Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN, UK1
Author for correspondence: Anne Moir. Tel: +44 114 2224418. Fax: +44 114 2728697. e-mail: a.moir{at}sheffield.ac.uk
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: germination-specific cortex-lytic enzymes, ypeB, yaaH
Abbreviations: GSLE, germination-specific cortex-lytic enzyme; RP-HPLC, reverse-phase HPLC
a Present address: Institute of Molecular Physiology, University of Sheffield, Alfred Denny Building, Sheffield S10 2TN, UK.
b Present address: Department of Biochemistry, Wellcome Trust Centre, Level 1, University of Dundee, Dundee DD1 4HN, UK.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Spore cortex-lytic enzymes have been purified from spores of Bacillus megaterium (Foster & Johnstone, 1987 ), Bacillus cereus (Makino et al., 1994
; Moriyama et al., 1996b
; Chen et al., 2000a
, b
) and Clostridium perfringens (Miyata et al., 1995
), and were reviewed by Atrih & Foster (1999)
. Based on structural analysis of spore peptidoglycan and its dynamics during germination, Atrih et al. (1998
, 1999
) have suggested that there are at least three different types of enzyme activities involved in spore-cortex hydrolysis and modification during germination: glucosaminidase, lytic transglycosylase, and a possible non-hydrolytic epimerase. SleB and CwlJ proteins have been recognized as important enzymes in normal spore germination of B. subtilis. The SleB protein (Moriyama et al., 1996a
) has apparent lytic transglycosylase activity (Boland et al., 2000
). The CwlJ protein is required for rapid germination in otherwise wild-type spores, and is strictly essential for cortex hydrolysis if the SleB protein is absent (Ishikawa et al., 1998
); recent genetic experiments suggest that it is activated by dipicolinic acid or its calcium chelate (Paidhungat et al., 2001
).
SleB has been shown to be responsible for the germination-assocated lytic transglycosylase activity (Boland et al., 2000 ), and spores of an sleB mutant are slow to complete late stages of germination. A ypeB mutant, defective in the gene downstream of sleB, shows the same germination defect as an sleB mutant (Boland et al., 2000
).
A cwlJ single mutant is blocked in late stages of germination, failing to phase-darken completely; Chen et al. (2000a) described a cortex-lytic enzyme of B. cereus (SleL) that is found in an active form in the germination exudate. It requires disrupted rather than intact spore peptidoglycan for its activity, and is therefore likely to act on a substrate that has already been cleaved by another lytic enzyme, such as SleB. Its role in germination, however, has not been tested by mutation. The YaaH, YdhD and YvbX proteins of B. subtilis are homologues of SleL (49, 30 and 26% amino acid identity, respectively), the homology extending throughout the protein. All have putative cell-wall-binding motifs. A yaaH mutant was reported as slow to germinate (Kodama et al., 1999
), and a ydhD-overexpressing strain made spores some of which germinated spontaneously, and others of which were reluctant to germinate (Kodama et al., 2000
).
In the work described below, we have characterized and localized the CwlJ, SleB and YpeB proteins in fractions of dormant and germinating spores. We have also analysed the function of the uncharacterized B. subtilis homologues of cortex-lytic enzymes, by making null mutations in each gene. This has shown that the likely epimerase activity during germination is dependent on YaaH.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Reverse-phase (RP)-HPLC analysis of spore peptidoglycan.
Preparation of peptidoglycan from spore cortex and vegetative cells of B. subtilis, muropeptide separation by RP-HPLC, and amino acid analysis were performed as described by Atrih et al. (1996 , 1998
).
ß-Galactosidase assay.
Levels of ß-galactosidase activity were measured using methylumbelliferyl ß-D-galactoside as substrate, as described by Horsburgh & Moir (1999) .
DNA isolation and cloning.
Standard cloning technologies (Sambrook et al., 1989 ) were used. PCR was carried out using Expand (Boehringer Mannheim). Transformation of B. subtilis was as described by Kunst & Rapoport (1995)
.
Construction of insertionally inactivated cwlJ and ykvT mutants.
