positive bacteria

JOURNAL OF BACTERIOLOGY, Oct. 1982, p. 315-3220021-9193/82/100315-08$02.00/0Copyright 0 1982, American Society for Microbiology

Vol. 152, No. 1

Glyceride-Cysteine Lipoproteins and Secretion by Gram-Positive Bacteria

JENNIFER B. K. NIELSEN AND J. 0. LAMPEN*Waksman Institute of Microbiology, Rutgers University, Piscataway, New Jersey 08854

Received 5 April 1982/Accepted 1 June 1982

The membrane penicillinases of Bacillus licheniformis and Bacillus cereus arelipoproteins with N-terminal glyceride thioether modification identical to that ofthe Escherichia coli outer membrane lipoprotein. They are readily labeled with[3H]palmitate present during exponential growth. At the same time, a few otherproteins in each organism become labeled and can be detected by fluorographyafter sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total mem-brane proteins. We distinguish these proteins from the 0-acyl proteolipids bydemonstrating the formation of glyceryl cysteine sulfone after performic acidoxidation and hydrolysis of the protein. By this criterion, B. licheniformis and B.cereus contain sets of lipoproteins larger in average molecular weight than that ofE. coli. Members of the sets probably are under a variety of physiologicalcontrols, as indicated by widely differing relative labeling intensity in differentmedia. The set in B. licheniformis shares with membrane penicillinase a sensitiv-ity to release from protoplasts by mild trypsin treatment, which suggests similarorientation on the outside of the membrane. At least one protein is the membrane-bound partner of an extracellular hydrophilic protein, the pair being related asmembrane and exopenicillinases are. We propose that the lipoproteins of gram-positive organisms are the functional equivalent of periplasmic proteins in E. coliand other gram-negative bacteria, prevented from release by anchorage to themembrane rather than by a selectively impermeable outer membrane.

Covalent modification of a cysteine residuewith a glyceride thioether group was first de-scribed by Hantke and Braun (11) in 1973 for themajor outer membrane protein of Escherichiacoli. It is responsible for the hydrophobic prop-erties of the mature lipoprotein, ensuring itsanchorage in the outer membrane. It is nowevident that E. coli possesses a class of suchproteins, about 10 in number, that are lessabundant than the Braun lipoprotein, but similarin structure, as shown by radioisotopic labelingwith glycerol and long-chain fatty acids and bysimilar responses to the cyclic peptide antibioticglobomycin (12). Lai et al. (19) noted that theapparently identical modification could be foundon Bacillus licheniformis membrane penicillin-ase when expressed in E. coli after cloning on agamma vector. Simultaneously, we reported thesame finding (20) and, furthermore, that thesame glyceride thioether modification was pres-ent in the membrane-bound form of B. licheni-formis penicillinase in the gram-positive orga-nism itself, accounting for the hydrophobicproperties of the cell-bound form. We haverecently extended this observation (21) to themembrane penicillinases of two other class Apenicillinases (1), those of Bacillus cereus (type

I) and Staphylococcus aureus (types A and C).In all cases so far described, a cysteine residuenear the junction of the hydrophobic membrane-spanning (10) stretch of the signal peptide and ahydrophilic bridge of conserved amino acids (21)become modified by an acyl glyceride residue.The signal peptidase of B. licheniformis cleavesthe in vitro translation product of the penP genejust before this cysteine residue (5) and, byanalogy with the well-characterized E. coli lipo-protein, functions at the same site in vivo torender this cysteine N-terminal in the maturemembrane protein. In E. coli, the membraneform appears to be the final destination of mostlipoproteins. In the three gram-positive orga-nisms, however, the membrane-bound penicil-linase is a stage of the secretory process in thatthe modified protein is largely cleaved from itssite on the outside of the membrane late in thegrowth cycle and released as a hydrophilic prod-uct into the medium. Several lines of evidencepoint to the membrane-attached form not beingan obligatory intermediate, but on an alternativepathway to secretion (7, 20).

In examining the penicillinases of B. licheni-formis, B. cereus, and S. aureus, we noticed thatsimilarly to E. coli, each of these organisms

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316 NIELSEN AND LAMPEN

possesses a set of apparent lipoproteins. In thispaper, we describe the properties of these sets inthe two bacilli.

