Synthetic Lethality of the lytE cwlO Genotype in Bacillus subtilis Is Caused by Lack of d,l-Endopeptidase Activity at the Lateral Cell Wall (original) (raw)

Abstract

Bacterial peptidoglycan acts as an exoskeleton to protect the bacterial cell. Although peptidoglycan biosynthesis by penicillin-binding proteins is well studied, few studies have described peptidoglycan disassembly, which is necessary for a dynamic structure that allows cell growth. In Bacillus subtilis, more than 35 genes encoding cell wall lytic enzymes have been identified; however, only two d,l-endopeptidases (lytE and cwlO) are involved in cell proliferation. In this study, we demonstrated that the d,l-endopeptidase activity at the lateral cell wall is essential for cell proliferation. Inactivation of LytE and CwlO by point mutation of the catalytic residues caused cell growth defects. However, the forced expression of LytF or CwlS, which are paralogs of LytE, did not suppress lytE cwlO synthetic lethality. Subcellular localization studies of these d,l-endopeptidases showed LytF and CwlS at the septa and poles, CwlO at the cylindrical part of the cell, and LytE at the septa and poles as well as the cylindrical part. Furthermore, construction of N-terminal and C-terminal domain-swapped enzymes of LytE, LytF, CwlS, and CwlO revealed that localization was dependent on the N-terminal domains. Only the chimeric proteins that were enzymatically active and localized to the sidewall were able to suppress the synthetic lethality, suggesting that the lack of d,l-endopeptidase activity at the cylindrical part of the cell leads to a growth defect. The functions of LytE and CwlO in cell morphogenesis were discussed.

INTRODUCTION

Autolysins are bacterial cell wall lytic enzymes found in all bacteria that possess peptidoglycan. In the Bacillus subtilis genome, more than 35 definite or probable autolysin genes have been identified and shown to be involved in cell morphogenesis, cannibalism, sporulation, and germination (22, 25). The bacterial peptidoglycan sacculus requires a dynamic structure for cell elongation and separation; therefore, a balance between peptidoglycan synthesis and disassembly is essential for cell proliferation. Although a number of autolysins are thought to be involved in peptidoglycan disassembly, none have been found to be essential for cell growth, perhaps due to their functional redundancy. However, it was recently reported that disruption of both lytE and cwlO in B. subtilis is lethal (4). To date, this is the sole report of an autolysin mutant of B. subtilis with a serious growth defect. Bisicchia et al. (4) also demonstrated that cwlO depletion in a _lytE_-disrupted background strain impairs cell elongation.

LytE and CwlO are d,l-endopeptidases that hydrolyze the linkage of d-γ-glutamyl-_meso_-diaminopimelic acid in peptidoglycan (13, 27). The B. subtilis genome contains seven d,l-endopeptidase genes. The mature forms of LytE, LytF, and CwlS all contain N-terminal LysM repeats, although the number of LysM domains differs, and C-terminal d,l-endopeptidase domains belonging to the NlpC/P60 family. Although phenotypes of single-gene knockout mutants were indistinguishable from that of the wild type, multiple gene disruptions led to a chained-cell morphology (10, 13, 19), suggesting that these proteins are involved in cell separation. In contrast, CwlO contains a domain with unknown function at the N terminus and a d,l-endopeptidase domain at the C terminus. The phenotype of the cwlO mutant was also indistinguishable from that of the wild type, but the lytE cwlO double disruption leads to synthetic lethality (4, 27). Two d,l-endopeptidase genes (pgdS and cwlT) are not likely to be involved in cell morphology, because the pgdS gene encodes a poly-γ-glutamic acid degradase, and the cwlT gene is part of an integrative and conjugative element (11, 23). The other gene is a function-unknown ykfC. Results of these previous studies indicate that LytE, LytF, and CwlS are cell separation enzymes, and LytE and CwlO are associated with cell growth. Thus, although their catalytic domains show high amino acid sequence similarity, these enzymes play different physiological roles in cell morphology. To elucidate the roles of LytE and CwlO in cell morphogenesis, we investigated the main factors causing synthetic lethality in B. subtilis.

MATERIALS AND METHODS

Bacterial strains and plasmids.

The bacterial strains, plasmids, and primers used in this study are listed in Table 1 and Tables S1 and S2 in the supplemental material, respectively. B. subtilis 168 was used as the parent strain throughout this study. The details of the strains and plasmids constructs used in this study are presented in the supplemental material. All constructed strains were confirmed by PCR.

Table 1.

Bacterial strains used in this study

General methods.

The B. subtilis and Escherichia coli strains were grown at 37°C in Luria broth (LB) (21). When required, antibiotics and chemical inducers were added in the following concentrations: ampicillin, 100 μg/ml; tetracycline, 5 μg/ml; kanamycin, 25 μg/ml; spectinomycin, 50 μg/ml; erythromycin, 0.3 μg/ml chloramphenicol, 5 μg/ml; and IPTG (isopropyl β-d-1-thiogalactopyranoside), 1 mM; and xylose, 1%.

