Vol. 15, No. 5
JouRNAL oF VIROLOGY, May 1975, p. 1141-1147 Copyright @ 1975 American Society for Microbiology
Printed in U.S.A.
Phospholipase Activity in Bacteriophage-Infected Escherichia coli II. Activation of Phospholipase by T4 Ghost Infection C. S. BULLER,* M. VANDER MATEN, D. FAUROT, AND E. T. NELSON Department of Microbiology, University of Kansas, Lawrence, Kansas 66045 Received for publication 9 December 1974
The release of free fatty acids from the phospholipids of Escherichia coli is initiated immediately after the attachment of T4 ghosts. A similar accumulation of free fatty acids is observed if the cells are infected with T4 phage in the presence of chloramphenicol or puromycin. An early accumulation of free fatty acids, however, is not observed in T4 infections in which chloramphenicol or puromycin are not present, nor does it occur if the E. coli are infected with T4 phage before ghost infection, suggesting that phage products can prevent the phospholipid deacylation. If E. coli is infected with T4 ghosts before T4 phage infection, the accumulation of free fatty acids is not suppressed. When phospholipase-deficient E. coli are infected with T4 ghosts the appearance of free fatty acids is not observed, suggesting that T4 ghost attachment can activate the phospholipase of wild-type E. coli. Although the formation of free fatty acid apparently is a consequence of activation of the detergent-resistant phospholipase of the outer membrane, it is not observed in mutants deficient in the detergent-sensitive phospholipase. A variety of agents, including heat, solvents, and detergents can be used to perturb bacterial membrane functions, and one of the consequences is the activation of phospholipase (4, 28), resulting in the release of free fatty acids (FFA) from the bacterial phospholipids. Colicins interact with specific receptors in the Escherichia coli cell surface and some can activate host phospholipase (4). The major phospholipase of E. coli is membrane bound (2, 31), and being latent normally it is likely that its activation signals some structural change or damage to the cell envelope. The attachment of T2 or T4 phage to E. coli results in a temporary efflux of cations from the cell (26, 27, 32, 33), also indicative of a change in the cell membrane. Although it has been demonstrated that phage infection leads to phospholipid deacylation before or at the time of lysis (3, 8, 17), it has not previously been shown that the phage attachment process itself can initiate phospholipid deacylation. That the attachment process can alter the membrane is indicated by the immediate block .in transport after T4 ghost attachment to E. coli (38). Since infection with intact T4 does not result in blocked transport (38), it is apparent that phage products modify or repair the damage incurred during attachment.
In this communication it is demonstrated that the attachment of T4 ghosts to E. coli results in the deacylation of host phospholipids, and that T4 phage gene expression is required to suppress the activity. MATERIALS AND METHODS Bacteria and phage. E. coli strains B and K-12 (A) IYMel and wild-type T4 phage were obtained from L. Astrachan. T4imm2 and T4imm2-s were received from H. Bernstein and T4s from J. Emrich. The imm locus specifies immunity against superinfecting ghosts and phage in T4-infected E. coli (35, 36), and the s gene specifies for resistance to lysis from without (14). E. coli B fad, a mutant which lacks the capability of ,-oxidation of fatty acids (3), was obtained from
J. Cronan and E. coli S/5,6 from W. Bode. E. coli
K-12 DR-DS-, DS-, DR-, and their wild-type parent were obtained from S. Nojima. DR and DS refer, respectively, to detergent-resistant phospholipase A and detergent-sensitive phospholipase A (9,21). High titer phage stocks were prepared from phage lysates either by differential centrifugation or by polyethyleneglycol sedimentation (39). T4 ghosts were prepared by an osmotic shocking procedure (10) which usually reduced plaque-forming ability by 99.9% or more, and titers were established as described by Duckworth and Bessman (13). Bacteriophage were assayed by the methods described by Adams (1). Media and reagents. Tryptone broth, containing 1% tryptone (Difco) and 0.1 M NaCl, was used as
1141
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BULLER ET AL.
