| Edited by Fujio Kawamura. Ryuichi Moriyama: Corresponding author. E-mail: moriyama@isc.chubu.ac.jp |
Bacterial spore germination, defined as the irreversible loss of spore dormancy, is triggered by specific germinants and proceeds through a set of sequential steps. Spore germination is essential to allow spore outgrowth and the formation of a new vegetative cell; once triggered, this process proceeds in the absence of germinants and germinant-stimulated metabolism. This fact indicates that spore germination is a process controlled by the sequential activation of a set of preexisting germination–related enzymes, but not by protein synthesis (Foster and Johnstone, 1990, Moir and Smith, 1990).
One of the key enzymes involved in germination process is a lytic enzyme specific to an intact spore cortex. Such enzymes had been identified in the spores of Bacillus megaterium KM (GSLE) (Foster and Johnstone, 1987), Bacillus cereus IFO13597 (SleB) (Makino et al., 1994, Moriyama et al., 1996b), Bacillus subtilis 168 (SleB) (Moriyama et al., 1996a) and Clostridium perfringens S40 (SleC) (Miyata et al., 1995a, Miyata et al., 1995b). B. cereus SleB degrades only spore cortex peptidoglycan that contains muramic δ-lactam unique to the cortex, but not the peptidoglycan of vegetative cell walls (Makino et al., 1994, Miyata et al., 1995b). B. subtilis cwlD spores which completely lack muramic δ-lactam in their cortex are unable to complete germination through the blocking of cortex degradation (Sekiguchi et al., 1995, Popham et al., 1996). These results suggest that muramic δ-lactam could be a substrate recognition determinant for SleB (Atrih et al., 1998, Makino and Moriyama, 2002). B. subtilis and B. cereus SleB and C. perfringens SleC contain the characteristic motif consisting of tandem repeat sequences at its N-terminal domain, which is conserved in noncatalytic regions of various peptidoglycan hydrolases with a diversity of substrate specificities (Moriyama et al., 1996b). The motif was suggested to be involved in the recognition of some repeated unit of peptidoglycan of the cell wall and spore cortex (Moriyama et al., 1996b).
The sleB gene, which was shown to be crucial in germination of B. subtilis spores (Moriyama et al., 1996a, Boland et al., 2000), is conserved among other Bacillus species, such as B. cereus, B. anthracis, B. halodurans, B. megaterium, and Oceanobacillus iheyensis (Makino and Moriyama, 2002). B. subtilis sleB is expressed as an operon with a 3’-adjacent ypeB gene in the forespore compartment of sporangia under the control of σG, a sporulation-specific sigma factor (Moriyama et al., 1999). Immunoelectron microscopic localization of B. subtilis and B. cereus SleB proteins just inside the spore coat layer in the dormant spores suggested that the enzymes are translocated across the inner forespore membrane by a secretion signal peptide, and deposited in spore cortex synthesized between the inner and outer forespore membranes during sporulation (Moriyama et al., 1999). However, further investigations should be conducted to obtain an understanding of the molecular mechanism of the subcellular localization of SleB in order to reveal the process by which the molecular construction of germination apparatus during sporulation takes place.
In the present study, we observed the localization patterns of B. subtilis SleB during sporulation by using fusions of N-terminal region of SleB to the green fluorescent protein (GFP), and examined the effects of sporulation defects and a deletion of the tandem repeat sequences of the enzyme on the localization of the fusion. The results suggested that SleB requires the interaction between the motif of the enzyme and muramic δ-lactam of the spore cortex for its proper localization.
The bacterial strains used in this study are listed in Table 1. Escherichia coli and B. subtilis strains were grown on Luria-Bertani broth or agar at 37°C. B. subtilis was transformed as previously described (Moriyama et al., 1999). If necessary, antibiotics were added in the following concentrations: ampicillin (100 μg ml–1), chloramphenicol (5 μg ml–1), tetracycline (20 μg ml–1).
 View Details | Table 1. B. subtilis strains and plasmids |
B. subtilis strains were sporulated at 30°C or 37°C by nutrient exhaustion in Difco sporulation medium (Schaeffer et al., 1965). The spores were harvested and purified by being washed with cold distilled water until the cell debris and vegetative cells had been removed.