DNA manipulation and E. coli transformation were performed using standard methods (21). B. subtilis transformation was performed by conventional transformation procedures (1).

Sample preparation for IFM.

Cells harvested from an overnight culture in LB medium were diluted 50-fold in 5 ml of fresh LB medium. The cells were grown to the late exponential growth phase (optical density at 600 nm [OD600] = 2.0), and then the precultured cells were inoculated into fresh LB medium to give an initial absorbance equivalent to an OD600 of 0.001. Cells corresponding to 0.3 of the OD600 unit for WECLytE6FL (LytE-6×FLAG), OH015 (CWBLytE-6×FLAG), WECO6FL (CwlO-6×FLAG), OH013 (overexpressed CwlO-6×FLAG), or OH018 (overexpressed NTDCwlO-6×FLAG) were collected when each culture reached an OD600 of 0.1. As described below, LytE-6×FLAG and CwlO-6×FLAG were functional for B. subtilis cell proliferation. Likewise, cells corresponding to 0.3 of the OD600 unit were collected for WECLytF6FL (LytF-6×FLAG) and OH014 (CWBLytF-6×FLAG) when the cultures reached an OD600 of 0.6. Similarly, cells corresponding to 0.3 of the OD600 unit were collected for WECS6FL (CwlS-6×FLAG) and OH016 (CWBCwlS-6×FLAG) when each culture reached an OD600 of 2.0. To determine the subcellular localization of the domain-swapped chimeric enzymes, cells were collected when the cultures reached an OD600 of 0.3 (for chimeric proteins transcribed from the lytE promoter) or an OD600 of 0.1 (for those transcribed from the cwlO promoter). Cell samples were prepared for immunofluorescence microscopy (IFM) as described previously (30).

Fluorescence microscopy.

Fluorescence microscopy was performed as described previously (29) with an Olympus BX61 microscope equipped with a BX-UCB control unit, a UPPlan Apo Fluorite phase-contrast objective (×100 magnification; numerical aperture, 1.3), and a standard rhodamine filter set for visualizing Cy3. Exposure times were 0.1 s for phase-contrast microscopy and 0.1 s (gain 2) for Cy3. The cells were photographed with a charge-coupled-device camera (CoolSNAP HQ; Nippon Roper) driven by MetaMorph software (version 4.6; Universal Imaging). For Cy3 imaging, out-of-focus light was removed using the two-dimensional deconvolution utility of the AutoDeblur software. All images were processed with Adobe Photoshop software.

Western blot analysis and zymography.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed with 14% (wt/vol) polyacrylamide gels as described previously (15). For Western blot analysis, the 6×FLAG-fused proteins were separated by 14% SDS-PAGE gels. After electrophoresis, the proteins were transferred to polyvinylidene fluoride membranes (Invitrogen) in a transfer buffer (25 mM Tris, 192 mM glycine, 20% [vol/vol] methanol, 0.1% SDS) by using a semidry blotting system (Bio-Rad). Immunoblot detection was carried out as described in the instruction manual for the ECL Plus Western blotting detection system (Invitrogen) using a mouse anti-FLAG M2 monoclonal antibody (Sigma) and horseradish peroxidase-labeled anti-mouse IgG antibody. Zymography was performed as described previously using 14% SDS-PAGE gels containing 0.5 mg/ml B. subtilis cell wall extract (17). The cell wall derived from B. subtilis 168 was prepared as described previously (8, 19). Renaturation was performed at 37°C in a renaturation solution (25 mM Tris-HCl [pH 7.2], 1% [vol/vol] Triton X-100) as described previously (10).

RESULTS

d,l-Endopeptidase activity of LytE or CwlO is essential for cell proliferation.

The catalytic domains of LytE and CwlO belong to the NlpC/P60 family, which hydrolyzes the γ-d-glutamyl-_meso_-diaminopimelic acid linkage or _N_-acetylmuramoyl-l-alanine linkage. In this superfamily of papain-like enzymes, a conserved cysteine residue was predicted to be a catalytic residue on amino acid sequence alignment (2, 16). Recently, the three-dimensional structures of NlpC/P60 enzymes were reported (Spr from E. coli, ABA23003 from Anabaena variabilis, and ACC79413 from Nostoc punctiforme) (3, 26). In these enzymes, the conserved cysteine residues are located at a predicted active site and are structurally conserved. To determine whether the conserved cysteine residues are involved in the catalytic activity of d,l-endopeptidases, we constructed point mutations in LytE and CwlO, replacing the conserved cysteine residue with a serine residue (LytEC247S and CwlOC377S). To evaluate the lytic activities of these mutated enzymes, the intact or mutated catalytic domains of LytE and CwlO were expressed in E. coli, and zymography was carried out with the cell lysates by using B. subtilis cell wall as a substrate (see Fig. S1B in the supplemental material). The intact catalytic domains of LytE and CwlO exhibited cell wall-degrading activity, but mutants in which the cysteine residue had been replaced appeared to be inactive. This finding suggests that the conserved cysteine residue is important for the catalytic activity of NlpC/P60 enzymes.

