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A Staphylococcus aureus ypfP mutant with strongly reduced lipoteichoic acid (LTA) content: LTA governs bacterial surface properties and autolysin activity.

Fedtke I, Mader D, Kohler T, Moll H, Nicholson G, Biswas R, Henseler K, Götz F, Zähringer U, Peschel A - Mol. Microbiol. (2007)

Bottom Line: The ypfP gene responsible for biosynthesis of a glycolipid found in LTA was deleted in Staphylococcus aureus SA113, causing 87% reduction of the LTA content.However, the autolytic activity of the mutant was strongly reduced demonstrating a role of LTA in controlling autolysin activity.Moreover, the hydrophobicity of the LTA mutant was altered and its ability to form biofilms on plastic was completely abrogated indicating a profound impact of LTA on physicochemical properties of bacterial surfaces.

View Article: PubMed Central - PubMed

Affiliation: Cellular and Molecular Microbiology Division, University of Tübingen, Department of Medical Microbiology and Hygiene, 72076 Tübingen, Germany.

ABSTRACT
Many Gram-positive bacteria produce lipoteichoic acid (LTA) polymers whose physiological roles have remained a matter of debate because of the lack of LTA-deficient mutants. The ypfP gene responsible for biosynthesis of a glycolipid found in LTA was deleted in Staphylococcus aureus SA113, causing 87% reduction of the LTA content. Mass spectrometry and nuclear magnetic resonance spectroscopy revealed that the mutant LTA contained a diacylglycerol anchor instead of the glycolipid, whereas the remaining part was similar to the wild-type polymer except that it was shorter. The LTA mutant strain revealed no major changes in patterns of cell wall proteins or autolytic enzymes compared with the parental strain indicating that LTA may be less important in S. aureus protein attachment than previously thought. However, the autolytic activity of the mutant was strongly reduced demonstrating a role of LTA in controlling autolysin activity. Moreover, the hydrophobicity of the LTA mutant was altered and its ability to form biofilms on plastic was completely abrogated indicating a profound impact of LTA on physicochemical properties of bacterial surfaces. We propose to consider LTA and its biosynthetic enzymes as targets for new antibiofilm strategies.

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Impact of ypfP deletion on cell wall proteins. A. Surface protein patterns of cells grown to logarithmic (left) or stationary (right) phase. SA113 wild-type and SA113 ΔypfP::ermB mutant strains did not differ in overall amounts of cell wall proteins. Ten micrograms of surface proteins of the indicated SA113 strains were separated in 10% polyacrylamide gels and stained with Coomassie brilliant blue R-250. B. Zymographic analyses. Surface proteins (20 μg) of SA113 log-phase bacteria were separated in 10% SDS-polyacrylamide gels containing heat-killed Micrococcus luteus (M. luteus), S. aureus SA113 wild type (wt) or S. aureus SA113 ΔypfP::ermB mutant cells (ΔypfP). Active bacteriolytic enzymes appear as clear zones in the opaque gels. The inverse pictures are shown. C. Spontaneous bacterial autolysis. Data represent the means ± SD of three independent experiments.
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fig07: Impact of ypfP deletion on cell wall proteins. A. Surface protein patterns of cells grown to logarithmic (left) or stationary (right) phase. SA113 wild-type and SA113 ΔypfP::ermB mutant strains did not differ in overall amounts of cell wall proteins. Ten micrograms of surface proteins of the indicated SA113 strains were separated in 10% polyacrylamide gels and stained with Coomassie brilliant blue R-250. B. Zymographic analyses. Surface proteins (20 μg) of SA113 log-phase bacteria were separated in 10% SDS-polyacrylamide gels containing heat-killed Micrococcus luteus (M. luteus), S. aureus SA113 wild type (wt) or S. aureus SA113 ΔypfP::ermB mutant cells (ΔypfP). Active bacteriolytic enzymes appear as clear zones in the opaque gels. The inverse pictures are shown. C. Spontaneous bacterial autolysis. Data represent the means ± SD of three independent experiments.

Mentions: Lipoteichoic acid has been assumed to serve as a scaffold structure for anchoring surface proteins non-covalently to the cell wall (Navarre and Schneewind, 1999; Neuhaus and Baddiley, 2003). This hypothesis prompted us to compare the overall patterns of SA113 wild-type and mutant cell wall-associated proteins. Surface proteins were isolated by gentle treatment of osmotically stabilized bacterial cells with the cell wall-degrading enzyme lysostaphin and subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Only minor changes were detected while the major protein bands were unaffected, irrespective of whether proteins from logarithmic or stationary growth phases were compared (Fig. 7A).