For the construction of a cwlJ mutant, a 1270 bp DNA fragment spanning the cwlJ region was synthesized by PCR using B. subtilis chromosomal DNA as template. Primers 5'-CGCGAATTCAGACCAAGCACCAGATACA-3' and 5'-GATCGAGCTCTTCGCAGCACGTCCTTCAT-3' were used as forward and reverse primers, respectively (the chromosomally derived sequence is italicized and the EcoRI and SacI restriction sites added are underlined). The PCR products were cloned into plasmid pBluescript IIKS (Stratagene) after appropriate restriction digestion and ligation. The recombinant plasmid, pHCJ1, was isolated from a transformant of E. coli DH5 and was used as a template DNA for an inverse-PCR using primers 5'-GACAGGATCCGCAGCCGAATTAATTCCA-3' and 5'-TGACAGGCCTCGGCTCGATTAATGGTGA-3' as forward and reverse primers, respectively (the italicized chromosomal sequences are 120 bp apart in the cwlJ gene; the BamHI and StuI restriction sites added are underlined). The PCR product was then restricted with BamHI and StuI and ligated with a kanamycin cassette obtained by restriction digestion of pDG792 (Guerout-Fleury et al., 1995
) with the same enzymes. E. coli DH5
was transformed with the ligation mixture, and a recombinant plasmid, pHCJ2, containing the insertion of the kanamycin cassette in the cwlJ gene, was isolated. This plasmid DNA was then linearized with ScaI, gel-purified, and used for transforming B. subtilis. HC101, a kanamycin-resistant transformant in which the insertionally inactivated copy had replaced the wild-type gene by a double crossover (confirmed by Southern hybridization) was isolated.
A mutant with a spectinomycin-resistance gene insertionally inactivating ykvT was constructed in an analogous fashion. A 1248 bp DNA fragment spanning the ykvT region was synthesized by PCR using B. subtilis chromosomal DNA as template. Primers 5'-CGCGAATTCACGAACTGAGAGTCCAGA-3' and 5'-GACCGAGCTCATTCAAGGCATTCAGCGA-3' were used as forward and reverse primers, respectively. A recombinant plasmid, pHYT1, containing the PCR fragment in pBluescript KS was used as a template DNA for an inverse-PCR using primers 5'-ATGTGGATCCGTGTGTGCTGATAGCTTA-3' and 5'-ACGCAGGCCTAAGGTCGCTGTAGCAAG-3' as forward and reverse primers, respectively, to leave a 124 bp gap within the ykvT gene. The PCR product was then restricted with BamHI and StuI and ligated with a spectinomycin cassette obtained by restriction digestion of pDG1727 (Guerout-Fleury et al., 1995 ) with the same enzymes. E. coli DH5
was transformed with the ligation mixture and a recombinant plasmid, pHYT2, containing the insertion of the spectinomycin cassette in the ykvT gene, was isolated. This plasmid was then linearized by ScaI, gel-purified, and used for transforming B. subtilis. A transformant with the correct insertion of the spectinomycin cassette in the ykvT gene, checked by Southern hybridization, was named HC201.
Construction of a ykvTlacZ fusion strain.
A lacZ transcriptional fusion of ykvT was constructed by cloning a 655 bp fragment generated by PCR in pMUTIN4 (Vagner et al., 1998 ) to yield pHCYT2. Primers 5'-CGCGAATTCACGAACTGAGAGTCCAGA-3' and 5'-ATGTGGATCCGTGTGTGCTGATAGCTTA-3' were used as upstream and downstream primers, respectively. The plasmid was used to transform B. subtilis; a transformant (HC202) contained the ykvTlacZ fusion integrated into the B. subtilis chromosome by single crossover, as confirmed by Southern hybridization.
Overexpression and purification of proteins (CwlJ, SleB and YpeB).
The CwlJ, SleB and YpeB proteins were overexpressed by cloning the respective genes in E. coli overexpression vector pET24d (Novagen). The primers were designed to make C-terminal histidine-tagged fusions for affinity purification of the overexpressed proteins.
