/. Biochem., 80, 1247-1258 (1976)
Nature of Escherichia coli Mutants Deficient in DetergentResistant and/or Detergent-Sensitive Phospholipase A Osamu DOI and Shoshichi NOJIMA 1 Department of Chemistry, National Institute of Health, Kamiosaki, Shinagawa-ku, Tokyo 141 Received for publication, June 7, 1976
1. Escherichia coli K-12 mutants deficient in detergent-resistant (DR) and detergent-sensitive (DS) phospholipases A and deficient in DS phospholipase A were isolated. 2. The growth, compositions of phospholipids and fatty acids and turnover of phospholipids of three mutants (DR~, DS", DR~DS~) were compared with those of their parent (DR" was isolated in a previous study). 3. Autodegradations of membrane phospholipids of 18,000xj supernatants and precipitates of the homogenates of these three mutants were also compared with those of the parent, and the effects of various detergents and organic solvents on these activities were examined. 4. We could not identify any significant physiological role for DR or DS phospholipase A.
Recently, we described the isolation of a mutant deficient in a detergent-resistant (DR) phospholipase A from E. coli K-12 (1). During the characterization of this mutant, another kind of phospholipase A, detergent-sensitive (DS) phospholipase A, was found in the mutant as well as in the parent (2). A double mutant deficient in both DR and DS enzymes has been isolated from the mutant deficient in DR enzyme by mutagenesis. A mutant deficient in only DS enzyme has also been isolated from the double mutant by conjugation. Thus, in our laboratory, three mutants, DR", DS", and D R " D S " are now available. In a separate paper, we reported the determination of DR phospholipase A gene on the E. coli chromosome (J). The present paper deals with a comparison of the growth, lipid composition, phospholipid turnover, and other characteristics of the three 1
Present address: Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113. Vol. 80, No. 6, 1976
mutants with those of the parent. A preliminary report has already been published (4). MATERIALS AND METHODS Organisms—E. coli K-12 strain 23 (leu~thr~thi~lac~pldA~f) derived from Wl, the isolation of which had already been reported (7), was used as a starting strain in this study. N912 (JabB strR), an unsaturated fatty acid auxotroph derived from 1
We proposed in a previous paper (5) that mutants specifically defective in phospholipid degradation be given the standard genetic symbol pld (for phospho/ipid degradation), following the proposal by Cronan (5) of the genetic symbol pis for phospho/ipid synthesis. The mutant in detergent-resistant phospholipase A was called pld A. However, in this paper, we use the original phenotypic expression, DR±, since it is easy to see its relation to DS, the location of which is not yet established.
1247
1248 P678 (Jacob and Wollman), which was also obtained in the previous study (/), was used as an indicator for the isolation of DS phospholipase A-negative mutant. Media—/i-Broth, which contains 10 g of polypeptone (Daigo Eiyo Co. Tokyo) and 2.5 g of NaCl per liter, Penassay broth (Difco) and minimum synthetic medium, which contains 7 g of K 2 HPO 4 , 2 g of KH.PO,, 0.5 g of sodium citrate2H,O, 1 g of (NH^SO,, 0.1 ml of 20% MgSO47H,O, 0.1 ml of Bx (10 mg/ml) and 5 g of glucose per liter, were used. Agar was prepared by supplementing broth with 1% agar; soft agar was prepared by supplementing with 0.5 % agar. Isolation of Mutants—The DR~DS" mutant was isolated from the parent strain 23 (DR~). The principle for selection of the mutant was similar to that described previously (7); endogenous phospholipids in the mutagenized cells were hydrolyzed by autolysis to form free fatty acids, probably by the combined action of phospholipase A and lysophospholipase [EC. 3.1.1.5]. The fatty acids released were assayed using the unsaturated fatty acid auxotroph of E. coli as an indicator strain. In the case of isolation of the DR~ mutant (7), colonies of mutagenized cells on the plate were lyzed with sodium dodecyl sulfate. However, for the isolation of a detergent-sensitive phospholipase A-negative mutant, some other procedure for the lysis of cells on the plate was required. After several trials, exposure of a spot of the culture to chloroform vapor followed by incubation at 37° was found to be suitable for the assay of DS phospholipase A on the plate. Practically, cells of the strain 23 (DR~) in the logarithmic phase of growth (2 x 10s cells/ml) were harvested and suspended in 0.2 M acetate buffer (pH 5.0) containing N-methyl-N'-nitro-N-nitrosoguanidine (700 fig/ ml). After incubation for 60 min at 37°, bacteria were washed once with saline-phosphate buffer, suspended in 5 ml of Penassay broth and incubated at 28° for 20 hr. The bacteria were washed once and resuspended in 5 ml of ,1-broth. One ml of the suspension was again mixed with 3 ml of /{-broth containing 900 fig of penicillin G, and the mixture was incubated at 28° for 24 hr without shaking. Aliquots were spread on /}-agar plates and incubated at 28° for 48 hr. Cells of each colony on the plate were transferred to 2 ml of /l-broth and incubated at 28° for 20 hr with shaking. 0.02-0.04 ml of each
O. DOI and S. NOJIMA culture was spotted on a /i-agar plate, and the plates were incubated at 42° for 2 hr. The cells on the plates were exposed to chloroform vapor at room temperature and further incubated at 37° for 30-60 min in order to increase autolysis of the cells. The sensitivity of the assay was increased by making use of spots of the culture instead of colonies on the plate. Three to four ml of soft agar, maintained at 50±l°, was carefully poured over the spots on the plate; the soft agar contained 5 x 10s cells of N912 (unsaturated fatty acid-auxotrophic and streptomycin-resistant) and streptomycin (300 /ig/ml). The plate was incubated at 42°. After incubation for 2 days, spots on which no or only slight growth of the unsaturated fatty acid auxotroph was present were sought. The detergent-sensitive phospholipase A-negative mutant was derived from this double mutant strain 17 (DR~DS~) by conjugation with Hfr P4X6. Details of the genetic experiments have been reported elsewhere (3). DR Phospholipase A Assay—Preparation of substrates was as described previously (2). Cells were grown to S x l O ^ l x l O " cells/ml at 37° in Penassay broth (Difco) medium with shaking. A culture (100 ml) was then chilled and centrifuged at 7,000-8,500 x g for 10 min at 0°. The cells were washed with cold saline. The pellet was suspended in 2 ml of 5 mM of Tris-HCl buffer (pH 7.5) and sonicated (Umeda Sonicator, 20 kc) for 2 min at 0°. The sonicate was centrifuged at 13,000 x g for 15 min at 0°. The supernatant solution was used as the enzyme preparation (4.2-8.6 mg protein/ml). The incubation mixture contained 0.25 ml of ["CJphospholipids (0.2 pmoles, approx. 8,000 cpm), 0.05 ml of 0.1 M CaCls, 0.50 ml of 0.2 M Tris-HCl buffer (pH 8.2), 0.20 ml of enzyme solution (in 5 mM Tris-HCl buffer, pH 7.5) and 1.5 ml of methanol, made up to a final volume of 3.0 ml with deionized water. Incubation was carried out at 37° with shaking in a 10 ml centrifuge tube with a glass stopper. The reaction period was usually 30 min. After the reaction, the incubation mixture was immediately chilled to 0° and 1.5 ml of methanol plus 6.0 ml of chloroform was added to the incubation mixture. The mixture was shaken for 1 min and centrifuged at 2,000 xg for 10 min to separate it into two layers. To the upper layer, 6.0 ml of chloroform was added, and the mixture was again shaken for 30 sec and centrifuged. The
