- Email: [email protected]
Autolysins during sporulation of Bacillus subtilis 168
FEMS Microbiology Letters 157 (1997) 141^147
Autolysins during sporulation of
Bacillus subtilis
168
Thomas J. Smith 1 , Simon J. Foster * Department of Molecular Biology and Biotechnology, University of She¤eld, Firth Court, Western Bank, She¤eld S10 2TN, UK
Received 22 September 1997; accepted 6 October 1997
Abstract
Conditions for zymographic detection of a 41-kDa spore cortex hydrolysis-specific autolysin, A6, from Bacillus subtilis 168 were optimised. A6 was present during sporulation from stages II^IV and remained active in the dormant spore. Its expression was controlled by the mother cell-specific early-sporulation sigma factor cE . The characteristic muramic acid N-lactam of spore cortical peptidoglycan was not necessary for cortex hydrolysis by A6, but it may be important in the inability of the major vegetative autolysin LytC to digest wild-type cortex. Two other minor autolysins were also observed during sporulation. The possible physiological significance of these observations is discussed. Keywords : Bacillus subtilis
; Autolysin; Sporulation; Renaturing SDS-PAGE; Sigma E
1. Introduction
Autolysins, bacterial enzymes that digest cell wall peptidoglycan, are believed to be essential for eubacterial growth and di¡erentiation. In Bacillus subtilis, it has been suggested that autolysins have a wide range of functions in cell wall metabolism during vegetative growth and sporulation [1,2]. During the di¡erentiation process of sporulation, it has been proposed that autolysins are involved in asymmetric septum digestion to permit prespore engulfment, cortex maturation, mother cell lysis and cortex hydrolysis during germination [3]. Mother
* Corresponding author. Tel.: +44 (114) 222 4411; Fax: +44 (114) 272 8697; E-mail: [email protected] 1 Present address: Department of Biological Sciences, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, UK.
cell lysis, which causes the mature spore to be released into the environment, has been shown to involve two autolysins, CwlC, a 30-kDa mother cell wall-speci¢c amidase, and LytC, the major amidase expressed during vegetative growth, which remains active until the end of sporulation. CwlC and LytC have mutually compensatory roles in mother cell lysis, and so the mutant phenotype, in which the mother cells do not lyse, is seen only in a cwlC lytC double mutant [4]. The spore cortex is a specialised layer of peptidoglycan with a unique structure, which surrounds the spore cytoplasm and is essential for maintenance of spore dormancy and heat resistance [5^7]. It has been suggested that selective, limited peptidoglycan hydrolysis is required for synthesis and maturation of the cortex. The putative sporulation-specific autolysin gene cwlD [8] has a role in formation of the characteristic muramic acid N-lactam moiety since this structure is absent from the cortex of a cwlD mutant [5,9]. Other possible roles for autolysins
0378-1097 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 4 6 7 - 9
FEMSLE 7895 20-11-97
142
T.J. Smith, S.J. Foster / FEMS Microbiology Letters 157 (1997) 141^147
during cortex maturation exist, for example, selective breaking of peptide crosslinks, perhaps to allow expansion of the cortex along the radial axis to maintain pressure on the spore cytoplasm and prevent its taking up water [7,10]. By using renaturing SDS-PAGE, we previously detected a 41-kDa enzyme associated with mature spores, which we named A6. A6 digested cortical peptidoglycan but not vegetative cell walls [11]. In this paper, we report optimisation of the conditions for zymographic detection of A6, which have enabled more detailed study of this enzyme and have led to observations that are consistent with A6 having a role in cortex synthesis or maturation. Two other minor sporulation-associated autolysins were also observed. 2. Materials and methods
2.1. Bacterial strains and growth conditions
Strains of B. subtilis 168 used in this study are shown in Table 1. Vegetative cells of B. subtilis were grown in nutrient broth. Micrococcus luteus ATCC 4698 was grown in LB broth. Puri¢ed spores of B. subtilis and Bacillus megaterium KM were prepared from cultures grown in CCY medium as described previously [5]. Sporulation of B. subtilis was achieved by the resuspension method [15]. Where appropriate, chromosomal resistance markers in B. subtilis were selected with chloramphenicol (3 Wg ml31 ), erythromycin (1 Wg ml31 ) and lincomycin (25 Wg ml31 ). All bacterial cultures were grown at 37³C. Table 1 Strains of B. subtilis 168 used in this study Strain Genotype Source or constructiona HR trpC2 Laboratory stock SH103 trpC2 cwlC: :cat [4] 1.5 trpC2 spoIIAC1 (sigF) [12] SH140 trpC2 sigE: :erm EU8701 [13]CHR SH170 trpC2 sigK: :cat 618 [14]CHR SH171 trpC2 sigG: :cat PS1129 (P. Setlow)CHR AA107 trpC2 cwlD : :cat [5] a C indicates construction by transformation of chromosomal DNA into HR.
2.2. Puri¢cation of bacterial cell wall substrates
Cell wall substrates to detect peptidoglycan hydrolase activity in renaturing SDS-PAGE gels were puri¢ed from vegetative cells of B. subtilis and M. luteus and from spores of B. megaterium and B. subtilis as described previously [11]. 2.3. Preparation of autolysin-containing extracts for SDS-PAGE
Autolysin-containing extracts were made from sporulating cultures of B. subtilis by breakage with glass beads, as follows, to ensure maximal release of cellular protein by breakage of the highly resistant cortical peptidoglycan. The sporulating cells (up to 50 mg dry weight) were harvested by centrifugation (14 000Ug, room temperature, 10 min), washed with 50 mM Tris-HCl (pH 7.5) and resuspended in 0.9 ml of the same bu¡er containing 0.5 mM phenylmethylsulfonyl £uoride (PMSF). The suspension was added to 4.2 g of ice-cold glass beads (diameter 0.1 mm and ¢ner; Sigma) in a glass test tube (12 cmU1 cm) and the mother cells and forespores were broken by vortex mixing (6U30 s, alternated with periods of 30 s on ice). The lysate was ¢ltered through a sintered glass ¢lter to remove the beads, and the beads were washed with about 7 ml of ice-cold 50 mM Tris-HCl (pH 7.5). Proteins from the combined ¢ltrate and washings were precipitated with trichloroacetic acid (TCA). TCA precipitation of proteins was performed as follows. TCA (to 10%, w/v) was added to the sample, which was incubated for 20 min on ice. The particulate material was pelleted by centrifugation (27 000Ug, 4³C, 15 min) and washed three times with 1 ml of ice-cold acetone by centrifugation (14 000Ug, 4³C, 5 min) and resuspension. The pellet was resuspended in SDS sample bu¡er (giving ¢nal concentrations of 62.5 mM Tris-HCl (pH 6.8) 1 mM EDTA, 1% (w/v) SDS, 5% (v/v) L-mercaptoethanol, 0.0025% (w/v) bromophenol blue, 10% (v/v) glycerol) and heated at 100³C for 5 min. The suspension was centrifuged (14000Ug, room temperature, 5 min) and the supernatant, which contained total protein from the original sample, was removed and stored at 320³C.