The cwlJ gene was amplified by PCR using 5'-CGTGCCATGGCGGTCGTGAGAGCA-3' and 5'-ACTGCTCGAGAAATGTGTTATATACATTTTCACACG-3' as forward and reverse primers, respectively. The sleB gene was amplified, without its signal sequence, by PCR using 5'-CATGCCATGGCCTTTTCGAATCAGGTC-3' and 5'-CGGCTCGAGCTCACAGAAAATGTGTTTACC-3' as forward and reverse primers, respectively. The ypeB gene was amplified by PCR using 5'-CATGCCATGGTCAGAGGAATTTTAATCG-3' and 5'-CCGGCTCGAGTAGGTCTTTATATATAGGTTCTGCAT-3' as forward and reverse primers, respectively (for both primer pairs, the restriction sites added, NcoI and XhoI, are underlined, and the gene sequences are italicized). The PCR products were gel-purified, restriction-digested with NcoI and XhoI and ligated with pET24d DNA, restricted with the same enzymes. The ligation mixtures were used to transform E. coli DH5 to kanamycin resistance. Recombinant plasmids, pETHCJ1, pETMOR3 and pETMOR2, carrying the cloned cwlJ, sleB and ypeB genes, respectively, were isolated. E. coli BL21(DE3) was transformed with pETHCJ1, pETMOR3 and pETMOR2, and expression was induced by the addition of 0·4 mM IPTG. Inclusion bodies were collected, and solubilized in 8 M urea. The overexpressed proteins were purified using a HisTrap (Amersham Pharmacia) affinity column in urea-containing buffers, and dialysed against PBS. The purity of the preparations was assessed on Coomassie-blue-stained gels as >95%.
Raising of antibodies and Western blot analysis.
Polyclonal antibodies against CwlJ, SleB and YpeB were raised in rabbits by injecting purified proteins as previously described (Hudson et al., 2001 ). Antisera were collected and contaminating E. coli antibodies were removed on an immobilized E. coli lysate column (Pierce). Proteins were detected after Western blotting by chemiluminescence (Amersham Pharmacia). Primary antibody was generally used at 1/500 dilution, and secondary antibody (horseradish-peroxidase-linked anti-rabbit IgG F(Ab)2 fragment) at 1/5000 dilution.
Spore fractionation and protein separation.
Spores were broken by using a Fast-Prep blue kit (BIO-101) with a Fast-Prep system reciprocal shaker (BIO-101), and fractions prepared from 50 mg dry weight of spores as described by Hudson et al. (2001) . In one experiment, the fractionation process was limited to separation of the integument fraction from the membrane plus soluble material, by omitting the high-speed centrifugation normally used to separate the latter components. Samples for analysis by SDS-PAGE were adjusted to give equivalent loading of protein (usually 10 µg protein per lane). Biotinylated markers (from Sigma or Bio-Rad) were detected with horseradish peroxidase-linked streptavidin.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Spores were isolated, washed and fractionated into soluble, inner-membrane and integument fractions by the method already described and verified (Hudson et al., 2001 ). Briefly, washed spores were broken by shaking with silica beads in a PMSF-containing buffer. Fractionation involved low-speed centrifugation to recover a pellet containing the integument fraction (which contains coats, cortex and outer membrane), then at higher centrifugal force to pellet a membrane fraction, which contains inner membrane, leaving the remaining soluble material (Hudson et al., 2001
). In equivalent fractions, the GerAA and GerAC proteins were found exclusively in the inner-membrane fraction. Whole spore extracts (Hudson et al., 2001
) of protease-negative strain WB600, wild-type HR, or of specific mutants were used as positive and negative controls. Western blot analysis of overexpressed CwlJ protein in E. coli, and of spore fractions probed with anti-CwlJ antibody, are shown in Fig. 1
. The predicted molecular mass of CwlJ is 16 kDa, and a protein of this size is detected in E. coli extracts, along with minor additional bands at twice this molecular mass and larger. This suggests that in E. coli the overexpressed CwlJ protein may form some oligomers, not entirely dissociated in standard SDS-PAGE loading buffer. In spore fractions, no cross-reaction was seen with proteins in either the soluble or the inner-membrane fraction, but strong signals were obtained from the integument fraction (Fig. 1a
). The specificity of the antiserum was verified by its reaction with overexpressed protein in E. coli extracts (Fig. 1a
, b
) with purified CwlJ protein (Fig. 1b
), and by the absence of cross-reactive bands in extracts from a cwlJ mutant (Fig. 1b
). Purified CwlJ from E. coli runs as a monomer and in higher molecular mass forms, but, under the conditions used, CwlJ in spore extracts ran as a diffuse band of 32 kDa. This suggests that CwlJ may be present in the spore in dimeric and larger forms, or alternatively, that its mobility is modified, possibly by association with another component. This higher apparent molecular mass was not the consequence of covalent binding to peptidoglyan, as lysozyme treatment neither released the enzyme from the integument, nor altered the apparent molecular size (Fig. 1b
).
|
|
YpeB is required for SleB localization
Anti-SleB serum was used to probe a Western blot of total spore extracts prepared from strain FB102 (ypeB); in this strain, the upstream sleB gene remains intact. No SleB protein was detected in either dormant or germinated spores (compare Fig. 3a with Fig. 3b
). The absence of SleB from these spores indicates that expression of YpeB is important for the presence of SleB in the spore; it may be required for the localization of the protein, and/or for its stabilization against proteolysis. Expression of the sleBypeB operon, as measured by a lacZ transcriptional fusion, remains high in a ypeB mutant background (Boland et al., 2000
).
|
|
|
Testing of other potential lytic enzymes for their role in germination
Spore suspensions of null mutants in cwlJ and in potential lytic enzyme genes ykvT, yaaH, ydhD and yvbX (Table 1) were prepared. For each one, germination behaviour, as measured by OD600 loss and by the muropeptide pattern during germination, was examined.
YaaH. YaaH is 49% identical to SleL of B. cereus, an enzyme demonstrated to have N-acetylglucosaminidase activity (Chen et al., 2000a ). Although Chen et al. (2000a
) reported a germination defect, spore preparations of a yaaH mutant constructed by transfer of their null mutation into our standard laboratory strain (HR) background showed wild-type germination kinetics in L-alanine, and outgrew normally (data not shown). The composition of dormant-spore peptidoglycan, as revealed by RP-HPLC analysis of muropeptides after Cellosyl digestion, was identical to wild-type traces published previously (Atrih et al., 1998
). The germination exudate, however, contained altered muropeptides (Fig. 6
). Muropeptides G1G4 and G6G7 are absent from the profile. These muropeptides have been previously identified in B. subtilis and in B. megaterium, and have been suggested to be generated by an epimerase (Atrih et al., 1998
, 1999
). In contrast, muropeptides generated by glucosaminidase activity (G8, G8AG8C) or lytic transglycosylase activity (G9G10 and G12G13), and those already identified in dormant spores (G10G11 and G20G21), are found in the yaaH-mutant exudate. The total amount of peptidoglycan released into the germination exudate of the mutant was similar to that of the wild-type. The activity of YaaH is therefore not required for the hydrolysis of the cortex in germination, but is required for the epimerase-like activity.
|
YdhD and YvbX. Both YdhD and YvbX are homologues of YaaH. Like yaaH, both ydhD and yvbX are expressed during sporulation (Kodama et al., 2000 ). Spores made from ydhD and yvbX null mutants germinated apparently normally. The RP-HPLC profiles of dormant-spore muropeptides, germinating spores, or germination exudate, showed no difference from wild-type (data not shown). Therefore these homologues appear to have no individually unique involvement in cortex peptidoglycan synthesis during sporulation, or in cortex breakdown during germination.
YkvT. SleB has strong similarity to two other proteins in the B. subtilis database: CwlJ and YkvT. The YkvT protein sequence shows 31% identity to the equivalent region of SleB. It has a shorter putative N-terminal wall-binding domain, and, as in SleB, this is connected to the predicted catalytic domain by a linker region. The ykvT gene was inactivated by insertion of a spectinomycin-resistance cassette by a double crossover event. Germination of mutant spores was identical to that of the wild-type (data not shown), and its combination with cwlJ and/or sleB mutations produced the same phenotypes as expected for those mutants without a ykvT mutation. Measurements of the ß-galactosidase activity of cells carrying a ykvT::lacZ fusion (strain HC203) showed that the gene is expressed maximally during vegetative growth, and expression falls in post-exponential phase cells (data not presented). Muropeptide analysis of the vegetative and spore peptidoglycan showed that there was no difference between the ykvT mutant and the wild-type parent (data not shown). The YkvT protein is therefore a vegetatively expressed protein of apparently redundant function.
