Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK1
Author for correspondence: Simon J. Foster. Tel: +44 114 222 4411. Fax: +44 114 272 8697. e-mail: S.Foster{at}sheffield.ac.uk
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ABSTRACT |
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Keywords: Bacillus subtilis, endospores, germination, peptidoglycan, cortex hydrolysis
Abbreviations: GSLE, germination-specific lytic enzyme; MUG, methylumbelliferyl ß-D-galactoside; RP-HPLC, reverse phase HPLC
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INTRODUCTION |
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A number of spore-cortex-lytic enzymes have been isolated from spores of different organisms. Two lytic enzymes have been isolated from Clostridium perfringens S40 spores a 31 kDa enzyme encoded by the gene sleC (Miyata et al., 1995 ) and a 38 kDa enzyme encoded by the gene sleM (Chen et al., 1997
). The Bacillus cereus IFO 13597 gene sleB encodes a 24 kDa enzyme (Makino et al., 1994
; Moriyama et al., 1996b
), and its homologue has been identified and inactivated in Bacillus subtilis. The resulting mutant germinates slower than the wild-type (Moriyama et al., 1996a
).
Foster & Johnstone (1987) isolated a germination-specific lytic enzyme (GSLE) that was capable of cortex hydrolysis from the spores of Bacillus megaterium KM. The enzyme was activated in vivo during germination and has a high specificity for intact spore cortex. Western blot analysis revealed cross-reactivity with proteins from spore fractions of other species and that the enzyme was capable of germinating permeabilized spores of other species (Foster & Johnstone, 1988
).
The recent application of reverse phase HPLC (RP-HPLC) to monitor peptidoglycan structural dynamics during the germination of B. subtilis revealed that the mechanism of cortex hydrolysis is complex and involves several hydrolases of differing specificity (Atrih et al., 1998 ).
In B. subtilis, the sleB and adjacent downstream ypeB gene form a bicistronic operon (Moriyama et al., 1999 ). In this study, we have shown that a defect in either the sleB or the ypeB genes results in incomplete germination. Analysis of cortex dynamics during germination revealed the likely hydrolytic bond specificity of SleB.
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METHODS |
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(ii) Transformation of E. coli and B. subtilis.
Transformation of E. coli was performed as described by Hanahan (1983) . Transformation of B. subtilis 168 with pFB101 and pFB102 was performed by the competent cell method (Anagnostopoulos & Spizizen, 1961
). Disruption of the sleB and ypeB genes by means of Campbell-type recombination was confirmed by Southern blot analysis, using the appropriate plasmid as probe. Hybridization, probe labelling and detection were done with the Boehringer Mannheim nonradioactive DNA labelling and detection kit.
Construction of sleB in-frame deletion mutant.
To construct an in-frame deletion of sleB, a 5·6 kb DNA fragment spanning the sleB and ypeB coding sequence was synthesized by PCR using B. subtilis HR chromosomal DNA as template. Primers 5'-ATGCGAGCTC2640CGGCTAAGAAGAGAGGCATC2621 and 5'-CTGAGTCGACC2995GCCTGATGCAGTATTGAAG3014 were used as forward and reverse primers, respectively (the chromosomal sequences are italicized and the SacI and SalI restriction sites added are underlined; the numbering is with respect to the A of the translational start codon of sleB). The PCR product was cloned in E. coli DH5 using plasmid pGEM3Z, after appropriate restriction digestion and ligation. A recombinant plasmid, pGSB1 containing the cloned DNA was isolated and this was used as template DNA for inverse-PCR using primers 5'-ATGCGGATCCG843ATACGGCTACAAGTCCGTG824 and 5 '-ATGAGGATCCC444TGCCGTGCTTCTGCTTGTTG464 as forward and reverse primers, respectively (the chromosomal sequences are italicized and the restriction site for BamHI is underlined). The PCR product from this experiment resulted in a deletion of 399 bases in the coding region of the sleB gene, removing amino acid residues 148281 of SleB. The PCR product was then restricted with BamHI and religated before transformation into E. coli DH5
. A plasmid, pGSB21, carrying the sleB deletion was isolated and verified by restriction analysis. The sleB deletion was then transferred into B. subtilis HR by congression using pGSB21 linearized with ScaI and trp+ DNA. Congressant colonies (trp+) were selected on SS minimal agar and then screened by PCR for the sleB deletion. One congressant, which showed the correct deletion, was verified by Southern blot and named HC145.
