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PG composition can vary considerably between species. The Gram-positive pathogen\n                    <jats:italic>Staphylococcus aureus<\/jats:italic>\n                    possesses highly crosslinked PG due to the presence of cross bridges containing five glycines, which are synthesised by the FemXAB protein family. FemX adds the first glycine of the cross bridge, while FemA and FemB add the second and the third, and the fourth and the fifth glycines, respectively. Of these, FemX was reported to be essential. To investigate the essentiality of FemAB, we constructed a conditional\n                    <jats:italic>S. aureus<\/jats:italic>\n                    mutant of the\n                    <jats:italic>femAB<\/jats:italic>\n                    operon. Depletion of\n                    <jats:italic>femAB<\/jats:italic>\n                    was lethal, with cells appearing as pseudomulticellular forms that eventually lyse due to extensive membrane rupture. This deleterious effect was mitigated by drastically increasing the osmolarity of the medium, indicating that pentaglycine crosslinks are required for\n                    <jats:italic>S. aureus<\/jats:italic>\n                    cells to withstand internal turgor. Despite the absence of canonical membrane targeting domains, FemA has been shown to localise at the membrane. To study its mechanism of localisation, we constructed mutants in key residues present in the putative transferase pocket and the \u03b16 helix of FemA, possibly involved in tRNA binding. Mutations in the \u03b16 helix led to a sharp decrease in protein activity\n                    <jats:italic>in vivo<\/jats:italic>\n                    and\n                    <jats:italic>in vitro<\/jats:italic>\n                    but did not impair correct membrane localisation, indicating that FemA activity is not required for localisation. Our data indicates that, contrarily to what was previously thought,\n                    <jats:italic>S. aureus<\/jats:italic>\n                    cells do not survive in the absence of a pentaglycine cross bridge.\n                  <\/jats:p>","DOI":"10.1038\/s41598-019-41461-1","type":"journal-article","created":{"date-parts":[[2019,3,21]],"date-time":"2019-03-21T07:12:55Z","timestamp":1553152375000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":54,"title":["The pentaglycine bridges of Staphylococcus aureus peptidoglycan are essential for cell integrity"],"prefix":"10.1038","volume":"9","author":[{"given":"Jo\u00e3o M.","family":"Monteiro","sequence":"first","affiliation":[]},{"given":"Gon\u00e7alo","family":"Covas","sequence":"additional","affiliation":[]},{"given":"Daniela","family":"Rausch","sequence":"additional","affiliation":[]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4485-832X","authenticated-orcid":false,"given":"S\u00e9rgio R.","family":"Filipe","sequence":"additional","affiliation":[]},{"given":"Tanja","family":"Schneider","sequence":"additional","affiliation":[]},{"given":"Hans-Georg","family":"Sahl","sequence":"additional","affiliation":[]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7132-8842","authenticated-orcid":false,"given":"Mariana G.","family":"Pinho","sequence":"additional","affiliation":[]}],"member":"297","published-online":{"date-parts":[[2019,3,21]]},"reference":[{"key":"41461_CR1","doi-asserted-by":"publisher","first-page":"629","DOI":"10.1038\/nrmicro2200","volume":"7","author":"HF Chambers","year":"2009","unstructured":"Chambers, H. 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