CwlJ. As expected from previous work (Ishikawa et al., 1998 ), spores of HC101 (cwlJ::kan) germinated more slowly than the wild-type spores in both L-alanine and AGFK, as estimated by a late-germination parameter, i.e. loss of OD600 (data not shown). Although the rate of OD600 loss in spore suspensions was slow in the first 30 min (28% fall by this time rather than 53%), by 60 min the proportion of the initial OD600 lost was almost as high as for the wild-type, and the spores were phase-dark.
The cortex-lysis products from this mutant were examined after germination at different pH values, as it has been demonstrated that the activity of cortex-lytic enzymes is affected by the pH of the germination buffer (Atrih & Foster, 2001 ). The RP-HPLC profiles of Cellosyl-digested muropeptides, both of germination exudate and of the remaining spore-associated material of cwlJ spores germinated for up to 2 h at pH 5, 7 and 9, were all identical to the profiles seen in the equivalent wild-type control (results not shown).
The possibility remains that CwlJ is a muramidase, whose activity would be masked by the overall muramidase digestion mediated by Cellosyl as part of the muropeptide analysis protocol. To address this, spore-associated material and exudates from spores of AM1596 (cwlJ yaaH) and AM1597 (yaaH) were compared (inclusion of the yaaH mutation simplifies the pattern of peaks observed). Muramidase activity would result in the production of new reducing groups which would be labelled with sodium borohydride. Thus, samples from spores germinated at pH 5 and 10 were borohydride-treated prior to Cellosyl digestion and the muropeptide profiles compared. In no case was there any difference in the reduced muropeptide profile between the strain bearing the cwlJ mutation and its respective parent. Thus CwlJ is unlikely to have significant muramidase activity.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have shown previously that sleB is expressed during sporulation and that the expression starts between 2 and 3 h after the onset of sporulation (Boland et al., 2000 ). Expression in the forespore, the possession of a signal sequence, and evidence of a peptidoglycan-binding domain would together suggest a likely location of SleB either on the outside of the inner spore membrane, or in the cortex. Our data show its presence in both inner-membrane and integument fractions of spores. SleB has been shown to be more resistant to spore damage, in particular heat and alkali, than the other germination-specific cortex-lytic enzyme (GSLE) activities (Atrih & Foster, 2001
). In fact, spores of a sleB mutant are relatively sensitive to alkali. SleB also has a different pH optimum from the other GSLEs. The present study suggests that the localization of GSLEs in the dormant spore may be involved in their resistance properties. SleB colocalizes, at least in part, with YpeB, which may therefore be responsible for SleB protection in the dormant spore. YpeB has a sequence at its N-terminus likely to represent either a signal sequence or a membrane anchor, and at least some of this protein is located in the inner-membrane fraction.
A dual location of SleB and YpeB is unexpected. Moriyama et al. (1999) used immunoelectron microscopy to demonstrate that the SleB enzymes from B. subtilis and B. cereus are located just inside the spore-coat layer in the dormant spore, but our study provides strong evidence for a second, additional localization in the inner spore membrane. In a cwlJ mutant, the function of SleB protein during germination is essential for colony formation on rich agar medium. Chemical decoating of cwlJ mutant spores makes very little difference to their ability to germinate to form colonies (Paidhungat et al., 2001
). Therefore chemical decoating, while it removes CwlJ from spores, does not remove enough SleB to interfere with its role in germination. It is possible, therefore, that the location of some SleB protein at the inner membrane is functionally important. The location of YpeB and SleB in the inner membrane also places them in an appropriate position for their activation by receptor-mediated signal transduction, as the receptor proteins are also inner membrane-located.
How GSLE activity is triggered during germination is still unknown. As SleB may be stabilized by YpeB, destabilization of their association, perhaps related to the proteolytic processing of YpeB, may result in activation of heat-sensitive SleB. The recent observation that CwlJ can be activated by calcium dipicolinate still does not explain how physiological, nutrient germinants act to initiate the process. The question of how germinant binding to receptors triggers activation of these lytic enzymes is currently being addressed.
The other enzymes recognized as germination-specific cortex-modifying enzymes, CwlJ and YaaH, do not require YpeB protein for their localization or function. The role of YaaH is uncertain, but it may alter the activity of other enzymes by the substrate modification it mediates. The enzyme(s) responsible for the glucosaminidase activity during germination and the activity of CwlJ remains elusive. It is only after identification of all the cortex hydrolysis/modification activities during germination that their individual and combined roles can be determined.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Atrih, A. & Foster, S. J. (2001). In vivo roles of the germination-specific lytic enzymes of Bacillus subtilis 168. Microbiology 147, 57-64.
Atrih, A., Zollner, P., Allmaier, G. & Foster, S. J. (1996). Structural analysis of Bacillus subtilis 168 endospore peptidoglycan and its role during differentiation. J Bacteriol 178, 6173-6183.[Abstract]
Atrih, A., Zollner, P., Allmaier, G., Williamson, M. & Foster, S. J. (1998). Peptidoglycan structural dynamics during germination of Bacillus subtilis 168 endospores. J Bacteriol 180, 4603-4612.
Atrih, A., Bacher, G., Korner, R., Allmaier, G. & Foster, S. J. (1999). Structural analysis of Bacillus megaterium KM spore peptidoglycan and its dynamics during germination. Microbiology 145, 1033-1041.[Abstract]
Bagyan, I., Noback, M., Bron, S., Paidhungat, M. & Setlow, P. (1998). Characterisation of yhcN, a new forespore-specific gene of Bacillus subtilis. Gene 212, 179-188.[Medline]
Boland, F., Atrih, A., Chirakkal, H., Foster, S. J. & Moir, A. (2000). Complete spore cortex hydrolysis during germination of Bacillus subtilis 168 requires SleB and YpeB. Microbiology 146, 57-64.
Chen, Y., Fukuoka, S. & Makino, S. (2000a). A novel spore peptidoglycan hydrolase of Bacillus cereus: biochemical characterisation and nucleotide sequence of the corresponding gene, sleL. J Bacteriol 182, 1499-1506.
Chen, Y., Miyata, S., Makino, S. & Moriyama, R. (2000b). Molecular characterisation of a germination specific muramidase from Clostridium perfringens S40 spores and nucleotide sequence of the corresponding gene. J Bacteriol 197, 3181-3187.
Foster, S. J. & Johnstone, K. (1987). Purification and properties of a germination-specific cortex lytic enzyme from spores of Bacillus megaterium KM. Biochem J 242, 573-579.[Medline]
Guerout-Fleury, A., Shazand, K., Frandsen, N. & Stragier, P. (1995). Antibiotic resistance cassettes for Bacillus subtilis. Gene 167, 335-336.[Medline]
Horsburgh, M. J. & Moir, A. (1999). Sigma M, an ECF RNA polymerase sigma factor of Bacillus subtilis 168, is essential for growth and survival in high concentrations of salt. Mol Microbiol 32, 4150.[Medline]
Hudson, K. D., Corfe, B. M., Kemp, E. H., Feavers, I. M., Coote, P. J. & Moir, A. (2001). Localization of GerAA and GerAC proteins in the Bacillus subtilis spore. J Bacteriol 183, 4317-4322.
Ishikawa, S., Yamane, K. & Sekiguchi, J. (1998). Regulation and characterisation of a newly deduced cell wall hydrolase gene (cwlJ) which affects germination of Bacillus subtilis spores. J Bacteriol 180, 1375-1380.
Kodama, T., Takamatsu, H., Asai, K., Kobayashi, K., Ogasawara, N. & Watabe, K. (1999). The Bacillus subtilis yaaH gene is transcribed by SigE RNA polymerase during sporulation, and its product is involved in germination of spores. J Bacteriol 181, 4584-4591.