growth media. Soft and hard agar for plating phage and bacteria contained 0.6 and 1.5% agar (Difco), respectively. All reagents used in phospholipid extraction and thin layer chromatography were analytical grade. Silica gel, Camag-type DO, without binder, was obtained from Arthur H. Thomas (Philadelphia, Pa.). [1,2-140 ]sodium acetate (56.2 mCi/mM) was obtained from New England Nuclear (Boston, Mass.). Chloramphenicol and puromycin were purchased from Sigma Chemical Co. (St. Louis, Mo.). Labeling of phospholipids and assay for FFA. The acyl groups of E. coli phospholipids were labeled by growth of the organisms in tryptone broth containing 0.4 MCi of [1C4Jlacetate/ml. The cultures were incubated at 37 C, with aeration, until mid-log phase growth was reached. The cells were collected by centrifugation, and after washing to remove unincorporated [140Jacetate, were suspended in tryptone broth prewarmed to 37 C. These cells were then infected with either T4 phage or T4 ghosts and incubated at 37 C with aeration. At various times after infection, 1.0-ml samples were removed for estimation of FFA by using a modification of the procedure of Cronan and Wulff (8). These samples were transferred to screw-cap tubes containing 6 ml of chloroform-methanol (1:2, vol/vol) and 0.6 ml of carrier cells (10 mg dry weight/ml). After incubation for at least 1 h at room temperature, the mixture was centrifuged, and the residue was again extracted with 6 ml of chloroform-methanol (2:1, vol/vol). The supernatants from the two extractions, containing the E. coli phospholipids and FFAs, were combined and separated into two phases by the addition of 3.8 ml of water, and the chloroform phase was removed by aspiration. After concentration to dryness in a stream of N2, the lipids were redissolved in 100 Ml of chloroform and were used for thin layer chromatography. Thin layer chromatography was performed by the methods described by Skipski and Barclay (34). End plates (5 by 20 cm) were coated with Camag gel type DO, without binder. For separation of phospholipid classes, plates were developed in chloroformmethanol-water (65:25:4, vol/vol). The FFA were separated from phospholipids by developing the plates in a solvent system containing isopropyl ether/ acetic acid (96:4, vol/vol). Lipids were detected by exposing the developed plates to iodine vapors. The radioactivity in the individual spots was determined by quantitatively transferring the gel to vials for counting in a Tri-Carb liquid scintillation spectrometer. To assure complete recovery, all of the gel in a lane was routinely assayed by this procedure. The scintillation fluid used contained 5 g of 2,5diphenyloxazole and 0.1 g of dimethyl 1,4-bis-2(5-phenyloxazolyl)-benzene in 1 liter of toluene. The identity of the individual phospholipids was established as previously described (24). In the FFA assays, the lipid extracts applied to the thin layer chromatography plates contained between 10,000 and 15,000 counts/min of 14C radioactivity. The extent of FFA formation was expressed as the percent of total radioactivity in the free-lipid fraction converted to FFA. In these experiments no attempts were made to assay for the presence of lysophospholipid products.