Next, we examined whether the d,l-endopeptidase activities of LytE and CwlO are involved in the synthetic lethality of the lytE cwlO double mutants (Fig. 1A and B). OH004 (_lytE-6×FLAG P_xyl-cwlO) grew normally without xylose induction of CwlO, indicating that LytE-6×FLAG was functional. In contrast, the growth of OH005 [lytE(C247S)_-_6×FLAG P_xyl-cwlO_] was normal in the presence of xylose but was arrested in the absence of xylose. Similarly, CwlO-6×FLAG was functional, but OH007 [cwlO(C377S)_-_6×FLAG P_spac-lytE_] showed growth arrest without LytE induction by IPTG. These results indicate that the d,l-endopeptidase activity of either LytE or CwlO is essential for cell proliferation.

Fig 1.

d,l-Endopeptidase activity of LytE and CwlO is important for cell proliferation, and LytF or CwlS induction could not suppress lytE cwlO synthetic lethality. Strains were precultured with the appropriate inducer until late exponential phase (OD600 = 2.0). An aliquot of each culture was washed and inoculated into fresh medium with or without the inducer to an OD600 of 0.01. The × symbol in panels A to D indicates the wild-type 168 strain. (A) Growth of OH005 [lytE(C247S)-6×FLAG P_xyl-cwlO; open circles] and OH004 (lytE_-6×FLAG P_xyl-cwlO; closed circles). Xylose (1%) was added to the preculture, but CwlO expression was not induced by xylose in the main culture. (B) Growth of OH007 [cwlO(C377S)-6×FLAG P_spac-lytE; open circles] and OH006 (cwlO_-6×FLAG P_spac-lytE; closed circles). IPTG (1 mM) was added to the preculture, but LytE expression was not induced by IPTG in the main culture. (C) Growth of OH009 (Δ_lytE P_xyl-cwlO P_spac-lytF_). The strain was cultured with 1 mM IPTG to induce LytF expression and with 1% xylose to induce CwlO induction (closed circles) or without xylose (open circles). (D) Growth of OH012 (Δ_lytE_ P_xyl-cwlO_ P_spac-cwlS_). The strain was cultured with 1 mM IPTG to induce CwlS expression and with 1% xylose to induce CwlO expression (closed circles) or without xylose (open circles).

As described above, LytE, LytF, and CwlS exhibit similar domain structures. However, lytE expression is regulated by σA and σH, cwlO expression is regulated by σA, and lytF and cwlS are regulated by σD and σH, respectively (5, 13, 19, 27). The σD and σH regulons are induced later than the σA regulon. Therefore, although LytF and CwlS can suppress the synthetic lethality, the LytE CwlO double-depleted cells may be dead before LytF or CwlS can be expressed. Consequently, OH009 (Δ_lytE_ P_xyl-cwlO_ P_spac-lytF_) and OH012 (Δ_lytE_ P_xyl-cwlO_ P_spac-cwlS_) were constructed to determine whether induction of LytF or CwlS could suppress the synthetic lethality. These strains were cultured in the presence of 1 mM IPTG to induce LytF or CwlS and in the presence or absence of 1% xylose to induce CwlO (Fig. 1C and D). Both strains grew normally when CwlO was expressed; however, growth was arrested by CwlO depletion, even though LytF or CwlS was expressed. The hydrolytic activities of induced LytF and CwlS were confirmed by zymography with B. subtilis cell wall as a substrate (see Fig. S2 in the supplemental material). We found that LytF and CwlS are not able to suppress the LytE CwlO-depleted synthetic lethality, even though their domain structures are similar to that of LytE.

Subcellular localization of B. subtilis d,l-endopeptidases.

