Vol. 174, No. 2

Analysis of the Autolysins of Bacillus subtilis 168 duringVegetative Growth and Differentiation by UsingRenaturing Polyacrylamide Gel Electrophoresis

SIMON J. FOSTER

Department of Molecular Biology and Biotechnology, P.O. Box 594, Firth Court,Western Bank, University of Sheffield, Sheffield, S10 2UH, United Kingdom

Received 22 July 1991/Accepted 11 November 1991

The autolysins of Bacillus subtilis 168 were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gelelectrophoresis with substrate-containing gels. Four bands of vegetative autolytic activity of 90, 50, 34, and 30kDa (bands Al to A4) were detected in SDS and LiCl extracts and in native cell walls by using B. subtilis 168vegetative cell walls as the substrate incorporated in the gel. The four enzyme activities showed differentsubstrate specificities and sensitivities to various chemical treatments. The autolysin proffle was not mediumdependent and remained constant during vegetative growth. During sporulation, band A4 greatly increased inactivity just prior to mother-cell lysis. No germination-associated changes in the profile were observed,although a soluble 41-kDa endospore-associated cortex-lytic enzyme was found. By using insertionallyinactivated mutants, bands Al and A2 were positively identified as the previously characterized 90-kDaglucosaminidase and 50-kDa amidase, respectively. The common filamentous phenotype of various regulatorymutants could not be correlated to specific changes in the autolysin profile.

Despite abundant speculation, the role of bacterial autol-ysins during cell growth and division has remained elusive.All bacteria apparently possess a complement of potentiallylethal autolysins capable of hydrolyzing the cell wall pepti-doglycan (13). Bacillus subtilis 168 has two major vegetativeautolysins, a 50-kDa amidase and a 90-kDa glucosaminidase,which have been purified and characterized (17, 31). Duringsporulation, two distinct lytic enzymes, an amidase and anendopeptidase, have been identified but not studied in detail(14). A 30-kDa amidase has also been identified, and the genehas been cloned and studied at the molecular level (9, 22).The specific function of all of these enzymes is at presentunknown, as is the total number of B. subtilis 168 autolyticenzymes. Work mainly using regulatory mutants with re-duced autolysin levels (lyt) has implicated them in severalimportant cellular functions, including cell wall turnover,cell separation, flagellation, competence, and the lytic actionof penicillin (8, 28, 30). The regulatory mutants sigD and sinshare a common filamentous and Lyt- phenotype, and thelack of cell separation has been proposed to be due toreduced expression of autolysin genes (12, 16, 25, 32). Insome lyt mutants, however, the rate of wall turnover wasdependent on the salt concentration, and the mutantsshowed levels of wall turnover comparable to that of the wildtype when they were grown in high salt concentrations (38).Because of the possible functional redundancy (9) and com-pensatory effect of multiple autolysins, it is important toidentify and characterize the total set of such enzymes

present during growth and development in order to be able toassess their involvement in any given cellular process.The ability of autolysins to renature after sodium dodecyl

sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) hasallowed their activity to be studied by using renaturing gelelectrophoresis in substrate-containing gels. This has provedvery useful in the determination of the autolytic profile ofgram-positive organisms (19, 23, 29, 35). B. subtilis ATCC6633 and Staphylococcus aureus ATCC 6538 have beenshown to have at least 7 and 13 potential autolysins, respec-

tively (23). Using renaturing gel electrophoresis, one is ableto test the substrate specificity and effect of physical andchemical treatments on autolysin activity (35). This paperdescribes the use of renaturing gel electrophoresis to studythe autolysins of B. subtilis 168 trpC2 during growth anddifferentiation, to biochemically characterize the activities,and to relate them to the previously identified autolyticenzymes of this organism.

MATERIALS AND METHODS

Bacteria and growth conditions. The strains of B. subtilis168 used in this study are shown in Table 1. Vegetative cellswere grown in Penassay broth (antibiotic medium 3; DifcoLaboratories) at 37°C unless otherwise stated. Purified,cleaned Bacillus megaterium KM and B. subtilis 168 endo-spores were prepared in CCY medium and stored as previ-ously described (34). Synchronous sporulation of B. subtilis168 in 400-ml volumes was performed by the Sterlini andMandelstam resuspension method (33). Micrococcus luteusATCC 4698 was grown at 37°C in Luria-Bertani (LB) me-dium.

