Glycerol Auxotroph of Staphylococcus aureus

Transcription

1 JOURNAL OF BACTERIOLOGY, Oct. 1972, p Copyright i 1972 Americn Society for Microbiology Vol. 112, No. 1 Printed in U.S.A. Consequences of Glycerol Deprivtion on the Synthesis of Membrne Components in Glycerol Auxotroph of Stphylococcus ureus PAUL H. RAY, THOMAS T. LILLICH, AND DAVID C. WHITE' Deprtment of Biochemistry, University of Kentucky Medicl School, Lexington, Kentucky 456 Received for publictioh 3 April 1972 In glycerol uxotroph of Stphylococcus ureus, the deprivtion of glycerol ffected the formtion of certin membrne components. (i) There ws synthesis of ftty cids t the predeprivtion rte even though the ftty cids synthesized ccumulted s free ftty cids rther thn s esterified ftty cids; (ii) there ws complete cesstion of phospholipid nd vitmin K isoprenologue biosynthesis; (iii) there ws conservtion of the glycerol esters of the complex phospholipids nd glucolipids; (iv) there ws n immedite decrese in the rte of synthesis of monoglucoslydiglyceride (3%) nd diglucosyldiglyceride (6%); (v) there ws 5% decrese in the rte of synthesis of the polr nd nonpolr crotenoids; (vi) there ws synthesis of protoheme, heme, nd nonspecific membrne protein t the predeprivtion rte; nd (vii) there ws n brupt cesstion in the formtion of new, functionl glycine trnsport ctivity. Glycerol nd olete uxotrophs of severl bcteril species hve been used to study the structure nd function of bcteril membrnes (4, 7, 8, 1, 11, 14). These mutnts hve been prticulrly useful becuse glycerol is n essentil component of both phospholipids nd glycolipids, nd these compounds re loclized in the bcteril membrnes (5, 2). We hve extended the observtions of Mindich (9, 11) in previous communictions (8, 14) nd hve shown tht when glycerol uxotrophs of Bcillus subtilis or Stphylococcus ureus re deprived of glycerol, there is n immedite cesstion of net phospholipid synthesis, wheres protein synthesis continues for bout two genertions. It hs lso been reported (9, 1) tht proteins re incorported into the cell membrne in the bsence of net phospholipid synthesis, resulting in n increse in the buoynt density of the membrne. This suggested tht phospholipid synthesis exerted no pprent control over the incorportion of proteins into the membrne. We hve lredy shown (8, 14) tht, lthough there is no net synthesis of phospholipids in the bsence of glycerol, there is ctive metbolism of the individul phospholipids nd chnge in the overll phospholipid composition in these uxotrophs. Furthermore, s first I Present ddress: Deprtment of Biologicl Science, Florid Stte University, Tllhssee, Fl noted by Mindich (personl communiction), there is n increse in the free ftty cid content of the bcteri nd shift from the usul C-15 to C-17 iso- nd nteiso-brnched-chin ftty cids to C-19 or C-21 brnched-chin ftty cids. Mindich subsequently showed (12) tht in B. subtilis the ftty cid synthetse is not inhibited by the presence of free ftty cids nd tht the rte of ftty cid synthesis is the sme when the cells re resuspended in the presence nd bsence of glycerol. In this study, we confirm the results of Mindich concerning the synthesis of ftty cids in glycerol uxotrophs of S. ureus nd show tht the synthesis of neutrl lipids (vitmin K isoprenologues, crotenoids, nd glycolipids) of this strin re in some wy regulted by the rte of phospholipid biosynthesis. It is shown tht formtion of new trnsport system for the mino cid glycine is impired during the deprivtion of glycerol. The mino cid trnsport system in these cells involves the electron trnsport system (16). MATERIALS AND METHODS Growth of bcteri. The medium nd the growth conditions used hve been described previously (14). The bcteri were grown in synthetic medium contining glycerol until they reched mid-log phse. They were then hrvested by filtrtion (14), wshed with t lest two volumes of medium without glycerol, nd resuspended in medium, with or without 413