FEMSLE 7895 20-11-97
T.J. Smith, S.J. Foster / FEMS Microbiology Letters 157 (1997) 141^147
2.4. SDS-PAGE and renaturing SDS-PAGE
Protein samples were analysed by SDS-PAGE [16]; all gels contained 11% (w/v) acrylamide. Autolysin activity was detected by renaturing gel electrophoresis, using substrate-containing gels as described by Foster [11]. The molecular masses of peptidoglycan hydrolases were estimated by comparison with standards (Dalton Mark VII-L; Sigma), which were run on the same gel and stained with Coomassie blue. During the work reported in this paper, a range of postelectrophoresis renaturation bu¡er conditions was used, so as to optimise the conditions for renaturation and activity of the 41-kDa sporulation autolysin A6. 2.5. Endospore fractionation
All operations during the handling of the spores and spore fractions were performed at 0^4³C to minimise protein and peptidoglycan degradation. B. subtilis SH103 (cwlC) spores (500 mg, dry weight) were resuspended to a ¢nal volume of 7.0 ml in 50 mM Tris-HCl (pH7.5) containing PMSF (0.5 mM). The spore suspension was added to 50 g of ice-cold glass beads (diameter 0.1 mm and ¢ner; Sigma) in a Braun homogeniser bottle, and the spores were broken by six 30 s bursts of rapid shaking in the Braun homogeniser, alternated with periods of 30 s on ice. The sample was cooled by occasional spraying with liquid CO2 . Phase contrast microscopy con¢rmed v99% spore breakage. The beads were washed with ice-cold 50 mM Tris-HCl (pH 7.5) in a sintered glass funnel, and the washings (30 ml), containing the broken spores, were centrifuged (30000Ug, 15 min). The pellet was washed by resuspension in 50 mM Tris-HCl pH 7.5 and then extracted (100³C, 5 min) with SDS sample bu¡er, to give fraction S1 (SDS extract of spore integuments). The supernatant fraction was centrifuged again (30000Ug, 15 min) to remove remaining particulate material, and then the membrane fragments were sedimented by centrifugation (105 000Ug, 2 h). The membrane pellet was washed with 50 mM Tris-HCl (pH 7.5) and centrifuged again under the same conditions. The washed pellet was resuspended in 0.4 ml of 50 mM Tris-HCl (pH 7.5) to give fraction S2 (spore membranes). The supernatant from the ¢rst ultracentrifugation was
143
centrifuged again under the same conditions to remove remaining membranous material. The protein from the ¢nal supernatant was precipitated with TCA (Section 2.3). This gave fraction S3 (spore cytoplasm). Separate aliquots of spore membrane suspension S2 (0.1 ml, each corresponding to 125 mg dry weight of spores) were added to 5 ml of 4 M LiCl, 50 mM Tris-HCl (pH 7.5) and 5 ml of 1% (v/v) Triton X100, 100 mM NaCl, 2 mM EDTA, 50 mM Tris-HCl (pH 7.5). The suspensions were mixed gently at 4³C for 1 h; then membranes were pelleted by centrifugation (105 000Ug, 2 h) and the supernatants were removed and centrifuged again under the same conditions. The LiCl extract was dialysed overnight against several changes of 50 mM Tris-HCl (pH 7.5) and the dialysed supernatant was centrifuged (34 000Ug, twice for 10 min each) to remove any precipitate and then concentrated to 50 Wl with a Centricon 10 centrifugal concentrator (Amicon), to give fraction S4 (LiCl extract of spore membranes). The proteins from the Triton X-100 extract were precipitated with TCA (Section 2.3), yielding fraction S5 (Triton X-100 extract of spore membranes). Another sample of spore membranes was similarly prepared and extracted with Triton X-100-containing bu¡er. 2.8 ml of the Triton extract, corresponding to material from 160 mg dry weight of SH103 spores, was made 10 mM with MgCl2 and inverted at 4³C for 1 h with 2 mg dry weight of puri¢ed B. megaterium spore cortex. The cortex was then pelleted by centrifugation (14 000Ug, 10 min) and washed twice with 1 ml of 1% (v/v) Triton X-100, 100 mM NaCl, 10 mM MgCl2 , 50 mM Tris-HCl (pH 7.5). The cortex-bound proteins were removed by extraction with SDS sample bu¡er (100³C, 5 min) and analysed by renaturing SDS-PAGE. 3. Results and discussion
3.1. Optimisation of zymographic detection of the 41-kDa spore-associated autolysin A6
The bu¡er used for autolysin renaturation during renaturing SDS-PAGE was varied to optimise detection of the 41-kDa spore-associated autolysin A6. Spores of B. subtilis SH103 (cwlC) were broken in
FEMSLE 7895 20-11-97
144
T.J. Smith, S.J. Foster / FEMS Microbiology Letters 157 (1997) 141^147
Table 2 E¡ect of incubation conditions on autolytic activity as judged by renaturing SDS-PAGE Renaturation bu¡er Autolysin activitya A3 (34 kDa) A6 (41 kDa) A7 (30 kDa) b pH 5.0 3 3 3 pH 5.5b 3 ++ 3 pH 6.0b + +++ + pH 6.5b ++ ++ ++ pH 7.0b +++ + +++ pH 7.5b ++ 3 ++ 10 mM MgCl2 ; pH 6.0c 3 3 3 10 mM CaCl2 ; 0.1% (v/v) Triton X-100c 3 + 3 0.1% (v/v) Triton X-100c 3 + 3 3 ++ 3 1 mM EDTA; 0.1% (v/v) Triton X-100c a Autolytic activity with B. megaterium spore cortex as substrate. 3, no activity detected; +, ++, +++, increasing amounts of activity. b Bu¡er contained 25 mM potassium phosphate, 10 mM MgCl2 , 0.1% (v/v) Triton X-100, at the pH shown. c Basic bu¡er contained 25 mM potassium phosphate (pH 6.0).
the Braun homogeniser and the soluble fraction (spore membranes plus cytoplasm) was analysed by renaturing SDS-PAGE using B. megaterium KM spore cortex as the substrate (Table 2). A6 renaturation and activity showed a sharp pH optimum around pH 6.0. A6 required the presence of the nonionic detergent Triton X-100, but not added divalent cations. It was only slightly inhibited by an excess of EDTA. The optimal conditions for A6 detection (25 mM potassium phosphate (pH 6.0), 10 mM MgCl2 , 0.1% (v/v) Triton X-100) were used in all subsequent experiments, except where stated otherwise. Two additional autolysins of about 30 kDa and 34 kDa, respectively, both with pH optima of about 7.0, were also observed (Table 2). The 30-kDa enzyme, termed A7, was distinct from the 30-kDa sporulation-speci¢c amidase encoded by the cwlC gene [17], in which the strain used in this experiment was de¢cient.
sporulation stages II^IV in these experiments. Its activity increased as sporulation continued and it remained active in the released spores. There was also a 30-kDa autolysin band that appeared late during sporulation, and a 34-kDa activity that is present between 1^2 h and 6^7.5 h after the start of sporulation (around stages II^VI in these experiments). The 30-kDa activity is probably largely accounted for by the major late-sporulation autolysin CwlC [17], which has a role in mother cell lysis [4]. The 34-kDa lytic band may be due to one of a number of autolysins comprising the 34-kDa activity A3, previously observed throughout sporulation [11].