J. Biochem.
NATURE OF E. coli PHOSPHOLIPASE A MUTANTS combined chloroform extract was evaporated to dryness in vacua at 30° and the residue was redissolved in 0.30 ml of chloroform-methanol ( 2 : 1 , v/v). Next, 0.1 ml of the solution was spotted on a silica gel thin-layer plate and developed with chloroform-methanol-water (65 : 25 : 4, by vol.). The spots corresponding to diacylphospholipids, Iysophospholipids, fatty acids, and fatty acid methylesters were scraped off and counted with a Beckman liquid scintillation counter using 10 ml of Bray's scintillator. DS Phospholipase A and Lysophospholipase Assay—Cell extracts were prepared as described above. The incubation mixture contained 0.25 ml of [14C]phospholipids or [14C]lysophospholipids (0.2 //moles, approx. 8,000 cpm), 0.05 ml of 0.1 M CaCl,, 0.50 ml of 0.2 M Tris-HCl (pH 7.0), and 0.20 ml of enzyme solution, made up to a final volume of 1.50 ml with deionized water. With 2-acyl-glyceryl-phosphorylethanolamine as a substrate, 0.50 ml of 0.1 M borate buffer (pH 6.5) was employed instead of Tris-HCl buffer. Incubation was carried out at 37° and the reaction period was usually 15 min. After the reaction, the incubation mixture was immediately chilled at 0°, 3.0 ml of methanol plus 6.0 ml of chloroform was added, and the mixture was treated as described for DR. phospholipase A assay. Preparation of 18,000 x g Supernatant and Precipitate—Cells were cultured at 37° in Penassay broth (Difco) medium overnight. Each of these cultures were added to 50 ml of the same medium at a level of 1 % (v/v), and an aqueous solution of sodium [l-"C]acetate was added (2.0 fid per ml of the culture). Each of these cultures was incubated at 37° for 2.5-3.5 hr with shaking to about 2 ~ 5 X 10 • cells/ml. Each culture was then chilled and centrifuged at 7,000-8,500 x g for 10 min at 0°. The cells were washed with cold saline. The pellet was resuspended in 1.5 ml of Tris-HCl buffer (5 mM, p H 7.0), and sonicated (20 kc) for 2 min at 0°. The sonicate was centrifuged at 7,000 xg for 10 min. The supernatant was recentrifuged at 18,000 x g for 15 min. The 18,000xg supernatant and precipitate, suspended in 1.5 ml of the same buffer, were used for the autolysis experiments. Preparation of Spheroplast Membrane—The cells were suspended in 4.5 ml of Tris-HCl buffer (10 mM, pH 7.6) containing 0.5 M sucrose and 10 mM 2-mercaptoethanol, and the suspension was Vol. 80, No. 6, 1976
1249
mixed with 200 /*g/ml of lysozyme [EC 3.2.1.17] (10,000 u/mg, Worthington Biochemical Corporation) dissolved in 0.5 ml of the same Tris-HCl buffer solution. The mixture was incubated at 25° for 10 min, and diluted with 5 ml of Tris-HCl buffer (10 mM, pH 7.6). To the diluted mixture, 0.1 ml of 100 mM EDTA solution was added and the solution was incubated at 25° for 10 min. The spheroplasts, sedimented by centrifugation at 27,000xg for 15 min, were suspended in 3 ml of Tris-HCl buffer (10 mM, pH 7.6) containing 0.5 M sucrose and 10 mM 2-mercaptoethanol. The suspension was diluted rapidly about 10-fold with a mixture of Tris-HCl buffer (10 mM, pH 7.6), 3 mM MgCl,, 10 mM 2mercaptoethanol, and 1 /ig/ml of DNase (2,200 u/mg, Worthington Biochemical Corporation). The solution was centrifuged at 69,000xg for 40 min at 0°, and the precipitate was resuspended in 30 ml of a mixture of Tris-HCl buffer (10 mM, pH 7.6), 10 mM 2-mercaptoethanol, and 3 mM EDTA and recentrifuged. The precipitate, suspended in 1.5 ml of Tris-HCl buffer (5 mM, pH 7.0), was used for assay of autolytic activity of the spheroplast membrane. Preparation of Toluenized Cells—Toluene was added to the cell suspension to 0.1 % (v/v) and the mixture was incubated at 25° for 10 min. The toluenized cell suspension was centrifuged at 3,000xg for 10 min at 0° and the toluene was discarded. Assay of Autolytic Activity—The reaction mixture, containing 0.2 ml of the labeled fraction, 0.4 ml of Tris-HCl buffer (0.2 M, pH 7.5) and 0.1 ml of 10 mM CaCl 2 , was made up to a final volume of 1.0 ml with distilled water. The incubation was carried out at 37° with shaking in a 10 ml centrifuge tubs with a glass stopper. After the reaction, the incubation mixture was immediately chilled with ice and 9 ml of chloroform-methanol ( 2 : 1 , v/v) was added. The mixture was shaken for 1 min and centrifuged at 4,000xg for 10 min. The chloroform extract was evaporated to dryness in vacuo at 30° and the residue was dissolved in 0.3 ml of chloroform-methanol (2 : 1). One-tenth ml of the solution was spotted on a silica gel thin-layer plate and developed with chloroform-methanolacetic acid (65 : 25 : 10, by vol.). The spots corresponding to various lipid fractions were scraped off and counted with a Beckman liquid scintillation counter using 10 ml of Bray's scintillator.
O. DOI and S. NOJIMA
1250
RESULTS
Characterization of DR~DS- and DS~ Mutants—The DR~DS~ mutant: In a previous report (2), we described the presence of two kinds of phospholipases A (DR and DS) in E. coli K-12 wild strains. The properties of DS phospholipase A are quite different from those of the DR enzyme. The DS enzyme hydrolyzes only phosphatidylglycerol. It seemed likely that the DS enzyme might play an important role in bacterial physiology. We also attempted to isolate from the DR~ mutant (parent) a double mutant lacking both DR and DS enzymes in order to investigate the role of the DS enzyme. Sixty colonies, which were possible double mutants, were selected on plates from 1658 colonies of the mutagenized parent. Only a single strain out of
sixty, strain 17, showed no activity of either DS phospholipase A or DR phospholipase A, having only lysophospholipase activity, as shown in Table I. Strain 17 could grow at 42° as well as at 28°. Lysophospholipase activity against both lysophosphatidylglycerol and lysophosphatidylethanolamine was as active as that of the parent. Loss of DS enzyme activity in the extract of the mutant was seen at both 28° and 42°. Preincubation of the extract of mutant 17 at 65° for 5 min (pH 7.0 in 5 mM borate buffer) did not cause recovery of DS enzyme activity. Figure 1 shows the results of experiments in which extracts of mutant 17 (DR~DS~) were mixed with extracts of the parent 23 (DR"DS+) at a protein concentration ratio of 9 to 1 or 49 to 1. In either case, the amount of fatty acids released was nearly proportional to the concentration of the
TABLE I. Distribution of DR phospholipase A, DS phospholipase A, and lysophospholipase in extracts of Escherichia coli K-12 strains. Activities are given as cpm of the unhydrolyzed substrate or reaction products. Assay procedures were as described in "MATERIALS AND METHODS." The protein content of the enzyme preparations in individual tubes for S15, 23, 21, and 17 were 6.3, 5.4, 6.5, and 6.0 mg, respectively. After the reaction, one-third of the total organic solvent extract of each reaction mixture was counted. E. coli K-12 strains
Incubation system for
SI 5
DR phospholi- Phosphatidylpase A assay ethanolamine Phosphatidylglycerol DS phospholi- Phosphatidylpase A ethanolamine Phosphatidylglycerol Cardiolipin
5827 Phosphatidylethanolamine Fatty acids+fatty acid methyl ester 211 Lysophosphatidylethanolamine 179 Phosphatidylglycerol 5663 Fatty acids + fatty acid methyl ester 238 111 Lysophosphatidylglycerol Phosphatidylethanolamine Fatty acids Lysophosphatidylethanolamine Phosphatidylglycerol Fatty acids Lysophosphatidylglycerol Cardiolipin Fatty acids Lysocardiolipin
Lysophospholi- Lysophosphatidyl- Lysophosphatidylethanolamine pase ethanolamine Fatty acids Lysophosphatidyl- Lysophosphatidylglycerol glycerol Fatty acids
23(DR") 21(DS") 17(DR"DS-) 6037
4812
5675
0 0
316 267
0 0
6011
5075
5631
0 0
227 192
0 0
6036
5250
5940
6038
0 0
0 0
0 0
0 0
5665
5451
5970
5137
310 0
658 0
30 0
0 0
N.D>
862 6 4
N.D.«
929 0 0
2721
3458
2675
3371
301
277
250
286
1030
960
1019
731
712
1130
745
1056
Not determined /. Biochem.
NATURE OF E. coli PHOSPHOLIPASE A MUTANTS
1251
shown in Fig. 2. Doubling times at the logarithmic phase of growth of the parent 15, 21 (DS"), 23 (DR-), and 17 (DR-DS") were 23, 27, 27, and 23 min, respectively, at 37° in Penassay medium, and 102, 90, 84, and 90 min, respectively, at 37° in the synthetic medium. No difference in growth at 28° or 42° among the mutants and parent was observed. If the fatty acid composition of cells of the bacteria changes after shift-down of the temperature (6) and phospholipase A participates in the redistribution of the membrane phospholipids, it is possible that some effect might occur on the growth of mutants after shift-down of the temperature when compared with that of the parent. However, no difference in the growth of mutants and parent was observed after shift-down from 37° to 19°. Thus, 30 60 90 Incubation Time (min) cultures of the parent 15 and mutant 17 (DR-DS"), Fig. 1. Hydrolysis of ["Qphosphatidylglycerol by cell grown at 37° in Penassay medium to concentraextracts of strain 17, strain 23, a mixture of 90% strain tions at 1.7 x 10s cells and 1.6 xlO8 cells/ml, re17 and 10% strain 23 and a mixture of 98% strain 17 spectively, were transferred to a water-bath (19° ± and 2% strain 23. Assay procedures were as described 0.5°) and shaken for 6 hr. Both strains showed a for DS phospholipase A assay in "MATERIALS AND similar lag period (about 55 min) and then multiMETHODS." The protein concentrations of the ex- plied with almost the same doubling time (parent: tracts were 10 mg/ml. o , strain 23 (DR"DS+); • , 240 min, mutant: 265 min). It may be concluded strain 17 (DR-DS-); A, 1:9 mixture; x, 1 :49 from these experiments that both DR and DS mixture. phospholipases A are not required for the growth of E. coli cells, even when the growth temperature is parent extract added. These results indicate that shifted down during the logarithmic phase of the inability of the mutant to hydrolyze phosphati- growth. dylglycerol is not due to the accumulation of an The Compositions of Phospholipids and Fatty inhibitory substance(s) within the mutant cell. Acids—Tables II and DI shows, respectively, comThe DS~ mutant: During mapping of the parisons of phospholipid and fatty acid composipldA gene, strain 21 was obtained after mating tions of the mutants and parent. There was no strain 17 (DR"DS") with Hfr P4X6. The DR and significant difference among the mutants and parent DS phospholipase A activities of strain 21 are in this case either. shown in Table I. The DR enzyme activity was Degradation of Phosphatidylglycerol In Vivo— fully recovered in the extract of strain 21, while the Extensive turnover of phosphatidylglycerol during DS enzyme activity was less than 10% of that of the the logarithmic phase of growth occurs in various extract of P4X6. bacteria, including E. coli (7-12). It was also Comparison of the mutants and the parent— shown that the phosphate moiety and the acyl Growth, fatty acid and phospholipid compositions, moiety of phosphatidylglycerol turn over at different and turnover of phospholipids were compared rates (11). The possible role of phospholipase A, especially DS-phospholipase A, which is specific to among the three mutants and parent. Growth—Growth characteristics were com- phosphatidylglycerol, in the mechanism of the in pared among mutants and parent by means of vivo degradation of phosphatidylglycerol, may be viable cell counts. Although the start of growth considered in relation to the turnover of the acyl seemed to be retarded just after transfer to a fresh moiety in phosphatidylglycerol. However, alternamedium, especially in the case of mutant 17 (DR-- tive possibilities may also exist. DS~), almost no difference of growth rates among In any event, cells in the logarithmic phase of the three mutants and parent was observed, as growth were labeled with ["Qacetate and the loss Vol. 80, No. 6, 1976
O. DOI and S. NOJIMA
1252
21 (DS")
S15(Parent )
0
30
60 90 120 Tlm»( ml n )
0
150
30
60
90
1 20 1 50
10'
Timt ( m i n )
23(DR')
17(DRDS") ..-o-o
10 9
10'
10'
s
10'
10' 30
60
90
1 20 150
Time ( min )
30
60 9 0 1 20 1 50 Time tmln )
Fig. 2. Comparison of growth and turnover of phospholipids between mutants of E. coli K-12 defective in phospholipase A and the parent. The cells of E. coli K-12 strains SI 5 (parent), 21 (DS-), 23 (DR"), and 17 (DR-DS") were cultured overnight in Penassay broth medium at 37°. Each of these cultures was added to 15 ml of the same medium as 0.5% (v/v) and an aqueous solution of [l-^CJacetate (Na) was added to the culture (2.5 /iCi per ml of the culture). Each of these cultures was incubated at 37° for 3 hr and centrifuged at room temperature for 5 min at 4000 x g. The pellet of cells from each sample was suspended in 5 ml of Penassay broth, the resulting suspension was filtered through a Millipore filter at room temperature and the washing was repeated once more. The labeled cells on the filter were resuspended in 40 ml of Penassay broth and cultured at 37° for 165 min. At appropriate times, a sample of 0.05 ml was assayed for viable cell count on i-agar plate and 2 samples of 1.0 ml were removed for analysis of phospholipids (2). O, cell growth; • , phosphatidylethanolamine; x , phosphatidylglycerol; A , cardiolipin.