J. VIROL.
RESULTS Phospholipid hydrolysis by T4 ghost-infected E. coli. Some of the consequences of T4 ghost infection of E. coli are suggestive of membrane alterations. It is well known that ghost attachment results in a immediate block in active transport by the host (38) and that the attachment process results in an efflux of cations from the cell (32). Additionally, host cell respiration and macromolecular synthesis are impaired (10, 15, 18). Since many of the enzymes of the membrane are thought to be allotopic (30), it could be expected that degradation of membrane phospholipids might result in membrane dysfunction. Figure 1 illustrates that T4 ghost attachment to E. coli results in the accumulation of FFA, whereas cells infected with T4 at multiplicities sufficient to cause lysis inhibition do not demonstrate FFA formation. Although the accumulation of FFA can occur in T4-infected cells, it is not observed until just before or during lysis from within (8, 17), or if E. coli B is infected with T4 rapid-lysis mutants the FFA appearance occurs substantially before lysis (3). In contrast, Fig. 1 illustrates that the appearance of FFA can be observed within 2 min after infection by T4 ghosts. The assay is based on the appearance of '4C-labeled FFA in cells containing phospholipids with acyl groups labeled before infection by growth in media containing [14C ]acetate. Because unincorporated [14C]acetate is removed before infection, the accumulation of [14C ]FFA cannot be attributed to de novo synthesis after ghost infection. An alternate explanation that [4I IFFA are synthesized from acyl thioester precursors derived by p3-oxidation of degradation products of phospholipids can be ruled out by the inability of T4 ghost-infected E. coli to 10. 84 CL
IL -
2
5
10 15 20 25 Minutes after Infection
30
FIG. 1. FFA formation in T4 ghost-infected E. coli. E. coli B cells with "4C-labeled phospholipids were infected with either T4 phage or T4 ghosts, at input multiplicities of three. At the indicated times samples were removed and FFA assayed, as described in Materials and Methods. Symbols: 0, ghosts; *, T4.
VOL. 15, 1975
incorporate ["CC]acetate into cellular lipids (unpublished data). Effect of chloramphenicol and puromycin on FFA accumulation in T4-infected E. coli. The results shown in Fig. 1 indicate that unlike ghost infection, T4 infection of E. coli does not result in an early formation of FFA. Since T4 phage and ghosts attach to the host cell surface by the same mechanism, and in doing so induce the same initial type of damage (32), this difference suggests that phage gene expression can result in a modification in the events which are triggered by attachment and with ghosts lead to FFA formation. Alternatively, it is possible that the passage of DNA and/or internal proteins through the membrane in some way prevents FFA formation. T4 ghosts are devoid of both DNA and internal protein. To distinguish between these alternatives, host cells were incubated with chloramphenicol or puromycin before and during infection with T4. Figure 2 indicates that under these conditions an early initiation of FFA formation, similar to that observed in ghost-infected cells (Fig. 1), occurs in T4-infected cells. Since the initiation of FFA formation in ghost-infected cells must occur as a consequence of cell surface alterations, these results suggest that phage attachment can produce the same change, but that it can be repaired or modulated by phage gene expression. I
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T4 GHOST ACTIVATION OF E. COLI PHOSPHOLIPASE
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T4 phage suppression of T4-ghost induced FFA formation. E. coli cells infected with T4 ghosts, and subsequently with T4 phage, are unable to produce progeny (15). If the cells are first infected with T4, they become resistant to the killing action of ghosts and progeny phage are produced (12, 22, 35, 36). The effect of T4-phage gene expression on the ghost-induced activation of FFA formation in E. coli is shown in Fig 3. As expected from the results of experiments shown in Fig.2, the primary infection with T4 prevented early FFA accumulation after ghost infection. These results indicate that the phage products responsible for the suppression are synthesized within 5 min after T4 infection. The ghost activated release of FFA, however, cannot be suppressed by superinfection with T4. It has been reported that mutations in the imm (35, 36) and s (6, 22) genes of T4 result in decreased immunity of T4-infected E. coli against the lethal effects of T4 ghost attachment. E. coli infected with T4imm2 do not form FFA before the time of lysis, indicating that the imm gene product is not directly involved in the suppression of early FFA formation (Fig. 4). If E. coli is infected with a double mutant T4imm2-s, FFA formation is observed beginning at about 10 min after infection. In other experiments it was observed that T4s-infected cells also demonstrated initiation of FFA formation at 10 min (data not shown). FFA formation has been observed at about 10 min after infection of E. coli by T4rll (3). T4s mutants, in addition to being unable to cause resistance to
8 LL IL 4-,
10
6
4)
U L.