MATERIALS AND METHODSBacterial stris. B. licheniformis 749 (penicillinase

inducible) and 749/C (ATCC 25972; penicillinase con-stitutive) were obtained from M. R. Pollock. B. cereus569 (ATCC 27348; type I, penicillinase inducible) wasobtained from N. Citri.Media. CH/S medium contained 10 g of acid-hydro-

lyzed Casamino Acids per liter (Difco Laboratories,Detroit, Mich.), 20 mM KH2PO4, 10 mM MgCl2, 0.1%Pollock's salts (23), pH 6.5. Low-phosphate Casitonemedium was prepared by precipitation with magnesia,using a modification of the method of Takeda andTsugita (26). Magnesia was prepared by adding con-centrated NH40H to a solution of 10 g ofMgCl2 * 6H20 and 20 g of NH4Cl in 100 ml of wateruntil alkaline. After standing at 4°C overnight, thesolution was ifitered, adjusted to pH 6.0 with HCI, anddiluted to 200 ml. For 1 liter of phosphate-free medi-um, 20 ml of this solution was added to 10 g ofCasitone (Difco) in 10 ml of water, stored at 4°C for 2days, and centrifuged at 25,000 x g for 1 h. Thesupernatant was carefully decanted, 6 g of Tris and 3 gof NaCl were added, and the solution was centrifugedagain as described before. 1-ml salt solution containing(in 100 ml) FeSO4 H20 (100 mg), ZnSO4 * H20 (100mg), MnSO4 * 4H20 (10 mg), K2CrO7 (0.2 mg), andCuSO4 - H20 (5 mg), was added. The pH was adjustedto 7.0 and the volume was adjusted to 1 liter beforeautoclaving. Separately autoclaved MgSO4 (1.2 mM),CaC12 (0.8 mM), and glucose (0.1%) were added. Theinoculum was grown in the same medium as above,except that glucose was omitted and 2 mM KH2PO4was added. The cells were pelleted and washed beforeuse for inoculation. Without this treatment, the phos-phate level in Casamino Acid medium is about 0.5mM, sufficient for substantial repression; after treat-ment, it is less than 0.05mM and allows the productionof more than 200 U of alkaline phosphatase per mg(dry weight) of cells. The assay was that described byGhosh et al. (9). In this low-phosphate medium, peni-cillinase production was about 30%o lower than in CH/S medium containing 20 mM phosphate.

L-broth contained (per liter) 10 g of tryptone, 5 g ofyeast extract (Difco), and 5 g of NaCl, pH 7.2. Brainheart infusion broth contained 37 g/liter of the BBLproduct.

Materials. [9,10-3H]palmitic acid (11.8 Ci/mmol),and L-["S]cysteine (1,108.5 Ci/mmol) were purchasedfrom New England Nuclear Corp., Boston, Mass., andL-[355]methionine (1,100 Ci/mmol) was purchasedfrom Amersham Corp., Arlington Heights, Ill. Globo-mycin was a gift of M. Arai (Sankyo, Tokyo, Japan),antibody to B. licheniformis 749/C alkaline phospha-tase was a gift of B. K. Ghosh (Rutgers MedicalSchool). Penicillin-binding proteins from B. lichenifor-mis 749 labeled with [14C]benzylpenicillin were pre-pared at the Squibb Institute for Medical Research,Lawrenceville, N.J., by N. Georgopapadakou (8).

Antibodies. Antibody to purified exopenicillinasewas prepared as previously described (14). The 63-kilodalton (kd) lipoprotein was obtained from partiallypurified membrane penicillinase preparations (through

DEAE-Sephadex chromatography [25]) that were sub-jected to sodium dodecyl sulfate-gel electrophoresis.Unstained bands were excised with a stained 63-kdband on the edge of the gel as a guide. About 100 ,ug ofthe protein could be isolated from one 3-mm gel. Atotal of 400 ,ug was used to prepare antibodies byinjecting the macerated gel intradermally (27).

Isotopic labeling. Palmitate labeling was performedin 2.5-ml cultures by adding isotope at 60 ,uCi/ml.Palmitic acid in benzene was dried in a nitrogen streamand dissolved in 2% Tween 20. In phosphate-freemedium, the usual level of Tween (0.016%) causedlysis and had to be reduced to 0.004%. Several rinseswith the culture were made to ensure complete trans-fer of the palmitate. Different media were used asnoted in the figure legends. [355]cysteine labeling forthe subsequent isolation of glyceryl cysteine sulfonewas performed in CH/S medium containing acid-hy-drolyzed casein, because acid hydrolysis yields anamino acid mixture virtually free of cysteine. After 2generation times,ilabeling in this case was terminatedby the addition of 20 mM cysteine, which was presentalso in all subsequent steps of the isolation. Cells wereharvested and converted to protoplasts as previouslydescribed for B. licheniformis and B. cereus (20). Theprotoplasts were disrupted by boiling in 2x electro-phoresis buffer, and the extracts were electrophoresedon 10%o sodium dodecyl sulfate gels (17). Extractscontaining [3H]palmitate and those with [35S]cysteinewere never analyzed in the same gel, because [355]cys-teine binds nonspecifically to proteins in low amounts.The slightest contamination with 3IS overburdens theweaker 3H signal in autoradiography. [3H]palmitate-containing gels were enhanced with sodium salicylate(4) before being dried, autoradiographed, and scannedon a Schoeffel Spectrodensitometer SD3000. Gels with[35S]cysteine were dried without enhancement andwere autoradiographed ovemight. The dried laneswere cut into 0.5-cm strips, the protein was eluted, andsodium dodecyl sulfate was removed. The protein wasthen oxidized with performic acid, hydrolyzed with 6N HCI, and subjected to high-voltage electrophoresisas previously described (21). Six 1-cm strips were cutfrom the paper in the regions of glyceryl cysteinesulfone and of cysteic acid for assay of 35S as anindicator of the presence of the substituted sulfone.

RESULTSLabding of membrane proteins with pahmitate.

When [3H]palmitate is present during exponen-tial growth of B. licheniformis or B. cereus,several proteins become covalently labeled withfatty acid. Autoradiograms of sodium dodecylsulfate gels of membrane proteins from theseorganisms (grown in CH/S medium) are shownin Fig. 1. With B. licheniformis 749 induced toform penicillinase, the labeled band correspond-ing to the membrane-bound enzyme can be seenat 32 kd (Fig. 1, lane A; compare with extract ofuninduced cells in lane B); mutant 749/C, mag-noconstitutive for penicfllinase synthesis, pro-duces more of this band (Fig. 1, lane D). Globo-mycin, a peptide antibiotic (13) whose effectappears limited to those proteins bearing a glyc-

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LIPOPROTEINS OF GRAM-POSITIVE BACTERIA 317

A B C D EFIG. 1. Labeling of membrane proteins in B. li-

cheniformis and B. cereus cells with [3H]palmitate.Cultures (2.5 ml) in CH/S medium were incubated with[3H]palmitate at 60 ,uCi/ml for two generations. Cellswere harvested, and membrane extracts were electro-phoresed and fluorographed as described in Materialsand Methods. Lane A, membrane proteins from B.licheniformis 749 induced for penicillinase synthesisby 5 ,uM 2-(2'-carboxyphenyl)benzoyl-6-aminopenicil-lanic acid; lane B: same as lane A but without inducer;lane C, extract of B. licheniformis 749/C cells incubat-ed with globomycin (50 ,ug/ml); lane D, same as lane Cwithout globomycin; and lane E, extract, of B. cereus

569 cells grown without inducer. Sizes are shown inkilodaltons deduced from Coomassie blue-stainedstandards and from membrane penicillinase at 32 kd.

eryl cysteine modification, depresses the accu-mulation of membrane penicillinase (20) and thelabeling of the 32-kd band by palmitate (Fig. 1,lanes C and D). In constrast, its presence in-creased palmitate labeling of a 63-kd protein.The related B. cereus penicillinase, type I (1),behaves in an analogous manner (21). It isevident from the intensity of labeling in inducedcells ofB. licheniformis 749 and in 749/C that themost abundant fatty acid-substituted membraneprotein is penicillinase, but that there are severalother minor but discrete bands in addition to thephospholipid band which runs near the front inthe gel system used. The [3H]palmitate is notextractable from these bands (20 to 63 kd) byCHCl3/CH30H (3:1) at pH 9.0 (3). With bothbacilli, several bands are clustered around 35 kd,more in B. cereus (Fig. 1, lane E) than in B.licheniformis, and with each organism there is aprominent band at 63 kd. In B. licheniformis,none of the minor protein bands above or belowpenicillinase (32 kd) is immunologically relatedto penicillinase (20). S. aureus, whose mem-brane penicillinase we have shown to be alipoprotein, also yielded a cluster of minor bands

around 35 kd (21), unrelated immunologically topenicillinase, but there were very few palmitate-labeled bands of higher molecular weights.

Thloether nature of lipoprotein bands. To de-termine whether the sets of palmitate-labeledbands represent a class of lipoproteins of the E.coli type, cultures of B. licheniformis 749 wereseparately labeled with [35S]cysteine and[3H]palmitate in the absence of an inducer forpenicillinase. (Without the dominant penicillin-ase band, other bands in the 32-kd region of thegel may be examined more readily.) The extract-ed membrane proteins were subjected to electro-phoresis. Serial segments of the 35S-labeled gelwere analyzed for [35S]glyceryl cysteine sulfoneafter oxidation and acid hydrolysis. The recov-ery as the substituted sulfone, plotted againstmobility in a 10% gel and superimposed on adensitometer scan of the 3H-labeled proteins, isshown in Fig. 2 (upper panel). A similar experi-ment with an uninduced B. cereus 569 culture isshown in the lower panel. It can be seen thatwith ever5 palmitate band from both bacilli,there is [3 S]glyceryl cysteine sulfone.

Factors affecting formation of lipoproteins. Thewide variations in intensity of palmitate labeling(Fig. 2) suggest that the bands fall into at leastseveral classes of physiological control. Figure 3compares densitometer scans of palmitate radio-activity incorporated into membrane proteins ofB. licheniformis 749 in three media providingincreasing growth rates. Labeling of the bandsincreased in the series from CH/S medium to L-broth to brain heart infusion broth; the oneexception (63-kd band) showed much less label-ing in rich media than in CH/S. No new bandsemerged in the rich media, except for the ap-pearance of [3H]palmitate labeling at 20 kd as asingle broad peak in L-broth and a doublet inbrain heart infusion broth. However, even inCH/S, this 20-kd protein(s) showed glycerolsubstitution of the cysteine despite the lack oflabeling from the palmitate pool (Fig. 2).We examined one specific growth medium

with the possibility in mind that the membrane-bound alkaline phosphatase of B. licheniformis(9) or one of its accessory proteins (A. Ghoshand B. K. Ghosh, personal communication) maybe a lipoprotein. Bacilli secrete a large portion oftheir total alkaline phosphatase activity into themedium and, as in the case of penicillinase,retain the rest as a membrane-bound form. Thisform differs, however, from penicillinase in be-ing extractable by 1 M MgSO4 rather than bydetergents. Under conditions derepressive foralkaline phosphatase synthesis (Fig. 4), the pal-mitate labeling of most bands other than penicil-linase (the prominent band at 32 kd; the constitu-tive penicillinase producer 749/C was used) ismarkedly reduced. The bands thus affected are

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RELATIVE MOBILITYFIG. 2. Congruence of palmitate labeling andglyc-

eryl cysteine thioether in membrane proteins fromcultures of B. lichenjformis and B. cereus uninducedfor penicillinase. The techniques used are described inMaterials and Methods. [3H]palmitate labeling wascarried out as described in the legend to Fig. 1 and[35S]cysteine was added to 50 FCi/ml; both were inCH/S medium. The densitometer scan of 3H activity isrepresented by the solid line, and 35S activity isrepresented by the shaded bars.

not phospholipids because no covalently at-tached 32p, was incorporated into any of thesebands (results not shown). Cells grown in 0.05mM phosphate medium did not release palmitatelabel during incubation with 1 M MgSO4, al-though a complex of alkaline phosphatase andassociated proteins was extracted from themembrane. Thus, none of the lipoproteins we

observe in B. licheniformis appears to have anyconnection to the alkaline phosphatase system.

Cellular location of lipoproteins. Membranepenicillinase is located on the outside of the cellmembrane and is readily released from proto-plasts by trypsin as a soluble exoenzyme withthe N-terminal, lipid-containing peptide cleavedoff and presumably remaining attached to themembrane (2). The released soluble penicillinase

is resistant to further proteolysis, and the proto-plasts sustain very little damage. The effects ofsuch mild trypsinolysis are illustrated in Fig. Sa.Trypsin released about 75% of the penicillinase(32-kd band) from the membrane and even great-er proportions of all the other lipoproteins withthe possible exception of a small peak at 0.8mobility. The palmitate label, now on shortchains, has migrated off the gel. It is obviousthat most of the lipoprotein class of strain 749/C,as identified by palmitate labeling and gel elec-trophoresis, are at least as susceptible to mildtrypsin treatment as is penicillinase and thusprobably have a similar orientation on the out-side of the cell membrane.Nature of 63-kd Upoprotein. The striking re-

semblance of the 63-kd protein to membranepenicillinase can be shown by immunoprecipita-tion and characterization of the released, tryp-sin-stable core molecule (Fig. 5b). B. lichenifor-mis 749/C cells were labeled in CH/S for onegeneration with [35S]methionine, and a mem-

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FIG. 3. Incorporation of [3H]palmitate into mem-brane proteins of B. licheniformis 749 (uninduced) inthree media. The experimental procedures are de-scribed in the legend to Fig. 1. Densitometer scans offluorographed gels are shown.

aCH/S

63 4842 3533 20Size 4 44 44 4

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RELATIVE MOBILITYFIG. 4. Effect of phosphate concentration on pal-

mitate labeling of B. licheniformis 749/C. As in Fig. 3,2.5-ml cultures contained 60 ,uCi/ml [3H]palmitate inCH/S medium (20 mM P.) or in phosphate-free CH/S(see Materials and Methods) which is derepressive foralkaline phosphatase synthesis. AP, Alkaline phospha-tase.

brane extract was prepared. One half was treat-ed with a mixture of antibody to penicillinaseand antibody to the 63-kd protein, and theremainder was treated with trypsin before im-munoprecipitation with the mixed antibodies. Itcan be seen that for both molecules, the coreresistant to further trypsin digestion (Fig. 5b,lane B) is 2 to 3 kd smaller than the intactmolecule (Fig. Sb, lane A). A similar experimentusing [3H]palmitate labeling showed the totalelimination of high-molecular-weight acylgroups from both molecules after trypsin treat-ment (data not shown). The 63-kd protein hadattracted our attention during purification ofmembrane penicillinase because it is the lastprotein to be removed and is very similar topenicillinase in all the properties examined. Ongel ifitration in detergents, the two proteins,despite their size difference, copurify in a ixedmicelle. They are not immunologically cross-reactive, and there is no evidence for the largerbeing a penicillinase dimer. Peptide patternsfollowing digestion of the two proteins with S.

aureus V8 protease (6) showed no obviousbands in common between the two proteins.A hydrophilic protein of about 60-kd appear-

ing in the medium toward the end of growth isprecipitable by the antibody prepared to the 63-kd lipoprotein. These two proteins then arepresumably the released and membrane-boundforms of a secreted enzyme, related just as exo-and membrane-bound penicillinase. Two dissim-ilarities in behavior between penicillinase andthe 63-kd protein point to different mechanismscontrolling their synthesis or fatty acid acyla-tion. As mentioned earlier, whereas labeling of

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FIG. 5. (a) Cleavage of palmitate-labeled proteinson protoplasts of B. licheniformis 749/C by trypsin.Protoplasts prepared from 2.5-ml cultures, labeled aspreviously described, were suspended in osmoticallysupported medium (2) and incubated with and without40 =ag of trypsin per ml for 30 min at 37°C. Trypsincleavage was terminated by the addition of 20 11l of 0.1M diisopropyl fluorophosphate. Membrane extractswere prepared, electrophoresed, and fluorographed asdescribed in the legend to Fig. 1. (b) The 63-kdlipoprotein has a trypsin-resistant core analogous tothat of membrane penicillinase. A culture ofB. licheni-formis 749/C was incubated with [35S]methionine (50,uCi/ml) for one generation, and membranes wereprepared and solubilized in Triton X-100 as describedpreviously (20). To one half of the culture, antibody topenicillinase plus antibody to the 63-kd protein wereadded. The other half was digested with trypsin at 100Fg/ml for 2 h at 37°C and treated with excess diisopro-pyl fluoro-phosphate before the addition of both anti-bodies. After 1 h at room temperature, excess S.aureus cells (Cowan strain) were added (15). Afteranother 1 h at room temperature, the cells werewashed, and antibody and antigen were extracted andelectrophoresed. Lane A shows pencillinase (32 kd)and the 63-kd protein before trypsin treatment, andlane B, shows their trypsin-resistant cores at approxi-mately 30 and 60 kd, respectively.

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membrane penicillinase with palmitate was de-pressed by globomycin, that of the 63-kd pro-teins was enhanced. In addition, rich media,which increase penicillinase labeling by palmi-tate (data not shown), depress that of the 63-kdprotein.

DISCUSSIONWe have demonstrated that a class of lipopro-

teins with glyceride-cysteine thioether structurecan be found in gram-positive bacteria. Thisstructure, hitherto considered unique- to a groupof outer membrane proteins from gram-negativeorganisms, has recently been established (19-21)for the membrane bound forms of a major se-creted protein, penicillinase, of B. licheniformis,B. cereus, and S. aureus. This is far from anisolated case, as the lipoprotein structure can befound in a discrete number of other proteins, 6 to10 or so, in each of the two bacilli. S. aureus alsocontains at least a few of these lipoproteins (Fig.1 and 3 of reference 21).The easiest way to detect potential glyceride-

cysteine liporpoteins is to label cultures with[3H]palmitate. We show here electrophoreticproffles of membrane proteins extracted fromcultures labeled in this way, and we furthershow that for every peak of palmitate-labeledprotein, a corresponding band of glyceryl-substi-tuted cysteine can be found. Thus, the lipopro-teins detected by palmitate labeling in the twobacilli are most likely the glyceride-cysteinetype rather than the proteolipid type describedby Schlesinger (24). The latter proteins carrylong-chain fatty acid residues directly acylatedto amino acids, probably serine, in the mem-brane-binding region and have been found inseveral viruses and in mammalian cells in tissueculture. From our results, we cannot rule out theexistence of proteolipids in bacilli. We can onlysay that there is no significant palmitate band forwhich we must invoke an 0-acyl serine structurerather than an acyl glyceryl cysteine.

Palmitate labeling then is indicative of glycer-yl substitution of a cysteine residue, but is onlypoorly related to quantitative ratios of glycerylcysteine residues in individual members of theset. This can be seen in the widely varying ratioof [3H]palmitate to [35S]glyceryl cysteine sul-fone for different proteins in Fig. 2. Variations inthe intensity of palmitate labeling of individualbands are also obvious in different media (Fig.3). We can only comment that for most proteins,labeling was greater in richer medium and mightbe related to greater abundance of this class ofproteins at higher growth rates or to more effi-cient labeling of the phospholipid pool, the prob-able acyl donor to these lipoproteins, as is thecase in fatty acid labeling of the major E. colilipoproteins (18). Furthermore, only in rich me-

dium could we label the amide-linked fatty acylresidue on the amino group of the N-terminalcysteine of B. licheniformis 749 penicillinase.(0-linked fatty acyl groups are released by treat-ment with 0.1 N NaOH at 37°C for 1 h [19],whereas N-linked residues are resistant to re-lease.) Because of these factors and the com-plexities of the specific activities of pools con-tributing differentially to 0- and N-linked fattyacids, labeling with [3H]palmitate is not usefulfor quantitation of the abundance of these lipo-proteins. Nevertheless, in conjunction with iso-lation of glyceryl cysteine sulfone, palmitatelabeling qualitatively demonstrates sets of lipo-proteins in B. licheniformis and B. cereus.We have been unable to identify any of the

