pseudomonas BAL-31 with bacteriophage PM2 led to an increase in the rate of PG biosynthesis and a decrease in the rate of biosynthesis of PE. This was determined by examining the net uptake of 32p into PE and PG in infected and uninfected cultures. Examination of the data of Tsukagoshi and Franklin (11), however, showed that the increase in the rate of PG biosynthesis occurred early in infection, whereas the decrease in the rate of PE biosynthesis occurred late in infection. This would be expected to lead to an increase in the total lipid phosphorus of an infected culture when compared with an uninfected culture during the latent period of the phage. We examined the increase in total lipid phosphorus by extracting the lipids from an infected and uninfected culture at intervals and determining the amount of inorganic phosphorus by the Bartlett procedure (2). The results of this experiment, in Fig. 1, show that phage infection did not alter the net biosynthesis or amount of lipid until the onset of culture lysis. At this time, there was a net loss of lipid phosphorus. Therefore, if PG is synthesized at a faster rate in infected cultures, the net decrease in the rate of PE biosynthesis must occur early in infection and must be of the same magnitude. Net biosynthesis of PE could be decreased by a slower rate of synthesis or by an enhanced rate of turnover. To examine the rates of synthesis of PE and PG and the relationship of these rates to phage assembly and culture lysis, an experiment was performed that examined the instantaneous rate of PE and PG synthesis. This experiment was performed according to Materials and Methods by removing samples from infected and uninfected control cultures and pulsing the samples for 5 min with 32P. At 1-min intervals samples were removed from the pulsed samples, and the lipids were extracted and fractionated. The rate of PE and PG biosynthesis was calculated over these 5-min intervals, and the rates were plotted versus time. It can be seen in Fig. 2 that the rates of PE
VOL. 19, 1976
BACTERIOPHAGE PM2 LIPIDS 0
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105 40 60 80 TI M E ( M N ) FIG. 2. Rateoj 32P incorporation into the phospholipids PE and PGrof bacteriophage PM2-infected and uninfected cultur res of Pseudomonas BAL-31. Each point represents t he rate of isotope incorporation calculated from plot 'sof the net incorporation of32P over a5-min interval. Symbols: Incorporation oflabel into PG in uninfectedctculture (@); incorporation of label culture (-); incorporationof into PE of uninf label into PG ofi nfected culture (0); and incorporacul ture (O). tion of label into PE of infected 20
Iected
and PG biosyntthesis in the infected culture are the same as the ir respective rates in uninfected cultures for the period of time corresponding to the latent period of the phage. At 60 min postinfection there is a dramatic increase in the rates of synthesis of PE and PG. This corresponds to
449
the lysis time of the culture. The increase in the rates of synthesis of PE and PG in both the infected and uninfected cultures for the first 60 min is a reflection of growth, since the data were not normalized for growth. These data are in contrast to the findings of Tsukagoshi and Franklin (11) and suggest that PM2 infection does not lead to a change in the rate of PE and PG biosynthesis until the onset of culture lysis, a time when lipid synthesis should be incidental to the maturation of the phage. We have observed that the intracellular phage are at full titer at 50 to 55 min infection. In the event that the increase in the rate of phospholipid biosynthesis at the onset of lysis may play some role in the late maturation stages of the phage, we examined the significance of these increased rates by pulsing an infected culture at 60 min postinfection with 32p (5 gCi per ml). At 90 min postinfection, when there is a decline in the rate of phospholipid biosynthesis, half of the culture was extracted and the specific activity of the lipid phosphorus was determined. The other half of the culture was allowed to lyse completely, and the phage were isolated and purified on sucrose gradients. The lipids were extracted from the phage, and the specific activity of the lipid phosphorus was determined. The first sample would show the specific activity of the phage and the host membrane. The second sample would show the specific activity of only the phage lipids. It was found that the phage isolated possessed lipid phosphorus at a specific activity of 6.8 ,uCi per,umol of lipid phosphorus, whereas the lysate lipids had a specific activity of 12.0 ,uCi per,Lmol of lipid phosphorus. The specific activity of the lysate would represent a minimum specific activity of the host membrane since the presence of phage lipid would dilute the radioactivity of the membrane. The dilution factor would depend on the amount of the phage lipid in the lysate lipid. The conclusion deduced from this experiment is that the increase in the rate of phospholipid biosynthesis at 50 min postinfection is of no significance to the assembly of the phage, and this lipid consists primarily of host membrane lipid.
Turnover studies. Tsukagoshi and Franklin previously showed that PG which was synthesized before infection was stable and did not turn over after infection. On the other hand, they showed that PE synthesized before infection was degraded at the onset of culture lysis, although they claimed that the rate of PE biosynthesis had declined before detectable turnover of PE. They employed [ 3P]serine and [14C]serine to label PE in two separate experiments, and these two experiments showed a
450
DIEDRICH AND COTA-ROBLES
difference of 30 min in the onset of culture lysis and PE turnover. We reexamined the turnover of the phosphatides in infected and uninfected cultures of Pseudomonas BAL-31 to determine if the phosphorus, glycerol, fatty acid, and polar moieties turned over at the same rates and if the onset of tumover could be closely related to culture lysis under conditions in which the lysis times of the cultures were reliable. Furthermore, these experiments would be used to evaluate the data of Snipes et al. (10), who interpreted their data to mean that PM2 infection leads to elevated levels of PG by effecting a PE to PG interconversion. If their hypothesis was correct, we predicted that radioactivity lost from PE labeled in the glycerol and perhaps fatty acid and phosphorus moieties would be recovered in PG. The data in Fig. 3 show that PE is not turned over in the uninfected culture during the time period examined. PE turnover in infected cultures commences at the onset of culture lysis, and the turnover of the glycerol, fatty acids, phosphorus, and ethanolamine (Fig. 4) is coincident. This suggests that the entire molecule is degraded simultaneously. The suitability of employing these radioactive isotopes to specifically label the different moieties in Pseudomonas BAL-31 has been discussed in Materials and Methods. Tsukagoshi and Franklin (11) previously observed that PG did not turn over in infected cultures. Our examination of the turnover of PG is given in Fig. 5. It can be seen that PG does not turn over during the period of time of phage eclipse; however, at the time of culture lysis, there is a slow turnover of the PG phosphorus and glycerol, along with an increase in the radioactivity associated with the PG fatty acids. Although the turnover of PG is more complex than that of PE, the events observed are postlytic phenomena. The turnover of the PG glycerol observed in the center panel of Fig. 5 represents the net turnover of the diacyl and nonacylated moieties. For this reason, we attempted to examine the turnover of each glycerol moiety by isolating the PG from turnover experiments and cleaving it with cabbage phospholipase D as described in Materials and Methods. Figure 6 shows that infection of Pseudomonas BAL-31 with bacteriophage PM2 results in turnover of diacyl-glycerol with an opposite and equal turnover of the unacylated glycerol. Radioactivity lost from the diacyl-glycerol is completely regained in the nonacylated glycerol, thus accounting for no net change in total radioactivity of the PG molecule seen in Fig. 5. In addition to a change in the distribution of radio-
J. VIROL.
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activity in PG, it can also be seen from Fig. 6
that at zero time there is approximately fourfold more radioactivity in the nonacylated glycerol than in the diacyl-glycerol. This is observed in both infected and uninfected cultures (see Discussion). Furthermore, the turnover of the two PG glycerol moieties occurs during the latent period of the phage, and represents to this point the only detectable influence of bacteriophage PM2 infection on host lipid metabolism before culture lysis.
VOL. 19, 1976
BACTERIOPHAGE PM2 LIPIDS
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