The C-terminal d,l-endopeptidase domains of LytE, LytF, CwlS, and CwlO show strong sequence similarity. In contrast, the N-terminal domains of LytE, LytF, and CwlS contain different numbers of the LysM repeats, and the N terminus of CwlO contains a COG3883 domain. Although the d,l-endopeptidase activity of either LytE or CwlO is essential for cell proliferation, forced expression of LytF or CwlS did not suppress the lytE cwlO synthetic lethality. These results suggest that the N-terminal domains are important for the function of the d,l-endopeptidases. Previously, we reported that B. subtilis WE1, a strain with defects in extracellular proteases WprE and Epr, accumulates d,l-endopeptidases on the cell surface (29). Therefore, we evaluated the subcellular localization of FLAG-tagged LytE, LytF, CwlS, and CwlO (full-length proteins and N-terminal domains) by IFM with _wprE epr_-deleted WEC background strains. Because these d,l-endopeptidases are regulated by different σ factors, we also evaluated the localization of these enzymes during different growth phases. Full-length LytE and CwlO and their N-terminal domains (CWBLytE and NTDCwlO, respectively) were observed during early exponential growth phase (OD600 = 0.1), full-length LytF and its N-terminal domain (CWBLytF) were observed in mid-exponential growth phase (OD600 = 0.6), and full-length CwlS and its N-terminal domain (CWBCwlS) were observed in early stationary phase (OD600 = 2.0). The results showed that LytE is localized at the cell septa, poles, and sidewall (Fig. 2A). LytF-6×FLAG and CwlS-6×FLAG were localized at the cell septa and poles, but neither was detected at the lateral cell wall (Fig. 2C and E). CwlO-6×FLAG expressed from the intact promoter was weakly detected at the lateral cell wall but not at the septa or poles (Fig. 2G). To better assess CwlO localization, we then used a CwlO-6×FLAG-overexpressing strain (Fig. 2H), which increased cell surface CwlO-6×FLAG expression to 2.4 times that of normal, as determined by Western blot analysis (data not shown). The overexpressed CwlO-6×FLAG was more clearly visualized at the sidewall but not detected at the cell septa or poles. To determine whether the localization of these d,l-endopeptidases depends on the N-terminal domain, we investigated the subcellular localization of the N-terminal domains under the same conditions used for the full-length proteins (Fig. 2B, D, F, and I). The localization pattern of each N-terminal domain was identical to that of the corresponding full-length protein, indicating that these d,l-endopeptidases localized on the cell surface through their N-terminal domains.

Fig 2.

Subcellular localization of full-length d,l-endopeptidases and their N-terminal domains. Phase-contrast and immunofluorescence microscopy analysis of FLAG-tagged proteins. The OD600 values at the sampling times were 0.1 for LytE and CwlO and their N-terminal domains (CWBLytE and NTDCwlO, respectively), 0.6 for LytF and its N-terminal domain (CWBLytF), and 2.0 for CwlS and its N-terminal domain (CWBCwlS). (A) WECLytE6FL (LytE-6×FLAG); (B) OH015 (CWBLytE-6×FLAG); (C) WECLytF6FL (LytF-6×FLAG); (D) OH014 (CWBLytF-6×FLAG); (E) WECS6FL (CwlS-6×FLAG); (F) OH016 (CWBCwlS-6×FLAG); (G) WECO6FL (CwlO-6×FLAG); (H) OH013 (overexpressed CwlO-6×FLAG); and (I) OH018 (overexpressed NTDCwlO-6×FLAG). Bars = 5 μm.

Characterization of domain-swapped d,l-endopeptidases.

IFM analysis demonstrated that LytF and CwlS (involved in cell separation) localize to the septa and poles, CwlO (involved in cell elongation) localizes to the lateral cell wall, and LytE (involved both in cell separation and elongation) localizes to the septa, poles, and lateral cell wall. These results suggest that the functions of these d,l-endopeptidases depend on their subcellular localization. To test this hypothesis, we generated domain-swapped d,l-endopeptidases and examined their ability to suppress the lytE cwlO synthetic lethality.

