Abstract
Sacculus is a peptidoglycan (PG) matrix that protects bacteria from osmotic lysis. In Gram-positive organisms, the sacculus is densely functionalized with glycopolymers important for survival, but the way in which assembly occurs is not known. In Staphylococcus aureus, three LCP (LytR-CpsA-Psr) family members have been implicated in attaching the major glycopolymer wall teichoic acid (WTA) to PG, but ligase activity has not been demonstrated for these or any other LCP proteins. Using WTA and PG substrates produced chemoenzymatically, we show that all three proteins can transfer WTA precursors to nascent PGs, establishing that LCP proteins are PG–glycopolymer ligases. Although all S. aureus LCP proteins have the capacity to attach WTA to PG, we show that their cellular functions are not redundant. Strains lacking lcpA have phenotypes similar to those of WTA-null strains, indicating that this is the most important WTA ligase. This work provides a foundation for studying how LCP enzymes participate in cell wall assembly.
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Acknowledgements
We would like to thank V. Haunreiter (Department of Molecular and Cellular Biology, Harvard University) for her generous gift of the strains J126, JH100, RH53, RH72, PS60, PS109, and PS47 (refs. 19, 42), S. Trauger and the Bauer Core Facilities for assistance with MS, and the T. Pang (Harvard Medical School, Department of Microbiology and Immunology) and the Bernhardt lab for their gift of the plasmid pTP63. This research was supported by the NIH (P01AI083214 and R01 AI099144 to S.W.; R01 GM066174 to D.K.; R01 GM076710 to D.K. and S.W.).
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S.W., D.K., K.S., and L.M.M. designed experiments. K.S. prepared wall teichoic acid substrates, and peptidoglycan substrates were prepared with help from Y.Q.; L.M.M. performed the Tn-seq experiments; K.S. and L.M.M. prepared the spot dilution assays and the MIC experiments; K.S. and L.M.M. made strains used in this study except those received from others as noted (Supplementary Table 5); K.S. cloned, expressed, and purified all LCP proteins and performed reconstitution experiments; S.W. designed and supervised the project; S.W., D.K., and K.S. wrote the manuscript with input from all authors.
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Schaefer, K., Matano, L., Qiao, Y. et al. In vitro reconstitution demonstrates the cell wall ligase activity of LCP proteins. Nat Chem Biol 13, 396–401 (2017). https://doi.org/10.1038/nchembio.2302
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DOI: https://doi.org/10.1038/nchembio.2302
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