JouRNAL oF VIROLOGY, May 1975, p. 1141-1147 Copyright @ 1975 American Society for Microbiology
Printed in U.S.A.
Phospholipase Activity in Bacteriophage-Infected Escherichia coli II. Activation of Phospholipase by T4 Ghost Infection C. S. BULLER,* M. VANDER MATEN, D. FAUROT, AND E. T. NELSON Department of Microbiology, University of Kansas, Lawrence, Kansas 66045 Received for publication 9 December 1974
The release of free fatty acids from the phospholipids of Escherichia coli is initiated immediately after the attachment of T4 ghosts. A similar accumulation of free fatty acids is observed if the cells are infected with T4 phage in the presence of chloramphenicol or puromycin. An early accumulation of free fatty acids, however, is not observed in T4 infections in which chloramphenicol or puromycin are not present, nor does it occur if the E. coli are infected with T4 phage before ghost infection, suggesting that phage products can prevent the phospholipid deacylation. If E. coli is infected with T4 ghosts before T4 phage infection, the accumulation of free fatty acids is not suppressed. When phospholipase-deficient E. coli are infected with T4 ghosts the appearance of free fatty acids is not observed, suggesting that T4 ghost attachment can activate the phospholipase of wild-type E. coli. Although the formation of free fatty acid apparently is a consequence of activation of the detergent-resistant phospholipase of the outer membrane, it is not observed in mutants deficient in the detergent-sensitive phospholipase. A variety of agents, including heat, solvents, and detergents can be used to perturb bacterial membrane functions, and one of the consequences is the activation of phospholipase (4, 28), resulting in the release of free fatty acids (FFA) from the bacterial phospholipids. Colicins interact with specific receptors in the Escherichia coli cell surface and some can activate host phospholipase (4). The major phospholipase of E. coli is membrane bound (2, 31), and being latent normally it is likely that its activation signals some structural change or damage to the cell envelope. The attachment of T2 or T4 phage to E. coli results in a temporary efflux of cations from the cell (26, 27, 32, 33), also indicative of a change in the cell membrane. Although it has been demonstrated that phage infection leads to phospholipid deacylation before or at the time of lysis (3, 8, 17), it has not previously been shown that the phage attachment process itself can initiate phospholipid deacylation. That the attachment process can alter the membrane is indicated by the immediate block .in transport after T4 ghost attachment to E. coli (38). Since infection with intact T4 does not result in blocked transport (38), it is apparent that phage products modify or repair the damage incurred during attachment.
In this communication it is demonstrated that the attachment of T4 ghosts to E. coli results in the deacylation of host phospholipids, and that T4 phage gene expression is required to suppress the activity. MATERIALS AND METHODS Bacteria and phage. E. coli strains B and K-12 (A) IYMel and wild-type T4 phage were obtained from L. Astrachan. T4imm2 and T4imm2-s were received from H. Bernstein and T4s from J. Emrich. The imm locus specifies immunity against superinfecting ghosts and phage in T4-infected E. coli (35, 36), and the s gene specifies for resistance to lysis from without (14). E. coli B fad, a mutant which lacks the capability of ,-oxidation of fatty acids (3), was obtained from
J. Cronan and E. coli S/5,6 from W. Bode. E. coli
K-12 DR-DS-, DS-, DR-, and their wild-type parent were obtained from S. Nojima. DR and DS refer, respectively, to detergent-resistant phospholipase A and detergent-sensitive phospholipase A (9,21). High titer phage stocks were prepared from phage lysates either by differential centrifugation or by polyethyleneglycol sedimentation (39). T4 ghosts were prepared by an osmotic shocking procedure (10) which usually reduced plaque-forming ability by 99.9% or more, and titers were established as described by Duckworth and Bessman (13). Bacteriophage were assayed by the methods described by Adams (1). Media and reagents. Tryptone broth, containing 1% tryptone (Difco) and 0.1 M NaCl, was used as
1141
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BULLER ET AL.
growth media. Soft and hard agar for plating phage and bacteria contained 0.6 and 1.5% agar (Difco), respectively. All reagents used in phospholipid extraction and thin layer chromatography were analytical grade. Silica gel, Camag-type DO, without binder, was obtained from Arthur H. Thomas (Philadelphia, Pa.). [1,2-140 ]sodium acetate (56.2 mCi/mM) was obtained from New England Nuclear (Boston, Mass.). Chloramphenicol and puromycin were purchased from Sigma Chemical Co. (St. Louis, Mo.). Labeling of phospholipids and assay for FFA. The acyl groups of E. coli phospholipids were labeled by growth of the organisms in tryptone broth containing 0.4 MCi of [1C4Jlacetate/ml. The cultures were incubated at 37 C, with aeration, until mid-log phase growth was reached. The cells were collected by centrifugation, and after washing to remove unincorporated [140Jacetate, were suspended in tryptone broth prewarmed to 37 C. These cells were then infected with either T4 phage or T4 ghosts and incubated at 37 C with aeration. At various times after infection, 1.0-ml samples were removed for estimation of FFA by using a modification of the procedure of Cronan and Wulff (8). These samples were transferred to screw-cap tubes containing 6 ml of chloroform-methanol (1:2, vol/vol) and 0.6 ml of carrier cells (10 mg dry weight/ml). After incubation for at least 1 h at room temperature, the mixture was centrifuged, and the residue was again extracted with 6 ml of chloroform-methanol (2:1, vol/vol). The supernatants from the two extractions, containing the E. coli phospholipids and FFAs, were combined and separated into two phases by the addition of 3.8 ml of water, and the chloroform phase was removed by aspiration. After concentration to dryness in a stream of N2, the lipids were redissolved in 100 Ml of chloroform and were used for thin layer chromatography. Thin layer chromatography was performed by the methods described by Skipski and Barclay (34). End plates (5 by 20 cm) were coated with Camag gel type DO, without binder. For separation of phospholipid classes, plates were developed in chloroformmethanol-water (65:25:4, vol/vol). The FFA were separated from phospholipids by developing the plates in a solvent system containing isopropyl ether/ acetic acid (96:4, vol/vol). Lipids were detected by exposing the developed plates to iodine vapors. The radioactivity in the individual spots was determined by quantitatively transferring the gel to vials for counting in a Tri-Carb liquid scintillation spectrometer. To assure complete recovery, all of the gel in a lane was routinely assayed by this procedure. The scintillation fluid used contained 5 g of 2,5diphenyloxazole and 0.1 g of dimethyl 1,4-bis-2(5-phenyloxazolyl)-benzene in 1 liter of toluene. The identity of the individual phospholipids was established as previously described (24). In the FFA assays, the lipid extracts applied to the thin layer chromatography plates contained between 10,000 and 15,000 counts/min of 14C radioactivity. The extent of FFA formation was expressed as the percent of total radioactivity in the free-lipid fraction converted to FFA. In these experiments no attempts were made to assay for the presence of lysophospholipid products.