3.2. Time-course of autolysin expression during sporulation
The timing of expression of the enzyme A6 during sporulation was investigated by taking samples at various time points during the sporulation of B. subtilis 168 HR. The sporulating cells were broken completely by vortexing with glass beads, and the total cell protein was analysed by renaturing SDS-PAGE (Fig. 1). A6 activity appeared between 2 and 4 h after the initiation of sporulation, corresponding to
Fig. 1. Time course of autolysin expression during sporulation. Samples were taken during sporulation of B. subtilis 168 HR initiated by resuspension. Extracts of whole broken sporangia were prepared and analysed by renaturing SDS-11% PAGE using B. megaterium spore cortex as substrate (Section 3.1). Lanes are labelled with the time in hours after the initiation of sporulation. Each lane contains material from 1 ml of original culture. Molecular masses of standards (in kDa) are indicated.
FEMSLE 7895 20-11-97
145
T.J. Smith, S.J. Foster / FEMS Microbiology Letters 157 (1997) 141^147
Fig. 2. E¡ect of sporulation-speci¢c regulatory mutations on autolysin expression. Sporulation was initiated by resupension. Samples of whole broken sporangia were analysed by renaturing SDS-11% PAGE using B. megaterium spore cortex as substrate (Section 3.1). Each lane contains about 30 Wg of total protein. Lanes 1, 2 and 3 are samples taken from cultures of HR (wild type), 1.5 (sigF) and SH140 (sigE), respectively, taken 4 h after the initiation of sporulation. Lanes 4, 5 and 6 are samples taken from cultures of HR, SH171 (sigG) and SH170 (sigK), respectively, taken 7.5 h after the initiation of sporulation. Molecular masses of standards (in kDa) are indicated. 3.3. Dependence of A6 expression on the sporulation sigma factors
Sporulation is controlled by a cascade of gene expression that includes four minor sigma factors, cF , cE , cG and cK , which regulate sporulation genes temporally and in a compartment-speci¢c manner [18,19]. In order to investigate the genetic control of the 41-kDa autolysin A6, resuspension cultures were prepared of the wild-type and mutants inactivated in the each of the four sporulation sigma factor genes. Samples were taken 4 h after resuspension from the early sigma factor (sigF and sigE) mutants, 7.5 h after resuspension from the late sigma factor (sigG and sigK) mutants and at both time points
from the wild-type. Renaturing SDS-PAGE (Fig. 2) showed that A6 activity was abolished by inactivation of sigF or sigE. Since activation of SigE from its inactive proform is dependent on sigF [19], these results can be most simply explained if transcription of the gene encoding the 41-kDa peptidoglycan hydrolase A6 proceeds from a single sigE-dependent promoter. SigE activity is speci¢c to the mother cell during early sporulation [18,19]. This suggests that A6 is a mother cell-speci¢c, early-sporulation enzyme. It was observed that a band of peptidoglycan hydrolase at about 30 kDa was dependent on the mother cell-speci¢c, late-sporulation sigma factor encoded by sigK (Fig. 2). This activity was probably largely due to the late-sporulation autolysin CwlC, which is sigK-controlled [17]. 3.4. Localisation of autolysins in mature endospores and further fractionation of the membrane-associated enzymes
Mature endospores of B. subtilis SH103 (cwlC) were broken in the Braun homogeniser and separated by di¡erential centrifugation into cytoplasmic, membrane and insoluble fractions (Section 2.5). The cwlC mutant strain was used in order to minimise the background of peptidoglycan hydrolase activity in the spores and so aid subsequent possible puri¢cation of minor autolysins. Renaturing SDS-PAGE (Fig. 3, lanes S1^S3) revealed that the 41- and 30kDa peptidoglycan hydrolases A6 and A7, respectively, were present at detectable levels in all three fractions, but were both located predominantly ( s 80%) in the membrane fraction.
Table 3 Substrate speci¢city of B. subtilis autolysins Substrate
Autolysin activitya 41-kDa (A6)b B. subtilis 168 HR (parental) spore cortex + B. megaterium spore cortex + B. subtilis AA107 (cwlD) spore cortex + B. subtilis 168 HR vegetative cell walls 3 M. luteus cell walls 3 a Autolysin activity detected by renaturing SDS-PAGE. +, activity detected; 3, no activity detected. b Activity in 25 mM potassium phosphate (pH 6.0), 10 mM MgCl2 , 0.1% (v/v) Triton X-100. c Activity in 25 mM Tris-HCl (pH 7.5), 10 mM MgCl2 , 0.1% (v/v) Triton X-100.
FEMSLE 7895 20-11-97
50-kDa (LytC)c 3 3 + + +
146
T.J. Smith, S.J. Foster / FEMS Microbiology Letters 157 (1997) 141^147
3.6. Conclusions
Fig. 3. Localisation of autolysins in mature endospores and extracts of endospore membranes. B. subtilis SH103 (cwlC) spores were broken and fractionated (Section 2.5). Samples, each containing material from 10 mg dry weight of spores, were analysed by renaturing SDS-11% PAGE using B. megaterium spore cortex as substrate (Section 3.1). Lanes: S1, insoluble fraction; S2, membranes; S3, cytoplasm; S4, LiCl extract of membranes; S5, Triton X-100 extract of membranes. Molecular masses of standards (in kDa) are indicated.
It was found that A6 and A7 could be partially solubilised by extraction of the spore membrane fractions with solutions containing LiCl or the nonionic detergent Triton X-100 (Fig. 3, lanes S4 and S5). The Triton-X100-solubilised enzymes both bound to puri¢ed spore cortex and were eluted with SDS-PAGE loading bu¡er (data not shown). All fractions were analysed by SDS-PAGE and renaturing SDS-PAGE, but none contained protein bands of su¤cient intensity comigrating with A6 or A7 activity to permit N-terminal sequencing. 3.5. Substrate speci¢city of peptidoglycan hydrolases present in spores
Renaturing SDS-PAGE of a soluble (membranes plus cytoplasm) extract of B. subtilis SH103 (cwlC) was performed using a variety of puri¢ed bacterial integuments as substrates (Table 3). From the ability of A6 to digest even cortex from the cwlD mutant, AA107, which lacks muramic acid N-lactam residues, it appears that recognition of cortical peptidoglycan by A6 does not depend on the N-lactam moiety. On the other hand, removal of the N-lactam may be suf¢cient to allow cortical peptidoglycan to be hydrolysed by the major vegetative autolysin LytC [20,21], which has little or no activity against wild-type cortex.