Domain-swapped d,l-endopeptidases (other than NLytFCLytE) were generated by C-terminal domain substitution at the original genetic loci of the N-terminal domains. For example, NLytECCwlS was constructed by substituting the C-terminal domain of LytE with that of CwlS at the lytE locus. Thus, the chimeric genes were transcribed from the promoters of the gene encoding the N-terminal domain. However, NLytFCLytE was constructed by substituting the N-terminal domain of LytE with that of LytF at the lytE locus; the chimeric gene was transcribed from the lytE promoter. All chimeric proteins were fused to a 6×FLAG tag at the C terminus to evaluate their expression and localization. Expression was confirmed by Western blot analysis, and the chimeric proteins were detected at positions corresponding to the predicted molecular sizes (Fig. 3A). Enzyme activity was assessed by zymography using the B. subtilis cell wall as a substrate (Fig. 3B). The results show that the chimeric enzymes containing the CwlO N-terminal domain did not retain cell wall-degrading activity. The C-terminal d,l-endopeptidase regions of NCwlOCLytF and NCwlOCCwlS are the same as those of NLytECLytF and NLytECCwlS, respectively. Since NLytECLytF and NLytECCwlS exhibited cell wall-degrading activity, it was assumed that the C-terminal d,l-endopeptidase domains of NCwlOCLytF and NCwlOCCwlS would exhibit enzyme activity as well; however, it is possible that the N-terminal region of CwlO interfered with the C-terminal d,l-endopeptidase domain activity in NCwlOCLytF and NCwlOCCwlS. Next, the subcellular localization of these domain-swapped d,l-endopeptidases was visualized by IFM (Fig. 4). The chimeric proteins containing the LytE N-terminal domain (NLytECLytF and NLytECCwlS) localized to the cell septa, poles, and lateral cell wall, similar to the localization of LytE-6×FLAG and CWBLytE-6×FLAG. However, NLytFCLytE localized only to the cell septa and poles, like LytF-6×FLAG and CWBLytF-6×FLAG. Only weak fluorescence of the chimeric enzymes containing the N-terminal domain of CwlO (NCwlOCLytF and NCwlOCCwlS) was detected. However, enhancing the signal intensity of IFM images revealed that these chimeric enzymes were localized to the sidewall, similar to full-length CwlO and its N-terminal domain. These results demonstrate that the N-terminal domains of d,l-endopeptidases determine their subcellular localization. Finally, we assessed whether these domain-swapped d,l-endopeptidases were able to suppress the lytE cwlO synthetic lethality (Fig. 4). The transcription of cwlO was induced by xylose in strains expressing LytE or LytF N-terminal domain-containing chimeric enzymes (NLytECLytF, NLytECCwlS, or NLytFCLytE), whereas lytE gene transcription was induced by IPTG in strains expressing the CwlO N-terminal domain-containing chimeric enzymes (NCwlOCLytF and NCwlOCCwlS). After exposure to the appropriate inducer, an aliquot of each culture was washed to remove the inducer, and the cells were inoculated into fresh medium with or without the inducer. OH019 (lytE::NLytECLytF P_xyl-cwlO_) and OH020 (lytE::NLytECCwlS P_xyl-cwlO_) were found to partially suppress the lytE cwlO synthetic lethality without xylose induction of cwlO. As described above, these chimeric proteins were enzymatically active and detected at the cell septa, poles, and sidewall. However, strains expressing chimeric proteins containing the CwlO N-terminal domain (OH023 [cwlO::NCwlOCLytF P_spac-lytE_] and OH024 [cwlO::NCwlOCCwlS P_spac-lytE_]), which were not enzymatically active, were localized at the lateral cell wall but not able to grow without IPTG induction of lytE. Furthermore, lack of xylose caused the growth arrest of OH022 (lytE::NLytFCLytE P_xyl-cwlO_). This strain expressed NLytFCLytE, which retained enzymatic activity but was not localized at the cellular sidewall.

Fig 3.

Expression and activity of domain-swapped d,l-endopeptidases. Strains were exposed to 1% xylose or 1 mM IPTG for 2 h to induce P_xyl-cwlO_ and P_spac-lytE_ expression, respectively. Lanes: 1, OH019 (NLytECLytF P_xyl-cwlO_, 41 kDa); 2, OH020 (NLytECCwlS P_xyl-cwlO_, 40 kDa); 3, OH022 (NLytFCLytE P_xyl-cwlO_, 53 kDa); 4, OH023 (NCwlOCLytF P_spac-lytE_, 55 kDa); and 5, OH024 (NCwlOCCwlS P_spac-lytE_, 56 kDa). (A) Domain-swapped d,l-endopeptidases were evaluated by Western blot analysis with an anti-FLAG antibody. Degraded products of the chimeric enzymes appear in lanes 4 and 5. (B) Zymography of the chimeric enzymes using B. subtilis cell wall as a substrate. Asterisks indicate clear zones produced by the chimeric enzymes.

Fig 4.

Subcellular localization of domain-swapped d,l-endopeptidases and suppression of the lytE cwlO synthetic lethality by these proteins. For microscopic imaging, OH019 (lytE::NLytECLytF P_xyl-cwlO_), OH020 (lytE::NLytECCwlS P_xyl-cwlO_), and OH022 (lytE::NLytFCLytE P_xyl-cwlO_) were cultured with 1% xylose to induce CwlO, and OH023 (cwlO::NCwlOCLytF P_spac-lytE_) and OH024 (cwlO::NCwlOCCwlS P_spac-lytE_) were cultured with 1 mM IPTG to induce LytE. For suppression assays, the strains were grown under the same conditions as those described in Fig. 1. They were cultured with xylose (closed circles) or without xylose (open circles) for P_xyl-cwlO_ and IPTG for P_spac-lytE_. The × symbol indicates the wild-type 168 strain. Bars = 5 μm.

Taken together, our findings show that only strains expressing at least one active d,l-endopeptidase localized at the lateral cell wall were able to proliferate. Therefore, we conclude that localization of d,l-endopeptidase activity at the lateral cell wall is essential for cell proliferation.

DISCUSSION

Peptidoglycan forms a network on the outer surface of bacterial cells. The dynamic structure of the peptidoglycan sacculus allows cell growth; therefore, maintaining the balance of peptidoglycan synthesis and disassembly is important. To the best of our knowledge, the synthetic lethality of lytE cwlO in B. subtilis is the only report of an autolysin mutant with a serious growth defect (4). In this study, we found that subcellular localization of these enzymes is determined by their N-terminal domains and that synthetic lethality is caused by the lack of d,l-endopeptidase activity at the lateral cell wall. The d,l-endopeptidases required for cell separation (LytE, LytF, and CwlS) were detected at the septa and poles, and the enzymes involved in cell elongation (LytE and CwlO) were detected at the cylindrical part of the cell. These results strongly suggest that the function of these autolysins depends on their subcellular localization. Our findings are consistent with a previous study reporting that a lytF cwlO double mutant and a lytE lytF cwlS triple mutant were not defective in cell growth (10, 27).

LytE and CwlO may participate in loosening the peptidoglycan sacculus of B. subtilis during growth. The cell wall of B. subtilis is comprised of multilayered thick peptidoglycan. Electron microscopy images show that the thick peptidoglycan consists of three distinct parts (18). Results of pulse-labeling studies revealed a delay between the incorporation of new material into the cell wall and its eventual appearance in the culture (12, 20). These results suggest that the inner zone of the thick peptidoglycan contains the newly synthesized layers and that the outer zone consists of old peptidoglycan (i.e., inside-to-outside peptidoglycan sacculus formation) (12, 18, 20). Peptidoglycan-synthesizing enzymes are anchored to cytoskeleton proteins (MreB homologs and FtsZ) and localize to the outside surface of the cytoplasmic membrane (6). Thus, the peptidoglycan-synthesizing enzymes are accessible to the inner zone of peptidoglycan. Degradation of the outer zone loosens the cell wall, enabling construction of a new peptidoglycan layer inside the preexisting peptidoglycan sacculus (22). Since lytE cwlO double disruption leads to synthetic lethality and impaired cell elongation, these autolysins are strong candidates for participation in the peptidoglycan dynamics. Consistent with this hypothesis, our results show that the cell elongation defect due to the lytE cwlO disruption is caused by the absence of d,l-endopeptidase activity at the lateral cell wall. However, results of a pulse-labeling experiment show that the rate of _N_-acetylglucosamine incorporation is not the same for lytE and cwlO mutants, demonstrating that LytE behavior differs from that of CwlO (4). LytE and CwlO differ in their subcellular localizations and specific activities (28). In addition, CwlO was rapidly degraded and released into culture medium, whereas most of LytE adsorbed to cell surface (27). Taken together, these findings demonstrate that although these two enzymes possess similar d,l-endopeptidase domains, they appear to have different functions in cell growth.

A previous study reported that LytE-3×FLAG transcribed from the lytE original promoter was observed at the septa and poles (29). However, slightly overexpressed LytE fused to a green fluorescent protein localized in a helical manner along the cylindrical wall of growing cells in addition to the poles and septa (7). In the present study, we observed the localization of 6 ×FLAG-tagged LytE transcribed from the original lytE promoter by IFM (Fig. 2A). The fluorescence intensity of the 6×FLAG fusion protein is greater than that of the 3×FLAG fusion protein, which may be the reason we were able to detect LytE-6×FLAG at the sidewall. The work of Carballido-Lopez et al. (7) strongly suggests that LytE-GFP is localized at the sidewall in a helical manner, similar to the localization pattern of MreB homologs. CwlO-6×FLAG also localized to the lateral cell wall but was not detected at the cell poles or septa (Fig. 2G). Although the fluorescence of the 6×FLAG-tagged CwlO was weak, staggered spots around the sidewall suggested a helical localization pattern. We then investigated whether MreB homologs are involved in the lateral localization of CwlO; however, the mutation of MreB homologs did not alter CwlO localization (data not shown).

Subcellular localization of the N-terminal domains of the four d,l-endopeptidases was similar to that of the corresponding full-length protein, suggesting that localization was determined by their N-terminal domains. This finding was supported by the localization of chimeric enzymes, which was similar to that of their N-terminal domains. The localization of the LytF N-terminal domain at the cell poles and septa was previously reported (30). As expected, the localization of LytE and CwlS was dependent on their N-terminal domains, which contained LysM repeats like that of LytF. Yamamoto et al. (30) also reported a helical localization of LytF-6×FLAG at the sidewall after partial removal of wall teichoic acid, suggesting that the cylindrical localization of N-terminal domains of LytE and CwlS are regulated by wall teichoic acid. Carballido-Lopez et al. (7) reported that LytE localization at the sidewall is dependent on MreBH, indicating that MreBH may regulate wall teichoic acid localization. It was reported that the helical localization of the major wall teichoic acid synthesis proteins was not altered in three mreB homolog single mutants (9). However, we note that these cells were cultured with 20 mM MgCl2, which suppresses mreB homolog deficiency (14).

The CwlO N terminus contains a COG3883 domain, which is an uncharacterized conserved domain in bacteria. According to Teng et al. (24), a secreted antigen (SagA) from Enterococcus faecium containing a COG3883 domain showed broad-spectrum binding to extracellular matrix proteins such as fibrinogen, collagen type I, collagen type IV, fibronectin, and laminin. However, full-length CwlO and its N-terminal domain did not bind some of the matrix proteins evaluated in this study (data not shown). The SagA protein migrated more slowly on cell wall-containing PAGE than on SDS-PAGE, suggesting an interaction between SagA and the cell wall (24); however, the purified CwlO protein did not bind to the cell wall in vitro (27). In the present study, we demonstrated the involvement of the CwlO N-terminal domain in cell surface localization. Taken together, these results suggest that CwlO interacts directly, but weakly, with the cell wall or a cell surface protein.

In this study, we found that the subcellular localization of LytE, LytF, CwlS, and CwlO is dependent on their N-terminal domains and that d,l-endopeptidase activity at the lateral cell wall is essential for cell proliferation. These results strongly suggest that LytE and CwlO are involved in cell elongation and support the inside-to-outside model for peptidoglycan sacculus formation. A more detailed study is necessary to clarify the role of d,l-endopeptidases in peptidoglycan dynamics and to characterize the localization mechanisms of these proteins.

Supplementary Material

Supplemental material

ACKNOWLEDGMENTS

We thank the members of our group, particularly Hiroki Yamamoto and Tatsuya Fukushima, for the helpful advice and discussion. We also thank N. Hariyama and Y. Miyake for technical assistance with strain construction and microscopy analysis.

This work was supported by Grants-in-Aid for Scientific Research (B) (19380047) and (A) (22248008), the New Energy and Industrial Department Organization (NEDO), the Global COE programs (J.S.), and the Program for Dissemination of Tenure-Track System funded by the Ministry of Education and Science, Japan (M.H.).

Footnotes

Published ahead of print 2 December 2011

Supplemental material for this article may be found at http://jb.asm.org/.