lipoproteins other than penicillinase in theseorganisms. That the others are not rare proteinsis clear when it is remembered that B. lichenifor-mis 749 and B. cereus 569, and in particular theirmagnoconstitutive mutants., have been selectedfor high penicillinase production; 1 to 2% of theirtotal protein and up to 50% of that secreted ispenicillinase. The others, although less abun-dant, are labeled in substantial amounts (see Fig.4 and 5, where labeling of 749/C, the constitutivepenicillinase producer, is shown). It seemedpossible that one or more of the observed lipo-proteins was a penicillin-binding protein becauseamino acid homologies between the major peni-cillin-binding protein (45 kd) of B. subtilis andclass A penicillinases suggest that they all derivefrom a common ancestral gene (30). However,there was no congruence between the lipopro-tein profile (palmitate labeling) of B. lichenifor-mis 749 (Fig. 3c) and that of the penicillin-binding proteins from comparable cells labeledin vitro with [14C]benzylpenicillin (8). The peni-cillin-binding proteins, which usually remain cellbound, apparently developed a different mode ofmembrane attachment by means of a hydropho-bic region near the carboxyl terminus (30).As we have shown (Fig. 4 and text), neither

the structural proteins for the membrane alka-line phosphatase of B. licheniformis 749 nor thebinding proteins appear to be lipoproteins. a-Amylase, another major secretory product of B.licheniformis (28), does not have a true mem-brane-bound form (H. Kuhn and J. 0. Lampen,unpublished results). Moreover, the closely ho-mologous a-amylase of B. amyloliquefaciens(16), in which DNA was recently sequenced byPalva et al. (22), does not have a modifiablecysteine residue in its signal sequence.

Despite the fact that we have not identified anenzymatic or structural role for any of this groupof lipoproteins other than penicillinase, we havedemonstrated their release from B. licheniformisprotoplasts by mild trypsin treatment. Suchtreatment causes very little damage to osmoti-

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cally supported protoplasts. They continue syn-thesizing proteins for at least 3 h, replacing thereleased membrane penicillinase before resump-tion of release (2). The location of the 20% or soof penicillinase not readily released is notknown; this enzyme is completely accessible tosubstrate. The 80% released is located on theoutside of the cytoplasmic membrane and acces-sible to trypsin digestion. The other lipoproteinsare similarly exposed to trypsin in protoplastsand resemble penicillinase in being largely butnot entirely released by trypsin.The 63-kd protein, one we have examined

more closely because we were able to isolateenough of it to prepare antibodies, shares atleast two further properties with penicillinase. Ithas a trypsin-resistant core that is 2 to 3 kdsmaller than the lipoprotein form (25, 29) and ahydrophilic exo form that appears in the mediumlate in growth. The 63-kd protein, then, is mostcertainly the membrane-bound partner of a re-leased protein. As far as we have been able toexamine the other lipoproteins of the two bacilli,they also appear to be related to the secretoryprocess. Their average size is about 35 kd,approximately the same as the average size of allprotein subunits in bacteria and considerablylarger than the mean size of E. coli lipoproteins,where the major outer membrane lipoprotein hasa mass of 7 kd and the minor lipoproteins rangeup to 52 kd (12). Those identified, including the25-kd product of the traT gene (N. B. Perumaland E. G. Minkley Jr., personal communication)are integral outer membrane proteins.The lipoprotein modification offers the gram-

positive bacterium an efficient way of retaining avariable portion of its secreted proteins in anactive releasable form. Gram-negative orga-nisms have a periplasmic space bounded by aselectively permeable outer membrane and aremuch less dependent on such a structure. Mostof their lipoproteins appear to be small structuralproteins associated mainly with the outer mem-brane, although small amounts of larger mole-cules are present (12). We propose that the setsof larger lipoproteins in B. licheniformis and B.cereus and to some extent in S. aureus are thefunctional equivalent of the free periplasmicproteins in E. coli. It will be interesting todetermine whether a gram-negative organismsuch as Pseudomonas aeruginosa, which pro-duces a variety of extracellular proteins, alsopossesses corresponding lipoproteins.

ACKNOWLEDGMENTSWe thank M. Arai for his generous gift of globomycin and J.

Sohm for expert technical assistance.This work was supported by Public Health Service grants

AI-04572 from the National Institutes of Health and grantsfrom Miles Laboratories and the Charles and Johanna BuschFund.

LITERATURE CITED1. Ambler, R. P. 1980. The structure of 1-lactamases. Phil.

Trans. R. Soc. London Ser. B 289:321-331.2. Betflnger, G. E., and J. 0. Lampen. 1975. Further evi-

dence for a partially folded intermediate in penicillinasesecretion by Bacillus licheniformis. J. Bacteriol. 121:83-90.

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