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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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S. aureus starved for WalK/WalR display thickened cell walls and aberrant division septa.Strain ST1000 carrying a chromosomal Pspac-walRK fusion was grown in TSB +/− 1 mM IPTG, harvested at 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. Panel A: ST1000 grown with 1 mM IPTG. Panel B: ST1000 grown without IPTG. Bars represent 0.5 µm for the upper panels and 2 µm for the lower panels. Black arrows indicate aberrant division septa.
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pone-0017054-g001: S. aureus starved for WalK/WalR display thickened cell walls and aberrant division septa.Strain ST1000 carrying a chromosomal Pspac-walRK fusion was grown in TSB +/− 1 mM IPTG, harvested at 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. Panel A: ST1000 grown with 1 mM IPTG. Panel B: ST1000 grown without IPTG. Bars represent 0.5 µm for the upper panels and 2 µm for the lower panels. Black arrows indicate aberrant division septa.

Mentions: In order to identify morphological changes associated with WalKR-depletion, we used electron microscopy to examine S. aureus cells where the walRK operon is placed under the control of an IPTG-dependent inducible promoter (strain ST1000, Pspac-walRK) [12], grown in the presence or absence of IPTG. Although no gross morphological differences were observed by scanning electron microscopy (data not shown), ultrastructural analysis by transmission electron microscopy (TEM) showed significant changes. Indeed, when WalKR is produced, the ST1000 strain displays the typical diplococcal S. aureus morphology, with a single central division septum (Fig. 1A). However, as shown in Fig. 1B, WalKR-depleted cells exhibit abnormal and misplaced division septa, with the formation of several new septa before separation of the daughter cells (indicated by arrows, Fig. 1B). WalKR-depleted cells also displayed a rougher cell surface, with amorphous “fuzzy” extracellular material, and increased cell wall thickness, almost twice that of cells producing WalKR (58.56 +/− 12.05 nm vs. 32.64 +/− 4.54 nm, respectively, Student t test P-value <0.05) (Fig. 1A and 1B). Interestingly, all of these phenotypes are strikingly similar to those described for S. aureus atlA sle1 mutants [21] and VISA (vancomycin intermediate S. aureus) strains with reduced susceptibility to vancomycin [22].


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)

S. aureus starved for WalK/WalR display thickened cell walls and aberrant division septa.Strain ST1000 carrying a chromosomal Pspac-walRK fusion was grown in TSB +/− 1 mM IPTG, harvested at 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. Panel A: ST1000 grown with 1 mM IPTG. Panel B: ST1000 grown without IPTG. Bars represent 0.5 µm for the upper panels and 2 µm for the lower panels. Black arrows indicate aberrant division septa.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3046168&req=5

pone-0017054-g001: S. aureus starved for WalK/WalR display thickened cell walls and aberrant division septa.Strain ST1000 carrying a chromosomal Pspac-walRK fusion was grown in TSB +/− 1 mM IPTG, harvested at 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. Panel A: ST1000 grown with 1 mM IPTG. Panel B: ST1000 grown without IPTG. Bars represent 0.5 µm for the upper panels and 2 µm for the lower panels. Black arrows indicate aberrant division septa.
Mentions: In order to identify morphological changes associated with WalKR-depletion, we used electron microscopy to examine S. aureus cells where the walRK operon is placed under the control of an IPTG-dependent inducible promoter (strain ST1000, Pspac-walRK) [12], grown in the presence or absence of IPTG. Although no gross morphological differences were observed by scanning electron microscopy (data not shown), ultrastructural analysis by transmission electron microscopy (TEM) showed significant changes. Indeed, when WalKR is produced, the ST1000 strain displays the typical diplococcal S. aureus morphology, with a single central division septum (Fig. 1A). However, as shown in Fig. 1B, WalKR-depleted cells exhibit abnormal and misplaced division septa, with the formation of several new septa before separation of the daughter cells (indicated by arrows, Fig. 1B). WalKR-depleted cells also displayed a rougher cell surface, with amorphous “fuzzy” extracellular material, and increased cell wall thickness, almost twice that of cells producing WalKR (58.56 +/− 12.05 nm vs. 32.64 +/− 4.54 nm, respectively, Student t test P-value <0.05) (Fig. 1A and 1B). Interestingly, all of these phenotypes are strikingly similar to those described for S. aureus atlA sle1 mutants [21] and VISA (vancomycin intermediate S. aureus) strains with reduced susceptibility to vancomycin [22].

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