J. VIROL.
RESULTS Phospholipid hydrolysis by T4 ghost-infected E. coli. Some of the consequences of T4 ghost infection of E. coli are suggestive of membrane alterations. It is well known that ghost attachment results in a immediate block in active transport by the host (38) and that the attachment process results in an efflux of cations from the cell (32). Additionally, host cell respiration and macromolecular synthesis are impaired (10, 15, 18). Since many of the enzymes of the membrane are thought to be allotopic (30), it could be expected that degradation of membrane phospholipids might result in membrane dysfunction. Figure 1 illustrates that T4 ghost attachment to E. coli results in the accumulation of FFA, whereas cells infected with T4 at multiplicities sufficient to cause lysis inhibition do not demonstrate FFA formation. Although the accumulation of FFA can occur in T4-infected cells, it is not observed until just before or during lysis from within (8, 17), or if E. coli B is infected with T4 rapid-lysis mutants the FFA appearance occurs substantially before lysis (3). In contrast, Fig. 1 illustrates that the appearance of FFA can be observed within 2 min after infection by T4 ghosts. The assay is based on the appearance of '4C-labeled FFA in cells containing phospholipids with acyl groups labeled before infection by growth in media containing [14C ]acetate. Because unincorporated [14C]acetate is removed before infection, the accumulation of [14C ]FFA cannot be attributed to de novo synthesis after ghost infection. An alternate explanation that [4I IFFA are synthesized from acyl thioester precursors derived by p3-oxidation of degradation products of phospholipids can be ruled out by the inability of T4 ghost-infected E. coli to 10. 84 CL
IL -
2
5
10 15 20 25 Minutes after Infection
30
FIG. 1. FFA formation in T4 ghost-infected E. coli. E. coli B cells with "4C-labeled phospholipids were infected with either T4 phage or T4 ghosts, at input multiplicities of three. At the indicated times samples were removed and FFA assayed, as described in Materials and Methods. Symbols: 0, ghosts; *, T4.
VOL. 15, 1975
incorporate ["CC]acetate into cellular lipids (unpublished data). Effect of chloramphenicol and puromycin on FFA accumulation in T4-infected E. coli. The results shown in Fig. 1 indicate that unlike ghost infection, T4 infection of E. coli does not result in an early formation of FFA. Since T4 phage and ghosts attach to the host cell surface by the same mechanism, and in doing so induce the same initial type of damage (32), this difference suggests that phage gene expression can result in a modification in the events which are triggered by attachment and with ghosts lead to FFA formation. Alternatively, it is possible that the passage of DNA and/or internal proteins through the membrane in some way prevents FFA formation. T4 ghosts are devoid of both DNA and internal protein. To distinguish between these alternatives, host cells were incubated with chloramphenicol or puromycin before and during infection with T4. Figure 2 indicates that under these conditions an early initiation of FFA formation, similar to that observed in ghost-infected cells (Fig. 1), occurs in T4-infected cells. Since the initiation of FFA formation in ghost-infected cells must occur as a consequence of cell surface alterations, these results suggest that phage attachment can produce the same change, but that it can be repaired or modulated by phage gene expression. I
1143
T4 GHOST ACTIVATION OF E. COLI PHOSPHOLIPASE
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T4 phage suppression of T4-ghost induced FFA formation. E. coli cells infected with T4 ghosts, and subsequently with T4 phage, are unable to produce progeny (15). If the cells are first infected with T4, they become resistant to the killing action of ghosts and progeny phage are produced (12, 22, 35, 36). The effect of T4-phage gene expression on the ghost-induced activation of FFA formation in E. coli is shown in Fig 3. As expected from the results of experiments shown in Fig.2, the primary infection with T4 prevented early FFA accumulation after ghost infection. These results indicate that the phage products responsible for the suppression are synthesized within 5 min after T4 infection. The ghost activated release of FFA, however, cannot be suppressed by superinfection with T4. It has been reported that mutations in the imm (35, 36) and s (6, 22) genes of T4 result in decreased immunity of T4-infected E. coli against the lethal effects of T4 ghost attachment. E. coli infected with T4imm2 do not form FFA before the time of lysis, indicating that the imm gene product is not directly involved in the suppression of early FFA formation (Fig. 4). If E. coli is infected with a double mutant T4imm2-s, FFA formation is observed beginning at about 10 min after infection. In other experiments it was observed that T4s-infected cells also demonstrated initiation of FFA formation at 10 min (data not shown). FFA formation has been observed at about 10 min after infection of E. coli by T4rll (3). T4s mutants, in addition to being unable to cause resistance to
8 LL IL 4-,
10
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4)
U L.
