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Peptidoglycan crosslinking relaxation plays an important role in Staphylococcus aureus WalKR-dependent cell viability.

Delaune A, Poupel O, Mallet A, Coic YM, Msadek T, Dubrac S - PLoS ONE (2011)

Bottom Line: Uncoupled expression of genes encoding lytic transglycosylases or amidases did not restore growth to a WalKR-depleted strain.Neither of these two genes are essential under our conditions and a ΔlytM ΔssaA mutant does not present any growth defect.Taken together, our results strongly suggest that peptidoglycan crosslinking relaxation through crossbridge hydrolysis plays a crucial role in the essential requirement of the WalKR system for cell viability.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Biology of Gram-Positive Pathogens, Department of Microbiology, Paris, France.

ABSTRACT
The WalKR two-component system is essential for viability of Staphylococcus aureus, a major pathogen. We have shown that WalKR acts as the master controller of peptidoglycan metabolism, yet none of the identified regulon genes explain its requirement for cell viability. Transmission electron micrographs revealed cell wall thickening and aberrant division septa in the absence of WalKR, suggesting its requirement may be linked to its role in coordinating cell wall metabolism and cell division. We therefore tested whether uncoupling autolysin gene expression from WalKR-dependent regulation could compensate for its essential nature. Uncoupled expression of genes encoding lytic transglycosylases or amidases did not restore growth to a WalKR-depleted strain. We identified only two WalKR-regulon genes whose expression restored cell viability in the absence of WalKR: lytM and ssaA. Neither of these two genes are essential under our conditions and a ΔlytM ΔssaA mutant does not present any growth defect. LytM is a glycyl-glycyl endopeptidase, hydrolyzing the pentaglycine interpeptide crossbridge, and SsaA belongs to the CHAP amidase family, members of which such as LysK and LytA have been shown to have D-alanyl-glycyl endopeptidase activity, cleaving between the crossbridge and the stem peptide. Taken together, our results strongly suggest that peptidoglycan crosslinking relaxation through crossbridge hydrolysis plays a crucial role in the essential requirement of the WalKR system for cell viability.

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Overexpression of lytM or ssaA restores cell viability but not morphology to cells lacking the WalK/WalR TCS.Bacteria were grown in TSB CdCl2 0.25 µM. To produce the WalKR system in derivatives of S. aureus strain ST1000 carrying a Pspac-walRK chromosomal fusion and the indicated plasmids, 1 mM IPTG was added to the culture (walRK on). In the absence of IPTG, cells were depleted for WalKR (walRK off). A) Transmission electron microscopy ultrastructure of strains overproducing SsaA or LytM. Cells were grown in TSB-CdCl2 +/− 1 mM IPTG, harvested at an OD600nm = 1 (corresponding to exponential phase for the culture with IPTG and cessation of growth for the culture without IPTG) and embedded in thin sections for ultrastructure examination by transmission electron microscopy. Bars represent 1 µm for the upper panels and 2 µm for the lower panels. B) Viability assay of strains overproducing SsaA or LytM using the LIVE/DEAD BacLightTM assay. Two hours after reaching stationary phase, 10 ml of culture were sampled for fluorescent staining. For the control ST1012 strain without IPTG, the sample was taken 4 hours after cessation of growth following WalKR-depletion. Live bacteria appear green, following SYTO-9 staining, and dead cells are stained in red due to penetration of propidium iodide through compromised membranes.
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pone-0017054-g005: Overexpression of lytM or ssaA restores cell viability but not morphology to cells lacking the WalK/WalR TCS.Bacteria were grown in TSB CdCl2 0.25 µM. To produce the WalKR system in derivatives of S. aureus strain ST1000 carrying a Pspac-walRK chromosomal fusion and the indicated plasmids, 1 mM IPTG was added to the culture (walRK on). In the absence of IPTG, cells were depleted for WalKR (walRK off). A) Transmission electron microscopy ultrastructure of strains overproducing SsaA or LytM. Cells were grown in TSB-CdCl2 +/− 1 mM IPTG, harvested at an OD600nm = 1 (corresponding to exponential phase for the culture with IPTG and cessation of growth for the culture without IPTG) and embedded in thin sections for ultrastructure examination by transmission electron microscopy. Bars represent 1 µm for the upper panels and 2 µm for the lower panels. B) Viability assay of strains overproducing SsaA or LytM using the LIVE/DEAD BacLightTM assay. Two hours after reaching stationary phase, 10 ml of culture were sampled for fluorescent staining. For the control ST1012 strain without IPTG, the sample was taken 4 hours after cessation of growth following WalKR-depletion. Live bacteria appear green, following SYTO-9 staining, and dead cells are stained in red due to penetration of propidium iodide through compromised membranes.

Mentions: As shown above, WalKR-depleted cells exhibit abnormal division septa and a rougher cell surface. We therefore examined the morphology of cells overproducing LytM or SsaA in the absence of WalKR, comparing strains ST1012 (pCN51), ST1002 (pSD3-13) and ST1123 (pAD12) by TEM. As shown in Fig. 5A, overexpression of lytM or ssaA only partially restored cell morphology: although cell wall thickness was diminished, cells presented a separation defect and some abnormal and misplaced division septa remained present in WalKR-depleted LytM- or SsaA-overproducing strains even though growth was indistinguishable from that of cells producing WalKR. In order to test the viability of these abnormal cells, we used fluorescence microscopy and the Live/Dead BacLight™ bacterial viability assay on stationary phase cultures of the ST1000 strain carrying either the pCN51 control plasmid or the pSD3-13 and pAD12 plasmids allowing overexpression of lytM or ssaA, respectively. As expected, viability of strain ST1000 carrying the pCN51 vector remains strictly dependent on WalKR, and almost all of the cells died when starved for WalKR (red staining, Fig. 5B upper panel). As shown in Fig. 5B, the ST1000/pSD3-13 or pAD12 strains were still viable in the absence of WalKR (green stain) but formed aggregates characteristic of bacteria with a cell separation defect (upper panel). This defect is not linked to lytM or ssaA overexpression since when the WalKR system was produced, the cells were well separated (lower panel).


Peptidoglycan crosslinking relaxation plays an important role in Staphylococcus aureus WalKR-dependent cell viability.

Delaune A, Poupel O, Mallet A, Coic YM, Msadek T, Dubrac S - PLoS ONE (2011)

Overexpression of lytM or ssaA restores cell viability but not morphology to cells lacking the WalK/WalR TCS.Bacteria were grown in TSB CdCl2 0.25 µM. To produce the WalKR system in derivatives of S. aureus strain ST1000 carrying a Pspac-walRK chromosomal fusion and the indicated plasmids, 1 mM IPTG was added to the culture (walRK on). In the absence of IPTG, cells were depleted for WalKR (walRK off). A) Transmission electron microscopy ultrastructure of strains overproducing SsaA or LytM. Cells were grown in TSB-CdCl2 +/− 1 mM IPTG, harvested at an OD600nm = 1 (corresponding to exponential phase for the culture with IPTG and cessation of growth for the culture without IPTG) and embedded in thin sections for ultrastructure examination by transmission electron microscopy. Bars represent 1 µm for the upper panels and 2 µm for the lower panels. B) Viability assay of strains overproducing SsaA or LytM using the LIVE/DEAD BacLightTM assay. Two hours after reaching stationary phase, 10 ml of culture were sampled for fluorescent staining. For the control ST1012 strain without IPTG, the sample was taken 4 hours after cessation of growth following WalKR-depletion. Live bacteria appear green, following SYTO-9 staining, and dead cells are stained in red due to penetration of propidium iodide through compromised membranes.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3046168&req=5

pone-0017054-g005: Overexpression of lytM or ssaA restores cell viability but not morphology to cells lacking the WalK/WalR TCS.Bacteria were grown in TSB CdCl2 0.25 µM. To produce the WalKR system in derivatives of S. aureus strain ST1000 carrying a Pspac-walRK chromosomal fusion and the indicated plasmids, 1 mM IPTG was added to the culture (walRK on). In the absence of IPTG, cells were depleted for WalKR (walRK off). A) Transmission electron microscopy ultrastructure of strains overproducing SsaA or LytM. Cells were grown in TSB-CdCl2 +/− 1 mM IPTG, harvested at an OD600nm = 1 (corresponding to exponential phase for the culture with IPTG and cessation of growth for the culture without IPTG) and embedded in thin sections for ultrastructure examination by transmission electron microscopy. Bars represent 1 µm for the upper panels and 2 µm for the lower panels. B) Viability assay of strains overproducing SsaA or LytM using the LIVE/DEAD BacLightTM assay. Two hours after reaching stationary phase, 10 ml of culture were sampled for fluorescent staining. For the control ST1012 strain without IPTG, the sample was taken 4 hours after cessation of growth following WalKR-depletion. Live bacteria appear green, following SYTO-9 staining, and dead cells are stained in red due to penetration of propidium iodide through compromised membranes.
Mentions: As shown above, WalKR-depleted cells exhibit abnormal division septa and a rougher cell surface. We therefore examined the morphology of cells overproducing LytM or SsaA in the absence of WalKR, comparing strains ST1012 (pCN51), ST1002 (pSD3-13) and ST1123 (pAD12) by TEM. As shown in Fig. 5A, overexpression of lytM or ssaA only partially restored cell morphology: although cell wall thickness was diminished, cells presented a separation defect and some abnormal and misplaced division septa remained present in WalKR-depleted LytM- or SsaA-overproducing strains even though growth was indistinguishable from that of cells producing WalKR. In order to test the viability of these abnormal cells, we used fluorescence microscopy and the Live/Dead BacLight™ bacterial viability assay on stationary phase cultures of the ST1000 strain carrying either the pCN51 control plasmid or the pSD3-13 and pAD12 plasmids allowing overexpression of lytM or ssaA, respectively. As expected, viability of strain ST1000 carrying the pCN51 vector remains strictly dependent on WalKR, and almost all of the cells died when starved for WalKR (red staining, Fig. 5B upper panel). As shown in Fig. 5B, the ST1000/pSD3-13 or pAD12 strains were still viable in the absence of WalKR (green stain) but formed aggregates characteristic of bacteria with a cell separation defect (upper panel). This defect is not linked to lytM or ssaA overexpression since when the WalKR system was produced, the cells were well separated (lower panel).

Bottom Line: Uncoupled expression of genes encoding lytic transglycosylases or amidases did not restore growth to a WalKR-depleted strain.Neither of these two genes are essential under our conditions and a ΔlytM ΔssaA mutant does not present any growth defect.Taken together, our results strongly suggest that peptidoglycan crosslinking relaxation through crossbridge hydrolysis plays a crucial role in the essential requirement of the WalKR system for cell viability.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Biology of Gram-Positive Pathogens, Department of Microbiology, Paris, France.

ABSTRACT
The WalKR two-component system is essential for viability of Staphylococcus aureus, a major pathogen. We have shown that WalKR acts as the master controller of peptidoglycan metabolism, yet none of the identified regulon genes explain its requirement for cell viability. Transmission electron micrographs revealed cell wall thickening and aberrant division septa in the absence of WalKR, suggesting its requirement may be linked to its role in coordinating cell wall metabolism and cell division. We therefore tested whether uncoupling autolysin gene expression from WalKR-dependent regulation could compensate for its essential nature. Uncoupled expression of genes encoding lytic transglycosylases or amidases did not restore growth to a WalKR-depleted strain. We identified only two WalKR-regulon genes whose expression restored cell viability in the absence of WalKR: lytM and ssaA. Neither of these two genes are essential under our conditions and a ΔlytM ΔssaA mutant does not present any growth defect. LytM is a glycyl-glycyl endopeptidase, hydrolyzing the pentaglycine interpeptide crossbridge, and SsaA belongs to the CHAP amidase family, members of which such as LysK and LytA have been shown to have D-alanyl-glycyl endopeptidase activity, cleaving between the crossbridge and the stem peptide. Taken together, our results strongly suggest that peptidoglycan crosslinking relaxation through crossbridge hydrolysis plays a crucial role in the essential requirement of the WalKR system for cell viability.

Show MeSH
Related in: MedlinePlus