/. Biochen.
1253
^NATURE OF E. coli PHOSPHOLIPASE A MUTANTS
TABLE II. Comparison of phospholipid compositions in parent and mutants of E. coli K-12 defective in phospholipase A. The cells of E. coli K-12 strains S15, 23, 21, and 17 were cultured in i-broth medium overnight at 37°. Each of these cultures was added to 15 ml of the same medium as 2% (v/v) and an aqueous solution of 1 -["CJacetate was added to the culture (5 /iCi per ml of the culture). Each of these cultures was incubated at 37° for 3.5 hr and the viable cells were counted. 3.1 X10*, 3.0x 10", 2.1 x 10», and 2.6x 10» cells per ml were taken for SI5, 23, 21, and 17, respectively. The culture was then centrifuged at 0° for 10 min at 10,000 xg. The pellet of cells was washed once with saline. Lipids were extracted from the cells and analyzed according to the procedures already described (2). PE, phosphatidylethanolamine; PG, phosphatidylglycerol; CL, cardiolipin. K-12 strain SI5 23 21 17
Total phospholipids ; of protein)
PE
4.6
78.3 79.9 75.5 78.3
(parent) (DR-) (DS-) (DR-DS-)
CL
PG /a
5.1 4.9 4.8
16.8 16.3 17.9 16.0
4.9 3.8 6.6 5.7
TABLE III. Comparison of fatty acid compositions in parent and mutants of E. coli K-12 defective in phospholipase A. Cells were cultured in 50 ml of Penassay broth for 3 h at 37°. The pellets obtained after centrifuga1ion of the culture were washed once with saline and lipids were extracted (2). The total lipids of each culture -were dissolved in 0.2 ml of methanol and 1.45 ml of water and hydrolyzed with 0.35 ml of 85% KOH by incubating ihe mixture at 70° for 30 min. After saponification, the mixture was cooled, neutralized with 6N HCl using thymol blue as an indicator, and extracted twice with diethylether. The combined ether extract was washed with concentrated aqueous KCl then evaporated to dryness. The residue was methylated with CH,N,. Analysis of the methyl esters was carried out on glass columns (1/8 x 10 inch) of Chromosorb W coated with 20% EGSS-X at 180° using a flame ionization detector (Varian Aerograph series 1800). The extracted cell residue of each strain was saponified under alkaline conditions and methyl esters were obtained as described above. The fatty acid patterns of each strain were very similar (C14:hydroxy; more than 50%, C l l : 0 , C 14;0 , C K : 0 and CU:L). Growth phase Strains
Logarithmic
Stationary
S15(parent) 23(DR") 21(DS-) 17(DR-DS") S15(parent) 23(DR") 21(DS") 17(DR-DS")
cells/ml(10-') Ci4 : o
5.6 3.5
6.0 5.3
6.0 5.1
6.1 5.0
34 4.9
30 6.8
24 8.5
13 6.3
•Cl«:0
48
46
50
48
50
49
53
50
•Ci.=i
29
33
35
32
23
28
27
26
•C17 (cyclopropane)
4.3 15
3.0 12
3.1 6.4
of the label in the acyl moieties of three phospholipids, phosphatidylethanolamine, phosphatidylgly•cerol and cardiolipin after chasing, was compared among the mutants and parent. As shown in Fig. 2, there was no marked difference between the patterns of phospholipid •degradation of the mutants and that of the parent. Phosphatidylethanolamine was stable, while phosphatidylglycerol was degraded. Cardiolipin was Vol. 80, No. 6, 1976
2.6 12
11 11
6.5 10
4.8 6.7
7.6 10
also degraded slowly. The percentage loss of phosphatidylglycerol in one doubling time was 11, 15, 11, and 11 in the parent 15 and the mutants 21 (DS-), 23 (DR-), and 17 (DR-SD"), respectively. Effects of Methanol and Detergents on the Hydrolysis of Phosphatidylglycerol by 18,000 X Q Supernatant of the Extracts of the Parent and Mutants—As shown in Fig. 3, the DS activity was
O. DOI andlS. NOJIMA
1254
2000
2000
8
1000
1000
20
40 60 Methanol 7.(v/v)
80
01
02 Q3 04 5DS 7 . ( w / v )
05
01
02 03 04 05 Cholate 7. ( w/v )
2000 3000 c
12000
Jhooo' •o
X
0 1000 < ffi
ai Q2 03 0A 05 Triton X-100 7.(w/v)
Fig. 3. Effects of methanol, SDS, Triton X-100 and sodium cholate on the hydrolysis of phosphatidylglycerol by extracts of E. coli K-12 strains. ["QPhosphatidylglycerol was used as a substrate. • , wild strain; O, DR+DS"; X, DR"DS+; A, DR-DS". suppressed by detergents and methanol while DR activity was seen only in the presence of methanol or detergents. In the case of cholate, the DS activity was slightly activated at lower concentrations but suppressed at higher concentrations. Autodegradation of Membrane Phospholipids—
As regards the growth of the unsaturated fatty acid auxotroph on an agar plate, some growth was noted even on the spot of mutant DR~DS". This indicates that autolyzed cells of this mutant still release
fatty acids, whatever the hydrolytic pathway may be. We have repeatedly confirmed the absence of phospholipase A activity in the extract of the mutant with respect to exogenous E. coli phospholipid,. provided incubation was carried out in an aqueousE. coli phospholipid. The following experiments were designed to compare, among mutants and parent, the hydrolysis of endogenous membrane phospholipids after incubation of their extracts or 18,000 xg precipitate. /. Biochem.
NATURE OF E. coli PHOSPHOLIPASE A MUTANTS -(DRD^f
DRDS) 20
40
60
1255
TABLE IV. Composition of fatty acids released from endogenous membrane lipids after incubation with the labeled 18,000xg supernatants of E. coli K-12 cell homogenates. After incubation of the 18,000 x g supernatant, the lipids were extracted and chromatographed on a silica gel thin-layer plate using chloroformmethanol-water (65 : 25 : 4, by vol.). The spot corresponding to fatty acids was scraped from the plate and extracted with chloroform-methanol ( 2 : 1 , v/v). The extracted lipids were saponified with alkali and the fatty acid fraction was methylated with CH.Ni. The methyl esters were analyzed by gas-liquid chromatography as described in Table HI, and the individual eluates from column were collected in glass U tubes and counted with a Beckman liquid scintillation counter using 10 ml of Bray's scintillator.
Incubation Time(mln)
Fig. 4. Time courses of endogenous membrane phospholipid degradation by 18,000xg supernatants of cell homogenates of E. coli K-12 mutants defective in phospholipase A. Experimental details are described in "MATERIALS AND METHODS." During incubation of the mixture of the labeled and non-labeled fractions, an equal amount of protein of each fraction was mixed. Usually 3.0 mg protein per ml was used, •denotes labeled membranes.
S15(DR+DS+)
Strain
Cll:0
Cn:o
C17 (cyclohropane)
3 32 40 5 15
17(DR-DS")
1 8 33 33 6 14
3 u
1! s
a.
1 —i-
25
50
100
200
400 800 1600 3200
Phosphatidylethandarnine ( uM )
Fig. 5. Inhibition of hydrolysis of phosphatidylglycerol by phosphatidylethanolamine. The incubation mixture contained 0.25 ml of ["Qphosphatidylglycerol (0.4 ^imoles, approx. 16,000 cmp), appropriate amounts of phosphatidylethanolamine as shown in thefigure,0.05 ml of 0.1 M CaCl,, 0.50 ml of 0.2 M Tris-HCl (pH 7.0), and 0.20 ml of enzyme solution, made up to a final volume of 1.5 ml with deionized water. Other procedures are described in "MATERIALS AND METHODS." Vol. 80, No. 6, 1976
10
0
30 60 90 Incubation Time ( min )
Fig. 6. Time courses of endogenous membrane phospholipid degradation by 18,000xg precipitates of cell homogenates of E. coli K-12 mutants defective in phospholipase A. The experimental procedures were as described in Fig. 4.