2 414 RAY, LILLICH, AND WHITE J. BACTERIOL. glycerol, s indicted in the figure legends. The experiments described were crried out in 125 ml (totl volume) of medium unless otherwise indicted. Isoltion nd seprtion of neutrl lipids. Totl lipids were extrcted by modifiction of the Bligh nd Dyer procedure (1, 21). Neutrl lipids were isolted from the totl lipid extrct by silicic cid, column chromtogrphy s previously described (15). A smple of the totl lipid ws pplied to 1 g silicic cid column. The neutrl lipids were then eluted with 6 ml of chloroform-cetone (1:1, v/v), nd the phospholipids were eluted with 1 ml of chloroform-methnol (1:1, v/v). One-dimensionl chromtogrphy on Whtmn silic gel-impregnted pper (SG-81) with chloroform-isooctne (2: 1, v/v) ws used to seprte the nonpolr crotenoids (RF 1.) nd the vitmin K isoprenologues (RF.7) from the polr crotenoids nd free ftty cids (RF to.2) (6). After chromtogrphy, the ppers were plced on Kodk no-screen X-ry film. Ares on the chromtogrms corresponding to drk spots on the film were cut out nd counted s previously described (8). Isoltion of ftty cids. Totl ftty cids were obtined by sponifying smples of whole cells with 3 N KOH for 4 hr t 1 C (19). Neutrl lipids were then recovered by extrction twice with.5 volumes of diethyl ether. The wter phse ws djusted to ph 2. with concentrted HCl, nd the ftty cids were extrcted with diethyl ether s described bove. Non-esterified ftty cids were isolted s follows. Before obtining the two-phse system in the Bligh nd Dyer procedure for extrcting totl lipids (14), NHCO, ws dded to give finl concentrtion of 1 M. After obtining two phses, the lipid phse ws removed, nd the queous phse ws djusted to ph 2 with concentrted HCl. This phse ws then extrcted twice with.5 volume of diethyl ether. Chromtogrphy of glycolipids. Glycolipids were seprted from the totl lipid extrct by chromtogrphy on Whtmn silic-gel-impregnted pper (SG-81) by using the first nd third solvents of Wuthier (22). Autordiogrms were prepred s described bove. The res of the chromtogrms corresponding to the glycolipids were cut out nd, in the cse of those smples lbeled with "4C-glucose, were eluted from the pper (21) nd decylted (15) before counting. Smples which were lbeled with 4C-cette were counted without further tretment. Rdioisotopes. The following rdioisotopes were used in the experiments reported here: cetic-1-"4c cid (59.2 mci/mmole); DL-mevlonic-2-"4C cid (6.33 mci/mmole); glycerol-1, 3-'4C (14.1 mci/mmole); D-glucose-U-"4C (15 mci/mmole); L- glycine-u-_4c (83 mci/mmole); 6-minolevulinic-4- "4C cid (24.5 mci/mmole). All of the isotopes were purchsed from New Englnd Nucler Corp. (Boston, Mss.), with the exception of 6-minolevulinic cid which ws supplied by Clbiochem (Los Angeles, Clif.). RESULTS Effect of glycerol deprivtion on ftty cid biosynthesis. The glycerol uxotroph S-2 of Stphylococcus ureus, when deprived of glycerol, did not incorporte H332Po into its phospholipids (Fig. 1). During the first 75 min of glycerol deprivtion, the incorportion of "4C-cette into its lipids continued t rte of 5 to 8% of tht in cultures supplemented with glycerol. When glycerol ws re-dded to the deprived cultures, the incorportion of both "IC-cette nd H332PO4 incresed t twice the predeprivtion rte. The increse in cette incorportion during glycerol deprivtion represented primrily the synthesis of free ftty cids (ftty cids not esterified to lipids) s shown in Fig. 2. These ftty cids tht were synthesized in the bsence of glycerol were four to six crbon toms longer thn the norml C-15 or C-17 brnchedchin ftty cids (14). The free ftty cid content of the cells incresed from the norml 1% level to 13% during the initil 6 min following the removl of glycerol. When cells were grown with "IC-cette, in medium plus nd minus glycerol, the mjority of the rdioctivity ws loclized in the free ftty cids in medium devoid of glycerol in contrst to esterified ftty cids in medium contining glycerol. After pulsing the mutnt cells with "4C-cette for one nd one-hlf genertions in medium contining glycerol nd 5POOI ACETATE W4CORPORATIN INTO LIPID () P INCORPORATION INTO LIPID (6) 11 i i i 1 (-)GLYCEROL TIME IN FIG. 1. Effect of glycerol deprivtion on the incorportion of "IC-cette nd H,"2PO4 into the lipids of Stphylococcus ureus. The cells were grown, wshed, nd resuspended in medium contining constnt-specific-ctivity 14C-cette nd H,32PO4. At the times indicted, 5-ml smples were withdrwn nd the lipids were extrcted (21). The neutrl lipids were seprted from the phospholipids on I g silicic cid column by elution with 6 ml of chloroform-cetone (1:1); the phospholipids were eluted with 1 ml of chloroform-methnol (1:1).