Kodama, T., Takamatsu, H., Asai, K., Sadie, Y. & Watabe, K. (2000). Synthesis and characterisation of the spore proteins of Bacillus subtilis YdhD, YkuD and YkvP, which carry a motif conserved among cell wall binding proteins. J Biochem 128, 655-663.[Abstract]
Kunst, F. & Rapoport, G. (1995). Salt stress is an environmental signal affecting degradative enzyme synthesis in Bacillus subtilis. J Bacteriol 177, 2403-2407.[Abstract]
Makino, S., Ito, N., Inoue, T., Miyata, S. & Moriyama, R. (1994). A spore-lytic enzyme released from Bacillus cereus spores during germination. Microbiology 140, 1403-1410.[Abstract]
Miyata, S., Moriyama, R., Miyahara, N. & Makino, S. (1995). A gene (sleC) encoding a spore-cortex-lytic enzyme from Clostridium perfringens S40 spores; cloning, sequence analysis and molecular characterisation. Microbiology 141, 2643-2650.[Abstract]
Moir, A., Corfe, B. M. & Behravan, J. (2002). Spore germination. Cell Mol Life Sci 59, 403-409.[Medline]
Moriyama, R., Hattori, A., Miyata, S., Kudoh, S. & Makino, S. (1996a). A gene (sleB) encoding a spore cortex lytic enzyme from Bacillus subtilis and its response to L-alanine mediated germination. J Bacteriol 178, 6059-6063.[Abstract]
Moriyama, R., Kudoh, S., Miyata, S., Nonobe, S., Hattori, A. & Makino, S. (1996b). A germination specific cortex lytic enzyme from Bacillus cereus spores: cloning and sequencing of the gene and molecular characterisation of the enzyme. J Bacteriol 178, 5330-5332.[Abstract]
Moriyama, R., Fukuoka, H., Miyata, S., Kudoh, S., Hattori, A., Kozuka, S., Yasuda, Y., Tochikubo, K. & Makino, S. (1999). Expression of a germination specific amidase, SleB, of bacilli in the forespore compartment of sporulating cells and its localisation on the exterior side of the cortex in dormant spores. J Bacteriol 181, 2373-2378.
Paidhungat, M. & Setlow, P. (1999). Isolation and characterization of mutations in Bacillus subtilis that allow spore germination in the novel germinant D-alanine. J Bacteriol 181, 3341-3350.
Paidhungat, M. & Setlow, P. (2000). Role of Ger proteins in nutrient and non-nutrient triggering of spore germination in Bacillus subtilis. J Bacteriol 182, 2513-2519.
Paidhungat, M. & Setlow, P. (2001). Localization of a germinant receptor protein (GerBA) to the inner membrane of Bacillus subtilis spores. J Bacteriol 183, 3982-3990.
Paidhungat, M., Ragkousi, K. & Setlow, P. (2001). Genetic requirements for induction of germination of spores of Bacillus subtilis by Ca2+-dipicolinate. J Bacteriol 183, 4886-4893.
Popham, D. J., Helin, J., Costello, C. E. & Setlow, P. (1996). Muramic lactam in peptidoglycan of Bacillus subtilis spores is required for spore outgrowth but not for spore dehydration or heat resistance. Proc Natl Acad Sci USA 93, 15405-15410.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Southworth, T. W., Guffanti, A. A., Moir, A. & Krulwich, T. A. (2001). GerN: an endospore germination protein of Bacillus cereus is a Na+/H+-K+ antiporter. J Bacteriol 183, 5896-5903.
Stewart, G. S. A. B., Johnstone, K., Hagelberg, E. & Ellar, D. (1981). Commitment of bacterial spores to germinate. Biochem J 198, 101-106.[Medline]
Thackray, P. D., Behravan, J., Southworth, T. W. & Moir, A. (2001). GerN, an antiporter homologue important in the germination of Bacillus cereus endospores. J Bacteriol 183, 476-482.
Vagner, V., Dervyn, E. & Ehrlich, S. D. (1998). A vector for systematic gene disruption in Bacillus subtilis. Microbiology 144, 3097-3104.[Abstract]
Received 1 February 2002;
revised 16 April 2002;
accepted 29 April 2002.