Construction of a mutant insertionally inactivated in the30-kDa amidase (cwlA) gene. A 341-bp NciI-HindIII fragmentof pSFP102 internal to the cwlA gene (9) was end filled andligated into SmaI-digested phosphatased integrational vectorpAZ106 (39) to create plasmid pSFP5 and was transformed(15) into Escherichia coli DH5a. Plasmid pSFP5 was thentransformed into B. subtilis 168 (1), and colonies of B.subtilis 168 SF2 were recovered by selection on LB agarcontaining erythromycin (1 ,ug/ml) and lincomycin (25 ,ug/ml).

Preparation of purified cell wall substrates. Exponentiallygrowing (A6. = 0.6) vegetative cells or purified spores werebroken and cell walls were prepared and stored at -20°C as

previously described (20).Preparation of autolysin-containing fractions. Unless oth-

erwise stated, autolysin-containing samples were prepared

464

JOURNAL OF BACTERIOLOGY, Jan. 1992, p. 464-4700021-9193/92/020464-07$02.00/0Copyright ©3 1992, American Society for Microbiology

B. SUBTILIS AUTOLYTIC ENZYMES 465

TABLE 1. B. subtilis 168 strains

Strain Genotype Source (reference)

HR trpC2 Laboratory stockSF2a trpC2 cwlA This studyL5047 pheA3 purAi6 hisA35 trpC2 D. Karamata (24)

metB5L16001a pheA3 purAJ6 hisA35 trpC2 D. Karamata (24)

metB5 lytDL16332a pheA3 purAJ6 hisA35 trpC2 D. Karamata (24)

metB5 lytCBG-2 trpC2 M. Chamberlin (16)DP-1 trpC2 sigD M. Chamberlin (16)IS75 metB5 hisAl leuA8 I. Smith (12)IS354 metB5 hisAl IeuA8 sin I. Smith (12)168 trpC2 G. Rapoport (26)QB127 trpC2 leuA8 degS200(Hy) G. Rapoport (26)QB136 trpC2 IeuA8 degU32(Hy) G. Rapoport (26)QB254 trpC2 hisAl sacA321 degS42 G. Rapoport (26)QB256 trpC2 hisAl sacA321 degUI22 G. Rapoport (26)1A138 trpC2 argF4 hag-i smo-J BGSCb1A675 trpC2 furBi sigB BGSCIS404 trpC2 pheAl spoOAA204 I. Smith

a Mutants insertionally inactivated in the structural genes for specificautolysins.

b BGSC, Bacillus Genetic Stock Center.

from cultures at an A600 of 1.0. Cell cultures (50 ml) were

harvested by centrifugation (4,000 x g, 10 min, 4°C), andvarious fractions and extracts were prepared as follows.Culture supernatant was concentrated by trichloroaceticacid (TCA) precipitation (10% [wt/vol], 4°C, 20 min), cen-

trifugation (20,000 x g, 15°C, 5 min), and three washes in 1:1(vol/vol) ethanol-ether. The dried pellet was prepared forSDS-PAGE by resuspension in 500 ,ul of SDS-PAGE samplebuffer to give final concentrations of 1% (wt/vol) SDS, 1 mMEDTA, 10% (vol/vol) glycerol, 5% (vol/vol) ,3-mercaptoeth-anol, 0.0025% (wt/vol) bromophenol blue, and 50 mM Tris-HCl (pH 7.5); the suspension was then boiled for 3 min at100°C and centrifuged to remove insoluble material (15,000x g, 5 min), and the supernatant was stored at -20°C. SDScell extracts were prepared by resuspending the cell pelletdirectly in 500 ,ul of SDS-PAGE sample buffer and wereextracted as described above. LiCl extracts were made byresuspending the cell pellet in 400 ,ul of 4 M LiCl-0.5 mMphenylmethylsulfonyl fluoride (PMSF; Sigma)-50 mM Tris-HCl (pH 7.5); they were then incubated with constantagitation at 4°C for 30 min. The cells were removed bycentrifugation (20,000 x g, 4°C, 5 min), and the supernatantwas dialyzed overnight at 4°C against 1,000 volumes of 100mM LiCl-50 mM Tris-HCl (pH 7.5). The dialyzed extractwas then boiled in SDS-PAGE sample buffer and stored asdescribed above. Native cell walls were prepared by disrupt-ing the cells with glass beads in 0.5 mM PMSF-10 mMMgCl2-50 mM Tris-HCl (pH 7.5) at 4°C (20). After filtering toremove the beads, the walls were recovered by centrifuga-tion (20,000 x g, 5 min, 4°C) and washed once in the abovebuffer before extraction in 500 Rl of SDS-PAGE samplebuffer. Cell membranes were recovered from the disruptedcell supernatant after removal of the native cell walls bycentrifugation at 100,000 x g (4°C, 1 h) to pellet the mem-brane fraction, which was washed once in the disruptionbuffer prior to denaturation in SDS-PAGE sample buffer.Protein concentration was measured in samples by using aprotein assay kit (Bio-Rad).

Sporulating cell samples were taken at intervals during

sporulation, and autolysin-containing extracts were pre-pared as for vegetative cells. After the 8-h sample was taken,the culture was cooled slowly to 4°C by placing it in a coldroom, and the final sample was taken after static overnightincubation.

Prior to germination, cleaned, dormant spores at 5 mg/ml(wt/vol) were heat activated at 70°C for 60 min (37). Heat-shocked spore suspensions were germinated at 5 mg/ml at37°C with constant shaking in 1 mM L-alanine-100 p.g ofchloramphenicol per ml-50 mM potassium phosphate buffer(pH 7.0). The extent of germination was determined by theloss of Awo by the spore suspension. Samples (10 ml) wereremoved at intervals for autolysin analysis and immediatelydiluted into 10 ml of ice-cold 20 mM D-alanine-1 mMPMSF-50 mM Tris-HCl (pH 7.5). The spores were recov-ered by centrifugation (20,000 x g, 4°C, 5 min) and broken,and autolysin-containing samples were prepared as de-scribed above.Renaturing gel electrophoresis. Renaturing gel electropho-

resis was essentially as previously described (9). SDS-polyacrylamide gels (11% [wt/vol] acrylamide) containing0.05% (wt/vol) purified B. subtilis 168 vegetative cell walls orB. megaterium KM spore cortex were used for the detectionof lytic activity. Following electrophoresis (20 mA of con-stant current), gels (7.1 cm by 10.2 cm by 0.75 mm) weresoaked for 30 min in 250 ml of distilled water at roomtemperature with gentle agitation. The gels were then trans-ferred to 250 ml of renaturation solution (unless otherwisestated, 0.1% Triton X-100 [BDH], 10 mM MgCl2, and 25 mMTris-HCl [pH 7.5]), gently agitated for 30 min at roomtemperature, and then transferred to 250 ml of the samesolution and incubated for 16 h at 37°C. After incubation, thegels were rinsed in distilled water, stained in 0.1% methyleneblue in 0.01% KOH for 3 h, and destained in distilled water(19). Autolytic activity appeared as zones of clearing in theblue background. Molecular masses were determined bycomparison to standards (Sigma) of known sizes which wererun on the same gel and stained with Coomassie blue. Theresults shown are representative photographs of gels; allobservations were confirmed in at least two independentexperiments.The effect of incubation conditions on autolytic activity

was studied by renaturing identical samples in gel slicesincubated in different solutions, each containing 0.1% TritonX-100.

RESULTS

Vegetative autolytic enzyme profile. Following renaturationof samples and incubation in gels containing B. subtilis 168vegetative cell walls as substrate, several bands of autolyticactivity could be identified in B. subtilis 168 trpC2 vegetativecell extracts and fractions from cultures grown in Penassaybroth (Fig. 1). Four bands appeared consistently (Fig. 1,bands Al to A4, which are 90, 50, 34, and 30 kDa, respec-tively). Band A2 was generally the most prominent zone ofautolytic activity in vegetative cell extracts. Several minorbands which varied between independent experiments werealso present. Samples from SDS and LiCl cell extracts andnative cell walls prepared from equivalent amounts of cells(2 ml; A6. = 1.0) gave an essentially similar profile in termsof the number of autolytic bands (Fig. 1, lanes 1, 2, and 3),although the LiCl extract had less overall activity. Cellmembranes and supernatant contained only autolytic bandsA2 and A4 in low amounts (Fig. 1, lanes 4 and 5). SDS cellextracts were subsequently used routinely for sample prep-

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FIG. 1. B. subtilis 168 vegetative cell autolysin profile. Variousvegetative cell extracts and fractions (2-ml original culture volume)were analyzed by renaturing SDS-PAGE (11% [wt/vol] acrylamide)as described in Materials and Methods. The gel contained 0.05%(wt/vol) B. subtilis 168 vegetative cell walls as the substrate. Themolecular masses (in kilodaltons) of standards separated on thesame gel are indicated on the right. Autolytic bands are identified onthe left. Lanes: 1, SDS cell extract; 2, LiCl cell extract; 3, native cellwalls; 4, cell membranes; 5, culture supernatant; 6, cell cytoplasm.

aration. The absence of P-mercaptoethanol in the SDS-PAGE sample buffer did not change the profile. Samplesfrom cells grown to an A600 of 1.0 in minimal salts medium(1) supplemented with 0.5% glucose, 0.3% sodium gluta-mate, and 25 jig of tryptophan per ml, in LB medium, or innutrient broth (Oxoid) also gave the same pattern. If SDScell extract samples containing equal amounts of proteinfrom cells harvested at different stages during exponentialcell growth and stationary phase were analyzed by renatur-ing SDS-PAGE, no changes in the profile were detected. Anidentical autolysin pattern was observed with either M.luteus ATCC 4698 or B. subtilis 168 vegetative cell walls asthe gel substrate.

Effect of incubation conditions on autolytic enzyme activity.The effect of various chemical treatments on autolytic en-

zyme activity in SDS vegetative cell extracts was studied byincubating gel slices in different solutions. The effects onbands Al to A4 are shown in Table 2. All four autolyticbands showed different characteristics. Treatment with 10mM CuCl2 or 1 mM HgCl2 completely inhibited all autolyticactivity. Band A2 was activated by Ca2+ and Mg2+ ions andwas greatly enhanced by incubation in Tris-HCl at pH 8.8 or

higher. Band A3, conversely, was inhibited by Tris-HCl atpH 8.8. Inclusion of penicillin G or EDTA in the renaturingbuffer had no effect on enzyme activity as measured by thistechnique.

Analysis of autolysin profile during differentiation. Sporu-lation and germination-associated changes in the autolysinprofile were investigated by renaturing SDS-PAGE. Sporu-lating cells were prepared by the Sterlini-Mandelstam resus-pension method (33). Both SDS cell extracts and TCAprecipitates of the culture supernatants were made at thetimes indicated (Fig. 2). By 6 h after the induction ofsporulation (T = 6), the mother cells could be seen to containphase bright spores (by phase-contrast microscopy), but<1% free spores were visible. Lysis of the mother cell wasessentially complete by T = 8 (>95% spores released). Theautolytic enzyme profile of sporulating cells from 2 ml ofculture is shown in Fig. 2. Figure 2A shows sporulating-cellautolysin profiles of SDS extracts obtained by using vegeta-

TABLE 2. Effect of incubation conditions on autolytic activity

Autolysin activityaChemical treatment

Al A2 A3 A4

No additionb +- +- +- +-NaCl (100 mM)b +- +- +- +-KC1(100mM)b +- +- +- +-LiCl (200 mM)b +- - +- +-CaCl2 (10mM)b +- + +- +-MgCl2 (10 mM)b +- + +- +-ZnC12 (10 mM)b +- + .CuCl2 (10 mM)bHgCl2 (1 mM)bEDTA (10mM)b +- +- +- +-Penicillin G (1 mg/ml)b + - +- +- + -Sodium acetate (pH 5.2, 25 mM)c +- +- +- +-Potassium phosphate (pH 6.8, 25 mM)C + - +- +- + -Tris-HCI (pH 8.8, 25 mM)C +- ++ - +-Tris-HCl (pH 9.5, 25 mM)' +- ++ - +-

a -, completely inhibited; -, some inhibition; +-, no effect; +, somestimulation; ++, greatly stimulated.

b Basic buffer contained 0.1% (vol/vol) Triton X-100 and 25 mM Tris-HCl(pH 7.5).

c Buffer of indicated pH contained 0.1% (vol/vol) Triton X-100 and 10 mMMgCI2.

tive cell walls as substrate. The profile remains essentiallyconstant until T = 6 (Fig. 2A, lane 7), when band A4 showsa great increase in activity. Band A2 also shows someincrease, but to a much lesser extent. The only activity in theculture supernatants which hydrolyzes vegetative cell wallsis a small amount of A4 (Fig. 2B, lanes 1 to 7) until T = 8,when, concomitant with mother-cell lysis, a large burst ofA4and a lesser amount of A2 activity appear in the supernatant(Fig. 2B, lane 8). The substrate specificity of the sporulation-associated autolytic enzyme activities was studied by usinggels containing purified B. megaterium KM spore cortex asthe substrate (Fig. 2C). Only bands A3 and A4 showed sporecortex lytic activity, which they both hydrolyzed to a greaterextent than vegetative cell walls (Fig. 2A and C). Band A4activity increased greatly at T = 6 (Fig. 2C, lane 7). A newlower-molecular-mass band (A5) of 23 kDa appeared at T =5, only to disappear after T = 6 (Fig. 2C, lanes 6 and 7). A5was also present when vegetative cell walls were used as thesubstrate (Fig. 2A, lanes 6 and 7). Band A2 showed nocortex-lytic activity. If sporulating cells were disrupted withglass beads and the soluble and insoluble cell fractions wereanalyzed by renaturing SDS-PAGE, the autolysin profileobtained by using both vegetative cell walls and spore cortexas substrates did not show any bands anomalous in compar-ison with those in Fig. 2 (results not shown). Thus, there areno detectable forespore-associated autolysins which are notpresent in the mother-cell SDS extracts.

Germination-associated changes in autolysins obtained byusing spore cortex and vegetative cell walls as substrates areshown in Fig. 3. Purified spores were germinated by usingL-alanine as the germinant. The loss of A600 by the sporesuspension and the times at which samples were taken forautolysin analysis are shown in Fig. 3A. No significantchanges can be seen to occur in the autolysin profile duringgermination with either substrate (Fig. 3B and C). However,in the soluble fraction from cleaned, disrupted spores, a41-kDa autolysin which specifically hydrolyzes spore cortexcan be seen (Fig. 3C, lanes 5 to 8). This activity (A6) ismasked in sporulating cells by the high levels of A4 (Fig.2C). Small amounts of A4 activity in the germination exudate

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FIG. 2. Analysis of sporulation-associated autolysins of B. sub-tilis 168. Samples were taken during sporulation and analyzed byrenaturing SDS-PAGE as described in Materials and Methods. (Aand C) SDS cell extracts; (B) TCA precipitate of culture superna-tants. Gels contained either B. subtilis 168 vegetative cell walls (Aand B) or B. megaterium KM spore cortex (C) as the enzymesubstrate. Molecular masses of standards (in kilodaltons) are shownon the right, and autolytic bands are indicated on the left. Sampleswere taken at various times after the induction of sporulation.Lanes: 1, T = 0 h; 2, T = 1 h; 3, T = 2 h; 4, T = 3 h; 5, T = 4 h; 6,T= 5h;7, T= 6h;8, T= 8h;9, T= 22h.

could be identified 20 min after the addition of L-alanine(results not shown).

Identification of autolysin bands by the use of specificautolysin mutants. Bands Al, A2, and A4 have molecularmasses which correspond to those of the three extensivelycharacterized autolysins of B. subtilis 168 (90-kDa glu-cosaminidase, 50-kDa amidase, and 30-kDa amidase) (9, 17,31). The autolysin activity profiles of mutants specificallyinactivated in each of the structural genes encoding theseenzymes were examined (Fig. 4). The profiles of B. subtilis

C

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FIG. 3. Analysis of germination-associated autolysins. Sampleswere prepared from 10 ml (5 mg/ml [wt/vol]) of germinating sporesuspension, as described in Materials and Methods, and analyzed byrenaturing SDS-PAGE with either B. subtilis 168 vegetative cellwalls (B) or B. megaterium KM spore cortex (C) as the substrate.The loss ofAwo by the germinating spore population was measured,and the autolysin samples were taken 0, 20, 50, and 90 min after theaddition of L-alanine (indicated by arrows in panel A). Lanes: 1 to 4,insoluble material after centrifugation (20,000 x g, 4°C) of disruptedspores; 5 to 8, soluble material from 2.5 mg of disrupted spores.Lanes 1 and 5, 2 and 6, 3 and 7, and 4 and 8 are samples taken at 0,20, 50, and 90 min, respectively, after the initiation of germination.Molecular masses of standards (in kilodaltons) are indicated, as arethe autolysin bands.

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FIG. 4. Analysis of mutants specifically inactivated in autolysingenes. SDS vegetative cell extracts (2-ml culture; A6w = 1.0) wereprepared and analyzed as in Fig. 1. (A) Lane 1, L5047 (wild-typeparent); lane 2, L16332 (50-kDa amidase mutant); lane 3, L16001(90-kDa glucosaminidase mutant). (B) Lane 1, SF2 (30-kDa amidasemutant); lane 2, E. coli DH5a (pSFP102) (cloned and expressed30-kDa amidase; 0.1-ml culture). Molecular masses (in kilodaltons)of the standards are indicated, as are the autolysin bands.

168 L16001 (90-kDa glucosaminidase mutant [24]) andL16332 (50-kDa amidase mutant [24]) lack bands Al and A2,respectively (Fig. 4A, lanes 2 and 3), whereas the parentfrom which these mutants were made (B. subtilis 168 L5047)shows an identical profile in terms of numbers of autolysinsto B. subtilis 168 trpC2 (Fig. 1 and Fig. 4A, lane 1). Figure4B, lane 1, shows the profile of B. subtilis 168 SF2, whichhas been insertionally inactivated in the gene for the 30-kDaamidase. This mutant has a profile identical to that of theparent strain (B. subtilis 168 trpC2; Fig. 1, lane 1); band A4is therefore not the previously studied 30-kDa amidase. AnSDS cell extract (A6. = 0.6) of E. coli DH5a containingplasmid pSFP102 and grown in LB medium containing 50 ,ugof ampicillin per ml (9) shows the size of the protein whenexpressed in E. coli (Fig. 4B, lane 2) and confirms itsrenaturability under these conditions.

Effect of regulatory mutations on autolysin profile. A selec-tion of regulatory mutants was analyzed for changes inautolysin profile. Of all of the mutants tested, only DP-1(sigD) and IS354 (sin) showed any significant changes inautolysin profile compared with those of their parent strains(Fig. 5). DP-1 had no Al and reduced levels ofA2 to A4 (Fig.5A, lane 2). In both BG-2 (parent strain) and DP-1, band A3appears as a doublet (Fig. 5, lanes 1 and 2). In this experi-ment, the levels ofAl in both strains IS75 (parent) and IS354(sin) were too low to be detected (Fig. 5, lanes 1 and 2).IS354 shows greatly reduced bands A2 and A3 (doublet) anda partial reduction in A4 (Fig. 5, lane 2). Strains QB127[degS200(Hy)], QB136 [degU32(Hy)], QB254 (degS42),QB256 (degUJ22), 1A138 (smo-i), lA675 (sigB) and IS404(spoOAA204) all had autolysin profiles essentially identical tothat of the wild-type strain (results not shown).

DISCUSSION

Using renaturing SDS-PAGE, I have identified and char-acterized the SDS-stable autolysins of B. subtilis 168 trpC2.Vegetative cell extracts show four reproducible autolyticbands, a relatively simple profile compared with that of S.

FIG. 5. Effect of regulatory mutations on autolysin profile. SDSvegetative cell extracts (2-ml culture; A600 = 1.0) were prepared andanalyzed as in Fig. 1. (A) Lane 1, BG-2 (wild type); lane 2, DP-1(sigD). (B) Lane 1, IS75 (wild type); lane 2, IS354 (sin). Molecularmasses (in kilodaltons) of standards are indicated, as are theautolysin bands; A3 refers to the doublet.

aureus (35). It is unlikely that this method will be able toidentify all B. subtilis 168 autolysins because of the possibleheat and SDS sensitivity and stringent substrate specificityof some enzymes. Also, any protein comprising nonidenticalsubunits will not be able to renature. The number of bandsfound in each profile may not reflect the absolute total ofdistinct autolysins present, since different proteolyticallyprocessed forms of the same enzyme may be visualized.Proteolytic processing of autolytic enzymes has been shownto occur as a means of regulation by activation from latentprecursors (11, 21). Also, cloned and expressed gram-posi-tive autolysins in E. coli have more than one active form (9,23, 29). The minor variable bands of autolytic activitypresent in the extracts may be due to processing, but bandsAl to A4 did not change in activity after incubation of thesamples at 37°C. Also, bands A3 and A4 could not beprocessed forms of Al and A2 because mutants specificallyinactivated in Al or A2 did not show any changes in theselower-molecular-mass forms. A3 could in fact be a doublet,as seen in strains BG-2 and DP-1 (Fig. 5A); however,because of the high levels of A3 in strain HR, the two bandsappear as one (Fig. 1). Since bands Al to A4 showeddifferent sensitivities to various chemical treatments, it islikely that they represent distinct autolysins. All the en-zymes were inhibited by Hg2+ ions, to which many autol-ysins are sensitive (10, 27). Band A2 showed an increase inactivity in the presence of Mg2+ ions, especially at high pH,which is consistent with A2 being the 50-kDa amidasepreviously purified and characterized in detail (17, 31). Ifresidual autolysin-cell wall peptidoglycan complexes are stillpresent after denaturation, they would be expected eithernot to enter the gel or to appear as a smear of retardedenzyme without distinct bands and thus not to affect theresults found in this work.

In contrast to a previous study (2), the growth medium didnot affect the relative amounts of autolysin present. Also,there was no specific increase in the amount of autolysin asthe cells entered stationary phase, merely an increase in thetotal autolysin activity in the culture, compared with that ofexponentially growing cells, due to an increase in total cellmass.

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B. SUBTILIS AUTOLYTIC ENZYMES 469

Differentiation of B. subtilis 168 has been intensivelystudied at the genetic level, and many differentially ex-pressed genes have been identified (5). During sporulation, a

thick peptidoglycan layer known as the spore cortex, with a

chemical structure distinct from that of vegetative cell pep-tidoglycan, is laid down (36). The cortex is apparentlyresponsible for the maintenance of spore dormancy (7). Thelast characteristic morphological event to occur duringsporulation is the lysis of the mother cell, which allowsrelease of the mature spore (5). At the onset of germination,the cortex is selectively hydrolyzed, leaving a thin layer ofvegetative cell peptidoglycan which forms the basis of thenew vegetative cell wall (4, 10). Such dramatic changes inwall structure must require the action of autolysins, somepresumably specific to these processes. On the basis of theirsubstrate specificities, three sporulation-associated autolyticactivities of Bacillus cereus T have been identified as spo-rangial, spore surface, and spore core-associated lytic en-