1256 18,000 x g Supernatant—-The 18,000 x g supernatants of the sonicates of ["Qacetate-labeled cells (the parent and three mutants) were incubated at 37° and the amounts of free fatty acids released were compared (Fig. 4). After incubation for 20 min, the extents of fatty acid release from endogenous substrate in the cases of mutants 23 (DR~) and 17 (DR-DS-) were about 10% of that in the case of the parent 15. After 1 hr, they increased to 14 and 19 %, respectively. As already pointed out (4), the difference between the parent and the three mutants, or that between mutant 21 (DS~) and the other two mutants (DRr, DR~DS") is clear. However, virtually no difference between mutant 23 (DR~) and mutant 17 (DR"DS~) was observed. These experimental results might be explained as follows: DS enzyme may be more latent than DR enzyme, while the DS enzyme activity is not manifested in the presence of excess phosphatidylethanolamine (Fig. 5). This accounts for the very slight difference between mutant 23 (DR") and mutant 17 (DR-DS"). The activity of DS enzyme could be observed in these experiments only when DS enzyme hydrolyzed phosphatidylglycerol released from the membrane after the initial attack of DR enzyme on membrane phospholipids. Mixing of the extract of mutant 23 or 17 (DR- or DR"DS") with that of mutant 21 (DS~) was expected to clarify the situation. [uC]Acetate-labeled extract of the mutant (DR~ or DR"DS") was incubated with or without non-labeled extract of mutant DS" and the labeled fatty acid release was observed. However, as shown in Fig. 4, no significant increase of fatty acid release from the labeled extract in the presence of the extract of mutant DS~ was observed. Thus, the above hypothesis was not supported by this experiment. A comparison of the composition of the fatty acids released from the extract of mutant D R ' D S " with that of the parent is shown in Table IV. No significant difference between mutant DR~DS~ and the parent is apparent. J8,000xg Precipitate—The 18,000X0 precipitate of the sonicate of ["Qacetate-labeled cells of the parent or mutants was also incubated at 37° and the amount of free fatty acids released was determined. As shown in Fig. 6, after incubation for 30 min, the amount of fatty acid released from endogenous substrate in the parent or mutant DS~ was more than 20-23% of that of the prelabeled
O. DOI and S. NOJIMA membrane phospholipids. On the other hand,, only 1 % of the endogenous phospholipids washydrolyzed in the other mutants (DR~, DR-DS"). As in the case of 18,000 x g supernatant, mixing of the extract of mutant DR" or DR~DS" with that of mutant DS~ was carried out, but the results were the same as those with 18,000 Xg supernatant. The autolytic activity in the 18,000xg precipitate fraction was about 6-7 times higher than that in the 18,000xg supernatant fraction (Figs. 4 and. 6). When cells of the parent or mutant DS~ wereused, strong membrane phospholipid degradation activity was also detected in the toluenized cell fraction, spheroplast cell membrane fraction,, polymyxin B-treated cell fraction and other cellsin which the membrane had been damaged by chemical reagents. However, these autodegradations were completely inhibited by the addition of EDTA. In every case, only slight degradatioa activity was detected in the cells of strains defective in DR enzyme (DS~, DR"DS"). Comparison of the Effects of Detergents and Organic Solvents on the Hydrolysis of Exogenous Substrate, Phosphatidylethanolamine, by 18,000 X q Supernatant and on the Release of Fatty Acids from the Supernatant after Incubation—The effects of detergents (0.1 %, w/v), such as sodium deoxycholate, sodium dodecyl sulfate, Tween 80, Triton X-100, and BRIJ 58, or organic solvents (20%, v/v), such as methanol, ethanol, and diethyl ether on the release of fatty acids from ["Qphosphatidylglycerol by extracts of the parent and the three mutants were examined. These compounds stimulated the release of fatty acids from the exogenous substrate by extracts of the parent and mutant DS~ while they suppressed the activities of the extracts of DR~. This result parallels the difference in the behavior of phospholipases A with detergents. The D R enzyme activity was apparently stimulated while the DS enzyme was inactivated when exogenoussubstrate was used. The effects of detergents on endogenous membrane hydrolysis were somewhat different from the effects obtained with exogenous substrates. At a concentration of 0.5%, all the detergents examined suppressed the endogenous substrate hydrolysis activity of any of the extracts of the parent and mutant DS" as well as mutants DR~ and DR"DS"; In order to clarity this discrepancy, we examined in. /. Biochem.
NATURE OF E. coli PHOSPHOLIPASE A MUTANTS 200
£
0
20 40 Methanol %(v/v)
60
1257
detail the effects of methanol, SDS, and Triton X-100 on the activity of the extract of mutant DS~ against endogenous substrate as well as exogenous substrate (Fig. 7a, b, c). As shown in Fig. 7a, methanol markedly activated DR activity against exogenous phosphatidylethanolamine, while it activated the endogenous phospholipid hydrolysis by 122% at 10-20% (v/v) but inactivated it at higher concentrations. In the case of SDS, this tendency is more marked. At a concentration of 0.005 %, SDS activated the endogenous activity by 149%, but the activity fell off sharply at higher concentrations and no activity was detected at 0.025%. On the other hand, exogenous phosphatidylethanolamine was hydrolyzed only in the presence of SDS, using 18,000 xg supernatant. Triton X-100 showed the same tendency, but at higher concjntrations. No activation of endogenous phospholipid hydrolyzing activity was observed in the case of Triton X-100. DISCUSSION
005 SOS 7.(\Wv)
£
1.0
Fig. 7 Vol. 80, No. 6, 1976
Q2-Q5
Some properties of three mutants of E. coli K-12 with respect to phospholipase A (DR~, DS", and DR~DS") were investigated in the present study. In summary, no marked differences between the parent and the three mutants were found with respect to growth, lipid composition, and other properties. During genetic mapping of the locus for DR phospholipase A (pldA) (J), the cells with wild-type phospholipase A were transduced to pldA~ by phage PI grown on strain 17 (DR-DS~) and strain 17 transduced to pldA+ by Pic grown on cells with wild-type phospholipase A. The growth of these transductants was similar to that of the wild strain. Several investigators, including the present authors, have used these mutants to clarify the role of phospholipase A in the following phenomena: the growth of bacteriophages {13), transfection of Cas+-dependent phage DNA (14) and the action of colicin K (75). However, no evidence was obtained for the participation of Fig. 7. Effect of methanol, SDS or Triton X-100 concentration on DR phospholipase A activity and endogenous phospholipid-hydrolyzing activity of E. coli. x, fatty acid released from endogenous phospholipid; O, fatty acid released from exogenous phosphatidylethanolamine.
1258
O. DOI and S. NOJIMA
phospholipase A. Moreover, it was indicated that phage-induced lysis and the release of progeny phage are separable in T4s-infected strain 17 (DR~DS") (75). In general, the growth of E. coli cells is very slow when the medium contains fatty acids as a sole carbon source (17). If phospholipases A, especially DR phospholipase A, which is in the outer membrane of E. coli (18-20), play a role in the digestion of phospholipids in the medium, the cells would be expected to grow in the presence of phospholipids as a sole carbon source. However, no growth was observed in this laboratory under such conditions. In this respect, it is very interesting that Mycobacterium tuberculosis and Mycobacterium bovis grow well in liquid media containing egg lecithin or synthetic dipalmitoyllecithin as a sole carbon source (21). A highly active membrane-bound phospholipase Ax has been purified and characterized from cell of Mycobacterium phlei (22).
2. Doi, O., Ohki, M., & Nojima, S. (1972) Biochim. Biophys. Ada 260, 244-258 3. Abe, M., Okamoto, N., Doi, O., & Nojima, S. (1974) / . Bacteriol. 119, 64-69 4. Nojima, S., Doi, O., Okamoto, N., & Abe, M. (1972) in Membrane Research (Fox, C.F., ed.) pp. 135-144, Academic Press 5. Cronan, J.E. & Godson, G.N. (1972) Molec. Gen. Genet. 116, 199-210 6. Cronan, J.E., Jr. & Vagelos, P.R. (1972) Biochim. Biophys. Ada 265, 25-60 7. Kanfer, J. & Kennedy, E.P. (1963) / . Biol. Chem. 238, 2919-2922 8. Kanemasa, Y., Akamatsu, Y., & Nojima, S. (1967) Biochim. Biophys. Ada 144, 382-390 9. Ohki, M. (1972) /. Mol. Bio!. 68, 249-264 10. Ames, G.F. (1968) / . Bacteriol. 95, 833-843 11. White, D.C. & Tucker, A.N. (1969) / . Lipid Res. 10, 220-233 12. Short, S.A. & White, D.C. (1971) / . Bacteriol. 108, 219-226 13. Sakakibara, Y., Doi, O., & Nojima, S. (1972) Biochem. Biophys. Res. Commun. 46, 1434-1440 The homogenates of mutant DR~DS~ showed 14. Taketo, A. (1974) / . Biochem. 75, 895-904 less than one-hundredth (DR) or one-fiftieth (DS) 15. Lusk, J.E. & Park, M.H. (1975) Biochim. Biophys. Ada 394, 129-134 of the activities of the homogenate of the parent against exogenous substrates. Autolysis of labeled 16. Hardaway, K.L., Marten, M.V., & Buller, C.S. (1975) J. Virology 16, 867-871 homogenates of the mutant caused the release of 17. Overath, P., Pauli, G., & Schairer, H.U. (1969) } | free fatty acids amounting to less than 5 % of that Eur. J. Biochem. 7, 559-574 of the parent (Figs. 4 and 6). At present, the na18. Albright, F.R., White, D.A., & Lannarz, W.J. ture of the fatty acid-releasing activity observed in (1973) J. Biol. Chem. 248, 3968-3977 the mutant remains to be clarified. Further 19. Bell, R.M., Mavis, R.D., Osborn, M.J., & Vagelos, attempts to isolate a mutant which lacks this acP.R. (1971) Biochim. Biophys. Ada 249, 628-635 tivity have so far been unsuccessful. 20. Yamato, I., Anraku, Y., & Hirosawa, K. (1975) / . Biochem. 77, 705-718 21. Kondo, E. & Kanai, K. (1976) Jap. J. Med. Sci. REFERENCES Biol. 29, 109-121 1. Ohki, M., Doi, O., & Nojima, S. (1972) /. Bacteriol. 22. Nishijima, M., Akamatsu, Y., & Nojima, S. (1974) / . Biol. Chem. 249, 5658-5669 110, 864-869
/. Biochem.