Spore preparation and germination.
Sporulation was initiated in CCY medium and spores of B. subtilis were prepared as described by Stewart et al. (1981) . Spores were stored at a concentration of 10 mg dry weight ml-1 in distilled water at -20 °C. Purified spores were heat-activated at 70 °C for 30 min and cooled in ice. Germination was initiated by the addition of L-alanine to a final concentration of 1 mM, or by the addition of asparagine to 30 mM with glucose and fructose each to a concentration of 5·6 mM (AGF). Germinant was added to a 5 mg dry weight ml-1 spore suspension in 10 mM Tris/HCl pH 7 containing KCl (10 mg ml-1). Spores were germinated at 37 °C and the extent of germination was monitored by recording the decrease in OD600 (Foster & Johnstone, 1987
) over a 2 h period. Phase-darkening of spores was determined by phase-contrast microscopy.
(i) Determination of loss of heat resistance during germination.
Germinating spore samples were diluted serially in 10 mM D-alanine and incubated at 70 °C for 30 min. After cooling in ice, viability was measured by plate counting on nutrient agar.
(ii) Measurement of loss of dipicolinic acid during germination.
Samples of germination spore suspension (3 ml) were filtered through a 0·45 µm membrane and the dipicolinic acid content was measured as described by Scott & Ellar (1978) .
RP-HPLC analysis of spore peptidoglycan.
Cortex extraction from dormant and germinated spores, muropeptide separation by RP-HPLC, and amino acid and mass spectrometry analyses were performed as previously described (Atrih et al., 1996 , 1998
).
Analysis of gene expression
(i) Expression under the control of the Pspac promoter.
Induced expression of sigma factor genes under the control of Pspac was carried out by adding IPTG (400 µM final concentration) to cells growing in LB at an OD600 of 0·25 (Sun et al., 1989 ).
(ii) Expression during sporulation.
Synchronous sporulation was performed by the resuspension method of Sterlini & Mandelstam (1969) . Samples were harvested every hour after the initiation of sporulation (t0) for 8 h and sporulation morphology was monitored by microscopy.
(iii) Measurement of ß-galactosidase activity.
ß-Galactosidase assays, using MUG (methylumbelliferyl ß-D-galactoside) as the substrate, were performed as described by Youngman (1990) , except that cells were permeabilized by incubation with lysozyme on ice for 20 min, MUG was used at a final concentration of 600 µg ml-1 in DMSO and the assay was incubated at 28 °C. Fluorescence was measured on a fluorometer (Hoefer). One unit of ß-galactosidase activity was defined as the amount of enzyme which releases 1 pmol methylumbelliferone min-1 ml-1 (Zuberi et al., 1987
), normalized to a culture OD600 of 1·0.
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RESULTS |
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The germinated spores from the mutant strains, in germination buffer, remained phase-grey even 12 h after germination was triggered. Strains FB101 (sleB), FB102 (ypeB) and HC145 (sleB) were, however, capable of outgrowth to form colonies when plated on nutrient agar overnight at 37 °C, indicating that B. subtilis can overcome the loss of SleB and/or YpeB to outgrow to form a new vegetative cell.
Peptidoglycan structural analysis of dormant spores
To determine whether the germination defect in the sleB and ypeB mutants is caused by a structural modification of spore peptidoglycan, the RP-HPLC profiles of Cellosyl-digested peptidoglycan from these mutants were compared to that of the wild-type. No peptidoglycan structural defect could be detected in strains FB101 (sleB), FB102 (ypeB) and HC145 (sleB) compared to the wild-type (data not shown). The sleB and ypeB gene products are both likely therefore to be involved in the processes associated with cortex hydrolysis during germination.
(i) Peptidoglycan dynamics during germination.
RP-HPLC profiles of germinated-spore-associated peptidoglycan in the wild-type and FB101 (sleB) are shown in Fig. 2(a) and 2(b)
, respectively. After 2 h germination, the ratio of dormant spore muropeptides and germination-associated muropeptides was altered between the wild-type (HR; Fig. 2a
) and strain FB101 (sleB; Fig. 2b
). In strain FB101 (sleB) there were less germination muropeptides (G1G7) and correspondingly more dormant spore muropeptides (10, 11, 20, 21). Strains FB102 (ypeB) and HC145 (sleB
) showed identical profiles to strain FB101 (sleB) (data not shown). This result indicates a partial hydrolysis of the cortex in the sleB mutant. The amount of peptidoglycan released, calculated as the ratio of retained material to that of primordial cell wall (muropeptides 1 and 8) at time 0 and after 2 h germination, for strains FB101 (sleB), and FB102 (ypeB) was 44% and 38%, respectively. This amount is approximately 20% lower than that released from the wild-type, where 64% of peptidoglycan fragments were released in the germination exudate after 2 h.