A Staphylococcus aureus ypfP mutant with strongly reduced lipoteichoic acid (LTA) content: LTA governs bacterial surface properties and autolysin activity.

Fedtke I, Mader D, Kohler T, Moll H, Nicholson G, Biswas R, Henseler K, Götz F, Zähringer U, Peschel A - Mol. Microbiol. (2007)

Impact of ypfP deletion on cell wall proteins. A. Surface protein patterns of cells grown to logarithmic (left) or stationary (right) phase. SA113 wild-type and SA113 ΔypfP::ermB mutant strains did not differ in overall amounts of cell wall proteins. Ten micrograms of surface proteins of the indicated SA113 strains were separated in 10% polyacrylamide gels and stained with Coomassie brilliant blue R-250. B. Zymographic analyses. Surface proteins (20 μg) of SA113 log-phase bacteria were separated in 10% SDS-polyacrylamide gels containing heat-killed Micrococcus luteus (M. luteus), S. aureus SA113 wild type (wt) or S. aureus SA113 ΔypfP::ermB mutant cells (ΔypfP). Active bacteriolytic enzymes appear as clear zones in the opaque gels. The inverse pictures are shown. C. Spontaneous bacterial autolysis. Data represent the means ± SD of three independent experiments.
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Related In: Results  -  Collection

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fig07: Impact of ypfP deletion on cell wall proteins. A. Surface protein patterns of cells grown to logarithmic (left) or stationary (right) phase. SA113 wild-type and SA113 ΔypfP::ermB mutant strains did not differ in overall amounts of cell wall proteins. Ten micrograms of surface proteins of the indicated SA113 strains were separated in 10% polyacrylamide gels and stained with Coomassie brilliant blue R-250. B. Zymographic analyses. Surface proteins (20 μg) of SA113 log-phase bacteria were separated in 10% SDS-polyacrylamide gels containing heat-killed Micrococcus luteus (M. luteus), S. aureus SA113 wild type (wt) or S. aureus SA113 ΔypfP::ermB mutant cells (ΔypfP). Active bacteriolytic enzymes appear as clear zones in the opaque gels. The inverse pictures are shown. C. Spontaneous bacterial autolysis. Data represent the means ± SD of three independent experiments.
Mentions: Lipoteichoic acid has been assumed to serve as a scaffold structure for anchoring surface proteins non-covalently to the cell wall (Navarre and Schneewind, 1999; Neuhaus and Baddiley, 2003). This hypothesis prompted us to compare the overall patterns of SA113 wild-type and mutant cell wall-associated proteins. Surface proteins were isolated by gentle treatment of osmotically stabilized bacterial cells with the cell wall-degrading enzyme lysostaphin and subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Only minor changes were detected while the major protein bands were unaffected, irrespective of whether proteins from logarithmic or stationary growth phases were compared (Fig. 7A).

Bottom Line: The ypfP gene responsible for biosynthesis of a glycolipid found in LTA was deleted in Staphylococcus aureus SA113, causing 87% reduction of the LTA content.However, the autolytic activity of the mutant was strongly reduced demonstrating a role of LTA in controlling autolysin activity.Moreover, the hydrophobicity of the LTA mutant was altered and its ability to form biofilms on plastic was completely abrogated indicating a profound impact of LTA on physicochemical properties of bacterial surfaces.

View Article: PubMed Central - PubMed

Affiliation: Cellular and Molecular Microbiology Division, University of Tübingen, Department of Medical Microbiology and Hygiene, 72076 Tübingen, Germany.

ABSTRACT
Many Gram-positive bacteria produce lipoteichoic acid (LTA) polymers whose physiological roles have remained a matter of debate because of the lack of LTA-deficient mutants. The ypfP gene responsible for biosynthesis of a glycolipid found in LTA was deleted in Staphylococcus aureus SA113, causing 87% reduction of the LTA content. Mass spectrometry and nuclear magnetic resonance spectroscopy revealed that the mutant LTA contained a diacylglycerol anchor instead of the glycolipid, whereas the remaining part was similar to the wild-type polymer except that it was shorter. The LTA mutant strain revealed no major changes in patterns of cell wall proteins or autolytic enzymes compared with the parental strain indicating that LTA may be less important in S. aureus protein attachment than previously thought. However, the autolytic activity of the mutant was strongly reduced demonstrating a role of LTA in controlling autolysin activity. Moreover, the hydrophobicity of the LTA mutant was altered and its ability to form biofilms on plastic was completely abrogated indicating a profound impact of LTA on physicochemical properties of bacterial surfaces. We propose to consider LTA and its biosynthetic enzymes as targets for new antibiofilm strategies.

Show MeSH
Related in: MedlinePlus