Nature of Escherichia coli Mutants Deficient in DetergentResistant and/or Detergent-Sensitive Phospholipase A Osamu DOI and Shoshichi NOJIMA 1 Department of Chemistry, National Institute of Health, Kamiosaki, Shinagawa-ku, Tokyo 141 Received for publication, June 7, 1976
1. Escherichia coli K-12 mutants deficient in detergent-resistant (DR) and detergent-sensitive (DS) phospholipases A and deficient in DS phospholipase A were isolated. 2. The growth, compositions of phospholipids and fatty acids and turnover of phospholipids of three mutants (DR~, DS", DR~DS~) were compared with those of their parent (DR" was isolated in a previous study). 3. Autodegradations of membrane phospholipids of 18,000xj supernatants and precipitates of the homogenates of these three mutants were also compared with those of the parent, and the effects of various detergents and organic solvents on these activities were examined. 4. We could not identify any significant physiological role for DR or DS phospholipase A.
Recently, we described the isolation of a mutant deficient in a detergent-resistant (DR) phospholipase A from E. coli K-12 (1). During the characterization of this mutant, another kind of phospholipase A, detergent-sensitive (DS) phospholipase A, was found in the mutant as well as in the parent (2). A double mutant deficient in both DR and DS enzymes has been isolated from the mutant deficient in DR enzyme by mutagenesis. A mutant deficient in only DS enzyme has also been isolated from the double mutant by conjugation. Thus, in our laboratory, three mutants, DR", DS", and D R " D S " are now available. In a separate paper, we reported the determination of DR phospholipase A gene on the E. coli chromosome (J). The present paper deals with a comparison of the growth, lipid composition, phospholipid turnover, and other characteristics of the three 1
Present address: Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113. Vol. 80, No. 6, 1976
mutants with those of the parent. A preliminary report has already been published (4). MATERIALS AND METHODS Organisms—E. coli K-12 strain 23 (leu~thr~thi~lac~pldA~f) derived from Wl, the isolation of which had already been reported (7), was used as a starting strain in this study. N912 (JabB strR), an unsaturated fatty acid auxotroph derived from 1
We proposed in a previous paper (5) that mutants specifically defective in phospholipid degradation be given the standard genetic symbol pld (for phospho/ipid degradation), following the proposal by Cronan (5) of the genetic symbol pis for phospho/ipid synthesis. The mutant in detergent-resistant phospholipase A was called pld A. However, in this paper, we use the original phenotypic expression, DR±, since it is easy to see its relation to DS, the location of which is not yet established.
1247
1248 P678 (Jacob and Wollman), which was also obtained in the previous study (/), was used as an indicator for the isolation of DS phospholipase A-negative mutant. Media—/i-Broth, which contains 10 g of polypeptone (Daigo Eiyo Co. Tokyo) and 2.5 g of NaCl per liter, Penassay broth (Difco) and minimum synthetic medium, which contains 7 g of K 2 HPO 4 , 2 g of KH.PO,, 0.5 g of sodium citrate2H,O, 1 g of (NH^SO,, 0.1 ml of 20% MgSO47H,O, 0.1 ml of Bx (10 mg/ml) and 5 g of glucose per liter, were used. Agar was prepared by supplementing broth with 1% agar; soft agar was prepared by supplementing with 0.5 % agar. Isolation of Mutants—The DR~DS" mutant was isolated from the parent strain 23 (DR~). The principle for selection of the mutant was similar to that described previously (7); endogenous phospholipids in the mutagenized cells were hydrolyzed by autolysis to form free fatty acids, probably by the combined action of phospholipase A and lysophospholipase [EC. 3.1.1.5]. The fatty acids released were assayed using the unsaturated fatty acid auxotroph of E. coli as an indicator strain. In the case of isolation of the DR~ mutant (7), colonies of mutagenized cells on the plate were lyzed with sodium dodecyl sulfate. However, for the isolation of a detergent-sensitive phospholipase A-negative mutant, some other procedure for the lysis of cells on the plate was required. After several trials, exposure of a spot of the culture to chloroform vapor followed by incubation at 37° was found to be suitable for the assay of DS phospholipase A on the plate. Practically, cells of the strain 23 (DR~) in the logarithmic phase of growth (2 x 10s cells/ml) were harvested and suspended in 0.2 M acetate buffer (pH 5.0) containing N-methyl-N'-nitro-N-nitrosoguanidine (700 fig/ ml). After incubation for 60 min at 37°, bacteria were washed once with saline-phosphate buffer, suspended in 5 ml of Penassay broth and incubated at 28° for 20 hr. The bacteria were washed once and resuspended in 5 ml of ,1-broth. One ml of the suspension was again mixed with 3 ml of /{-broth containing 900 fig of penicillin G, and the mixture was incubated at 28° for 24 hr without shaking. Aliquots were spread on /}-agar plates and incubated at 28° for 48 hr. Cells of each colony on the plate were transferred to 2 ml of /l-broth and incubated at 28° for 20 hr with shaking. 0.02-0.04 ml of each
O. DOI and S. NOJIMA culture was spotted on a /i-agar plate, and the plates were incubated at 42° for 2 hr. The cells on the plates were exposed to chloroform vapor at room temperature and further incubated at 37° for 30-60 min in order to increase autolysis of the cells. The sensitivity of the assay was increased by making use of spots of the culture instead of colonies on the plate. Three to four ml of soft agar, maintained at 50±l°, was carefully poured over the spots on the plate; the soft agar contained 5 x 10s cells of N912 (unsaturated fatty acid-auxotrophic and streptomycin-resistant) and streptomycin (300 /ig/ml). The plate was incubated at 42°. After incubation for 2 days, spots on which no or only slight growth of the unsaturated fatty acid auxotroph was present were sought. The detergent-sensitive phospholipase A-negative mutant was derived from this double mutant strain 17 (DR~DS~) by conjugation with Hfr P4X6. Details of the genetic experiments have been reported elsewhere (3). DR Phospholipase A Assay—Preparation of substrates was as described previously (2). Cells were grown to S x l O ^ l x l O " cells/ml at 37° in Penassay broth (Difco) medium with shaking. A culture (100 ml) was then chilled and centrifuged at 7,000-8,500 x g for 10 min at 0°. The cells were washed with cold saline. The pellet was suspended in 2 ml of 5 mM of Tris-HCl buffer (pH 7.5) and sonicated (Umeda Sonicator, 20 kc) for 2 min at 0°. The sonicate was centrifuged at 13,000 x g for 15 min at 0°. The supernatant solution was used as the enzyme preparation (4.2-8.6 mg protein/ml). The incubation mixture contained 0.25 ml of ["CJphospholipids (0.2 pmoles, approx. 8,000 cpm), 0.05 ml of 0.1 M CaCls, 0.50 ml of 0.2 M Tris-HCl buffer (pH 8.2), 0.20 ml of enzyme solution (in 5 mM Tris-HCl buffer, pH 7.5) and 1.5 ml of methanol, made up to a final volume of 3.0 ml with deionized water. Incubation was carried out at 37° with shaking in a 10 ml centrifuge tube with a glass stopper. The reaction period was usually 30 min. After the reaction, the incubation mixture was immediately chilled to 0° and 1.5 ml of methanol plus 6.0 ml of chloroform was added to the incubation mixture. The mixture was shaken for 1 min and centrifuged at 2,000 xg for 10 min to separate it into two layers. To the upper layer, 6.0 ml of chloroform was added, and the mixture was again shaken for 30 sec and centrifuged. The
J. Biochem.
NATURE OF E. coli PHOSPHOLIPASE A MUTANTS combined chloroform extract was evaporated to dryness in vacua at 30° and the residue was redissolved in 0.30 ml of chloroform-methanol ( 2 : 1 , v/v). Next, 0.1 ml of the solution was spotted on a silica gel thin-layer plate and developed with chloroform-methanol-water (65 : 25 : 4, by vol.). The spots corresponding to diacylphospholipids, Iysophospholipids, fatty acids, and fatty acid methylesters were scraped off and counted with a Beckman liquid scintillation counter using 10 ml of Bray's scintillator. DS Phospholipase A and Lysophospholipase Assay—Cell extracts were prepared as described above. The incubation mixture contained 0.25 ml of [14C]phospholipids or [14C]lysophospholipids (0.2 //moles, approx. 8,000 cpm), 0.05 ml of 0.1 M CaCl,, 0.50 ml of 0.2 M Tris-HCl (pH 7.0), and 0.20 ml of enzyme solution, made up to a final volume of 1.50 ml with deionized water. With 2-acyl-glyceryl-phosphorylethanolamine as a substrate, 0.50 ml of 0.1 M borate buffer (pH 6.5) was employed instead of Tris-HCl buffer. Incubation was carried out at 37° and the reaction period was usually 15 min. After the reaction, the incubation mixture was immediately chilled at 0°, 3.0 ml of methanol plus 6.0 ml of chloroform was added, and the mixture was treated as described for DR. phospholipase A assay. Preparation of 18,000 x g Supernatant and Precipitate—Cells were cultured at 37° in Penassay broth (Difco) medium overnight. Each of these cultures were added to 50 ml of the same medium at a level of 1 % (v/v), and an aqueous solution of sodium [l-"C]acetate was added (2.0 fid per ml of the culture). Each of these cultures was incubated at 37° for 2.5-3.5 hr with shaking to about 2 ~ 5 X 10 • cells/ml. Each culture was then chilled and centrifuged at 7,000-8,500 x g for 10 min at 0°. The cells were washed with cold saline. The pellet was resuspended in 1.5 ml of Tris-HCl buffer (5 mM, p H 7.0), and sonicated (20 kc) for 2 min at 0°. The sonicate was centrifuged at 7,000 xg for 10 min. The supernatant was recentrifuged at 18,000 x g for 15 min. The 18,000xg supernatant and precipitate, suspended in 1.5 ml of the same buffer, were used for the autolysis experiments. Preparation of Spheroplast Membrane—The cells were suspended in 4.5 ml of Tris-HCl buffer (10 mM, pH 7.6) containing 0.5 M sucrose and 10 mM 2-mercaptoethanol, and the suspension was Vol. 80, No. 6, 1976
1249
mixed with 200 /*g/ml of lysozyme [EC 3.2.1.17] (10,000 u/mg, Worthington Biochemical Corporation) dissolved in 0.5 ml of the same Tris-HCl buffer solution. The mixture was incubated at 25° for 10 min, and diluted with 5 ml of Tris-HCl buffer (10 mM, pH 7.6). To the diluted mixture, 0.1 ml of 100 mM EDTA solution was added and the solution was incubated at 25° for 10 min. The spheroplasts, sedimented by centrifugation at 27,000xg for 15 min, were suspended in 3 ml of Tris-HCl buffer (10 mM, pH 7.6) containing 0.5 M sucrose and 10 mM 2-mercaptoethanol. The suspension was diluted rapidly about 10-fold with a mixture of Tris-HCl buffer (10 mM, pH 7.6), 3 mM MgCl,, 10 mM 2mercaptoethanol, and 1 /ig/ml of DNase (2,200 u/mg, Worthington Biochemical Corporation). The solution was centrifuged at 69,000xg for 40 min at 0°, and the precipitate was resuspended in 30 ml of a mixture of Tris-HCl buffer (10 mM, pH 7.6), 10 mM 2-mercaptoethanol, and 3 mM EDTA and recentrifuged. The precipitate, suspended in 1.5 ml of Tris-HCl buffer (5 mM, pH 7.0), was used for assay of autolytic activity of the spheroplast membrane. Preparation of Toluenized Cells—Toluene was added to the cell suspension to 0.1 % (v/v) and the mixture was incubated at 25° for 10 min. The toluenized cell suspension was centrifuged at 3,000xg for 10 min at 0° and the toluene was discarded. Assay of Autolytic Activity—The reaction mixture, containing 0.2 ml of the labeled fraction, 0.4 ml of Tris-HCl buffer (0.2 M, pH 7.5) and 0.1 ml of 10 mM CaCl 2 , was made up to a final volume of 1.0 ml with distilled water. The incubation was carried out at 37° with shaking in a 10 ml centrifuge tubs with a glass stopper. After the reaction, the incubation mixture was immediately chilled with ice and 9 ml of chloroform-methanol ( 2 : 1 , v/v) was added. The mixture was shaken for 1 min and centrifuged at 4,000xg for 10 min. The chloroform extract was evaporated to dryness in vacuo at 30° and the residue was dissolved in 0.3 ml of chloroform-methanol (2 : 1). One-tenth ml of the solution was spotted on a silica gel thin-layer plate and developed with chloroform-methanolacetic acid (65 : 25 : 10, by vol.). The spots corresponding to various lipid fractions were scraped off and counted with a Beckman liquid scintillation counter using 10 ml of Bray's scintillator.
O. DOI and S. NOJIMA
1250
RESULTS
Characterization of DR~DS- and DS~ Mutants—The DR~DS~ mutant: In a previous report (2), we described the presence of two kinds of phospholipases A (DR and DS) in E. coli K-12 wild strains. The properties of DS phospholipase A are quite different from those of the DR enzyme. The DS enzyme hydrolyzes only phosphatidylglycerol. It seemed likely that the DS enzyme might play an important role in bacterial physiology. We also attempted to isolate from the DR~ mutant (parent) a double mutant lacking both DR and DS enzymes in order to investigate the role of the DS enzyme. Sixty colonies, which were possible double mutants, were selected on plates from 1658 colonies of the mutagenized parent. Only a single strain out of
sixty, strain 17, showed no activity of either DS phospholipase A or DR phospholipase A, having only lysophospholipase activity, as shown in Table I. Strain 17 could grow at 42° as well as at 28°. Lysophospholipase activity against both lysophosphatidylglycerol and lysophosphatidylethanolamine was as active as that of the parent. Loss of DS enzyme activity in the extract of the mutant was seen at both 28° and 42°. Preincubation of the extract of mutant 17 at 65° for 5 min (pH 7.0 in 5 mM borate buffer) did not cause recovery of DS enzyme activity. Figure 1 shows the results of experiments in which extracts of mutant 17 (DR~DS~) were mixed with extracts of the parent 23 (DR"DS+) at a protein concentration ratio of 9 to 1 or 49 to 1. In either case, the amount of fatty acids released was nearly proportional to the concentration of the
TABLE I. Distribution of DR phospholipase A, DS phospholipase A, and lysophospholipase in extracts of Escherichia coli K-12 strains. Activities are given as cpm of the unhydrolyzed substrate or reaction products. Assay procedures were as described in "MATERIALS AND METHODS." The protein content of the enzyme preparations in individual tubes for S15, 23, 21, and 17 were 6.3, 5.4, 6.5, and 6.0 mg, respectively. After the reaction, one-third of the total organic solvent extract of each reaction mixture was counted. E. coli K-12 strains
Incubation system for
SI 5
DR phospholi- Phosphatidylpase A assay ethanolamine Phosphatidylglycerol DS phospholi- Phosphatidylpase A ethanolamine Phosphatidylglycerol Cardiolipin
5827 Phosphatidylethanolamine Fatty acids+fatty acid methyl ester 211 Lysophosphatidylethanolamine 179 Phosphatidylglycerol 5663 Fatty acids + fatty acid methyl ester 238 111 Lysophosphatidylglycerol Phosphatidylethanolamine Fatty acids Lysophosphatidylethanolamine Phosphatidylglycerol Fatty acids Lysophosphatidylglycerol Cardiolipin Fatty acids Lysocardiolipin
Lysophospholi- Lysophosphatidyl- Lysophosphatidylethanolamine pase ethanolamine Fatty acids Lysophosphatidyl- Lysophosphatidylglycerol glycerol Fatty acids
23(DR") 21(DS") 17(DR"DS-) 6037
4812
5675
0 0
316 267
0 0
6011
5075
5631
0 0
227 192
0 0
6036
5250
5940
6038
0 0
0 0
0 0
0 0
5665
5451
5970
5137
310 0
658 0
30 0
0 0
N.D>
862 6 4
N.D.«
929 0 0
2721
3458
2675
3371
301
277
250
286
1030
960
1019
731
712
1130
745
1056
Not determined /. Biochem.
NATURE OF E. coli PHOSPHOLIPASE A MUTANTS
1251
shown in Fig. 2. Doubling times at the logarithmic phase of growth of the parent 15, 21 (DS"), 23 (DR-), and 17 (DR-DS") were 23, 27, 27, and 23 min, respectively, at 37° in Penassay medium, and 102, 90, 84, and 90 min, respectively, at 37° in the synthetic medium. No difference in growth at 28° or 42° among the mutants and parent was observed. If the fatty acid composition of cells of the bacteria changes after shift-down of the temperature (6) and phospholipase A participates in the redistribution of the membrane phospholipids, it is possible that some effect might occur on the growth of mutants after shift-down of the temperature when compared with that of the parent. However, no difference in the growth of mutants and parent was observed after shift-down from 37° to 19°. Thus, 30 60 90 Incubation Time (min) cultures of the parent 15 and mutant 17 (DR-DS"), Fig. 1. Hydrolysis of ["Qphosphatidylglycerol by cell grown at 37° in Penassay medium to concentraextracts of strain 17, strain 23, a mixture of 90% strain tions at 1.7 x 10s cells and 1.6 xlO8 cells/ml, re17 and 10% strain 23 and a mixture of 98% strain 17 spectively, were transferred to a water-bath (19° ± and 2% strain 23. Assay procedures were as described 0.5°) and shaken for 6 hr. Both strains showed a for DS phospholipase A assay in "MATERIALS AND similar lag period (about 55 min) and then multiMETHODS." The protein concentrations of the ex- plied with almost the same doubling time (parent: tracts were 10 mg/ml. o , strain 23 (DR"DS+); • , 240 min, mutant: 265 min). It may be concluded strain 17 (DR-DS-); A, 1:9 mixture; x, 1 :49 from these experiments that both DR and DS mixture. phospholipases A are not required for the growth of E. coli cells, even when the growth temperature is parent extract added. These results indicate that shifted down during the logarithmic phase of the inability of the mutant to hydrolyze phosphati- growth. dylglycerol is not due to the accumulation of an The Compositions of Phospholipids and Fatty inhibitory substance(s) within the mutant cell. Acids—Tables II and DI shows, respectively, comThe DS~ mutant: During mapping of the parisons of phospholipid and fatty acid composipldA gene, strain 21 was obtained after mating tions of the mutants and parent. There was no strain 17 (DR"DS") with Hfr P4X6. The DR and significant difference among the mutants and parent DS phospholipase A activities of strain 21 are in this case either. shown in Table I. The DR enzyme activity was Degradation of Phosphatidylglycerol In Vivo— fully recovered in the extract of strain 21, while the Extensive turnover of phosphatidylglycerol during DS enzyme activity was less than 10% of that of the the logarithmic phase of growth occurs in various extract of P4X6. bacteria, including E. coli (7-12). It was also Comparison of the mutants and the parent— shown that the phosphate moiety and the acyl Growth, fatty acid and phospholipid compositions, moiety of phosphatidylglycerol turn over at different and turnover of phospholipids were compared rates (11). The possible role of phospholipase A, especially DS-phospholipase A, which is specific to among the three mutants and parent. Growth—Growth characteristics were com- phosphatidylglycerol, in the mechanism of the in pared among mutants and parent by means of vivo degradation of phosphatidylglycerol, may be viable cell counts. Although the start of growth considered in relation to the turnover of the acyl seemed to be retarded just after transfer to a fresh moiety in phosphatidylglycerol. However, alternamedium, especially in the case of mutant 17 (DR-- tive possibilities may also exist. DS~), almost no difference of growth rates among In any event, cells in the logarithmic phase of the three mutants and parent was observed, as growth were labeled with ["Qacetate and the loss Vol. 80, No. 6, 1976
O. DOI and S. NOJIMA
1252
21 (DS")
S15(Parent )
0
30
60 90 120 Tlm»( ml n )
0
150
30
60
90
1 20 1 50
10'
Timt ( m i n )
23(DR')
17(DRDS") ..-o-o
10 9
10'
10'
s
10'
10' 30
60
90
1 20 150
Time ( min )
30
60 9 0 1 20 1 50 Time tmln )
Fig. 2. Comparison of growth and turnover of phospholipids between mutants of E. coli K-12 defective in phospholipase A and the parent. The cells of E. coli K-12 strains SI 5 (parent), 21 (DS-), 23 (DR"), and 17 (DR-DS") were cultured overnight in Penassay broth medium at 37°. Each of these cultures was added to 15 ml of the same medium as 0.5% (v/v) and an aqueous solution of [l-^CJacetate (Na) was added to the culture (2.5 /iCi per ml of the culture). Each of these cultures was incubated at 37° for 3 hr and centrifuged at room temperature for 5 min at 4000 x g. The pellet of cells from each sample was suspended in 5 ml of Penassay broth, the resulting suspension was filtered through a Millipore filter at room temperature and the washing was repeated once more. The labeled cells on the filter were resuspended in 40 ml of Penassay broth and cultured at 37° for 165 min. At appropriate times, a sample of 0.05 ml was assayed for viable cell count on i-agar plate and 2 samples of 1.0 ml were removed for analysis of phospholipids (2). O, cell growth; • , phosphatidylethanolamine; x , phosphatidylglycerol; A , cardiolipin.
/. Biochen.
1253
^NATURE OF E. coli PHOSPHOLIPASE A MUTANTS
TABLE II. Comparison of phospholipid compositions in parent and mutants of E. coli K-12 defective in phospholipase A. The cells of E. coli K-12 strains S15, 23, 21, and 17 were cultured in i-broth medium overnight at 37°. Each of these cultures was added to 15 ml of the same medium as 2% (v/v) and an aqueous solution of 1 -["CJacetate was added to the culture (5 /iCi per ml of the culture). Each of these cultures was incubated at 37° for 3.5 hr and the viable cells were counted. 3.1 X10*, 3.0x 10", 2.1 x 10», and 2.6x 10» cells per ml were taken for SI5, 23, 21, and 17, respectively. The culture was then centrifuged at 0° for 10 min at 10,000 xg. The pellet of cells was washed once with saline. Lipids were extracted from the cells and analyzed according to the procedures already described (2). PE, phosphatidylethanolamine; PG, phosphatidylglycerol; CL, cardiolipin. K-12 strain SI5 23 21 17
Total phospholipids ; of protein)
PE
4.6
78.3 79.9 75.5 78.3
(parent) (DR-) (DS-) (DR-DS-)
CL
PG /a
5.1 4.9 4.8
16.8 16.3 17.9 16.0
4.9 3.8 6.6 5.7
TABLE III. Comparison of fatty acid compositions in parent and mutants of E. coli K-12 defective in phospholipase A. Cells were cultured in 50 ml of Penassay broth for 3 h at 37°. The pellets obtained after centrifuga1ion of the culture were washed once with saline and lipids were extracted (2). The total lipids of each culture -were dissolved in 0.2 ml of methanol and 1.45 ml of water and hydrolyzed with 0.35 ml of 85% KOH by incubating ihe mixture at 70° for 30 min. After saponification, the mixture was cooled, neutralized with 6N HCl using thymol blue as an indicator, and extracted twice with diethylether. The combined ether extract was washed with concentrated aqueous KCl then evaporated to dryness. The residue was methylated with CH,N,. Analysis of the methyl esters was carried out on glass columns (1/8 x 10 inch) of Chromosorb W coated with 20% EGSS-X at 180° using a flame ionization detector (Varian Aerograph series 1800). The extracted cell residue of each strain was saponified under alkaline conditions and methyl esters were obtained as described above. The fatty acid patterns of each strain were very similar (C14:hydroxy; more than 50%, C l l : 0 , C 14;0 , C K : 0 and CU:L). Growth phase Strains
Logarithmic
Stationary
S15(parent) 23(DR") 21(DS-) 17(DR-DS") S15(parent) 23(DR") 21(DS") 17(DR-DS")
cells/ml(10-') Ci4 : o
5.6 3.5
6.0 5.3
6.0 5.1
6.1 5.0
34 4.9
30 6.8
24 8.5
13 6.3
•Cl«:0
48
46
50
48
50
49
53
50
•Ci.=i
29
33
35
32
23
28
27
26
•C17 (cyclopropane)
4.3 15
3.0 12
3.1 6.4
of the label in the acyl moieties of three phospholipids, phosphatidylethanolamine, phosphatidylgly•cerol and cardiolipin after chasing, was compared among the mutants and parent. As shown in Fig. 2, there was no marked difference between the patterns of phospholipid •degradation of the mutants and that of the parent. Phosphatidylethanolamine was stable, while phosphatidylglycerol was degraded. Cardiolipin was Vol. 80, No. 6, 1976
2.6 12
11 11
6.5 10
4.8 6.7
7.6 10
also degraded slowly. The percentage loss of phosphatidylglycerol in one doubling time was 11, 15, 11, and 11 in the parent 15 and the mutants 21 (DS-), 23 (DR-), and 17 (DR-SD"), respectively. Effects of Methanol and Detergents on the Hydrolysis of Phosphatidylglycerol by 18,000 X Q Supernatant of the Extracts of the Parent and Mutants—As shown in Fig. 3, the DS activity was
O. DOI andlS. NOJIMA
1254
2000
2000
8
1000
1000
20
40 60 Methanol 7.(v/v)
80
01
02 Q3 04 5DS 7 . ( w / v )
05
01
02 03 04 05 Cholate 7. ( w/v )
2000 3000 c
12000
Jhooo' •o
X
0 1000 < ffi
ai Q2 03 0A 05 Triton X-100 7.(w/v)
Fig. 3. Effects of methanol, SDS, Triton X-100 and sodium cholate on the hydrolysis of phosphatidylglycerol by extracts of E. coli K-12 strains. ["QPhosphatidylglycerol was used as a substrate. • , wild strain; O, DR+DS"; X, DR"DS+; A, DR-DS". suppressed by detergents and methanol while DR activity was seen only in the presence of methanol or detergents. In the case of cholate, the DS activity was slightly activated at lower concentrations but suppressed at higher concentrations. Autodegradation of Membrane Phospholipids—
As regards the growth of the unsaturated fatty acid auxotroph on an agar plate, some growth was noted even on the spot of mutant DR~DS". This indicates that autolyzed cells of this mutant still release
fatty acids, whatever the hydrolytic pathway may be. We have repeatedly confirmed the absence of phospholipase A activity in the extract of the mutant with respect to exogenous E. coli phospholipid,. provided incubation was carried out in an aqueousE. coli phospholipid. The following experiments were designed to compare, among mutants and parent, the hydrolysis of endogenous membrane phospholipids after incubation of their extracts or 18,000 xg precipitate. /. Biochem.
NATURE OF E. coli PHOSPHOLIPASE A MUTANTS -(DRD^f
DRDS) 20
40
60
1255
TABLE IV. Composition of fatty acids released from endogenous membrane lipids after incubation with the labeled 18,000xg supernatants of E. coli K-12 cell homogenates. After incubation of the 18,000 x g supernatant, the lipids were extracted and chromatographed on a silica gel thin-layer plate using chloroformmethanol-water (65 : 25 : 4, by vol.). The spot corresponding to fatty acids was scraped from the plate and extracted with chloroform-methanol ( 2 : 1 , v/v). The extracted lipids were saponified with alkali and the fatty acid fraction was methylated with CH.Ni. The methyl esters were analyzed by gas-liquid chromatography as described in Table HI, and the individual eluates from column were collected in glass U tubes and counted with a Beckman liquid scintillation counter using 10 ml of Bray's scintillator.
Incubation Time(mln)
Fig. 4. Time courses of endogenous membrane phospholipid degradation by 18,000xg supernatants of cell homogenates of E. coli K-12 mutants defective in phospholipase A. Experimental details are described in "MATERIALS AND METHODS." During incubation of the mixture of the labeled and non-labeled fractions, an equal amount of protein of each fraction was mixed. Usually 3.0 mg protein per ml was used, •denotes labeled membranes.
S15(DR+DS+)
Strain
Cll:0
Cn:o
C17 (cyclohropane)
3 32 40 5 15
17(DR-DS")
1 8 33 33 6 14
3 u
1! s
a.
1 —i-
25
50
100
200
400 800 1600 3200
Phosphatidylethandarnine ( uM )
Fig. 5. Inhibition of hydrolysis of phosphatidylglycerol by phosphatidylethanolamine. The incubation mixture contained 0.25 ml of ["Qphosphatidylglycerol (0.4 ^imoles, approx. 16,000 cmp), appropriate amounts of phosphatidylethanolamine as shown in thefigure,0.05 ml of 0.1 M CaCl,, 0.50 ml of 0.2 M Tris-HCl (pH 7.0), and 0.20 ml of enzyme solution, made up to a final volume of 1.5 ml with deionized water. Other procedures are described in "MATERIALS AND METHODS." Vol. 80, No. 6, 1976
10
0
30 60 90 Incubation Time ( min )
Fig. 6. Time courses of endogenous membrane phospholipid degradation by 18,000xg precipitates of cell homogenates of E. coli K-12 mutants defective in phospholipase A. The experimental procedures were as described in Fig. 4.
