Synthetic lethality of the lytE cwlO genotype in Bacillus subtilis is caused by lack of D,L-endopeptidase activity at the lateral cell wall - PubMed (original) (raw)

Synthetic lethality of the lytE cwlO genotype in Bacillus subtilis is caused by lack of D,L-endopeptidase activity at the lateral cell wall

Masayuki Hashimoto et al. J Bacteriol. 2012 Feb.

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.

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Figures

Fig 1

Fig 1

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-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).

Fig 2

Fig 2

Subcellular localization of full-length

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-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.

Fig 3

Fig 3

Expression and activity of domain-swapped

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-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

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-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

Fig 4

Subcellular localization of domain-swapped

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-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.

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