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To test the sigma-factor specificity of RNA polymerase which could transcribe the sleB gene, the sleBlacZ fusion was introduced by transformation into strains FB107, FB108 and FB109 (Table 1), which contain plasmids having Pspac-inducible genes encoding spoIIGB (
E), spoIIAC (
F) and spoIIIG (
G), respectively (Popham & Stragier, 1991
; Oke & Losick, 1993
; Sun et al., 1989
). The sleBlacZ fusion was also introduced into strain FB110,which contains a chromosomally located
K coding sequence under Pspac control.
Expression of ß-galactosidase activity was detected on induction of G, showing that production of
G is sufficient to direct expression from the sleB promoter (Fig. 4
). A putative consensus sequence for
G binding is detectable upstream of the transcriptional start site of sleB. Expression of ß-galactosidase did not occur following induction of
K or
F, but expression was detected after induction of
E in vegetative cells, in three separate experiments (Fig. 4
). No expression of sleBlacZ was observed in vivo in a spoIIIG mutant, which would contain functional
E. The expression in vegetative cells in response to
E induction may be artefactual, or may reflect further levels of regulation.
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DISCUSSION |
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The sleB gene is the first in the operon and its inactivation is likely to have an effect on the downstream gene, ypeB. Insertion inactivation of ypeB or in-frame deletion of sleB produced mutants with similar germination defects. This indicates that both genes are necessary for cortex hydrolysis during germination and therefore are essential for the activity of the lytic transglycosylase encoded by sleB. SleB has recently been shown to be located just inside the spore coat layer in the dormant spore and to exist in mature form but lacking a signal sequence (Moriyama et al., 1999 ). The enzyme is translocated across the forespores inner membrane by a secretion signal peptide and is deposited in cortex layer synthesized between the forespore inner and outer membrane (Moriyama et al., 1999
).
The role of YpeB is still unclear. It appears to be required for either expression, localization, activation or function of SleB. The N-terminal region of YpeB could represent a hydrophobic anchor for the localization of the protein in the membrane or a signal peptide sequence involved in the translocation of the protein across the membrane. The ypeB gene has homologues in B. cereus and B. megaterium KM, all in the same operon organization, downstream of a sleB homologue. In B. cereus, the gene corresponding to ypeB encodes a protein that has 75% identity to YpeB from B. subtilis (Moriyama et al., 1996b ) and the equivalent gene in B. megaterium KM encodes a protein having 65% identity (Pettigrew, 1996
). These data suggest that ypeB may have the same role in B. cereus and B. megaterium as in B. subtilis.
There are two more homologues of SleB in B. subtilis, CwlJ and YkvT, which exhibit 28% and 30% identity, respectively, with the putative catalytic C-terminal domain of SleB. CwlJ, like SleB, is involved in the later stages of germination (Ishikawa et al., 1998 ), although its effects are less pronounced. The double mutant sleB cwlJ is blocked completely in later germination so that colony formation is not possible. Interestingly, the lack of cortex hydrolysis does not affect the loss of optical density or dipicolinic acid release, indicating that cortex hydrolysis and release of small solutes during germination are probably two separate events (Sekiguchi et al., 1995
; Atrih et al., 1996
, 1998
; Popham et al., 1996b
). However, unlike sleB, cwlJ and ykvT do not occur in an operon with a homologue of ypeB. The observation that the phenotype of the sleB or ypeB mutants are identical suggests that ypeB is not involved in the expression or the function of cwlJ. The genes sleB and cwlJ differ in their compartment-specific regulation during sporulation; cwlJ is transcribed by E
E RNA polymerase in the mother cell (Ishikawa et al., 1998
), and this work demonstrates that sleB expression is dependent on
G, a forespore-specific sigma factor. Expression of sleB has also recently been shown to be controlled by
G using primer extension analysis (Moriyama et al., 1999
).
The mechanism of SleB and CwlJ activation and their molecular interplay, as an integral part of the germination triggering response, is currently under investigation.
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ACKNOWLEDGEMENTS |
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Received 27 August 1999;
revised 20 September 1999;
accepted 23 September 1999.