1256 18,000 x g Supernatant—-The 18,000 x g supernatants of the sonicates of ["Qacetate-labeled cells (the parent and three mutants) were incubated at 37° and the amounts of free fatty acids released were compared (Fig. 4). After incubation for 20 min, the extents of fatty acid release from endogenous substrate in the cases of mutants 23 (DR~) and 17 (DR-DS-) were about 10% of that in the case of the parent 15. After 1 hr, they increased to 14 and 19 %, respectively. As already pointed out (4), the difference between the parent and the three mutants, or that between mutant 21 (DS~) and the other two mutants (DRr, DR~DS") is clear. However, virtually no difference between mutant 23 (DR~) and mutant 17 (DR"DS~) was observed. These experimental results might be explained as follows: DS enzyme may be more latent than DR enzyme, while the DS enzyme activity is not manifested in the presence of excess phosphatidylethanolamine (Fig. 5). This accounts for the very slight difference between mutant 23 (DR") and mutant 17 (DR-DS"). The activity of DS enzyme could be observed in these experiments only when DS enzyme hydrolyzed phosphatidylglycerol released from the membrane after the initial attack of DR enzyme on membrane phospholipids. Mixing of the extract of mutant 23 or 17 (DR- or DR"DS") with that of mutant 21 (DS~) was expected to clarify the situation. [uC]Acetate-labeled extract of the mutant (DR~ or DR"DS") was incubated with or without non-labeled extract of mutant DS" and the labeled fatty acid release was observed. However, as shown in Fig. 4, no significant increase of fatty acid release from the labeled extract in the presence of the extract of mutant DS~ was observed. Thus, the above hypothesis was not supported by this experiment. A comparison of the composition of the fatty acids released from the extract of mutant D R ' D S " with that of the parent is shown in Table IV. No significant difference between mutant DR~DS~ and the parent is apparent. J8,000xg Precipitate—The 18,000X0 precipitate of the sonicate of ["Qacetate-labeled cells of the parent or mutants was also incubated at 37° and the amount of free fatty acids released was determined. As shown in Fig. 6, after incubation for 30 min, the amount of fatty acid released from endogenous substrate in the parent or mutant DS~ was more than 20-23% of that of the prelabeled
O. DOI and S. NOJIMA membrane phospholipids. On the other hand,, only 1 % of the endogenous phospholipids washydrolyzed in the other mutants (DR~, DR-DS"). As in the case of 18,000 x g supernatant, mixing of the extract of mutant DR" or DR~DS" with that of mutant DS~ was carried out, but the results were the same as those with 18,000 Xg supernatant. The autolytic activity in the 18,000xg precipitate fraction was about 6-7 times higher than that in the 18,000xg supernatant fraction (Figs. 4 and. 6). When cells of the parent or mutant DS~ wereused, strong membrane phospholipid degradation activity was also detected in the toluenized cell fraction, spheroplast cell membrane fraction,, polymyxin B-treated cell fraction and other cellsin which the membrane had been damaged by chemical reagents. However, these autodegradations were completely inhibited by the addition of EDTA. In every case, only slight degradatioa activity was detected in the cells of strains defective in DR enzyme (DS~, DR"DS"). Comparison of the Effects of Detergents and Organic Solvents on the Hydrolysis of Exogenous Substrate, Phosphatidylethanolamine, by 18,000 X q Supernatant and on the Release of Fatty Acids from the Supernatant after Incubation—The effects of detergents (0.1 %, w/v), such as sodium deoxycholate, sodium dodecyl sulfate, Tween 80, Triton X-100, and BRIJ 58, or organic solvents (20%, v/v), such as methanol, ethanol, and diethyl ether on the release of fatty acids from ["Qphosphatidylglycerol by extracts of the parent and the three mutants were examined. These compounds stimulated the release of fatty acids from the exogenous substrate by extracts of the parent and mutant DS~ while they suppressed the activities of the extracts of DR~. This result parallels the difference in the behavior of phospholipases A with detergents. The D R enzyme activity was apparently stimulated while the DS enzyme was inactivated when exogenoussubstrate was used. The effects of detergents on endogenous membrane hydrolysis were somewhat different from the effects obtained with exogenous substrates. At a concentration of 0.5%, all the detergents examined suppressed the endogenous substrate hydrolysis activity of any of the extracts of the parent and mutant DS" as well as mutants DR~ and DR"DS"; In order to clarity this discrepancy, we examined in. /. Biochem.
NATURE OF E. coli PHOSPHOLIPASE A MUTANTS 200
£
0
20 40 Methanol %(v/v)
60
1257
detail the effects of methanol, SDS, and Triton X-100 on the activity of the extract of mutant DS~ against endogenous substrate as well as exogenous substrate (Fig. 7a, b, c). As shown in Fig. 7a, methanol markedly activated DR activity against exogenous phosphatidylethanolamine, while it activated the endogenous phospholipid hydrolysis by 122% at 10-20% (v/v) but inactivated it at higher concentrations. In the case of SDS, this tendency is more marked. At a concentration of 0.005 %, SDS activated the endogenous activity by 149%, but the activity fell off sharply at higher concentrations and no activity was detected at 0.025%. On the other hand, exogenous phosphatidylethanolamine was hydrolyzed only in the presence of SDS, using 18,000 xg supernatant. Triton X-100 showed the same tendency, but at higher concjntrations. No activation of endogenous phospholipid hydrolyzing activity was observed in the case of Triton X-100. DISCUSSION
005 SOS 7.(\Wv)
£
1.0
Fig. 7 Vol. 80, No. 6, 1976
Q2-Q5
Some properties of three mutants of E. coli K-12 with respect to phospholipase A (DR~, DS", and DR~DS") were investigated in the present study. In summary, no marked differences between the parent and the three mutants were found with respect to growth, lipid composition, and other properties. During genetic mapping of the locus for DR phospholipase A (pldA) (J), the cells with wild-type phospholipase A were transduced to pldA~ by phage PI grown on strain 17 (DR-DS~) and strain 17 transduced to pldA+ by Pic grown on cells with wild-type phospholipase A. The growth of these transductants was similar to that of the wild strain. Several investigators, including the present authors, have used these mutants to clarify the role of phospholipase A in the following phenomena: the growth of bacteriophages {13), transfection of Cas+-dependent phage DNA (14) and the action of colicin K (75). However, no evidence was obtained for the participation of Fig. 7. Effect of methanol, SDS or Triton X-100 concentration on DR phospholipase A activity and endogenous phospholipid-hydrolyzing activity of E. coli. x, fatty acid released from endogenous phospholipid; O, fatty acid released from exogenous phosphatidylethanolamine.
1258
O. DOI and S. NOJIMA
phospholipase A. Moreover, it was indicated that phage-induced lysis and the release of progeny phage are separable in T4s-infected strain 17 (DR~DS") (75). In general, the growth of E. coli cells is very slow when the medium contains fatty acids as a sole carbon source (17). If phospholipases A, especially DR phospholipase A, which is in the outer membrane of E. coli (18-20), play a role in the digestion of phospholipids in the medium, the cells would be expected to grow in the presence of phospholipids as a sole carbon source. However, no growth was observed in this laboratory under such conditions. In this respect, it is very interesting that Mycobacterium tuberculosis and Mycobacterium bovis grow well in liquid media containing egg lecithin or synthetic dipalmitoyllecithin as a sole carbon source (21). A highly active membrane-bound phospholipase Ax has been purified and characterized from cell of Mycobacterium phlei (22).
2. Doi, O., Ohki, M., & Nojima, S. (1972) Biochim. Biophys. Ada 260, 244-258 3. Abe, M., Okamoto, N., Doi, O., & Nojima, S. (1974) / . Bacteriol. 119, 64-69 4. Nojima, S., Doi, O., Okamoto, N., & Abe, M. (1972) in Membrane Research (Fox, C.F., ed.) pp. 135-144, Academic Press 5. Cronan, J.E. & Godson, G.N. (1972) Molec. Gen. Genet. 116, 199-210 6. Cronan, J.E., Jr. & Vagelos, P.R. (1972) Biochim. Biophys. Ada 265, 25-60 7. Kanfer, J. & Kennedy, E.P. (1963) / . Biol. Chem. 238, 2919-2922 8. Kanemasa, Y., Akamatsu, Y., & Nojima, S. (1967) Biochim. Biophys. Ada 144, 382-390 9. Ohki, M. (1972) /. Mol. Bio!. 68, 249-264 10. Ames, G.F. (1968) / . Bacteriol. 95, 833-843 11. White, D.C. & Tucker, A.N. (1969) / . Lipid Res. 10, 220-233 12. Short, S.A. & White, D.C. (1971) / . Bacteriol. 108, 219-226 13. Sakakibara, Y., Doi, O., & Nojima, S. (1972) Biochem. Biophys. Res. Commun. 46, 1434-1440 The homogenates of mutant DR~DS~ showed 14. Taketo, A. (1974) / . Biochem. 75, 895-904 less than one-hundredth (DR) or one-fiftieth (DS) 15. Lusk, J.E. & Park, M.H. (1975) Biochim. Biophys. Ada 394, 129-134 of the activities of the homogenate of the parent against exogenous substrates. Autolysis of labeled 16. Hardaway, K.L., Marten, M.V., & Buller, C.S. (1975) J. Virology 16, 867-871 homogenates of the mutant caused the release of 17. Overath, P., Pauli, G., & Schairer, H.U. (1969) } | free fatty acids amounting to less than 5 % of that Eur. J. Biochem. 7, 559-574 of the parent (Figs. 4 and 6). At present, the na18. Albright, F.R., White, D.A., & Lannarz, W.J. ture of the fatty acid-releasing activity observed in (1973) J. Biol. Chem. 248, 3968-3977 the mutant remains to be clarified. Further 19. Bell, R.M., Mavis, R.D., Osborn, M.J., & Vagelos, attempts to isolate a mutant which lacks this acP.R. (1971) Biochim. Biophys. Ada 249, 628-635 tivity have so far been unsuccessful. 20. Yamato, I., Anraku, Y., & Hirosawa, K. (1975) / . Biochem. 77, 705-718 21. Kondo, E. & Kanai, K. (1976) Jap. J. Med. Sci. REFERENCES Biol. 29, 109-121 1. Ohki, M., Doi, O., & Nojima, S. (1972) /. Bacteriol. 22. Nishijima, M., Akamatsu, Y., & Nojima, S. (1974) / . Biol. Chem. 249, 5658-5669 110, 864-869
/. Biochem.