REFERENCES

• 1.Anagnostopoulos C, Spizizen J. 1961. Requirements for transformation in Bacillus subtilis. J. Bacteriol. 81:741–746 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 2.Anantharaman V, Aravind L. 2003. Evolutionary history, structural features and biochemical diversity of the NlpC/P60 superfamily of enzymes. Genome Biol. 4:R11. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 3.Aramini JM, et al. 2008. Solution NMR structure of the NlpC/P60 domain of lipoprotein Spr from Escherichia coli: structural evidence for a novel cysteine peptidase catalytic triad. Biochemistry 47:9715–9717 [DOI] [PubMed] [Google Scholar]
• 4.Bisicchia P, et al. 2007. The essential YycFG two-component system controls cell wall metabolism in Bacillus subtilis. Mol. Microbiol. 65:180–200 [DOI] [PubMed] [Google Scholar]
• 5.Britton RA, et al. 2002. Genome-wide analysis of the stationary phase sigma factor (sigma-H) regulon of Bacillus subtilis. J. Bacteriol. 184:4881–4890 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 6.Cabeen MT, Jacobs-Wagner C. 2005. Bacterial cell shape. Nat. Rev. Microbiol. 3:601–610 [DOI] [PubMed] [Google Scholar]
• 7.Carballido-Lopez R, et al. 2006. Actin homolog MreBH governs cell morphogenesis by localization of the cell wall hydrolase LytE. Dev. Cell 11:399–409 [DOI] [PubMed] [Google Scholar]
• 8.Fein JE, Rogers HJ. 1976. Autolytic enzyme-deficient mutants of Bacillus subtilis 168. J. Bacteriol. 127:1427–1442 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 9.Formstone A, Carballido-Lopez R, Noirot P, Errington J, Scheffers DJ. 2008. Localization and interactions of teichoic acid synthetic enzymes in Bacillus subtilis. J. Bacteriol. 190:1812–1821 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 10.Fukushima T, et al. 2006. A new d,l-endopeptidase gene product, YojL (renamed CwlS), plays a role in cell separation with LytE and LytF in Bacillus subtilis. J. Bacteriol. 188:5541–5550 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 11.Fukushima T, et al. 2008. Identification and characterization of novel cell wall hydrolase CwIT: a two-domain autolysin exhibiting _N_-acetylmuramidase and dl-endopeptidase activities. J. Biol. Chem. 283:11117–11125 [DOI] [PubMed] [Google Scholar]
• 12.Graham LL, Beveridge TJ. 1994. Structural differentiation of the Bacillus subtilis 168 cell wall. J. Bacteriol. 176:1413–1421 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 13.Ishikawa S, Hara Y, Ohnishi R, Sekiguchi J. 1998. Regulation of a new cell wall hydrolase gene, cwlF, which affects cell separation in Bacillus subtilis. J. Bacteriol. 180:2549–2555 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 14.Kawai Y, Asai K, Errington J. 2009. Partial functional redundancy of MreB isoforms, MreB, Mbl and MreBH, in cell morphogenesis of Bacillus subtilis. Mol. Microbiol. 73:719–731 [DOI] [PubMed] [Google Scholar]
• 15.Laemmli UK. 1970. Cleavage of structural proteins during assembly of head of bacteriophage T4. Nature 227:680–685 [DOI] [PubMed] [Google Scholar]
• 16.Layec S, Decaris B, Leblond-Bourget N. 2008. Characterization of proteins belonging to the CHAP related superfamily within the firmicutes. J. Mol. Microbiol. Biotechnol. 14:31–40 [DOI] [PubMed] [Google Scholar]
• 17.Leclerc D, Asselin A. 1989. Detection of bacterial cell wall hydrolases after denaturing polyacrylamide gel electrophoresis. Can. J. Microbiol. 35:749–753 [DOI] [PubMed] [Google Scholar]
• 18.Merad T, Archibald AR, Hancock IC, Harwood CR, Hobot JA. 1989. Cell wall assembly in Bacillus subtilis visualization of old and new wall material by electron microscopic examination of samples stained selectively for teichoic acid and teichuronic acid. J. Gen. Microbiol. 135:645–655 [DOI] [PubMed] [Google Scholar]
• 19.Ohnishi R, Ishikawa S, Sekiguchi J. 1999. Peptidoglycan hydrolase LytF plays a role in cell separation with Cw1F during vegetative growth of Bacillus subtilis. J. Bacteriol. 181:3178–3184 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 20.Pooley HM. 1976. Layered distribution, according to age, within cell wall of Bacillus subtilis. J. Bacteriol. 125:1139–1147 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 21.Sambrook J, Fritch EF, Maniatis T. 1989. Molecular cloning: a laboratory manual, 2nd ed Cold Spring Harbor Laboratory, Cold Spring Harbor, NY [Google Scholar]
• 22.Smith TJ, Blackman SA, Foster SJ. 2000. Autolysins of Bacillus subtilis: multiple enzymes with multiple functions. Microbiology 146:249–262 [DOI] [PubMed] [Google Scholar]
• 23.Suzuki T, Tahara Y. 2003. Characterization of the Bacillus subtilis_ywtD_ gene, whose product is involved in gamma-polyglutamic acid degradation. J. Bacteriol. 185:2379–2382 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 24.Teng F, Kawalec M, Weinstock GM, Hryniewicz W, Murray BE. 2003. An Enterococcus faecium secreted antigen, SagA, exhibits broad spectrum binding to extracellular matrix proteins and appears essential for E. faecium growth. Infect. Immun. 71:5033–5041 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 25.Vollmer W, Joris B, Charlier P, Foster S. 2008. Bacterial peptidoglycan (murein) hydrolases. FEMS Microbiol. Rev. 32:259–286 [DOI] [PubMed] [Google Scholar]
• 26.Xu QP, et al. 2009. Structural basis of murein peptide specificity of a gamma-d-glutamyl-l-diamino acid endopeptidase. Structure 17:303–313 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 27.Yamaguchi H, Furuhata K, Fukushima T, Yamamoto H, Sekiguchi J. 2004. Characterization of a new Bacillus subtilis peptidoglycan hydrolase gene, yvcE (named cwlO), and the enzymatic properties of its encoded protein. J. Biosci. Bioeng. 98:174–181 [DOI] [PubMed] [Google Scholar]
• 28.Yamamoto H, et al. 2008. Post-translational control of vegetative cell separation enzymes through a direct interaction with specific inhibitor IseA in Bacillus subtilis. Mol. Microbiol. 70:168–182 [DOI] [PubMed] [Google Scholar]
• 29.Yamamoto H, Kurosawa S, Sekiguchi J. 2003. Localization of the vegetative cell wall hydrolases LytC, LytE, and LytF on the Bacillus subtilis cell surface and stability of these enzymes to cell wall bound or extracellular proteases. J. Bacteriol. 185:6666–6677 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 30.Yamamoto H, Miyake Y, Hisaoka M, Kurosawa SI, Sekiguchi J. 2008. The major and minor wall teichoic acids prevent the sidewall localization of vegetative dl-endopeptidase LytF in Bacillus subtilis. Mol. Microbiol. 70:297–310 [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental material