Vol. 165, No. 3JOURNAL OF BACTERIOLOGY, Mar. 1986, p. 923-9280021-9193/86/030923-06$02.00/0Copyright © 1986, American Society for Microbiology

Isolation and Properties of Streptomyces Spore MembranesLUIS M. QUIR6S, CARLOS HARDISSON, AND JOSE A. SALAS*

Departamento de Microbiologia, Universidad de Oviedo, 33006 Oviedo, SpainReceived 16 August 1985/Accepted 2 December 1985

A simple procedure for the isolation of membranes from Streptomyces spores is described which producesabout 12 mg of membrane protein per g of dry weight. The membrane fractions were contaminated by lowlevels of DNA, RNA, and hexosamines. The functional integrity of the membrane is conserved throught theisolation procedure, as evaluated by the presence of several activities of the membrane-bound electrontransport chain. This isolation procedure allowed the determination of the biosynthesis of proteins andphospholipids of the membrane. Both biosynthetic processes started in the first 5 min of germination andincreased progressively during spore germination. A stable mRNA fraction of the dormant spore encoded 44%of the membrane proteins synthesized early in germination, but most of the phospholipid biosynthesis was notdependent on this fraction.

The genus Streptomyces is now considered an interestingmodel for cell differentiation studies. In its cell cycle thisorganism is capable of forming spores specialized in survivaland reproduction functions. These spores, although not in acryptobiotic state, have several properties in common withtrue spores and are thus considered as spores in a dormant orresting state (19). Under adequate simuli, these spores losetheir typical features and undergo germination, resulting in avegetative organism (mycelium). Many general aspects ofthe germination process have been studied (9, 15, 17, 20). Inaddition, divalent cations are known to initiate germinationin Streptomyces antibioticus (15, 27) and in Streptomycesviridochromogenes (8), and early events occurring duringspore germination such as calcium release, loss of spore heatresistance (27), and the release of spore carbon and of agermination inhibitor (18) have been reported. Other inter-esting events such as macromolecular synthesis during ger-mination (13, 15, 16), the existence of a stable mRNAfraction in dormant spores (14, 16, 25), and changes in sporerespiration (15, 18), in the adenylate energy charge (10), andin the glucose catabolism pathways (28) have been reported.The cytoplasmic membrane of bacteria has been clearly

established as the site of many of the enzyme activities of thecell. Its importance is evident since many of the functionscarried out by mitochondria, chloroplasts, and endoplasmicreticulum in eucaryotic cells are located in the cytoplasmicmembrane of procaryotic cells. Streptomyces spp. are surelynot an exception among procaryotic organisms. However,as far as we know there is no report of the isolation ofStreptomyces spore membranes. This lack of informationcould be due at least partially to the fact that Streptomycesonly sporulates on solid medium. It is difficult to obtain anamount of spores which would allow the isolation of mem-branes. For this reason we have decided to develop aprocedure for the isolation of spore membranes with integralfunctionality which will allow future studies on the role ofthe spore membrane in the germination process. Based onthis membrane isolation procedure we have initiated a studyof the Streptomyces spore membrane, and in this communi-cation we report on the isolation procedure, some criteria ofits purity, and our preliminary results on the synthesis of

* Corresponding author.

the protein and phospholipid components of these mem-branes.

MATERIALS AND METHODS

Microorganism and culture conditions. S. antibioticusATCC 11891 spores were prepared from cultures grown onglucose-asparagine-yeast extract-salts medium (GAE) solid-ified with 2% agar as previously described (15). Sporegermination was carried out in a minimal medium (13) at 35°Con an orbital Gallenkamp incubator. The germination processwas monitored by phase-contrast microscopy. Spores indifferent stages of germination were collected by centrifuga-tion when the highest percentage of spores in the appropriatestage of germination was observed (0 h of germination fordormant spores; 1 to 1.5 h for dark spores; 4 to 4.5 h forswollen spores).Breakage of spores and membrane isolation. The entire

process was carried out at 4°C. The disruption of the sporeswas carried out by glass-bead homogenization as follows.After harvesting, the spores were washed twice with 50 mMTris hydrochloride (pH 7.5), suspended in the same buffer(108 spores per ml), mixed with cold glass beads (0.10 to 0.11mm in diameter) in a ratio of 3 g of glass beads per ml of cellsuspension, and stirred in a Vortex mixer. Ten bursts of 30s each, with intermittent cooling on ice-cold water, wereused to avoid enzyme inactivation. More than 95% of thecells were broken by this treatment. After cell breakage, theglass beads were removed by low-speed centrifugation, andthe unbroken cells and cellular debris were eliminated bycentrifugation at 8,000 rpm (7,740 x g) for 1 h (shorter timesof centrifugation lead to increased contamination by cell wallfragments in the ultimate step). The supernatant was thencentrifuged at 25,000 rpm (75,500 x g) for 1 h in a BeckmanSW 42.1 rotor. The sediment was suspended in 50 mM Trishydrochloride buffer (pH 7.5) by brief sonication in anUltramet II sonic cleaner water bath and washed by centrif-ugation at 25,000 rpm (75,500 x g) for 1 h. The pellet wassuspended in the buffer, and small aliquotes were stored at-200C.

Analytical methods. Protein was measured with the Folinphenol reagent (23) using crystalline bovine serum albuminas the standard and including 0.1 ml of 10% (wt/vol) sodiumdodecyl sulfate in the assay to aid solubilization of the

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membrane protein. DNA was estimated with the diphe-nylamine reagent (4) using 2-deoxyribose as the standard.RNA was determined by the orcinol procedure (30) usingribose as the standard. Hexosamine determinations followeda modification of the Morgan-Elson procedure (12), usingN-acetylglucosamine as the standard. Total lipid phosphoruswas determined by the method described by Ames (1).

Protein biosynthesis. For determination of protein biosyn-thesis, spores (80 ml; A580 = 0.7) were incubated in a minimalmedium consisting of (grams per liter): glucose, 10.0; as-paragine, 2.0; (NH4)2SO4, 2.0; MgSO4 * 7H20, 0.5; FeSO4 '

7H20, 0.01; K2HPO4, 4.3; KH2PO4, 0.82. At different timesof germination, [35S]methionine (0.5 ,uCi/ml, 0.45 ,uM) wasadded, and after a short labeling period (30 s to 5 min) thepulse-labeling was stopped by the addition of 1 ml of anice-cold nonlabeled methionine solution (1 mg/ml, final con-centration) and rapid immersion of the Erlenmeyer flask inice-cold water. After 10 min, samples (0.5 ml) were with-drawn and added to 1.5 ml of ice-cold trichloroacetic acid(TCA) (10%, wt/vol, final concentration) for determinationof spore protein biosynthesis as TCA-insoluble radioactivematerial as previously described (16). The cells in the labeledsuspension were then collected by centrifugation, washedthree times in 50 mM Tris hydrochloride (pH 7.5), andsuspended in the same buffer. Breakage of spores andisolation of membranes were then carried out as describedabove. The radioactivity incorporated into membrane pro-teins was then measured as TCA-insoluble material using atoluene-based scintillation fluid. Since a different amount ofmembrane was obtained in the different stages of germina-tion, the radioactivity values were corrected by the amountof protein in each individual membrane preparation.

Phospholipid biosynthesis and extraction. [2-3H]glycerolwas used as a precursor for phospholipid biosynthesis. Atdifferent stages of germination, spore suspensions (10 ml;A580 = 0.7) were incubated in minimal medium and pulse-labeled for 5 min in the presence of [2-3H]glycerol (1 RCi/ml;0.2 ,M). Incorporation of the labeled precursor was stoppedby addition of 1 ml of an ice-cold nonlabeled glycerolsolution (1 mg/ml, final concentration) and rapid immersionof the flask in ice-cold water. Spores were then sedimentedby centrifugation, washed twice in buffer, and broken withglass beads as previously described. After cell breakage,TCA (5%, wt/vol, final concentration) was added to thecell-free extracts, which were then incubated for 30 min at4°C. The samples were then centrifuged at 10,000 rpm(12,000 x g) for 5 min, and the phospholipids were extractedfrom the peliet as described (3) with some modifications. Thepellet was suspended by sonic treatment in 1 volume of 50mM Tris hydrochloride buffer (pH 7.5), and 2.2 volumes ofmethanol were added. After incubation at 55°C for 15 min, 1volume of chloroform was added, and the mixture wasincubated for a period of 2 h at room temperature withintermittent shaking. In a preliminary experiment comparingextraction times of up to 24 h, 2 h was found to be sufficientto extract most of the phospholipids. The one-phase solutionthus resulting was centrifuged at 10,000 rpm (12,000 x g) for5 min, and 0.25 volume each of chloroform and Tris hydro-chloride buffer were added to the supernatant. After thor-ough shaking, the two phases were separated; the upperphase was discarded, and the lower phase (containing chlo-roform) was recovered. The chloroform was removed byevaporation under a stream of nitrogen at 40°C, the extractedphospholipids were suspended by sonication in the scintilla-tion fluid, and the radioactivity was counted.

Polyacrylamide gel electrophoresis. The polypeptide com-

position of the isolated membrane fractions was analyzed bysodium dodecyl sulfate-polyacrylamide gel slab electropho-resis using the discontinuous buffer systems of Laemmli(22). The separating gel was made of 12% polyacrylamide,and a 2-cm 5% stacking gel was included. Protein solubili-zation was accomplished by suspending the membrane frac-tions in 60 mM Tris hydrochloride (pH 6.8)-2 mMEDTA-2% (wt/vol) sodium dodecyl sulfate-2% (vol/vol),B-mercaptoethanol. After heating for 5 min in a boiling-waterbath, 10% (vol/vol) glycerol and 0.001% (wt/vol) bromophe-nol blue were added. The solubilized membrane prepara-tions were applied to the gel, and electrophoresis was carriedout at room temperature at 45 mA of constant current perslab gel until the tracking dye migrated just to the bottom ofthe gel (total running time was approximately 7 h). Gels werefixed by immersion in 5% (wt/vol) TCA for 30 min, stainingovernight with Coomassie brilliant blue R-250, and destainedfor 15 h in 10% (vol/vol) acetic acid-20% (vol/vol) methanolwith gentle agitation. Molecular weights were estimated bycomparing the relative mobilities with a standard curveprepared with ,B-lactoglobulin (18,400), trypsinogen (24,000),egg albumin (45,000), and bovine serum albumin (66,000).Fluorography of the gels was carried out after electrophore-sis by the method described by Chamberlain (5).Enzyme assays. All enzyme assays were carried out at

35°C in a Unicam SP 1700 ultraviolet spectrophotometercoupled to a Unicam AR 25 linear recorder. In all cases,blanks were run without addition of the substrate and thesevalues were subtracted from those of the experiments.

(i) NADH oxidase. The assay mixture contained 1 ml of 50mM Tris hydrochloride buffer (pH 7.5) and 100 to 400 pLg ofmembrane protein. The oxidation ofNADH was followed at340 nm after the reaction was initiated by addition of70 nmolof NADH.

(ui) NADH-DCPIP reductase. The assay mixture contained1 ml of 50 mM Tris hydrochloride buffer (pH 7.5), 5 ,ug of 5mM, 2,6-dichlorophenol-indophenol (DCPIP), and 100 to 400,ug of membrane protein. The reaction was initiated by theaddition of 20 ,ul of 10 mM NADH, and the reduction ofDCPIP was measured at 600 nm.

(iii) DL-at-Glycerophosphate dehydrogenase. DL-a-Glycerophosphate dehydrogenase activity was assayed asdescribed above for NADH-DCPIP reductase, but the reac-tion was initiated with 30 ,ul of 1 M DL-a-glycerophosphateinstead of NADH.

(iv) Succinate dehydrogenase. The assay mixture contained1 ml of 50 mM Tris hydrochloride buffer (pH 7.5), 100 ,ul of40 mM sodium succinate, 10 p,l of 0.1 M KCN (freshlyprepared and neutralized), 25 p,l of 5 mM DCPIP, and 100 to400 p,g of membrane protein. The reaction was started byaddition of 50 ,ul of phenazine methosulfate, and the de-crease in A6w was registered.

(v) NADH-cytochrome c reductase. The assay mixturecontained 1 ml of 50 mM Tris hydrochloride buffer (pH 7.5),10 ,ul of 0.1 M KCN (freshly prepared and neutralized), 23 ,ulof cytochrome c (21 mg/ml), and 100 to 400 p,g of membraneprotein. The reaction was started by addition of 50 ,ul of 10mM NADH, and the reduction of cytochrome c was fol-lowed at 550 nm.

Chemicals. L-[3SJmethionine (specific activity, 1,026.9Ci/mmol) was from New England Nuclear Corp. [2-3H]glycerol (specific activity, 0.75 Ci/mmol) was fromAmersham International Ltd. Chloroform and methanolwere from Merck. NADH, NADPH, DCPIP, DL-a-glycerophosphate, succinic acid, phenazine methosulfate,cytochrome c, rifampin, chloramphenicol, and electrophore-

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sis standards were from Sigma Chemical Co. All otherreagents were of analytical grade.

RESULTS

Composition and functionality of spore membranes. Themembrane fractions of dormant spores of S. antibioticusobtained by the procedure described above had a transpar-ent reddish color. The chemical analysis of a representativepreparation is shown in Table 1. All bacterial phospholipidscontain one phosphate group per molecule (with the excep-tion of diphosphatidylglycerol, which contains two groups),and therefore the determination of the phosphorus content isa reasonable approximation of the phospholipid content ofmembranes. The ratio of phosphorus to proteins was about0.39, a value close to that reported for the cytoplasmicmembrane of several gram-positive (11, 21, 26) and gram-negative (29) bacteria. The membrane contained very smallamounts of DNA and hexosamines which could not beremoved, even after extensive washing. Low levels of RNAwere also present, and our attempts to lower the RNAcontent of membranes by washing the preparations withbuffer and treating them with RNase (40 jig/ml) were notsuccessful. In this sense, wide variations have been reportedfor the RNA content of cytoplasmic membranes of gram-positive bacteria (2).To test the functional integrity of the isolated membranes

and as an additional purity criterion, several segments of theelectron transport chain were assayed (Table 2). The sporemembranes were able to oxidize NADH, and dehy-drogenases for NADH, NADPH, succinate, and DL-a-glycerophosphate were found. The transfer of electrons fromNADH to cytochrome c was also detected.Membrane protein biosynthesis during germination. In a

time-course experiment, it was found that the incorporationof [35S]methionine into membrane-associated proteins beganbefore 5 min into germination and increased linearly in thefollowing 25 min of germination. Since the possibility existedthat the incorporation of the radioactive precursor did notreally represent membrane protein biosynthesis but that ofmembrane-bound ribosome proteins or proteins being syn-thesized by the membrane-bound ribosomes, an experimentwas designed to clarify this problem. Spores were labeledwith [35S]methionine for 15 min, and the membranes wereisolated as described above. The ribosomes in the superna-tant fraction were then sedimented by centrifugation at45,000 rpm (100,000 x g) for 4 h, and the radioactivity andRNA content of each fraction (membranes and ribosomes)were determined. On the assumption that the radioactiveprecursor was being incorporated into polypeptides differentfrom those of the membrane, it would be expected that thespecific labeling (counts per minute per microgram of RNA)

TABLE 1. Chemical composition of membranes of S.antibioticus dormant spores

Component Content" (mg/g

spore dry weight)

Proteinb........ 11.28 (10.98-11.58)

Phosphorusb........ 4.40 (3.80-5.00)DNA........ 0.07 (0.06-0.08)RNA........ 1.69 (1.54-1.84)

Hexosamine....... 1.06 (0.98-1.14)

aResults are expressed as the mean values, and the figures in parenthesesrepresent the range of values obtained in three independent determinations.

b Ratio of phosphorus to protein = 0.39.

TABLE 2. Assay of different segments of the spore electrontransport chain

Enzyme Sp acta

NADH oxidase ................................ 0.541.20bNADH-DCPIP reductase .............................29.35-33.50cNADPH-DCPIP reductase............................39.16 39.35cNADH-cytochrome c reductase ......................66.20-72.77"Succinate-DCPIP reductase...........................44.26-55.45cDL-a-Glycerophosphate-DCPIP reductase............10.19-26.30c

a Minimum and maximum limit values obtained in six determinations usingthree different batches of membranes. Activities expressed as given infootnotes b through d.bNanomoles of NADH oxidized per minute per milligram of protein.cNanomoles of DCPIP reduced per minute per milligram of protein.d Nanomoles of cytochrome c reduced per minute per milligram of protein.

would be similar in both fractions. In contrast, if the[35S]methionine incorporation was really labeling mainlymembrane protein biosynthesis, a clear difference in thespecific labeling values should be expected, with that of themembrane fraction being the highest. The results obtained(data not shown) showed that the specific label was 13.5times higher in the membrane fraction than in the ribosomefration, and therefore the [35S]methionine was actually beingincorporated into recently synthesized membrane proteins.The rate of membrane protein biosynthesis during germi-

nation was also studied. Preliminary experiments (data notshown) showed that the rate of [35S]methionine incorpora-tion into TCA-insoluble material was constant for at least 15min during the first 2 h of germination and for only 1 minlater in germination. Consequently, for the determination ofthe initial rates of protein biosynthesis, we selected a 5-minperiod during the first 2 h of germination and a 30-s periodduring h 3 and 4 of germination. During the first hour ofspore germination in minimal medium, the rate of membraneprotein biosynthesis was very low, and from that time on, acontinuous and sharp increase was observed (Fig. 1). Thepattern of membrane protein biosynthesis showed a clearcorrelation with the one found for spore protein biosynthesis(16). When both rates were compared, a practically constantvalue was obtained through all the germination process (datanot shown).

Since S. antibioticus spores are able to synthesize proteinsduring incubation in distilled water (16), some experimentswere run to determine whether any of these proteins wereincorporated into the spore membrane. The results (Table 3)showed that, upon incubation of spores for a short time (15min) with [35S]methionine in distilled water, incorporation ofthe precursor into membrane proteins was detected. How-ever, the ratio between the biosynthesis of membrane pro-teins and that of the total spore proteins was lower than thisvalue during germination in minimal medium (0.62 and 0.77,respectively).Membrane proteins encoded by stable mRNA of dormant

spores. Previous reports pointed out the existence of a stablemRNA fraction in dormant spores of S. antibioticus; thismRNA is translated from the onset of germination and, atleast partially, during incubation of spores in distilled water(14). Recently, we have also shown that this stable mRNAfraction is present in spores of several Streptomyces species(25). It was therefore interesting to test whether this mRNAfraction encoded some membrane proteins. In these exper-iments the additional precaution was taken of includingrifampin during all processes of spore harvesting to avoidany mRNA synthesis during this procedure. Spores were

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FIG. 1. Biosynthesis of membrane macromolecules during ger-mination of S. antibioticus spores. Spores were incubated in mini-mal medium and, at different times of germination, pulse-labeledwith [35S]methionine (open bars) or [3H]glycerol (closed bars) asprecursors of protein or phospholipid biosynthesis, respectively, asdescribed in the text. The membrane fraction was then isolated andthe incorporation of label was determined.

then pulse-labeled with [35S]methionine in minimal mediumin the presence and absence of rifampin (15 p,g/ml), and theincorporation of the radioactive precursor into membraneproteins was compared. The ratio of membrane proteinssynthesized in the presence (154 cpm/,jg of protein) andabsence (346 cpm/,lg of protein) of rifampin showed thatabout 44% of the membrane proteins synthesized in the first15 min of germination were encoded by the mRNA fraction.When this ratio was calculated for spore protein biosynthe-sis, it was found that 48% of the protein biosynthesis wasdependent on this fraction (Table 3). The pattern of mem-brane proteins encoded by the stable mRNA was alsoanalyzed by polyacrylamide gel electrophoresis andfluorography. Fluorography of the gels showed (Fig. 2) thatthe membrane polypeptides encoded by this fraction were ina wide range of molecular weights (10,000 to 100,000), andno important qualitative differences were found betweenthose synthesized in the presence or absence of rifampin.

Phospholipid biosynthesis during germination. Membranesof gram-positive bacteria contain most of the phospholipidsof the cell (2, 6, 24), and therefore we used the incorporation

TABLE 3. Influence of different incubation conditions onincorporation of [35S]methionine into membrane and

spore proteinscpm incorporated

Conditions per ,ug of proteina Ratio:Conditions ~~~~~membrane/sporeMembrane Spore

Distilled water 250 405 0.62Minimal medium 346 447 0.77Minimal medium + rifampin 154 214 0.71

(15 ,ug/ml)Minimal medium + 17 22 0.77chloramphenicol (100,ug/ml)a Mean values of three independent determinations. All the values were

within 10%6 of the mean value.

24K-

FIG. 2. Fluorography of the membrane proteins synthesized atthe initiation of germination of S. antibioticus spores. Spores werelabeled in a minimal medium with [35S]methionine for 15 min asdescribed in the text in the absence (A) or presence (B) of rifampin(15 ,ug/ml). Then the membrane fraction was isolated, and theproteins were separated by sodium dodecyl sulfate-polyacrylamidegel electrophoresis and subjected to fluorography as described in thetext. The arrows indicate the positions and molecular weights ofseveral standard proteins used as markers.

of a labeled phospholipid precursor to follow membranebiosynthesis. [2-3H]glycerol was selected as a precursorsince the label of the glycerol molecule in this position hasinteresting advantages. It is known (6) that this tritiatedcompound labels only phospholipids because if it is metab-olized after transport and phosphorylation the tritium labelin position 2 would be lost to the medium. However, theutilization of glycerol for teichoic acid biosynthesis producesnot the release of this tritium label but its incorporation intothe teichoic acid molecule. Consequently, in spite of the factthat the existence of teichoic acid in Streptomyces spores isnot known, the precaution was taken of extracting thephospholipids by a method which would not extract theteichoic acid fraction (see Materials and Methods). Somepreliminary experiments were run to select pulse-labelingtimes for the different stages of germination. These experi-ments (data not given) showed that the incorporation of[2-3H]glycerol into TCA-insoluble material was linear for atleast 15 min, so we chose a 5-min labeling period forphospholipid biosynthesis during germination. The incorpo-ration of [2-3H]glycerol into phospholipids started at leastbefore 5 min had elapsed in germination, and the rate ofphospholipid biosynthesis remained constant during the firsthour of germination, increasing progressively in the follow-ing 3 h (Fig. 1).The influence of two antibiotics on phospholipid biosyn-

thesis during germination was also studied. Spores werepulse-labeled with [2-3H]glycerol for 15 min in the presenceof rifampin (inhibitor ofRNA synthesis) or chloramphenicol(inhibitor of protein synthesis). After pulse-labeling, the

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MEMBRANES OF STREPTOMYCES SPORES 927

phospholipids were extracted, and the radioactive valueswere compared with those obtained from parallel samplesrun without antibiotic. Chloramphenicol and rifampin inhib-ited phospholipid biosynthesis by dormant spores by about20 and 18%, respectively, suggesting that dormant sporescontain the enzymes necessary to initiate phospholipid bio-synthesis. Similar inhibition was found in more advancedstages of germination.

DISCUSSIONTo our knowledge, this is the first report describing the

isolation of Streptomyces spore membranes and the study ofmacromolecular membrane changes occurring during sporegermination. The isolation of functional spore membranespresents some problems. Attempts to cause spore breakageby gentle methods were unsuccessful, since Streptomycesspores are resistant to ultrasound (19) and lysozyme treat-ment (7, 32) and, therefore, harsh treatments (i.e., glass-bead homogenization) are necessary to disrupt them. Themethod we report here for the isolation of Streptomycesspore membranes is simple, and the yield of spore membrane(about 12 mg of membrane protein per g dry weight) isenough to allow numerous studies of membrane-associatedphenomena. The purity of the membrane fractions wascomparable to that of others described for Bacillus spores(26), and further purification steps such as sucrose gradientcentrifugation were unnecessary. In fact, the chemical com-position and enzyme activities of membranes before andafter sucrose gradient centrifugation were very similar (datanot shown). The fractions showed a typical membranecomposition with very low levels of contaminating material.Such was also the case with RNA, which was present insmaller amounts than those reported for membranes ofvegetative cells (11) and spores of Bacillus species (26). It ispossible that most of this RNA belongs to membrane-boundribosomes. The purity of the membrane fractions was alsoexamined by electron microscope using negative stainingwith phosphotungstic acid. The preparations containedmembrane fragments and were comprised mainly of mem-brane vesicles (data not shown). No contamination by sporecell wall was detected. The functional integrity of the mem-branes was conserved, as evaluated by the determination ofseveral activities of the particulate electron transport chain.Using purified spore membrane fractions, it was possible

to study protein and phospholipid membrane biosynthesis. Itis interesting that at no time during germination was therepreferential protein biosynthesis either in the spore mem-brane or in the whole spore, since the ratio between bothrates of biosynthesis remained constant during germination.Protein synthesis occurring in the presence of rifampin mustbe the consequence of the translation of preexistent messen-gers in the dormant spore, since this antibiotic is an inhibitorof RNA polymerase (31) and has a dramatic effect on S.antibioticus spores and in other Streptomyces species, com-pletely blocking RNA synthesis in less than 90 s (16, 25).About half of the membrane proteins synthesized in the first15 min of germination were encoded by such a stable mRNAfraction, and these protein species were of a wide molecularweight range. This early membrane protein biosynthesis wasaccompanied by simultaneous phospholipid biosynthesis,and it is possible that this synthesis of membrane phos-pholipids may be required to include the new synthesizedproteins in the membrane structure and to adapt the mem-brane for the transition from the dormant state to the darkstate.

It has been suggested that some early events in the

initiation of germination of Streptomyces spores may occurin the vicinity of the spore membrane. Germination initiatedby calcium ions in S. viridochromogenes must occur exter-nally to the cytoplasmic membrane (8). In addition, the highcalcium content of Streptomyces spores is located outside ofthe spore cytoplasm (27), and it is possible that the reac-tion(s) producing its release may also take place internally orexternally to the membrane. Many studies on the earlyevents of germination will require a methodology to isolatethese fractions with adequate purity. Our work presentedhere will allow the initiation of these studies concerning thespore membrane.

ACKNOWLEDGMENTSWe thank S. G. Bradley for critical reading of the manuscript.L.M.Q. was the recipient of a predoctoral fellowship of the

Ministry of Education and Science, Spain.

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chemical composition of the cytoplasmic membranes of Bacillussubtilis. Eur. J. Biochem. 2:448-453.

3. Bligh, E. G., and W. J. Dyer. 1959. A rapid method of total lipidextraction and purification. Can. J. Biochem. Physiol. 37:911-917.

4. Burton, K. 1956. Study of the condition and mechanism of thediphenylamine reaction for the colorimetric estimation of DNA.Biochem. J. 62:315-323.

5. Chamberlain, J. P. 1979. Fluorographic detection of radioactiv-ity in polyacrylamide gels with the water-soluble fluor, sodiumsalicylate. Anal. Biochem. 98:132-135.

6. Daniels, M. J. 1%9. Lipid synthesis in relation to the cell cycleof Bacillus megaterium KM and Escherichia coli. Biochem. J.115:697-701.

7. De Jong, P. J., and E. McCoy. 1966. Qualitative analyses ofvegetative cell walls and spore walls of some representativespecies of Streptomyces. Can. J. Microbiol. 12:985-994.

8. Eaton, D., and J. C. Ensign. 1980. Streptomyces virido-chromogenes spore germination initiated by calcium ions. J.Bacteriol. 143:377-382.

9. Ensign, J. C. 1978. Formation, properties and germination ofactinomycete spores. Annu. Rev. Microbiol. 32:185-219.

10. Garcia Diaz, L., J. A. Salas, and C. Hardisson. 1983. Intracel-lular pool of Streptomyces spores: amino acids, nucleosides,adenine nucleotides and energy charge. FEMS Microbiol. Lett.19:215-219.

11. Gel'man, N. S., M. A. Lukoyanova, and D. N. Ostrovskii. 1975.The chemical composition of bacterial membranes. Bio-membranes 6:27-53.

12. Ghuysen, J., D. J. Tipper, and J. L. Strominger. 1966. Enzymesthat degrade bacterial cell walls. Methods Enzymol. 8:685-699.

13. GuUarro, J. A., J. E. Suairez, J. A. Salas, and C. Hardisson.1981. Characterization of RNA species synthesized during ger-mination and vegetative growth of Streptomyces antibioticus.FEMS Microbiol. Lett. 14:205-209.

14. GuUarro, J. A., J. E. Suairez, J. A. Salas, and C. Hardisson.1983. Pattern of protein degradation during germination ofStreptomyces antibioticus spores. Can. J. Microbiol. 29:637-643.

15. Hardisson, C., M. B. Manzanal, J. A. Salas, and J. E. Sufirez.1978. Fine structure, physiology and biochemistry of ar-throspore germination in Streptomyces antibioticus. J. Gen.Microbiol. 105:203-214.

16. Hardisson, C., J. A. Salas, J. A. Guijarro, and J. E. Suarez.1980. Macromolecular synthesis during the germination ofStreptomyces spores in a chemically defined medium. FEMSMicrobiol. Lett. 7:233-235.

17. Hirsch, C. F., and J. C. Ensign. 1975. Germination of Strepto-myces viridochromogenes spores, p. 28-35. In P. Gerhardt,

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