Plasmid pHYS108G and pHYS45G were constructed as follows. B. subtilis sleB was obtained by PCR amplification from pBS45H as a DNA segment containing the sleB promoter region and Met-1 to Val-108 or Met-1 to Val-45 of SleB, using as primers oligonucleotides that created a HindIII site at the 5’ end and a SalI site at the 3’ end. The PCR fragments were digested with HindIII and SalI and then ligated to pBluescriptII SK(–) (Stratagene) that had been digested with HindIII and SalI, yielding pBSL108 and pBSL45, respectively. These plasmids were digested with SacI and SalI, and the fragment was ligated to pHYG1 (a vector that contains the gfp gene) (Moriyama et al., 1999) that had been digested with SacI and SalI, yielding pHYS108G and pHYS45G, respectively, which contained the in-frame fusion of gfp to sleB.
To construct the cwlDgerE mutant, we used plasmid pBT374 (Sekiguchi et al., 1995). The plasmid was linearized with SalI and used to transform strain AD17 to chloramphenicol resistance (Cmr) by a double-crossover event, producing strain HF17.
The microscopic fluorescence observations for GFP visualization were performed as described previously (Moriyama et al., 1999).
To investigate the subcellular localization of SleB during sporulation, we fused the coding sequence for GFP in the frame to the 3’ terminus of the sleB gene. The gene for GFP was joined to the 3’ end of the 305-codon sleB open reading frame, and the resultant plasmid was transformed into B. subtilis PY79 with selection for tetracycline resistance. However, when the transformant was sporulated in Difco sporulation medium containing tetracycline at either 30°C or 37°C, no fluorescence was observed in the sporangia (data not shown), possibly because of impaired folding of the GFP moiety. Therefore, we constructed another fusion gene lacking the C-terminal region of the sleB open reading frame. The gene for GFP was joined to the 3’ end of the 108-codon sleB open reading frame, which contains a secretion signal peptide and putative peptidoglycan binding domains (Fig. 1A). The resultant plasmid, pHYS108G, was transformed into B. subtilis PY79 with selection for tetracycline resistance, yielding strain RM1. Strain RM1 was sporulated at 30°C because no fluorescence was observed in sporangia sporulated at 37°C, as reported previously (Moriyama et al., 1999).
 View Details | Fig. 1. (A) Schematic representation of pHYS108G (expressing SleB1-108-GFP) and pHYS45G (expressing SleB-GFP1-45). Shuttle vectors are derived from pHY300PLK (Takara) and constructed as described in Materials and Methods. SS (gray), signal sequence of SleB; gfp (black), gfp gene; SD (white), Shine-Dalgarno sequence. The tandem repeat sequences of SleB are shown in hatched boxes. (B) Protein sequence alignment of N-terminal regions of SleB homologues. B. subtilis (accession no. BAA11473), B. anthracis(accession no. NP_845098), B. cereus (accession no. BAA09800), B. halodurans (accession no. NP_242497), B. megaterium (accession no. BAE02885), Oceanobacillus iheyensis (accession no. P59105). |
As shown in Fig. 2, SleB1-108-GFP exhibited a distinct pattern of the subcellular localization. In sporangia producing the SleB1-108-GFP fusion protein, the fluorescence appeared to surround the forespore as a ring at t24 (24 h after the end of the exponential-growth phase). Phase-contrast microscopic observation suggested that sporulation stage of t24 cells under this condition correspond to that of B. subtilis t7 cells which were sporulated at 37°C without antibiotic (Moriyama et al., 1999, Fukushima et al., 2002). At the point, more than 80% of sporangia possessed refractile forespores, indicating that at least some cortex had formed and that the forespores had partially dehydrated. SleB1-108-GFP was also seen in the majority of free dormant spores at t48. The fluorescence of SleB1-108-GFP was localized around the spore’s periphery, as about 75% of the dormant spores were surrounded by SleB1-108-GFP. Previous works have shown that SleB is translocated by a secretion signal peptide from the forespore compartment and located in the spore cortex of the dormant spore (Moriyama et al., 1999, Chirakkal et al., 2002). Thus, the peripheral location of SleB1-108-GFP suggests the deposition of the fusion protein in the spore cortex synthesized between the inner and the outer forespore membranes, which is consistent with that of wild-type SleB.
 View Details | Fig. 2. Subcellular localization of SleB1-108-GFP in sporulating cells and dormant spores. Cells were sporulated by nutrient exhaustion as described in Materials and methods, and prepared for microscopy at approximately 0 h, 13 h, 24 h and 48 h after the onset of sporulation. Differential interference contrast micrographs (top panels) and fluorescence photographs (bottom panels) of sporulating cells and dormant spores of strain PY79 bearing sleB1-108-gfp (strain RM1) are shown. Phase-contrast microscopic observation of the same samples was also carried out separately (data not shown). The arrowheads indicate the developing forespores in the top panels. Bar, 1 μm. |
As described above, it has been reported that various peptidoglycan hydrolases contain the characteristic tandem repeat sequences in their primary structure known as a putative peptidoglycan binding motif which might be involved in the recognition of substrate (Ghuysen et al., 1994). The motif is located in the N-terminal region of B. cereus and B. subtilis SleBs (Moriyama et al., 1996a, Moriyama et al., 1996b), as well as in the SleB homologs of other Bacillus species (Fig. 1B). To investigate whether the motif of SleB is involved in its localization during sporulation, we made a construct in which gfp was fused to the N-terminal 45 residues of SleB lacking the motif (SleB1-45-GFP), and the resultant plasmid, pHYS45G, was transformed into B. subtilis PY79, yielding strain RM2. In the sporangia of strain RM2, fluorescence encircled the forespore as in the case of SleB1-108-GFP (Fig. 3A). The results suggested that the signal peptides of both fusions are functional during sporulation. Next, we observed the localization patterns of both SleB1-108-GFP and SleB1-45-GFP in the gerE mutant. Since GerE regulates the activation of the late expression of spore coat genes, mutation leads to the production of lysozyme-sensitive spores with aberrant coat ultrastructure (Moir, 1981). In the sporangia of the gerE mutant, the fluorescence of SleB1-108-GFP surrounded the forespore as a ring at t24 (Fig. 3B), and the localization pattern was similar to that in the wild-type strain. However, SleB1-45-GFP showed different localization patterns; the fusion protein diffused in the mother cell cytoplasm at t24, and no fluorescence was detected in the dormant spores (Fig. 3C). These results suggested that the tandem repeat sequences of SleB are crucial in the enzyme localization, and that the coat layer, if it is formed normally, prevents the fusion without the motif from diffusing into a mother cell compartment, possibly as a physical barrier.
 View Details | Fig. 3. Localization of SleB1-108-GFP and SleB1-45-GFP in sporulating cells and dormant spores of wild-type and gerE mutant strain. Sporulation was induced by nutrient exhaustion as described in Materials and methods, and samples were prepared for microscopy at approximately 24 h and 48 h after the onset of sporulation. Differential interference contrast micrographs (top panels) and fluorescence photographs (bottom panels) of sporulating cells and dormant spores of each strains bearing sleB-gfp are shown. (A) Sporulating cells and dormant spores of strain RM2 (sleB1-45-gfp). (B and C) Sporangia of the gerE mutant, strains RM3 (ΔgerE sleB1-108-gfp) (B) and RM4 (ΔgerE sleB1-45-gfp) (C), which synthesized the SleB-GFP fusion protein. Phase-contrast microscopic observation of the same samples was also carried out separately (data not shown). The arrowheads indicate the developing forespores in the top panels. Bar, 1 μm. |
In our previous study, it was shown that SleB degrades only intact cortex but not the muramic δ-lactam-deficient cortex (Makino et al., 1994), suggesting that muramic δ-lactam is a major specific determinant of SleB as a germination lytic enzyme. Therefore, we assumed that muramic δ-lactam might play an important role in the localization process of SleB as well as in the substrate specificity of the enzyme. To investigate this, we observed the localization pattern of SleB1-108-GFP in the cwlD mutant (strain ADD1) which completely lacks muramic δ-lactam in its spore cortex (Sekiguchi et al., 1995, Popham et al., 1996). As discussed above, however, there was a possibility that coat formation could prevent SleB1-108-GFP from dispersing into mother cell compartment, even if the fusion protein could not interact with the cortex peptidoglycan without δ-lactam. In fact, SleB1-108-GFP encircled the forespore at t24 and appeared as a ring around a cwlD spore (Fig. 4A). So we constructed cwlDgerE mutant (strain HF17) which lacks both δ-lactam structure in the spore cortex and many of spore coat proteins to evaluate a net importance of δ-lactam in the localization of SleB during sporulation. Despite the presence of the repeat sequences, the fluorescence of the fusion was only observed in 15% of the sporangia at t24 as a dot in the mother cell cytoplasm of the cwlDgerE mutant and no fluorescence was detected in the dormant spores (Fig. 4B). These results suggested that the synthesis of the muramic δ-lactam during sporulation is crucial for the localization of SleB as well as the putative peptidoglycan binding motif of the enzyme.
 View Details | Fig. 4. Effect of muramic δ-lactam defect on the localization pattern of SleB1-108-GFP. (A and B) Fluorescence of sporulating cells and dormant spores of strains RM5 (ΔcwlD sleB1-108-gfp) (A) and HF18 (ΔcwlDgerE sleB1-108-gfp) (B) that synthesized the fusion protein with the tandem repeat sequences. Phase-contrast microscopic observation of the same samples was also carried out separately (data not shown). Bar, 1 μm. |
In the present work, we demonstrated the subcellular localization of the spore cortex-lytic enzyme SleB during sporulation by using two fusions of N-terminal region of SleB to GFP (SleB1-108-GFP and SleB1-45-GFP). The N-terminal region of SleB contains the repeat sequence motif (Moriyama et al., 1996b). The motif is composed of three -helices (Ghuysen et al., 1994) and classified in the putative peptidoglycan binding domain-1 [Pfam database]. This motif is conserved among many peptidoglycan hydrolases (e.g., SleC from C. perfringens (Miyata et al., 1995a), CwlA from B. subtilis (Kuroda and Sekiguchi, 1990), CwlL from B. licheniformis (Oda et al., 1993), DD-carboxypeptidase from Streptmyces albus (Joris et al., 1983), and autolytic lysozyme from C. acetobutylicum (Croux and Garcia, 1991)). Interestingly, when the importance of the motif in SleB localization was examined, we could discriminate the localization patterns of two fusion proteins in the gerE mutant (Fig. 3B and C) but not in the wild-type. The fluorescence of SleB1-45-GFP was diffused in the mother cell compartment in the gerE mutant (Fig. 3C), so we proposed that the coat layer might serve as a certain physical barrier for the fusion protein, and fluorescent ring formation by SleB1-45-GFP around the forespore of the wild-type (Fig. 3A) could be the artifact. Previously, Moir and Smith described that presumably the coat of the gerE spore is permeable to any soluble enzyme in the cortex that is free to diffuse (Moir, 1981, Moir and Smith, 1990), which is consistent with our results and supports above supposition. It should be noted, however, that SleB1-45-GFP in the gerE mutant after translocation across the inner forespore membrane was not trapped between the inner and outer forespore membrane where the spore peptidoglycan is synthesized. The integrity of the outer forespore membrane, which is ill-defined in electron micrographs, has been questioned (Craft-Lighty and Ellar, 1980) and there are some reports that the outer forespore membrane would not be effective permeability barrier in coat-defective spores (Driks, 1999, Cowan et al., 2004). Taken together with these reports, the diffusion of the fusion protein in the mother cell could be due to the dysfunction of both the outer forespore membrane and the coat layer as a barrier in coat-defective mutant.
The SleB-GFP fusion protein, even though it contained the repeat sequence motif, failed to localize normally in the coat- and muramic δ-lactam-deficient strain (Fig. 4B), suggesting that the interaction between the peptidoglycan binding motif of SleB and the muramic δ-lactam structure in the spore cortex are crucial to the proper localization of SleB. The peptidoglycan binding motif of SleB is conserved many peptidoglycan hydrolases as mentioned above, while each hydrolase varies in the substrate specificity. The germination-related enzymes, such as SleB and SleC, recognize and hydrolyze only peptidoglycan with muramic δ-lactam moiety (Makino et al., 1994, Miyata et al., 1995a). The identification of the residues in the motif of the germination enzymes responsible for the recognition and/or binding to the muramic δ-lactam structure requires further study.
From the results that SleB could not be detected immunochemically in the spore ingredients of ypeB mutant, Chirakkal et al. concluded that YpeB might be required for the localization and/or stabilization of SleB (Chirakkal et al., 2002). However, our results suggested that SleB after translocation across the inner forespore membrane could be localized in the spore cortex mediated by the interaction between the peptidoglycan binding motif of SleB and the muramic δ-lactam in the spore cortex. In addition, SleB possesses a secretion Sec-type signal peptide (Moriyama et al., 1999, Tjalsma et al., 2000), and Imamura et al. have reported that a requisite cortex formation protein, SpoIVH, with a substitution of the SleB signal domain in place of its signal sequence was functional, suggesting that the translocation across the inner forespore membrane could be mediated only by the signal peptide (Imamura et al., 2004). Taken together, it might be possible that SleB could be localized properly in the spore cortex without any assistance from other spore proteins including YpeB. However, our results cannot exclude a possibility of the stabilization of SleB by YpeB. Further studies are needed to elucidate the roles of YpeB during sporulation. It will be also interesting in reference to the activation of SleB during spore germination that exists as a mature protein in the dormant spore.
We thank Adam Driks for providing B. subtilis strains PY79 and AD17, and Junichi Sekiguchi for providing B. subtilis strain ADD1 and plasmid pBT374.
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