3 VOL. 112, 1972 GLYCEROL DEPRIVATION AND MEMBRANE SYNTHESIS 415 TIME IN FIG. 2. Effect of glycerol deprivtion on ftty cid biosynthesis. The cells were grown in medium contining constnt-specific-ctivity "4C-cette. At the times indicted 15-mi smples were withdrwn, nd the totl lipids were extrcted by modifiction of the Bligh nd Dyer method (21). Free ftty cids were obtined by dding NHCO, to finl concentrtion of 1 M in the one-phse system. After dding chloroform nd buffer to mke two-phse system, the lipid phse ws withdrwn nd the free ftty cids were extrcted with.5 volume of diethyl ether twice fter djusting the ph to 2. with concentrted HC1. resuspending them in medium plus nd minus glycerol contining.1 M cette, there ws essentilly no turnover of the ftty cids (Fig. 3B). This suggested tht once ftty cids were esterified in this mutnt, there ws very little exchnge or degrdtion of the ftty cids. However, when cells were pulsed with 14C_ cette in medium devoid of glycerol, there ws considerble turnover of the free ftty cids, 4% decrese in the rdioctivity in one genertion (Fig. 3A). These results suggested tht upon glycerol deprivtion there ws free ftty cid degrdtion medited either by the induction or ctivtion of ftty cid oxidtion pthwy. More importnt, becuse the free ftty cids formed during the deprivtion of glycerol were degrded, the ctul rte of ftty cid synthesis in cells deprived of glycerol ws much higher thn indicted by the cette incorportion. Becuse the decrese in free ftty cid content per genertion mounted to 4% (Fig. 3A) nd the verge rte of free ftty cid synthesis during glycerol deprivtion ws 65% of the exponentil rte, free ftty cid synthesis ws t lest equl to the predeprivtion rte. This mens tht, even though free ftty cids ccumulted up to 13% of the totl, they did not pper to be feedbck inhibitors of the ftty cid synthetse, s ws lso noted by Mindich in B. subtilis (12). Effect of glycerol deprivtion on the turnover of glycerol in the phospholipids nd glucolipids. It ws previously shown (8, 1, 14) tht when cells were deprived of glycerol net phospholipid synthesis cesed immeditely; however, there ws turnover nd metbolism of the polr portions of the phospholipids (8, 14). After pulsing the cells grown in medium contining l4c-glycerol for two genertions, the turnover of the glycerol in the individul lipids ws mesured in medium plus nd minus glycerol (Fig. 4). In medium contining glycerol, the turnover of the "4C-glycerol incorported into the totl lipids ws bout 5% fster thn in cells deprived of glycerol (Fig. 4B). In medium supplemented with glycerol, 5% of the "4C-glycerol ws lost from phosphtidic cid (PA), 3% from phosphtidylglycerol (PG), nd 1% from lysylphosphtidylglycerol (LPG) in one genertion (Fig. 4C, F, nd G). In medium devoid of glycerol, 5% of the rdioctivity ws lost from PG; however, LPG nd PA ccumulted the glycerol lbel (Fig. 4C, F, nd G). The glucolipids, monoglucosyldiglyceride (MG) nd diglucosyldiglyceride (DG), ccumulted the "4C-glycerol in both plus nd minus glycerol medi; however, in the bsence of glycerol the ccumultion ws greter. This i l~I FATTY ACID TURNOVER A 8 PULSED (-) GLYCEROL PULSED I.) GLYCEROL A 3 2 tipplycerol (-)-GLYLER TIME IN FIG. 3. Turnover of the ftty cids lbeled in the presence nd bsence of glycerol. The cells were grown in 1 ml of medium contining glycerol to n bsorbncy of.45 t 75 nm, wshed with 2 ml of medium devoid of glycerol, nd resuspended in 1 ml of medium plus nd minus glycerol, ech contining 1 MUCi of "4C-cette. After 1.5 genertions (c 6 min) the cells were filtered, wshed s described bove, nd resuspended in medium plus nd minus glycerol contining nonrdioctive cette (.1 M). At the times indicted, 15-mI smples were removed nd the totl ftty cids were extrcted s before (14).