By optimising the conditions for renaturing SDSPAGE, we have been able to show that A6, the 41kDa endospore-associated autolysin of B. subtilis, is a mother cell-speci¢c enzyme that appears early during sporulation (around stages II^IV) and persists in the dormant spore. It had previously been reported that A6 hydrolysed B. megaterium spore cortex but not B. subtilis vegetative cell walls [11]. In this paper, we have shown that it also digests spore cortex from its parent organism B. subtilis. The substrate speci¢city, genetic control and timing of expression of A6 suggest that it may have a role in synthesis or maturation of the cortex, which are thought to be directed by the mother cell [2,22] from stage III of sporulation onwards [18]. A6 may therefore be involved in establishment of spore resistance and/or dormancy. The presence of A6 in the membrane fraction of broken spores may be because it is associated with fragments of the mother cellderived outer forespore membrane adhering to the mature spore. Alternatively, it could be located elsewhere in the intact spore and become redistributed upon spore breakage. Although A6 activity was clearly observed by renaturing SDS-PAGE, the protein giving rise to it eluded detection, showing that A6 is a highly active enzyme present at only a few molecules per cell. A6 may be homologous to the 43-kDa cortex hydrolysisspeci¢c hexosaminidase that was previously isolated from spores of Bacillus cereus T [23] and/or the 38kDa spore-associated muramidase SleM from Clostridium perfringens [24]. Alternatively, A6 could correspond to the endopeptidase that was observed to appear at around stage IV during sporulation of B. subtilis [25]. The mutational analysis necessary to determine the precise function of the sporulationspeci¢c autolysin A6 must await the identi¢cation of its gene from the B. subtilis genome sequencing project. Acknowledgments
We are grateful to Je¡ Errington, Charlie Moran, Junichi Sekiguchi and Peter Setlow for providing
FEMSLE 7895 20-11-97
T.J. Smith, S.J. Foster / FEMS Microbiology Letters 157 (1997) 141^147 strains. This work was funded by the BBSRC and
147
[13] Kenney, T.J. and Moran, C.P. Jr. (1987) Organisation and regulation of an operon that encodes a sporulation-essential
the Royal Society.
sigma factor in
Bacillus subtilis.
J. Bacteriol. 169, 3329^3339.
[14] Turner, S.M., Errington, J. and Mandelstam, J. (1986) Use of a
References
lacZ
gene fusion to determine the dependence pattern of
sporulation operon
spoIIIC
in
spo
mutants of
Bacillus subtilis :
a branched pathway of expression of sporulation operons. [1] Smith, T.J., Blackman, S.A. and Foster, S.J. (1996) Peptidoglycan hydrolases of
Bacillus subtilis
168. Microb. Drug Re-
J. Gen. Microbiol. 132, 2995^3003. [15] Sterlini, J.M. and Mandelstam, J. (1969) Commitment to sporulation in
sist. 2, 113^118. [2] Foster, S.J. (1994) The role and regulation of cell wall structural dynamics during di¡erentiation of endospore-forming
[3] Doi, R.H (1989) Sporulation and germination. In :
Bacillus
(Harwood, C.R., Ed.), pp. 169^215. Plenum Press, London. [4] Smith, T.J. and Foster, S.J. (1995) Characterisation of the involvement of two compensatory autolysins in mother cell
Bacillus subtilis
and its relationship to develop-
[16] Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227,
bacteria. J. Appl. Bacteriol. 76, 25S^39S.
lysis during sporulation of
Bacillus subtilis
ment of actinomycin resistance. Biochem. J. 113, 29^37.
168. J. Bacteriol.
680^685. [17] Kuroda, A., Asami, Y. and Sekiguchi, J. (1993) Molecular cloning of a sporulation-speci¢c cell wall hydrolase gene of
Bacillus subtilis.
J. Bacteriol. 175, 6260^6268.
[18] Errington, J. (1993)
Bacillus subtilis
sporulation : regulation of
gene expression and control of morphogenesis. Microbiol.
177, 3855^3862. [5] Atrih, A., Zo ë llner, P., Allmaier, G. and Foster, S.J. (1996) Structural analysis of
Bacillus subtilis
168 endospore peptido-
Rev. 57, 1^33. [19] Losick, R. and Stragier, P. (1992) Crisscross regulation of cell-
glycan and its role during di¡erentiation. J. Bacteriol. 178,
type-speci¢c gene expression during development in
6173^6183.
Nature 355, 601^604.
[6] Popham, D.L., Helin, J., Costello, C.E. and Setlow, P. (1996) Analysis of the peptidoglycan structure of
Bacillus subtilis
[20] Lazarevic, V., Margot, P., Soldo, B. and Karamata, D. (1992) Sequencing and analysis of the
[7] Warth, A.D. (1978) Molecular structure of the bacterial spore.
di-
the
N-acetyl
muramoyl-L-alanine amidase and its modi¢er.
J. Gen. Microbiol. 138, 1949^1961.
Adv. Microbiol. Physiol. 17, 1^47. [8] Sekiguchi, J., Akeo, K., Yamamoto, H., Khasnov, F.K.,
[21] Kuroda, A. and Sekiguchi, J. (1991) Cloning and sequencing
Alonso, J.C. and Kuroda, A. (1995) Nucleotide sequence
of a major
and regulation of a new putative cell wall hydrolase gene,
7304^7312.
which a¡ects germination in
Bacillus subtilis lytRABC
vergon : a regulatory unit encompassing the structural genes of
endospores. J. Bacteriol. 178, 6451^6458.
cwlD,
B. subtilis.
Bacillus subtilis.
J. Bacter-
Bacillus subtilis
autolysin gene. J. Bacteriol. 173,
[22] Tipper, D.J. and Linnett, P.E. (1976) Distribution of peptidoglycan synthetase activities between sporangia and forespores
iol. 177, 5582^5589. [9] Popham, D.L., Helin, J., Costello, C.E. and Setlow, P. (1996) Muramic lactam in peptidoglycan of
Bacillus subtilis
spores is
in sporulating cells of
Bacillus sphaericus.
J. Bacteriol. 126,
213^221.
required for spore outgrowth but not for spore dehydration or
[23] Brown, W.C., Vellom, D., Schnepf, E. and Greer, C. (1978)
heat resistance. Proc. Natl. Acad. Sci. USA 93, 15405^15410.
Puri¢cation of a surface-bound hexosaminidase from spores
[10] Alderton, G. and Snell, N. (1963) Base exchange and heat resistance in bacterial spores. Biochem. Biophys. Res. Com-
Bacillus sub-
168 during vegetative growth and di¡erentiation by using
renaturing gel electrophoresis. J. Bacteriol. 174, 464^470. [12] Errington, J. and Mandelstam, J. (1986) Use of a
lacZ
gene
fusion to determine the dependence pattern of sporulation operon
spoIIA
in
Bacillus cereus
T. FEMS Microbiol. Lett. 3, 247^251.
Molecular characterisation of a germination-speci¢c murami-
mun. 10, 139^143. [11] Foster, S.J. (1992) Analysis of the autolysins of
tilis
of
[24] Chen, Y., Miyata, S., Makino, S. and Moriyama, R. (1997)
spo
mutants of
Bacillus subtilis.
J. Gen.
dase from
Clostridium perfringens
S40 spores and nucleotide
sequence of the corresponding gene. J. Bacteriol. 179, 3181^ 3187. [25] Guinand, M., Michel, G. and Balassa, G. (1976) Lytic enzymes in sporulating
Bacillus subtilis.
Commun. 68, 1287^1293.
Microbiol. 132, 2967^2976.