zymes (2). The spore surface enzyme has been purified,although its specific function is unknown (3). A germination-specific cortex-lytic enzyme which is apparently responsiblefor the hydrolysis of the spore cortex during the germinationresponse has been purified from spores of B. megateriumKM (10, 11). It has previously proved difficult to solubilizethe autolysins from spores of B. subtilis (2), although twosporulation-specific lytic activities have been identified byusing synthetic substrates (14). Band A4 shows a massiveincrease in activity just prior to mother-cell lysis and aftercortex synthesis has occurred. Only after mother-cell lysis isA4 present in any appreciable quantity in the culture super-natant. Thus, A4 may be responsible for mother-cell lysisand release of the mature endospore, the final landmarkevent to occur during sporulation. The significance of theability of A3 and A4 to hydrolyze spore cortex is unknown,but it may relate to a role in cortex maturation or germina-tion. The 41-kDa minor autolysin (A6), which is only able tohydrolyze spore cortex, may be equivalent to the sporesurface-associated lytic enzyme of B. cereus T (3). Nosignificant activation of any autolysin occurred during ger-mination as measured by this technique. The germination-specific lytic enzyme of B. megaterium KM is heat sensitiveand has a very stringent substrate specificity (10); therefore,if such an enzyme has a role during the germination of B.subtilis 168, it is unlikely that it would be identified by thismethod.The use of insertionally inactivated mutants confirms that

Al and A2 are the previously characterized 90-kDa glu-cosaminidase and 50-kDa amidase, respectively (17, 31)(Fig. 4). Band A4 was not lost in a mutant lacking the 30-kDaamidase, although it has the same apparent molecular weightas the cloned gene product (9) (Fig. 4). B. subtilis SF2(30-kDa amidase mutant) shows normal sporulation in Ster-lini-Mandelstam resuspension medium, including the burstofA4 at T = 6 (results not shown). Thus, the 30-kDa amidasemay be expressed at levels too low to be detected by thismethod, may be masked by A4 of the same molecularweight, or may not be present under the growth conditionsused in this study.

Several independent regulatory mutations have beenshown to have reduced autolysin levels and to give rise to a

common filamentous phenotype (16, 32). The autolysinprofile of various mutants was examined to determinewhether there was any correlation between filamentationand lack of specific autolysins. Although both the sigD andsin mutants had reduced levels of autolysins in their profiles,the absence of one or more bands could not be associated

with filamentation. Also, none of the mutants specificallyinactivated in the 30-kDa amidase, 50-kDa amidase, or90-kDa glucosaminidase of B. subtilis are filamentous (9, 24).Thus, cell separation after septation involves componentsnot identified by this technique. The minor sigma factor (aD)may be responsible for the transcription of the 90-kDaglucosaminidase, as this enzyme is missing from the sigDmutant. Both the Sin and aD proteins may be involved in thetranscriptional regulation of the other autolysins of B. sub-tilis; however, these effects are subtle and may be incombination with other regulators. Regulatory mutationsinvolved in degradative enzyme synthesis (degU and degS),sporulation (spoOA), and a minor sigma factor (sigB) all hadno noticeable effect on the autolysin profile.The physiological role of the many autolysins purified and

studied biochemically remains a mystery (6, 18). If one is tounderstand the function of the autolysin complement of anyorganism, the total range of activities must first be deter-mined. Renaturing SDS-PAGE provides a convenient assayfor specific autolysins and thus is a useful tool in theiranalysis. I have identified at least four vegetative cell-associated autolytic activities (Al to A4), two of which are

novel (A3 and A4). During sporulation, two distinct new

autolysins (A5 and A6) appear and one of the vegetative cellactivities (A4) greatly increases. The roles of A3 and A4during vegetative growth and the possible involvement of A4in mother-cell lysis are currently under investigation.

ACKNOWLEDGMENTS

This work was carried out during the tenure of the J. G. GravesMedical Research Fellowship and was also supported by a grantfrom the University of Sheffield Medical Research Fund.

I am very grateful to Dimitri Karamata for providing results andstrains before publication. I also thank Georges Rapoport, IsaarSmith, and Mike Chamberlin for strains and Anne Moir for the giftof pAZ106 and for critical reading of the manuscript.

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