4 416 RAY, LILLICH, AND WHITE J. BACTERIOL. J EC 5 1 i..,. ti 6- GROWTH.4.1 le6r- 151 IO 3 Oj 144 oe IO TOTAL LIPS GUVEROL(S) LPG I + 7G TIME IN 9L Ie* er W o MG PA 2o2 5 F I G. 4. Turnover of the lipid glycerol in the presence nd bsence of glycerol. The turnover of 14Cglycerol ws mesured by growing the cells in 1 ml of medium contining 25 ACi of "4C-glycerol for two genertions. After hrvesting by filtrtion, the cells were wshed with medium devoid of glycerol nd resuspended in medium plus (3 ug/ml) nd minus glycerol. At the times indicted, smples were extrcted s described (21), nd the individul lipids were seprted by chromtogrphy on Whtmn SG- 81 pper by using the first nd third solvents of Wuthier (22). LPG refers to lysylphosphtidylglycerol (C), DG refers to diglucosyldiglyceride (D), CL refers to crdiolipin (E), PG refers to phosphtidylglycerol (F), PA refers to phosphtidic cid (G), nd MG refers to monoglucosyldiglyceride (H). suggested tht once these two compounds were synthesized they were metboliclly stble. Figure 4B illustrtes tht the turnover of the "4C-glycerol moiety in the phospholipids nd glucolipids ws slower in medium devoid of glycerol nd tht the glycerol moiety, once incorported into lipid, ws conserved when the cells were deprived of glycerol. In Fig. 4, the zero time points for cells incubted with nd without glycerol were not exctly equl, for the volumes of the originl culture collected on the membrne filter nd wshed into the medium were not exctly equl. Effect of glycerol deprivtion on glucolipid synthesis. The synthesis of the two glucolipids of S. ureus, MG nd DG, ws mesured by the incorportion of "4C-cette into the esterified ftty cids nd "4C-glucose into the polr portion of the glucolipids, s shown in Fig. 5. Studies with the isolted glucolipids hve estblished tht in the glycerol uxotroph 95% of the "4C-cette ws incorported into the ftty cids, nd the "4C-glycerol exclusively lbeled the glycerol portion of the glucolipids. In the decylted esters derived from the glucolipids, "4C-glucose lbeled only the glucose of these decylted lipids. Upon glycerol deprivtion, the rte of "4C-cette incorportion into DG immeditely decresed to 4% of the norml rte (Fig. 5A) nd cesed 6 min fter deprivtion. The incorportion of cette into MG decresed to 7% of the norml rte. Similr results were obtined for both glucolipids when uniformly lbeled glucose incorportion ws mesured in the bsence of glycerol (Fig. 5B). Effect of glycerol deprivtion on neutrl w ie iso I lipid biosynthesis. To study the reltionship between phospholipid nd neutrl lipid biosynthesis, cells were grown in complete medium with constnt-specific-ctivity 14C-cette, filtered, wshed, nd resuspended in medium devoid of glycerol. As in other experiio4r w *3!i IL IOF ACETATE INCORPORATION MG SO ( SO FIG. 5. Effect of glycerol deprivtion on the synthesis of monoglucosyldiglyceride (MG) nd diglucosyldiglyceride (DG). The cells were grown, wshed, nd resuspended in medium contining either 14Ccette or 14C-glucose t the sme specific ctivities in medium plus or minus glycerol. At vrious times fter glycerol deprivtion, smples were withdrwn nd the lipids were extrcted. The individul lipids were isolted by two-dimensionl chromtogrphy s described (22). The glucolipids lbeled with 14C-cette were counted without further tretment. The glucolipids lbeled with l 4C-glucose were decylted by mild lkline methnolysis (21), nd the wtersoluble frction ws dried under ir nd counted. The rte of glucolipid synthesis in cells filtered nd resuspended in medium with glycerol continued t the pretretment exponentil rte without fg. S GLUCOS G E INCORPORATION -IS - 1 w.