FEMSLE 7895 20-11-97
Biochem. Biophys Res.
Autolysins during sporulation of
Bacillus subtilis
168
Thomas J. Smith 1 , Simon J. Foster * Department of Molecular Biology and Biotechnology, University of She¤eld, Firth Court, Western Bank, She¤eld S10 2TN, UK
Received 22 September 1997; accepted 6 October 1997
Abstract
Conditions for zymographic detection of a 41-kDa spore cortex hydrolysis-specific autolysin, A6, from Bacillus subtilis 168 were optimised. A6 was present during sporulation from stages II^IV and remained active in the dormant spore. Its expression was controlled by the mother cell-specific early-sporulation sigma factor cE . The characteristic muramic acid N-lactam of spore cortical peptidoglycan was not necessary for cortex hydrolysis by A6, but it may be important in the inability of the major vegetative autolysin LytC to digest wild-type cortex. Two other minor autolysins were also observed during sporulation. The possible physiological significance of these observations is discussed. Keywords : Bacillus subtilis
; Autolysin; Sporulation; Renaturing SDS-PAGE; Sigma E
1. Introduction
Autolysins, bacterial enzymes that digest cell wall peptidoglycan, are believed to be essential for eubacterial growth and di¡erentiation. In Bacillus subtilis, it has been suggested that autolysins have a wide range of functions in cell wall metabolism during vegetative growth and sporulation [1,2]. During the di¡erentiation process of sporulation, it has been proposed that autolysins are involved in asymmetric septum digestion to permit prespore engulfment, cortex maturation, mother cell lysis and cortex hydrolysis during germination [3]. Mother
* Corresponding author. Tel.: +44 (114) 222 4411; Fax: +44 (114) 272 8697; E-mail: [email protected] 1 Present address: Department of Biological Sciences, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, UK.
cell lysis, which causes the mature spore to be released into the environment, has been shown to involve two autolysins, CwlC, a 30-kDa mother cell wall-speci¢c amidase, and LytC, the major amidase expressed during vegetative growth, which remains active until the end of sporulation. CwlC and LytC have mutually compensatory roles in mother cell lysis, and so the mutant phenotype, in which the mother cells do not lyse, is seen only in a cwlC lytC double mutant [4]. The spore cortex is a specialised layer of peptidoglycan with a unique structure, which surrounds the spore cytoplasm and is essential for maintenance of spore dormancy and heat resistance [5^7]. It has been suggested that selective, limited peptidoglycan hydrolysis is required for synthesis and maturation of the cortex. The putative sporulation-specific autolysin gene cwlD [8] has a role in formation of the characteristic muramic acid N-lactam moiety since this structure is absent from the cortex of a cwlD mutant [5,9]. Other possible roles for autolysins
0378-1097 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 4 6 7 - 9
FEMSLE 7895 20-11-97
142
T.J. Smith, S.J. Foster / FEMS Microbiology Letters 157 (1997) 141^147
during cortex maturation exist, for example, selective breaking of peptide crosslinks, perhaps to allow expansion of the cortex along the radial axis to maintain pressure on the spore cytoplasm and prevent its taking up water [7,10]. By using renaturing SDS-PAGE, we previously detected a 41-kDa enzyme associated with mature spores, which we named A6. A6 digested cortical peptidoglycan but not vegetative cell walls [11]. In this paper, we report optimisation of the conditions for zymographic detection of A6, which have enabled more detailed study of this enzyme and have led to observations that are consistent with A6 having a role in cortex synthesis or maturation. Two other minor sporulation-associated autolysins were also observed. 2. Materials and methods
2.1. Bacterial strains and growth conditions
Strains of B. subtilis 168 used in this study are shown in Table 1. Vegetative cells of B. subtilis were grown in nutrient broth. Micrococcus luteus ATCC 4698 was grown in LB broth. Puri¢ed spores of B. subtilis and Bacillus megaterium KM were prepared from cultures grown in CCY medium as described previously [5]. Sporulation of B. subtilis was achieved by the resuspension method [15]. Where appropriate, chromosomal resistance markers in B. subtilis were selected with chloramphenicol (3 Wg ml31 ), erythromycin (1 Wg ml31 ) and lincomycin (25 Wg ml31 ). All bacterial cultures were grown at 37³C. Table 1 Strains of B. subtilis 168 used in this study Strain Genotype Source or constructiona HR trpC2 Laboratory stock SH103 trpC2 cwlC: :cat [4] 1.5 trpC2 spoIIAC1 (sigF) [12] SH140 trpC2 sigE: :erm EU8701 [13]CHR SH170 trpC2 sigK: :cat 618 [14]CHR SH171 trpC2 sigG: :cat PS1129 (P. Setlow)CHR AA107 trpC2 cwlD : :cat [5] a C indicates construction by transformation of chromosomal DNA into HR.
2.2. Puri¢cation of bacterial cell wall substrates
Cell wall substrates to detect peptidoglycan hydrolase activity in renaturing SDS-PAGE gels were puri¢ed from vegetative cells of B. subtilis and M. luteus and from spores of B. megaterium and B. subtilis as described previously [11]. 2.3. Preparation of autolysin-containing extracts for SDS-PAGE
Autolysin-containing extracts were made from sporulating cultures of B. subtilis by breakage with glass beads, as follows, to ensure maximal release of cellular protein by breakage of the highly resistant cortical peptidoglycan. The sporulating cells (up to 50 mg dry weight) were harvested by centrifugation (14 000Ug, room temperature, 10 min), washed with 50 mM Tris-HCl (pH 7.5) and resuspended in 0.9 ml of the same bu¡er containing 0.5 mM phenylmethylsulfonyl £uoride (PMSF). The suspension was added to 4.2 g of ice-cold glass beads (diameter 0.1 mm and ¢ner; Sigma) in a glass test tube (12 cmU1 cm) and the mother cells and forespores were broken by vortex mixing (6U30 s, alternated with periods of 30 s on ice). The lysate was ¢ltered through a sintered glass ¢lter to remove the beads, and the beads were washed with about 7 ml of ice-cold 50 mM Tris-HCl (pH 7.5). Proteins from the combined ¢ltrate and washings were precipitated with trichloroacetic acid (TCA). TCA precipitation of proteins was performed as follows. TCA (to 10%, w/v) was added to the sample, which was incubated for 20 min on ice. The particulate material was pelleted by centrifugation (27 000Ug, 4³C, 15 min) and washed three times with 1 ml of ice-cold acetone by centrifugation (14 000Ug, 4³C, 5 min) and resuspension. The pellet was resuspended in SDS sample bu¡er (giving ¢nal concentrations of 62.5 mM Tris-HCl (pH 6.8) 1 mM EDTA, 1% (w/v) SDS, 5% (v/v) L-mercaptoethanol, 0.0025% (w/v) bromophenol blue, 10% (v/v) glycerol) and heated at 100³C for 5 min. The suspension was centrifuged (14000Ug, room temperature, 5 min) and the supernatant, which contained total protein from the original sample, was removed and stored at 320³C.
FEMSLE 7895 20-11-97
T.J. Smith, S.J. Foster / FEMS Microbiology Letters 157 (1997) 141^147
2.4. SDS-PAGE and renaturing SDS-PAGE
Protein samples were analysed by SDS-PAGE [16]; all gels contained 11% (w/v) acrylamide. Autolysin activity was detected by renaturing gel electrophoresis, using substrate-containing gels as described by Foster [11]. The molecular masses of peptidoglycan hydrolases were estimated by comparison with standards (Dalton Mark VII-L; Sigma), which were run on the same gel and stained with Coomassie blue. During the work reported in this paper, a range of postelectrophoresis renaturation bu¡er conditions was used, so as to optimise the conditions for renaturation and activity of the 41-kDa sporulation autolysin A6. 2.5. Endospore fractionation
All operations during the handling of the spores and spore fractions were performed at 0^4³C to minimise protein and peptidoglycan degradation. B. subtilis SH103 (cwlC) spores (500 mg, dry weight) were resuspended to a ¢nal volume of 7.0 ml in 50 mM Tris-HCl (pH7.5) containing PMSF (0.5 mM). The spore suspension was added to 50 g of ice-cold glass beads (diameter 0.1 mm and ¢ner; Sigma) in a Braun homogeniser bottle, and the spores were broken by six 30 s bursts of rapid shaking in the Braun homogeniser, alternated with periods of 30 s on ice. The sample was cooled by occasional spraying with liquid CO2 . Phase contrast microscopy con¢rmed v99% spore breakage. The beads were washed with ice-cold 50 mM Tris-HCl (pH 7.5) in a sintered glass funnel, and the washings (30 ml), containing the broken spores, were centrifuged (30000Ug, 15 min). The pellet was washed by resuspension in 50 mM Tris-HCl pH 7.5 and then extracted (100³C, 5 min) with SDS sample bu¡er, to give fraction S1 (SDS extract of spore integuments). The supernatant fraction was centrifuged again (30000Ug, 15 min) to remove remaining particulate material, and then the membrane fragments were sedimented by centrifugation (105 000Ug, 2 h). The membrane pellet was washed with 50 mM Tris-HCl (pH 7.5) and centrifuged again under the same conditions. The washed pellet was resuspended in 0.4 ml of 50 mM Tris-HCl (pH 7.5) to give fraction S2 (spore membranes). The supernatant from the ¢rst ultracentrifugation was
143
centrifuged again under the same conditions to remove remaining membranous material. The protein from the ¢nal supernatant was precipitated with TCA (Section 2.3). This gave fraction S3 (spore cytoplasm). Separate aliquots of spore membrane suspension S2 (0.1 ml, each corresponding to 125 mg dry weight of spores) were added to 5 ml of 4 M LiCl, 50 mM Tris-HCl (pH 7.5) and 5 ml of 1% (v/v) Triton X100, 100 mM NaCl, 2 mM EDTA, 50 mM Tris-HCl (pH 7.5). The suspensions were mixed gently at 4³C for 1 h; then membranes were pelleted by centrifugation (105 000Ug, 2 h) and the supernatants were removed and centrifuged again under the same conditions. The LiCl extract was dialysed overnight against several changes of 50 mM Tris-HCl (pH 7.5) and the dialysed supernatant was centrifuged (34 000Ug, twice for 10 min each) to remove any precipitate and then concentrated to 50 Wl with a Centricon 10 centrifugal concentrator (Amicon), to give fraction S4 (LiCl extract of spore membranes). The proteins from the Triton X-100 extract were precipitated with TCA (Section 2.3), yielding fraction S5 (Triton X-100 extract of spore membranes). Another sample of spore membranes was similarly prepared and extracted with Triton X-100-containing bu¡er. 2.8 ml of the Triton extract, corresponding to material from 160 mg dry weight of SH103 spores, was made 10 mM with MgCl2 and inverted at 4³C for 1 h with 2 mg dry weight of puri¢ed B. megaterium spore cortex. The cortex was then pelleted by centrifugation (14 000Ug, 10 min) and washed twice with 1 ml of 1% (v/v) Triton X-100, 100 mM NaCl, 10 mM MgCl2 , 50 mM Tris-HCl (pH 7.5). The cortex-bound proteins were removed by extraction with SDS sample bu¡er (100³C, 5 min) and analysed by renaturing SDS-PAGE. 3. Results and discussion
3.1. Optimisation of zymographic detection of the 41-kDa spore-associated autolysin A6
The bu¡er used for autolysin renaturation during renaturing SDS-PAGE was varied to optimise detection of the 41-kDa spore-associated autolysin A6. Spores of B. subtilis SH103 (cwlC) were broken in
FEMSLE 7895 20-11-97
144
T.J. Smith, S.J. Foster / FEMS Microbiology Letters 157 (1997) 141^147
Table 2 E¡ect of incubation conditions on autolytic activity as judged by renaturing SDS-PAGE Renaturation bu¡er Autolysin activitya A3 (34 kDa) A6 (41 kDa) A7 (30 kDa) b pH 5.0 3 3 3 pH 5.5b 3 ++ 3 pH 6.0b + +++ + pH 6.5b ++ ++ ++ pH 7.0b +++ + +++ pH 7.5b ++ 3 ++ 10 mM MgCl2 ; pH 6.0c 3 3 3 10 mM CaCl2 ; 0.1% (v/v) Triton X-100c 3 + 3 0.1% (v/v) Triton X-100c 3 + 3 3 ++ 3 1 mM EDTA; 0.1% (v/v) Triton X-100c a Autolytic activity with B. megaterium spore cortex as substrate. 3, no activity detected; +, ++, +++, increasing amounts of activity. b Bu¡er contained 25 mM potassium phosphate, 10 mM MgCl2 , 0.1% (v/v) Triton X-100, at the pH shown. c Basic bu¡er contained 25 mM potassium phosphate (pH 6.0).
the Braun homogeniser and the soluble fraction (spore membranes plus cytoplasm) was analysed by renaturing SDS-PAGE using B. megaterium KM spore cortex as the substrate (Table 2). A6 renaturation and activity showed a sharp pH optimum around pH 6.0. A6 required the presence of the nonionic detergent Triton X-100, but not added divalent cations. It was only slightly inhibited by an excess of EDTA. The optimal conditions for A6 detection (25 mM potassium phosphate (pH 6.0), 10 mM MgCl2 , 0.1% (v/v) Triton X-100) were used in all subsequent experiments, except where stated otherwise. Two additional autolysins of about 30 kDa and 34 kDa, respectively, both with pH optima of about 7.0, were also observed (Table 2). The 30-kDa enzyme, termed A7, was distinct from the 30-kDa sporulation-speci¢c amidase encoded by the cwlC gene [17], in which the strain used in this experiment was de¢cient.
sporulation stages II^IV in these experiments. Its activity increased as sporulation continued and it remained active in the released spores. There was also a 30-kDa autolysin band that appeared late during sporulation, and a 34-kDa activity that is present between 1^2 h and 6^7.5 h after the start of sporulation (around stages II^VI in these experiments). The 30-kDa activity is probably largely accounted for by the major late-sporulation autolysin CwlC [17], which has a role in mother cell lysis [4]. The 34-kDa lytic band may be due to one of a number of autolysins comprising the 34-kDa activity A3, previously observed throughout sporulation [11].
3.2. Time-course of autolysin expression during sporulation
The timing of expression of the enzyme A6 during sporulation was investigated by taking samples at various time points during the sporulation of B. subtilis 168 HR. The sporulating cells were broken completely by vortexing with glass beads, and the total cell protein was analysed by renaturing SDS-PAGE (Fig. 1). A6 activity appeared between 2 and 4 h after the initiation of sporulation, corresponding to
Fig. 1. Time course of autolysin expression during sporulation. Samples were taken during sporulation of B. subtilis 168 HR initiated by resuspension. Extracts of whole broken sporangia were prepared and analysed by renaturing SDS-11% PAGE using B. megaterium spore cortex as substrate (Section 3.1). Lanes are labelled with the time in hours after the initiation of sporulation. Each lane contains material from 1 ml of original culture. Molecular masses of standards (in kDa) are indicated.
FEMSLE 7895 20-11-97
145
T.J. Smith, S.J. Foster / FEMS Microbiology Letters 157 (1997) 141^147
Fig. 2. E¡ect of sporulation-speci¢c regulatory mutations on autolysin expression. Sporulation was initiated by resupension. Samples of whole broken sporangia were analysed by renaturing SDS-11% PAGE using B. megaterium spore cortex as substrate (Section 3.1). Each lane contains about 30 Wg of total protein. Lanes 1, 2 and 3 are samples taken from cultures of HR (wild type), 1.5 (sigF) and SH140 (sigE), respectively, taken 4 h after the initiation of sporulation. Lanes 4, 5 and 6 are samples taken from cultures of HR, SH171 (sigG) and SH170 (sigK), respectively, taken 7.5 h after the initiation of sporulation. Molecular masses of standards (in kDa) are indicated. 3.3. Dependence of A6 expression on the sporulation sigma factors
Sporulation is controlled by a cascade of gene expression that includes four minor sigma factors, cF , cE , cG and cK , which regulate sporulation genes temporally and in a compartment-speci¢c manner [18,19]. In order to investigate the genetic control of the 41-kDa autolysin A6, resuspension cultures were prepared of the wild-type and mutants inactivated in the each of the four sporulation sigma factor genes. Samples were taken 4 h after resuspension from the early sigma factor (sigF and sigE) mutants, 7.5 h after resuspension from the late sigma factor (sigG and sigK) mutants and at both time points
from the wild-type. Renaturing SDS-PAGE (Fig. 2) showed that A6 activity was abolished by inactivation of sigF or sigE. Since activation of SigE from its inactive proform is dependent on sigF [19], these results can be most simply explained if transcription of the gene encoding the 41-kDa peptidoglycan hydrolase A6 proceeds from a single sigE-dependent promoter. SigE activity is speci¢c to the mother cell during early sporulation [18,19]. This suggests that A6 is a mother cell-speci¢c, early-sporulation enzyme. It was observed that a band of peptidoglycan hydrolase at about 30 kDa was dependent on the mother cell-speci¢c, late-sporulation sigma factor encoded by sigK (Fig. 2). This activity was probably largely due to the late-sporulation autolysin CwlC, which is sigK-controlled [17]. 3.4. Localisation of autolysins in mature endospores and further fractionation of the membrane-associated enzymes
Mature endospores of B. subtilis SH103 (cwlC) were broken in the Braun homogeniser and separated by di¡erential centrifugation into cytoplasmic, membrane and insoluble fractions (Section 2.5). The cwlC mutant strain was used in order to minimise the background of peptidoglycan hydrolase activity in the spores and so aid subsequent possible puri¢cation of minor autolysins. Renaturing SDS-PAGE (Fig. 3, lanes S1^S3) revealed that the 41- and 30kDa peptidoglycan hydrolases A6 and A7, respectively, were present at detectable levels in all three fractions, but were both located predominantly ( s 80%) in the membrane fraction.
Table 3 Substrate speci¢city of B. subtilis autolysins Substrate
Autolysin activitya 41-kDa (A6)b B. subtilis 168 HR (parental) spore cortex + B. megaterium spore cortex + B. subtilis AA107 (cwlD) spore cortex + B. subtilis 168 HR vegetative cell walls 3 M. luteus cell walls 3 a Autolysin activity detected by renaturing SDS-PAGE. +, activity detected; 3, no activity detected. b Activity in 25 mM potassium phosphate (pH 6.0), 10 mM MgCl2 , 0.1% (v/v) Triton X-100. c Activity in 25 mM Tris-HCl (pH 7.5), 10 mM MgCl2 , 0.1% (v/v) Triton X-100.
FEMSLE 7895 20-11-97
50-kDa (LytC)c 3 3 + + +
146
T.J. Smith, S.J. Foster / FEMS Microbiology Letters 157 (1997) 141^147
3.6. Conclusions
Fig. 3. Localisation of autolysins in mature endospores and extracts of endospore membranes. B. subtilis SH103 (cwlC) spores were broken and fractionated (Section 2.5). Samples, each containing material from 10 mg dry weight of spores, were analysed by renaturing SDS-11% PAGE using B. megaterium spore cortex as substrate (Section 3.1). Lanes: S1, insoluble fraction; S2, membranes; S3, cytoplasm; S4, LiCl extract of membranes; S5, Triton X-100 extract of membranes. Molecular masses of standards (in kDa) are indicated.
It was found that A6 and A7 could be partially solubilised by extraction of the spore membrane fractions with solutions containing LiCl or the nonionic detergent Triton X-100 (Fig. 3, lanes S4 and S5). The Triton-X100-solubilised enzymes both bound to puri¢ed spore cortex and were eluted with SDS-PAGE loading bu¡er (data not shown). All fractions were analysed by SDS-PAGE and renaturing SDS-PAGE, but none contained protein bands of su¤cient intensity comigrating with A6 or A7 activity to permit N-terminal sequencing. 3.5. Substrate speci¢city of peptidoglycan hydrolases present in spores
Renaturing SDS-PAGE of a soluble (membranes plus cytoplasm) extract of B. subtilis SH103 (cwlC) was performed using a variety of puri¢ed bacterial integuments as substrates (Table 3). From the ability of A6 to digest even cortex from the cwlD mutant, AA107, which lacks muramic acid N-lactam residues, it appears that recognition of cortical peptidoglycan by A6 does not depend on the N-lactam moiety. On the other hand, removal of the N-lactam may be suf¢cient to allow cortical peptidoglycan to be hydrolysed by the major vegetative autolysin LytC [20,21], which has little or no activity against wild-type cortex.
By optimising the conditions for renaturing SDSPAGE, we have been able to show that A6, the 41kDa endospore-associated autolysin of B. subtilis, is a mother cell-speci¢c enzyme that appears early during sporulation (around stages II^IV) and persists in the dormant spore. It had previously been reported that A6 hydrolysed B. megaterium spore cortex but not B. subtilis vegetative cell walls [11]. In this paper, we have shown that it also digests spore cortex from its parent organism B. subtilis. The substrate speci¢city, genetic control and timing of expression of A6 suggest that it may have a role in synthesis or maturation of the cortex, which are thought to be directed by the mother cell [2,22] from stage III of sporulation onwards [18]. A6 may therefore be involved in establishment of spore resistance and/or dormancy. The presence of A6 in the membrane fraction of broken spores may be because it is associated with fragments of the mother cellderived outer forespore membrane adhering to the mature spore. Alternatively, it could be located elsewhere in the intact spore and become redistributed upon spore breakage. Although A6 activity was clearly observed by renaturing SDS-PAGE, the protein giving rise to it eluded detection, showing that A6 is a highly active enzyme present at only a few molecules per cell. A6 may be homologous to the 43-kDa cortex hydrolysisspeci¢c hexosaminidase that was previously isolated from spores of Bacillus cereus T [23] and/or the 38kDa spore-associated muramidase SleM from Clostridium perfringens [24]. Alternatively, A6 could correspond to the endopeptidase that was observed to appear at around stage IV during sporulation of B. subtilis [25]. The mutational analysis necessary to determine the precise function of the sporulationspeci¢c autolysin A6 must await the identi¢cation of its gene from the B. subtilis genome sequencing project. Acknowledgments
We are grateful to Je¡ Errington, Charlie Moran, Junichi Sekiguchi and Peter Setlow for providing
FEMSLE 7895 20-11-97
T.J. Smith, S.J. Foster / FEMS Microbiology Letters 157 (1997) 141^147 strains. This work was funded by the BBSRC and
147
[13] Kenney, T.J. and Moran, C.P. Jr. (1987) Organisation and regulation of an operon that encodes a sporulation-essential
the Royal Society.
sigma factor in
Bacillus subtilis.
J. Bacteriol. 169, 3329^3339.
[14] Turner, S.M., Errington, J. and Mandelstam, J. (1986) Use of a
References
lacZ
gene fusion to determine the dependence pattern of
sporulation operon
spoIIIC
in
spo
mutants of
Bacillus subtilis :
a branched pathway of expression of sporulation operons. [1] Smith, T.J., Blackman, S.A. and Foster, S.J. (1996) Peptidoglycan hydrolases of
Bacillus subtilis
168. Microb. Drug Re-
J. Gen. Microbiol. 132, 2995^3003. [15] Sterlini, J.M. and Mandelstam, J. (1969) Commitment to sporulation in
sist. 2, 113^118. [2] Foster, S.J. (1994) The role and regulation of cell wall structural dynamics during di¡erentiation of endospore-forming
[3] Doi, R.H (1989) Sporulation and germination. In :
Bacillus
(Harwood, C.R., Ed.), pp. 169^215. Plenum Press, London. [4] Smith, T.J. and Foster, S.J. (1995) Characterisation of the involvement of two compensatory autolysins in mother cell
Bacillus subtilis
and its relationship to develop-
[16] Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227,
bacteria. J. Appl. Bacteriol. 76, 25S^39S.
lysis during sporulation of
Bacillus subtilis
ment of actinomycin resistance. Biochem. J. 113, 29^37.
168. J. Bacteriol.
680^685. [17] Kuroda, A., Asami, Y. and Sekiguchi, J. (1993) Molecular cloning of a sporulation-speci¢c cell wall hydrolase gene of
Bacillus subtilis.
J. Bacteriol. 175, 6260^6268.
[18] Errington, J. (1993)
Bacillus subtilis
sporulation : regulation of
gene expression and control of morphogenesis. Microbiol.
177, 3855^3862. [5] Atrih, A., Zo ë llner, P., Allmaier, G. and Foster, S.J. (1996) Structural analysis of
Bacillus subtilis
168 endospore peptido-
Rev. 57, 1^33. [19] Losick, R. and Stragier, P. (1992) Crisscross regulation of cell-
glycan and its role during di¡erentiation. J. Bacteriol. 178,
type-speci¢c gene expression during development in
6173^6183.
Nature 355, 601^604.
[6] Popham, D.L., Helin, J., Costello, C.E. and Setlow, P. (1996) Analysis of the peptidoglycan structure of
Bacillus subtilis
[20] Lazarevic, V., Margot, P., Soldo, B. and Karamata, D. (1992) Sequencing and analysis of the
[7] Warth, A.D. (1978) Molecular structure of the bacterial spore.
di-
the
N-acetyl
muramoyl-L-alanine amidase and its modi¢er.
J. Gen. Microbiol. 138, 1949^1961.
Adv. Microbiol. Physiol. 17, 1^47. [8] Sekiguchi, J., Akeo, K., Yamamoto, H., Khasnov, F.K.,
[21] Kuroda, A. and Sekiguchi, J. (1991) Cloning and sequencing
Alonso, J.C. and Kuroda, A. (1995) Nucleotide sequence
of a major
and regulation of a new putative cell wall hydrolase gene,
7304^7312.
which a¡ects germination in
Bacillus subtilis lytRABC
vergon : a regulatory unit encompassing the structural genes of
endospores. J. Bacteriol. 178, 6451^6458.
cwlD,
B. subtilis.
Bacillus subtilis.
J. Bacter-
Bacillus subtilis
autolysin gene. J. Bacteriol. 173,
[22] Tipper, D.J. and Linnett, P.E. (1976) Distribution of peptidoglycan synthetase activities between sporangia and forespores
iol. 177, 5582^5589. [9] Popham, D.L., Helin, J., Costello, C.E. and Setlow, P. (1996) Muramic lactam in peptidoglycan of
Bacillus subtilis
spores is
in sporulating cells of
Bacillus sphaericus.
J. Bacteriol. 126,
213^221.
required for spore outgrowth but not for spore dehydration or
[23] Brown, W.C., Vellom, D., Schnepf, E. and Greer, C. (1978)
heat resistance. Proc. Natl. Acad. Sci. USA 93, 15405^15410.
Puri¢cation of a surface-bound hexosaminidase from spores
[10] Alderton, G. and Snell, N. (1963) Base exchange and heat resistance in bacterial spores. Biochem. Biophys. Res. Com-
Bacillus sub-
168 during vegetative growth and di¡erentiation by using
renaturing gel electrophoresis. J. Bacteriol. 174, 464^470. [12] Errington, J. and Mandelstam, J. (1986) Use of a
lacZ
gene
fusion to determine the dependence pattern of sporulation operon
spoIIA
in
Bacillus cereus
T. FEMS Microbiol. Lett. 3, 247^251.
Molecular characterisation of a germination-speci¢c murami-
mun. 10, 139^143. [11] Foster, S.J. (1992) Analysis of the autolysins of
tilis
of
[24] Chen, Y., Miyata, S., Makino, S. and Moriyama, R. (1997)
spo
mutants of
Bacillus subtilis.
J. Gen.
dase from
Clostridium perfringens
S40 spores and nucleotide
sequence of the corresponding gene. J. Bacteriol. 179, 3181^ 3187. [25] Guinand, M., Michel, G. and Balassa, G. (1976) Lytic enzymes in sporulating
Bacillus subtilis.
Commun. 68, 1287^1293.
Microbiol. 132, 2967^2976.
FEMSLE 7895 20-11-97
Biochem. Biophys Res.