5 VOL. 112, 1972 GLYCEROL DEPRIVATION AND MEMBRANE SYNTHESIS ments, there ws no cette incorportion into the ftty cids of the phospholipids in the period of glycerol deprivtion (Fig. 6B). The incorportion of "4C-cette into the nonpolr crotenoids (minly phytoene) nd the polr crotenoids (minly rubixnthin) ws bout hlf the rte of synthesis by cells grown in the presence of glycerol (Fig. 6C nd D). There ws complete cesstion of vitmin K isoprenologue biosynthesis during glycerol deprivtion (Fig. 5D). The immedite nd complete I 417 cesstion of vitmin K, biosynthesis ws lso detectble when "4C-mevlonte, direct precursor for the synthesis of the isoprenologue side chin, ws utilized s mesure of continued synthesis (Fig. 7). Effect of glycerol deprivtion on the incorportion of protein into the membrne. In order to investigte the effects of glycerol deprivtion on membrne function, the incorportion of protein into the membrne nd the trnsport of the mino cid glycine ws mesured. The mesurement of protein incorportion into the membrne ws ssyed by the incorportion of "4C-serine into nonspecific membrne protein nd by the incorportion of '4C-6-minolevulinic cid, precursor of the prosthetic groups of the cytochromes, into membrne-bound protein. 6-minolevulonic cid ws shown to be precursor of protoheme nd heme which re the prosthetic groups of the cytochromes in the membrne of S. ureus (17). From both ssys it ws cler tht proteins were incorported into the membrne t n undiminished rte for 9 min fter glycerol deprivtion (Fig. 8). These results were consistent with the continution of totl protein synthesis which ccumulted "4C-serine t the ILA, 4'IO t! 12 VITAAIN K SORENOLGUES PX 2 FIG. 6. Effect of glycerol deprivtion on the synthesis of neutrl lipids. The cells were grown in medium contining "4C-cette, filtered, nd wshed s before (14). After wshing, the cells were resuspended in medium devoid of glycerol nd contining the sme-specific-ctivity "4C-cette. At the times indicted, 5-ml smples were removed, nd the lipids were extrcted s described (21). After the lipids were extrcted from ech smple, the neutrl lipids nd phospholipids were seprted by silicic cid column chromtogrphy s described (15). The nonpolr crotenoids (RF.9 to 1.), vitmin K isoprenologues (RF.7 to.8), nd polr lipids (ftty cids nd polr crotenoids, RF to.2) were seprted by WVhtmn SG-81 pper chromtogrphy by using solvent of chloroform-isooctne (2:1). * PHYTOENE CAROTENES ' 4 4 VITAMIN K2 BIOSYNTHESIS +.(MEVALONATE) 13 FILTER ND WASH.5 GROWTH GLYCEROL -GLYCEROL +GLYCEROL -GLYCEROL go FIG. 7. Effect of glycerol deprivtion on the synthesis of vitmin K isoprenologues. The cells were grown, wshed, nd resuspended in medium plus nd minus glycerol contining the sme specific ctivity of 14C-DL-mevlonic cid (MEV). At the times indicted, 5-ml smples were withdrwn nd the lipids were extrcted. The vitmin K isoprenologues were seprted from the phospholipids, free ftty cids, nd crotenoids by chromtogrphy on Whtmn SG-81 pper by using solvent of chloroform-isooctne (2:1). The vitmin K isoprenologues were mesured, without further purifiction or seprtion, into their individul isoprenologues.

6 418 RAY, LILLICH, AND WHITE J. BACTERIOL. predeprivtion rte for 9 min nd then cesed (14). In order to study the effect of glycerol deprivtion on the functionlity of this membrne, the rte of trnsport of the mino cid glycine into the free mino cid pool of whole cells ws mesured (Fig. 9). Amino cids cn be trnsported into the free mino cid pool of whole cells in the presence of chlormphenicol (2). When glycerol uxotrophs were deprived of glycerol, the specific ctivity of the glycine trnsport, insted of remining constnt s ws the cse for cells grown in the presence of glycerol, decresed s function of time, s seen in Fig. 9. Thus, when cells were deprived of glycerol some component of the membrne involved in glycine trnsport ws either not synthesized or, if synthesized, ws nonfunctionl. DISCUSSION In glycerol uxotroph of S. ureus designted S-2, the deprivtion of glycerol effected certin chnges in the formtion of the membrne components. (i) There ws synthesis of ftty cids t the predeprivtion rte; however, these ftty cids ccumulted s free ftty cids, not s esters. (ii) There ws complete cesstion of phospholipid nd vitmin K biosynthesis. (iii) There ws conservtion of the glycerol esters of the complex lipids. (iv) There ws decrese in the rte of synthesis. of the polr nd nonpolr crotenoids. (v) There ws IV I I I I 7 I (+) GLYCEROL _ v, ~- > (-) GLYCEROL t zw Z s TIME FIG. 8. Incorportion of '4C-6-minolevulinic cid (ALA) nd '4C-serine into cell membrne protein. In both experiments the cells were grown with the isotope indicted, filtered, wshed with medium devoid of glycerol, nd resuspended in medium plus nd minus glycerol contining the sme specific ctivity of the isotope (ALA,.8 MCi/ml; serine, 1.2 MCi/mI). At the times indicted, 2-mI smples were withdrwn nd centrifuged. The smples were resuspended in 2 ml of 5 mm phosphte buffer (ph 7.4) contining lysostphin (8 Mg/ml), deoxyribonuclese (2 Mig/ml), nd ribonuclese (2 Mg/mI). After incubtion for 3 min t 37 C, 5 ml of buffer ws dded, nd the membrnes were centrifuged t 17, x g for 3 min. The membrnes were resuspended in 5 mm phosphte buffer, nd smples were tken for the determintion of rdioctivity. Iv O FIG. 9. Effect of glycerol deprivtion on the trnsport of 14C-glycine into whole cells. The cells were grown to n bsorbncy of.45 in 1 ml of medium, wshed with 15 ml of medium devoid of glycerol, nd resuspended in 1 ml of medium minus glycerol. At the times indicted, 1 ml smples were withdrwn nd centrifuged t 1, x g for 2 min t 37 C. The pellets were resuspended in 2 ml of medium, devoid of glycerol nd contining 5 ug of chlormphenicol per ml, nd incubted t 37 C. After 1 min,.1 MUCi of "4C-glycine ws dded, nd smples were withdrwn fter, 2, 5, 7, 1, nd 15 min of incubtion. Smples of.2 ml were diluted with 2. ml of cold 5 mm phosphte buffer (ph 7.4), filtered, nd wshed with 5 ml of buffer. The filters were dried nd counted. Smples were lso tken for the determintion of protein. Specific ctivities of uptke mesurements were clculted fter 2 min of incorportion. When uptke ws liner with time (16), the logrithm of the trnsport ctivity ws plotted on the bciss.

7 VOL. 112, 1972 GLYCEROL DEPRIVATION AND MEMBRANE SYNTHESIS synthesis of protoheme, heme, nd membrne protein t the predeprivtion rte. (vi) There ws n brupt cesstion in the formtion of new, functionl glycine trnsport ctivity. When exponentilly growing cells of S. ureus were deprived of glycerol, net phospholipid synthesis cesed immeditely; however, growth, RNA synthesis, nd protein synthesis continued for 3 to 9 min (14). During this period of glycerol deprivtion, pprent ftty cid synthesis continued t 65% of the norml rte for 9 min (Fig. 1). The ftty cids tht were synthesized during glycerol deprivtion ppered s free ftty cids contining four to six more crbon toms thn the norml ftty cids (14). During glycerol deprivtion there ws very little esterifiction of ftty cids into phospholipids or glucolipids (Fig. 1 nd 2). Becuse the pprent rte of ftty cid synthesis ws bout 65% of the norml rte immeditely fter glycerol deprivtion nd becuse the free ftty cids ccumulted to 13% of the totl ftty cids, the free ftty cids possibly exerted feedbck inhibitory effect on the ftty cid synthetse, s hs been suggested (3). However, Mindich (12) hs shown with glycerol uxotroph of B. subtilis tht the ctul rte of ftty cid synthesis ws the sme in medium plus nd minus glycerol, even though in the Bcillus system free ftty cids ccumulte to 2% of the totl in the bsence of glycerol. The free ftty cids, which were synthesized in the bsence of glycerol, hve much fster turnover rte thn the esterified ftty cids synthesized in the presence of glycerol. The mintennce of n pprent rte of ftty cid biosynthesis of 65% of the predeprivtion rte, when the free ftty cids were ctbolized t rte of 4% of the totl per genertion (Fig. 3A), indicted tht the ctul rte of ftty cid biosynthesis ws t lest equl to the predeprivtion rte. Thus, there ws no evidence for feedbck inhibition of the ftty cid synthetse by free ftty cids, s lso noted by Mindich (12). Also, there ws no loss of rdioctivity from the ftty cids of cells resuspended in medium, either with or without glycerol, if these ftty cids were esterified to the complex lipids (Fig. 3B). This suggested tht there ws very little exchnge or trnsesterifiction of the esterified ftty cids, even in the bsence of glycerol where the free ftty cids ccount for 13% of the totl. If there hd been exchnge, there would hve been turnover. Even though net phospholipid synthesis cesed immeditely fter glycerol deprivtion, 419 glucolipid synthesis continued t reduced rte for 6 to 9 min. The continued synthesis of MG nd DG cn be ccounted for by the turnover of the "4C-glycerol from the phospholipids s shown in Fig. 4. When cells were pulsed with "4C-glycerol nd resuspended in medium plus nd minus nonrdioctive glycerol, the rte of turnover of the lipid glycerol ws 5% fster in cells resuspended in medium contining glycerol. The rdioctive lbel ccumulted in the glucolipids both in the presence nd bsence of glycerol; however, in the bsence of glycerol the rte of ccumultion ws fster (Fig. 4). This suggested precursor role for the diglyceride portion of the phospholipid molecule in the synthesis of the glucolipids, s hs been indicted by Peringer (13). The turnover of "4C-glycerol from the phospholipids in cells deprived of glycerol ws consistent with chnges in the proportions of the individul phospholipids during glycerol deprivtion (14). The content of PG decresed with concomitnt increse in the content of LPG. During glycerol deprivtion the cells conserved the lipid glycerol to much greter extent thn cells supplemented with glycerol. The synthesis of the neutrl lipids ws ffected to vrious degrees by the deprivtion of glycerol. The crotenoids were continully synthesized t slower rte fter glycerol deprivtion throughout the experiment (Fig. 6). The synthesis of the vitmin K isoprenologues ws drsticlly ffected by the deprivtion of glycerol. By utilizing both "4C-cette nd 14C_ mevlonte to mesure the synthesis of vitmin K (Fig. 6 nd 7), it ws shown tht immeditely fter deprivtion synthesis cesed. During glycerol deprivtion it hs been shown tht totl protein synthesis continued unbted for 9 min, even though net phospholipid synthesis stopped immeditely (14). In Fig. 8 it ws shown tht protein incorportion into the cytoplsmic membrne continued t the predeprivtion rte for 9 min. During the induction of the membrne-bound electron trnsport system in S. ureus, it ws previously shown tht there ws simultneous increse in the rte of synthesis of phospholipids, polr crotenoids, vitmin K2 isoprenologues, glucolipids, hemes, nd cytochromes (5). However, even though crotenoid biosynthesis ws slowed nd vitmin K2 biosynthesis cesed bruptly, the incorportion of 8-minolevulinic cid into the hemes of the cytochromes continued t predeprivtion rte (Fig. 6, 7, nd 8). The synthesis of these components of the electron trnsport sysfem (18) did not

8 42 RAY, LILLICH, AND WHITE J. BACTERIOL. pper to be coordintely linked under these experimentl conditions. Mindich (1) hs shown tht in glycerol uxotroph of B. subtilis the buoynt density of the membrne incresed upon deprivtion nd hs suggested tht these findings plce some constrint on models of membrne ssembly, becuse it ppered tht proteins were not incorported into the cytoplsmic membrne s lipoprotein subunits. To look t the functionlity of these lipid-poor membrnes, the trnsport of the mino cid glycine ws investigted. These results, shown in Fig. 9, suggest tht upon glycerol deprivtion some importnt component of the glycine trnsport system is either not synthesized or, if synthesized, is not functionl, becuse the specific ctivity of the trnsport process decresed with the time of glycerol deprivtion. The results obtined from the trnsport of glycine in cells deprived of glycerol might be ccounted for by the cesstion of vitmin K biosynthesis, becuse Short et l. (16) hve shown tht the electron trnsport system ws obligtory for mino cid trnsport in vesicles. These results could lso be explined by the lck of the proper environment supplied by the phospholipids for the individul components involved with mino cid trnsport. Preliminry results with vesicles derived from this glycerol uxotroph hve shown tht the substrte for oxygen utiliztion nd mino cid trnsport is L-lctte nd tht the substrte vilble for mino cid trnsport is not lcking during glycerol deprivtion. ACKNOWLEDGMENTS We thnk L. Mindich for sending us the glycerol uxotroph, S. ureus S-2. This study ws supported by Public Helth Service grnt GM-1285 from the Ntionl Institute of Generl Medicl Sciences nd grnt GB from the Metbolic Biology Section of the Ntionl Science Foundtion. P.H.R. ws supported by Public Helth Service grnt 1-F2-GM from the Ntionl Institute of Generl Medicl Sciences. T.T.L. ws supported by Public Helth Service fellowship 1-F2-AM from the Ntionl Institute of Arthritis nd Metbolic Diseses. LITERATURE CITED 1. Bligh, E. G., nd W. J. Dyer A rpid method of totl lipid extrction nd purifiction. Cn. J. Biochem. Physiol. 37: Brock, T. D., nd G. Moo-Penn An mino cid trnsport system in Streptococcus fecium. Arch. Biochem. Biophys. 98: Esfhni, M., T. Ioned, nd S. J. Wkil Studies on the control of ftty cid metbolism. II. Incorportion of ftty cids into phospholipids nd regultion of ftty cid synthetse of Escherichi coli. J. Biol. Chem. 246: Fox, C. F A lipid requirement for induction of lctose trnsport in Escherichi coli. Biochemistry 63: Frermn, F. E., nd D. C. White Membrne lipid chnges during formtion of functionl electron trnsport system in Stphylococcus ureus. J. Bcteriol. 94: Hmmond, R K., nd D. C. White Seprtion of vitmin K, isoprenologues by reversed phse thinlyer chromtogrphy. J. Chromtogr. 45: Hsu, C. C., nd C. F. Fox Induction of the lctose trnsport system in lipid-synthesis-defective mutnt of Escherichi coli. J. Bcteriol. 13: Lillich, T. T., nd D. C. White Phospholipid metbolism in the bsence of net phospholipid synthesis in glycerol-requiring mutnt of Bcillus subtilis. J. Bcteriol. 17: Mindich, L Membrne synthesis in Bcillus subtilis. I. Isoltion nd properties of strins bering muttions in glycerol metbolism. J. Mol. Biol. 49: Mindich, L Membrne synthesis in Bcillus subtilis. II. Integrtion of membrne proteins in the bsence of lipid synthesis. J. Mol. Biol. 49: Mindich, L Induction of Stphylococcus ureus lctose permese in the bsence of lipid synthesis. Proc. Nt. Acd. Sci. U.S.A. 68: Mindich, L Studies on the control of ftty cid synthesis in bcteri. J. Bcteriol. 11: Pieringer, R A The metbolism of glyceride glycolipids. I. Biosynthesis of monoglucosyl diglyceride nd diglucosyl diglyceride by Streptococcus feclis. J. Biol. Chem. 243: Ry, P. H., nd D. C. White Effect of glycerol deprivtion on the phospholipid metbolism of glycerol uxotroph of Stphylococcus ureus. J. Bcteriol. 19: Ry, P. H., D. C. White, nd T. D. Brock Effect of growth temperture on the lipid composition of Thermus quticus. J. Bcteriol. 18: Short, S. A., D. C. White, nd H. R Kbck Active trnsport in isolted bcteril membrne vesicles. V. The trnsport of mino cids by membrne vesicles prepred from Stphylococcus ureus. J. Biol. Chem. 247: Sinclir, P., D. C. White, nd J. Brrett The conversion of protoheme to heme in Stphylococcus. Biochim. Biophys. Act 143: White, D. C The function of 2-demethyl vitmin K, in the electron trnsport system of Hemophilus prinfluenze. J. Biol. Chem. 24: White, D. C., nd R. H. Cox Identifiction nd locliztion of the ftty cids in Hemophilus prinfluenze. J. Bcteriol. 93: White, D. C., nd F. E. Frermn Extrction, chrcteriztion, nd cellulr locliztion of the lipids of Stphylococcus ureus. J. Bcteriol. 94: White, D. C., nd A. N. Tucker Phospholipid metbolism during bcteril growth. J. Lipid Res. 1: Wuthier, R. E Two-dimensionl chromtogrphy on silic gel-loded pper for the micronlysis of polr lipids. J. Lipid Res. 7: