{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,28]],"date-time":"2026-08-28T08:19:49Z","timestamp":1787905189351,"version":"build-2784847793"},"reference-count":73,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2026,8,28]],"date-time":"2026-08-28T00:00:00Z","timestamp":1787875200000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2026,8,28]],"date-time":"2026-08-28T00:00:00Z","timestamp":1787875200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"funder":[{"name":"Universit\u00e4tsklinikum Bonn"}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Appl Microbiol Biotechnol"],"abstract":"<jats:sec>\n                    <jats:title>Abstract<\/jats:title>\n                    <jats:p>\n                      The WalRK two-component system monitors cell wall biosynthesis, and its activity decreases when biosynthesis is inhibited. As WalRK is essential for many Gram-positive bacteria, including\n                      <jats:italic>Staphylococcus aureus<\/jats:italic>\n                      and\n                      <jats:italic>Bacillus subtilis<\/jats:italic>\n                      , directly inhibiting WalK would disrupt cell wall biosynthesis and result in cell death. This study presents the construction of new reporter strains with two WalRK-dependent promoters: P\n                      <jats:italic>iseA<\/jats:italic>\n                      and P\n                      <jats:italic>ssaA<\/jats:italic>\n                      . These reporter strains enable indirect in vivo measurement of WalRK activity based on luminescence. We evaluated these reporters in combination with the established P\n                      <jats:italic>liaI<\/jats:italic>\n                      reporter system, which is regulated by LiaRS, in order to improve the identification of cell wall inhibitors during the screening of natural products. The WalRK-dependent P\n                      <jats:italic>iseA<\/jats:italic>\n                      reporter yielded specific responses to almost all known cell wall biosynthesis inhibitors, including \u03b2-lactams, tunicamycin, glycopeptides, daptomycin, and fosfomycin, with no discernible response to other antibiotic classes. Furthermore, combining the P\n                      <jats:italic>liaI<\/jats:italic>\n                      reporter system with the P\n                      <jats:italic>iseA<\/jats:italic>\n                      reporter enhanced the range of potential cell wall inhibitors that could be detected. Deleting the gene of the eukaryotic-like Ser\/Thr kinase PrkC increased the P\n                      <jats:italic>iseA<\/jats:italic>\n                      signal obtained with certain antibiotics, as well as after a longer incubation period. Additionally, deletion of\n                      <jats:italic>prkC<\/jats:italic>\n                      lowered P\n                      <jats:italic>liaI<\/jats:italic>\n                      expression in the presence of lipid II-targeting antibiotics. Screening natural products with an unknown mode of action identified several compounds that elicited vancomycin-like patterns with the reporter systems, indicating potential cell wall inhibitory activity. Overall, combining WalRK and LiaRS reporters enables a rapid, complementary evaluation of cell wall stress responses, providing a powerful method for discovering new cell wall-targeting antibiotics.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Key points<\/jats:title>\n                    <jats:p>\n                      \u2022\n                      <jats:italic>The PiseA\/PssaA reporters enable continuous in vivo observation of WalRK activity<\/jats:italic>\n                    <\/jats:p>\n                    <jats:p>\n                      \u2022\n                      <jats:italic>Cell envelope stress responses are differentially sensed by WalRK and LiaRS<\/jats:italic>\n                    <\/jats:p>\n                    <jats:p>\n                      \u2022\n                      <jats:italic>The PiseA reporter strain detects nearly all cell wall biosynthesis inhibitors<\/jats:italic>\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1007\/s00253-026-14013-7","type":"journal-article","created":{"date-parts":[[2026,8,28]],"date-time":"2026-08-28T07:45:21Z","timestamp":1787903121000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Construction of a novel reporter strain indicating inhibition of cell wall biosynthesis"],"prefix":"10.1007","volume":"110","author":[{"ORCID":"https:\/\/orcid.org\/0009-0000-9804-8173","authenticated-orcid":false,"given":"Maike Laura","family":"Karcher","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Annette","family":"Siskowski","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chantal","family":"M\u00fcller","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Laura","family":"Engel","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vincent","family":"Franz","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1294-0182","authenticated-orcid":false,"given":"Susanne H.","family":"Kirsch","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Birthe","family":"Sandargo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tanja","family":"Schneider","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2933-8473","authenticated-orcid":false,"given":"Gabriele","family":"Bierbaum","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,8,28]]},"reference":[{"key":"14013_CR1","doi-asserted-by":"publisher","first-page":"180","DOI":"10.1111\/j.1365-2958.2007.05782.x","volume":"65","author":"P Bisicchia","year":"2007","unstructured":"Bisicchia P, Noone D, Lioliou E, Howell A, Quigley S, Jensen T, Jarmer H, Devine KM (2007) The essential YycFG two-component system controls cell wall metabolism in Bacillus subtilis. Mol Microbiol 65:180\u2013200. https:\/\/doi.org\/10.1111\/j.1365-2958.2007.05782.x","journal-title":"Mol Microbiol"},{"key":"14013_CR2","doi-asserted-by":"publisher","first-page":"223","DOI":"10.1016\/S0005-2736(99)00208-4","volume":"1462","author":"E Breukink","year":"1999","unstructured":"Breukink E, de Kruijff B (1999) The lantibiotic nisin, a special case or not? Biochim Biophys Acta 1462:223\u2013234. https:\/\/doi.org\/10.1016\/S0005-2736(99)00208-4","journal-title":"Biochim Biophys Acta"},{"key":"14013_CR3","doi-asserted-by":"publisher","first-page":"154","DOI":"10.1128\/aac.42.1.154","volume":"42","author":"H Br\u00f6tz","year":"1998","unstructured":"Br\u00f6tz H, Bierbaum G, Leopold K, Reynolds PE, Sahl HG (1998) The lantibiotic mersacidin inhibits peptidoglycan synthesis by targeting lipid II. Antimicrob Agents Chemother 42:154\u2013160. https:\/\/doi.org\/10.1128\/aac.42.1.154","journal-title":"Antimicrob Agents Chemother"},{"key":"14013_CR4","doi-asserted-by":"publisher","first-page":"734","DOI":"10.1016\/j.chom.2023.04.003","volume":"31","author":"A Bublitz","year":"2023","unstructured":"Bublitz A, Brauer M, Wagner S, Hofer W, M\u00fcsken M, Deschner F, Lesker TR, Neumann-Schaal M, Paul L-S, N\u00fcbel U, Bartel J, Kany AM, Z\u00fchlke D, Bernecker S, Jansen R, Sievers S, Riedel K, Herrmann J, M\u00fcller R, Fuchs TM, Strowig T (2023) The natural product chlorotonil A preserves colonization resistance and prevents relapsing Clostridioides difficile infection. Cell Host Microbe 31:734-750.e8. https:\/\/doi.org\/10.1016\/j.chom.2023.04.003","journal-title":"Cell Host Microbe"},{"key":"14013_CR5","doi-asserted-by":"publisher","first-page":"1267","DOI":"10.1046\/j.1365-2958.2002.03050.x","volume":"45","author":"M Cao","year":"2002","unstructured":"Cao M, Wang T, Ye R, Helmann JD (2002) Antibiotics that inhibit cell wall biosynthesis induce expression of the Bacillus subtilis sigma(W) and sigma(M) regulons. Mol Microbiol 45:1267\u20131276. https:\/\/doi.org\/10.1046\/j.1365-2958.2002.03050.x","journal-title":"Mol Microbiol"},{"key":"14013_CR6","doi-asserted-by":"publisher","first-page":"5879","DOI":"10.1038\/s41467-024-49629-8","volume":"15","author":"M-J Cheng","year":"2024","unstructured":"Cheng M-J, Wu Y-Y, Zeng H, Zhang T-H, Hu Y-X, Liu S-Y, Cui R-Q, Hu C-X, Zou Q-M, Li C-C, Ye W-C, Huang W, Wang L (2024) Asymmetric total synthesis of polycyclic xanthenes and discovery of a WalK activator active against MRSA. Nat Commun 15:5879. https:\/\/doi.org\/10.1038\/s41467-024-49629-8","journal-title":"Nat Commun"},{"key":"14013_CR7","doi-asserted-by":"publisher","first-page":"106","DOI":"10.1016\/j.phytol.2017.04.009","volume":"20","author":"C Chepkirui","year":"2017","unstructured":"Chepkirui C, Matasyoh JC, Decock C, Stadler M (2017) Two cytotoxic triterpenes from cultures of a Kenyan Laetiporus sp. (Basidiomycota). Phytochem Lett 20:106\u2013110. https:\/\/doi.org\/10.1016\/j.phytol.2017.04.009","journal-title":"Phytochem Lett"},{"key":"14013_CR8","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0017054","volume":"6","author":"A Delaune","year":"2011","unstructured":"Delaune A, Poupel O, Mallet A, Coic Y-M, Msadek T, Dubrac S (2011) Peptidoglycan crosslinking relaxation plays an important role in Staphylococcus aureus WalKR-dependent cell viability. PLoS ONE 6:e17054. https:\/\/doi.org\/10.1371\/journal.pone.0017054","journal-title":"PLoS ONE"},{"issue":"4","key":"14013_CR9","doi-asserted-by":"publisher","first-page":"586","DOI":"10.1016\/j.chembiol.2025.03.005","volume":"32","author":"F Deschner","year":"2025","unstructured":"Deschner F, Mostert D, Daniel JM, Voltz A, Schneider DC, Khangholi N, Bartel J, de Carvalho LP, Brauer M, Gorelik TE, Kleeberg C (2025) Natural products chlorotonils exert a complex antibacterial mechanism and address multiple targets. Cell chemical biology. 32(4):586\u2013602. https:\/\/doi.org\/10.1016\/j.chembiol.2025.03.005","journal-title":"Cell chemical biology."},{"key":"14013_CR10","doi-asserted-by":"publisher","first-page":"623","DOI":"10.1016\/j.bbrc.2015.02.159","volume":"459","author":"A Dhiman","year":"2015","unstructured":"Dhiman A, Gopalani M, Bhatnagar R (2015) WalRK two component system of Bacillus anthracis responds to temperature and antibiotic stress. Biochem Biophys Res Commun 459:623\u2013628. https:\/\/doi.org\/10.1016\/j.bbrc.2015.02.159","journal-title":"Biochem Biophys Res Commun"},{"key":"14013_CR11","doi-asserted-by":"publisher","unstructured":"Dietrich A, Gajdiss M, T\u00fcrck M, Monk I, Bierbaum G (2023) Bacterial two component systems: overexpression and purification: in vitro and in vivo inhibitor screens. Methods Mol Biol 2601:313\u2013333. https:\/\/doi.org\/10.1007\/978-1-0716-2855-3_17","DOI":"10.1007\/978-1-0716-2855-3_17"},{"key":"14013_CR12","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.52088","author":"GS Dobihal","year":"2019","unstructured":"Dobihal GS, Brunet YR, Flores-Kim J, Rudner DZ (2019) Homeostatic control of cell wall hydrolysis by the WalRK two-component signaling pathway in Bacillus subtilis. Elife. https:\/\/doi.org\/10.7554\/eLife.52088","journal-title":"Elife"},{"key":"14013_CR13","doi-asserted-by":"publisher","DOI":"10.1128\/jb.00533-21","volume":"204","author":"GS Dobihal","year":"2022","unstructured":"Dobihal GS, Flores-Kim J, Roney IJ, Wang X, Rudner DZ (2022) The WalR-WalK signaling pathway modulates the activities of both CwlO and LytE through control of the peptidoglycan deacetylase PdaC in Bacillus subtilis. J Bacteriol 204:e0053321. https:\/\/doi.org\/10.1128\/jb.00533-21","journal-title":"J Bacteriol"},{"key":"14013_CR14","doi-asserted-by":"publisher","first-page":"716","DOI":"10.1111\/mmi.12586","volume":"92","author":"J Dom\u00ednguez-Escobar","year":"2014","unstructured":"Dom\u00ednguez-Escobar J, Wolf D, Fritz G, H\u00f6fler C, Wedlich-S\u00f6ldner R, Mascher T (2014) Subcellular localization, interactions and dynamics of the phage-shock protein-like Lia response in Bacillus subtilis. Mol Microbiol 92:716\u2013732. https:\/\/doi.org\/10.1111\/mmi.12586","journal-title":"Mol Microbiol"},{"key":"14013_CR15","doi-asserted-by":"publisher","first-page":"1175","DOI":"10.1128\/jb.186.4.1175-1181.2004","volume":"186","author":"S Dubrac","year":"2004","unstructured":"Dubrac S, Msadek T (2004) Identification of genes controlled by the essential YycG\/YycF two-component system of Staphylococcus aureus. J Bacteriol 186:1175\u20131181. https:\/\/doi.org\/10.1128\/jb.186.4.1175-1181.2004","journal-title":"J Bacteriol"},{"key":"14013_CR16","doi-asserted-by":"publisher","first-page":"8257","DOI":"10.1128\/jb.00645-07","volume":"189","author":"S Dubrac","year":"2007","unstructured":"Dubrac S, Boneca IG, Poupel O, Msadek T (2007) New insights into the WalK\/WalR (YycG\/YycF) essential signal transduction pathway reveal a major role in controlling cell wall metabolism and biofilm formation in Staphylococcus aureus. J Bacteriol 189:8257\u20138269. https:\/\/doi.org\/10.1128\/jb.00645-07","journal-title":"J Bacteriol"},{"key":"14013_CR17","doi-asserted-by":"publisher","first-page":"1307","DOI":"10.1111\/j.1365-2958.2008.06483.x","volume":"70","author":"S Dubrac","year":"2008","unstructured":"Dubrac S, Bisicchia P, Devine KM, Msadek T (2008) A matter of life and death: cell wall homeostasis and the WalKR (YycGF) essential signal transduction pathway. Mol Microbiol 70:1307\u20131322. https:\/\/doi.org\/10.1111\/j.1365-2958.2008.06483.x","journal-title":"Mol Microbiol"},{"key":"14013_CR18","doi-asserted-by":"publisher","first-page":"6375","DOI":"10.1128\/jb.180.23.6375-6383.1998","volume":"180","author":"C Fabret","year":"1998","unstructured":"Fabret C, Hoch JA (1998) A two-component signal transduction system essential for growth of Bacillus subtilis: implications for anti-infective therapy. J Bacteriol 180:6375\u20136383. https:\/\/doi.org\/10.1128\/jb.180.23.6375-6383.1998","journal-title":"J Bacteriol"},{"key":"14013_CR19","doi-asserted-by":"publisher","first-page":"609","DOI":"10.1093\/jac\/20.4.609","volume":"20","author":"D Felmingham","year":"1987","unstructured":"Felmingham D, Solomonides K, O\u2019Hare MD, Wilson AP, Gr\u00fcneberg RN (1987) The effect of medium and inoculum on the activity of vancomycin and teicoplanin against coagulase-negative staphylococci. J Antimicrob Chemother 20:609\u2013610. https:\/\/doi.org\/10.1093\/jac\/20.4.609","journal-title":"J Antimicrob Chemother"},{"key":"14013_CR20","doi-asserted-by":"publisher","first-page":"90","DOI":"10.1101\/gr.1275704","volume":"14","author":"HP Fischer","year":"2004","unstructured":"Fischer HP, Brunner NA, Wieland B, Paquette J, Macko L, Ziegelbauer K, Freiberg C (2004) Identification of antibiotic stress-inducible promoters: a systematic approach to novel pathway-specific reporter assays for antibacterial drug discovery. Genome Res 14:90\u201398. https:\/\/doi.org\/10.1101\/gr.1275704","journal-title":"Genome Res"},{"key":"14013_CR21","doi-asserted-by":"publisher","first-page":"503","DOI":"10.1111\/j.1365-2958.2010.07464.x","volume":"79","author":"T Fukushima","year":"2011","unstructured":"Fukushima T, Furihata I, Emmins R, Daniel RA, Hoch JA, Szurmant H (2011) A role for the essential YycG sensor histidine kinase in sensing cell division. Mol Microbiol 79:503\u2013522. https:\/\/doi.org\/10.1111\/j.1365-2958.2010.07464.x","journal-title":"Mol Microbiol"},{"key":"14013_CR22","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-020-15257-1","volume":"11","author":"F Grein","year":"2020","unstructured":"Grein F, M\u00fcller A, Scherer KM, Liu X, Ludwig KC, Kl\u00f6ckner A, Strach M, Sahl H-G, Kubitscheck U, Schneider T (2020) Ca2+-Daptomycin targets cell wall biosynthesis by forming a tripartite complex with undecaprenyl-coupled intermediates and membrane lipids. Nat Commun 11:1455. https:\/\/doi.org\/10.1038\/s41467-020-15257-1","journal-title":"Nat Commun"},{"key":"14013_CR23","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1016\/j.ijmm.2016.12.001","volume":"307","author":"P Hardt","year":"2017","unstructured":"Hardt P, Engels I, Rausch M, Gajdiss M, Ulm H, Sass P, Ohlsen K, Sahl H-G, Bierbaum G, Schneider T, Grein F (2017) The cell wall precursor lipid II acts as a molecular signal for the Ser\/Thr kinase PknB of Staphylococcus aureus. Int J Med Microbiol 307:1\u201310. https:\/\/doi.org\/10.1016\/j.ijmm.2016.12.001","journal-title":"Int J Med Microbiol"},{"key":"14013_CR24","doi-asserted-by":"publisher","first-page":"1636","DOI":"10.1126\/science.1129818","volume":"313","author":"HE Hasper","year":"2006","unstructured":"Hasper HE, Kramer NE, Smith JL, Hillman JD, Zachariah C, Kuipers OP, Kruijff B, Breukink E (2006) An alternative bactericidal mechanism of action for lantibiotic peptides that target lipid II. Science 313:1636\u20131637. https:\/\/doi.org\/10.1126\/science.1129818","journal-title":"Science"},{"key":"14013_CR25","doi-asserted-by":"publisher","first-page":"165","DOI":"10.1016\/S1369-5274(00)00070-9","volume":"3","author":"JA Hoch","year":"2000","unstructured":"Hoch JA (2000) Two-component and phosphorelay signal transduction. Curr Opin Microbiol 3:165\u2013170. https:\/\/doi.org\/10.1016\/S1369-5274(00)00070-9","journal-title":"Curr Opin Microbiol"},{"key":"14013_CR26","doi-asserted-by":"publisher","first-page":"1639","DOI":"10.1046\/j.1365-2958.2003.03661.x","volume":"49","author":"A Howell","year":"2003","unstructured":"Howell A, Dubrac S, Andersen KK, Noone D, Fert J, Msadek T, Devine K (2003) Genes controlled by the essential YycG\/YycF two-component system of Bacillus subtilis revealed through a novel hybrid regulator approach. Mol Microbiol 49:1639\u20131655. https:\/\/doi.org\/10.1046\/j.1365-2958.2003.03661.x","journal-title":"Mol Microbiol"},{"key":"14013_CR27","doi-asserted-by":"publisher","first-page":"1199","DOI":"10.1111\/j.1365-2958.2005.05017.x","volume":"59","author":"A Howell","year":"2006","unstructured":"Howell A, Dubrac S, Noone D, Varughese KI, Devine K (2006) Interactions between the YycFG and PhoPR two-component systems in Bacillus subtilis: the PhoR kinase phosphorylates the non-cognate YycF response regulator upon phosphate limitation. Mol Microbiol 59:1199\u20131215. https:\/\/doi.org\/10.1111\/j.1365-2958.2005.05017.x","journal-title":"Mol Microbiol"},{"key":"14013_CR28","doi-asserted-by":"publisher","first-page":"2838","DOI":"10.1128\/AAC.48.8.2838-2844.2004","volume":"48","author":"B Hutter","year":"2004","unstructured":"Hutter B, Schaab C, Albrecht S, Borgmann M, Brunner NA, Freiberg C, Ziegelbauer K, Rock CO, Ivanov I, Loferer H (2004) Prediction of mechanisms of action of antibacterial compounds by gene expression profiling. Antimicrob Agents Chemother 48:2838\u20132844. https:\/\/doi.org\/10.1128\/AAC.48.8.2838-2844.2004","journal-title":"Antimicrob Agents Chemother"},{"issue":"10369","key":"14013_CR29","doi-asserted-by":"publisher","first-page":"2221","DOI":"10.1016\/S0140-6736(22)02185-7","volume":"400","author":"KS Ikuta","year":"2022","unstructured":"Ikuta KS, Swetschinski LR, Aguilar GR, Sharara F, Mestrovic T, Gray AP, Weaver ND, Wool EE, Han C, Hayoon AG, Aali A (2022) Global mortality associated with 33 bacterial pathogens in 2019: a systematic analysis for the Global Burden of Disease Study 2019. The Lancet. 400(10369):2221\u201348. https:\/\/doi.org\/10.1016\/S0140-6736(22)02185-7","journal-title":"The Lancet."},{"key":"14013_CR30","doi-asserted-by":"publisher","first-page":"4684","DOI":"10.1002\/j.1460-2075.1994.tb06792.x","volume":"13","author":"C Jacobs","year":"1994","unstructured":"Jacobs C, Huang LJ, Bartowsky E, Normark S, Park JT (1994) Bacterial cell wall recycling provides cytosolic muropeptides as effectors for beta-lactamase induction. EMBO J 13:4684\u20134694. https:\/\/doi.org\/10.1002\/j.1460-2075.1994.tb06792.x","journal-title":"EMBO J"},{"key":"14013_CR31","doi-asserted-by":"publisher","first-page":"2684","DOI":"10.1002\/ejoc.200200699","volume":"2003","author":"R Jansen","year":"2003","unstructured":"Jansen R, Kunze B, Reichenbach H, H\u00f6fle G (2003) Chondrochloren A and B, new \u03b2-amino styrenes from Chondromyces crocatus (Myxobacteria). Eur J Org Chem 2003:2684\u20132689. https:\/\/doi.org\/10.1002\/ejoc.200200699","journal-title":"Eur J Org Chem"},{"key":"14013_CR32","doi-asserted-by":"publisher","first-page":"5153","DOI":"10.1128\/jb.00310-06","volume":"188","author":"S Jordan","year":"2006","unstructured":"Jordan S, Junker A, Helmann JD, Mascher T (2006) Regulation of LiaRS-dependent gene expression in Bacillus subtilis: identification of inhibitor proteins, regulator binding sites, and target genes of a conserved cell envelope stress-sensing two-component system. J Bacteriol 188:5153\u20135166. https:\/\/doi.org\/10.1128\/jb.00310-06","journal-title":"J Bacteriol"},{"key":"14013_CR33","doi-asserted-by":"publisher","first-page":"2530","DOI":"10.1099\/mic.0.2007\/006817-0","volume":"153","author":"S Jordan","year":"2007","unstructured":"Jordan S, Rietk\u00f6tter E, Strauch MA, Kalamorz F, Butcher BG, Helmann JD, Mascher T (2007) LiaRS-dependent gene expression is embedded in transition state regulation in Bacillus subtilis. Microbiology (Reading) 153:2530\u20132540. https:\/\/doi.org\/10.1099\/mic.0.2007\/006817-0","journal-title":"Microbiology (Reading)"},{"key":"14013_CR34","doi-asserted-by":"publisher","first-page":"107","DOI":"10.1111\/j.1574-6976.2007.00091.x","volume":"32","author":"S Jordan","year":"2008","unstructured":"Jordan S, Hutchings MI, Mascher T (2008) Cell envelope stress response in Gram-positive bacteria. FEMS Microbiol Rev 32:107\u2013146. https:\/\/doi.org\/10.1111\/j.1574-6976.2007.00091.x","journal-title":"FEMS Microbiol Rev"},{"key":"14013_CR35","doi-asserted-by":"publisher","first-page":"121","DOI":"10.1007\/978-1-4939-6634-9_7","volume":"1520","author":"CM Kobras","year":"2017","unstructured":"Kobras CM, Mascher T, Gebhard S (2017) Application of a Bacillus subtilis whole-cell biosensor (PliaI-lux) for the identification of cell wall active antibacterial compounds. Methods Mol Biol 1520:121\u2013131. https:\/\/doi.org\/10.1007\/978-1-4939-6634-9_7","journal-title":"Methods Mol Biol"},{"key":"14013_CR36","doi-asserted-by":"publisher","first-page":"807","DOI":"10.1046\/j.1365-2958.2003.03599.x","volume":"49","author":"M Kuroda","year":"2003","unstructured":"Kuroda M, Kuroda H, Oshima T, Takeuchi F, Mori H, Hiramatsu K (2003) Two-component system VraSR positively modulates the regulation of cell-wall biosynthesis pathway in Staphylococcus aureus. Mol Microbiol 49:807\u2013821. https:\/\/doi.org\/10.1046\/j.1365-2958.2003.03599.x","journal-title":"Mol Microbiol"},{"key":"14013_CR37","doi-asserted-by":"publisher","first-page":"4665","DOI":"10.1128\/AAC.48.12.4665-4672.2004","volume":"48","author":"KL LaPlante","year":"2004","unstructured":"LaPlante KL, Rybak MJ (2004) Impact of high-inoculum Staphylococcus aureus on the activities of nafcillin, vancomycin, linezolid, and daptomycin, alone and in combination with gentamicin, in an in vitro pharmacodynamic model. Antimicrob Agents Chemother 48:4665\u20134672. https:\/\/doi.org\/10.1128\/AAC.48.12.4665-4672.2004","journal-title":"Antimicrob Agents Chemother"},{"key":"14013_CR38","doi-asserted-by":"publisher","first-page":"21","DOI":"10.1186\/s13036-020-00243-4","volume":"14","author":"N Lautenschl\u00e4ger","year":"2020","unstructured":"Lautenschl\u00e4ger N, Popp PF, Mascher T (2020) Development of a novel heterologous \u03b2-lactam-specific whole-cell biosensor in Bacillus subtilis. J Biol Eng 14:21. https:\/\/doi.org\/10.1186\/s13036-020-00243-4","journal-title":"J Biol Eng"},{"key":"14013_CR39","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pgen.1005275","volume":"11","author":"EA Libby","year":"2015","unstructured":"Libby EA, Goss LA, Dworkin J (2015) The eukaryotic-like Ser\/Thr kinase PrkC regulates the essential WalRK two-component system in Bacillus subtilis. PLoS Genet 11:e1005275. https:\/\/doi.org\/10.1371\/journal.pgen.1005275","journal-title":"PLoS Genet"},{"key":"14013_CR40","doi-asserted-by":"publisher","first-page":"1591","DOI":"10.1046\/j.1365-2958.2003.03786.x","volume":"50","author":"T Mascher","year":"2003","unstructured":"Mascher T, Margulis NG, Wang T, Ye RW, Helmann JD (2003) Cell wall stress responses in Bacillus subtilis: the regulatory network of the bacitracin stimulon. Mol Microbiol 50:1591\u20131604. https:\/\/doi.org\/10.1046\/j.1365-2958.2003.03786.x","journal-title":"Mol Microbiol"},{"key":"14013_CR41","doi-asserted-by":"publisher","first-page":"2888","DOI":"10.1128\/aac.48.8.2888-2896.2004","volume":"48","author":"T Mascher","year":"2004","unstructured":"Mascher T, Zimmer SL, Smith T-A, Helmann JD (2004) Antibiotic-inducible promoter regulated by the cell envelope stress-sensing two-component system LiaRS of Bacillus subtilis. Antimicrob Agents Chemother 48:2888\u20132896. https:\/\/doi.org\/10.1128\/aac.48.8.2888-2896.2004","journal-title":"Antimicrob Agents Chemother"},{"key":"14013_CR42","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1611173113","volume":"113","author":"A M\u00fcller","year":"2016","unstructured":"M\u00fcller A, Wenzel M, Strahl H, Grein F, Saaki TNV, Kohl B, Siersma T, Bandow JE, Sahl H-G, Schneider T, Hamoen LW (2016) Daptomycin inhibits cell envelope synthesis by interfering with fluid membrane microdomains. Proc Natl Acad Sci U S A 113:E7077-E7086. https:\/\/doi.org\/10.1073\/pnas.1611173113","journal-title":"Proc Natl Acad Sci U S A"},{"key":"14013_CR43","doi-asserted-by":"publisher","first-page":"768","DOI":"10.1021\/np200776v","volume":"75","author":"PW Okanya","year":"2012","unstructured":"Okanya PW, Mohr KI, Gerth K, Steinmetz H, Huch V, Jansen R, M\u00fcller R (2012) Hyaladione, an S-methyl cyclohexadiene-dione from Hyalangium minutum. J Nat Prod 75:768\u2013770. https:\/\/doi.org\/10.1021\/np200776v","journal-title":"J Nat Prod"},{"key":"14013_CR44","doi-asserted-by":"publisher","first-page":"1420","DOI":"10.1021\/np500145f","volume":"77","author":"PW Okanya","year":"2014","unstructured":"Okanya PW, Mohr KI, Gerth K, Kessler W, Jansen R, Stadler M, M\u00fcller R (2014) Hyafurones, hyapyrrolines, and hyapyrones: polyketides from Hyalangium minutum. J Nat Prod 77:1420\u20131429. https:\/\/doi.org\/10.1021\/np500145f","journal-title":"J Nat Prod"},{"key":"14013_CR45","doi-asserted-by":"publisher","first-page":"392","DOI":"10.1007\/s00018-002-8431-9","volume":"59","author":"ZEV Phillips","year":"2002","unstructured":"Phillips ZEV, Strauch MA (2002) Bacillus subtilis sporulation and stationary phase gene expression. Cell Mol Life Sci 59:392\u2013402. https:\/\/doi.org\/10.1007\/s00018-002-8431-9","journal-title":"Cell Mol Life Sci"},{"key":"14013_CR46","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-018-20145-2","volume":"8","author":"F Pompeo","year":"2018","unstructured":"Pompeo F, Byrne D, Mengin-Lecreulx D, Galinier A (2018) Dual regulation of activity and intracellular localization of the PASTA kinase PrkC during Bacillus subtilis growth. Sci Rep 8:1660. https:\/\/doi.org\/10.1038\/s41598-018-20145-2","journal-title":"Sci Rep"},{"key":"14013_CR47","doi-asserted-by":"publisher","first-page":"29","DOI":"10.1186\/1754-1611-7-29","volume":"7","author":"J Radeck","year":"2013","unstructured":"Radeck J, Kraft K, Bartels J, Cikovic T, D\u00fcrr F, Emenegger J, Kelterborn S, Sauer C, Fritz G, Gebhard S, Mascher T (2013) The Bacillus BioBrick Box: generation and evaluation of essential genetic building blocks for standardized work with Bacillus subtilis. J Biol Eng 7:29. https:\/\/doi.org\/10.1186\/1754-1611-7-29","journal-title":"J Biol Eng"},{"key":"14013_CR48","doi-asserted-by":"publisher","DOI":"10.1128\/msystems.00847-23","volume":"9","author":"J Ravi","year":"2024","unstructured":"Ravi J, Anantharaman V, Chen SZ, Brenner EP, Datta P, Aravind L, Gennaro ML (2024) The phage shock protein (PSP) envelope stress response: discovery of novel partners and evolutionary history. mSystems 9:e00847-23. https:\/\/doi.org\/10.1128\/msystems.00847-23","journal-title":"mSystems"},{"key":"14013_CR49","doi-asserted-by":"publisher","first-page":"1965","DOI":"10.1074\/mcp.m113.035949","volume":"13","author":"V Ravikumar","year":"2014","unstructured":"Ravikumar V, Shi L, Krug K, Derouiche A, Jers C, Cousin C, Kobir A, Mijakovic I, Macek B (2014) Quantitative phosphoproteome analysis of Bacillus subtilis reveals novel substrates of the kinase PrkC and phosphatase PrpC. Mol Cell Proteomics 13:1965\u20131978. https:\/\/doi.org\/10.1074\/mcp.m113.035949","journal-title":"Mol Cell Proteomics"},{"key":"14013_CR50","doi-asserted-by":"publisher","DOI":"10.1177\/1179916117703999","volume":"10","author":"PN Reddy","year":"2017","unstructured":"Reddy PN, Srirama K, Dirisala VR (2017) An update on clinical burden, diagnostic tools, and therapeutic options of Staphylococcus aureus. Infect Dis (Auckl) 10:1179916117703999. https:\/\/doi.org\/10.1177\/1179916117703999","journal-title":"Infect Dis (Auckl)"},{"key":"14013_CR51","doi-asserted-by":"publisher","first-page":"4671","DOI":"10.1128\/jb.00403-07","volume":"189","author":"LI Salzberg","year":"2007","unstructured":"Salzberg LI, Helmann JD (2007) An antibiotic-inducible cell wall-associated protein that protects Bacillus subtilis from autolysis. J Bacteriol 189:4671\u20134680. https:\/\/doi.org\/10.1128\/jb.00403-07","journal-title":"J Bacteriol"},{"key":"14013_CR52","doi-asserted-by":"publisher","first-page":"180","DOI":"10.1111\/mmi.12092","volume":"87","author":"LI Salzberg","year":"2013","unstructured":"Salzberg LI, Powell L, Hokamp K, Botella E, Noone D, Devine KM (2013) The WalRK (YycFG) and \u03c3(I) RsgI regulators cooperate to control CwlO and LytE expression in exponentially growing and stressed Bacillus subtilis cells. Mol Microbiol 87:180\u2013195. https:\/\/doi.org\/10.1111\/mmi.12092","journal-title":"Mol Microbiol"},{"key":"14013_CR53","doi-asserted-by":"publisher","DOI":"10.1128\/mbio.02262-23","volume":"14","author":"LKR Sharkey","year":"2023","unstructured":"Sharkey LKR, Guerillot R, Walsh CJ, Turner AM, Lee JYH, Neville SL, Klatt S, Baines SL, Pidot SJ, Rossello FJ, Seemann T, McWilliam HEG, Cho E, Carter GP, Howden BP, McDevitt CA, Hachani A, Stinear TP, Monk IR (2023) The two-component system WalKR provides an essential link between cell wall homeostasis and DNA replication in Staphylococcus aureus. Mbio 14:e02262-23. https:\/\/doi.org\/10.1128\/mbio.02262-23","journal-title":"Mbio"},{"key":"14013_CR54","doi-asserted-by":"publisher","first-page":"1072","DOI":"10.1073\/pnas.44.10.1072","volume":"44","author":"J Spizizen","year":"1958","unstructured":"Spizizen J (1958) Transformation of biochemically deficient strains of Bacillus subtilis by deoxyriobnucleate. Proc Natl Acad Sci U S A 44:1072\u20131078. https:\/\/doi.org\/10.1073\/pnas.44.10.1072","journal-title":"Proc Natl Acad Sci U S A"},{"key":"14013_CR55","doi-asserted-by":"publisher","first-page":"20676","DOI":"10.1021\/ja208080r","volume":"133","author":"F Squeglia","year":"2011","unstructured":"Squeglia F, Marchetti R, Ruggiero A, Lanzetta R, Marasco D, Dworkin J, Petoukhov M, Molinaro A, Berisio R, Silipo A (2011) Chemical basis of peptidoglycan discrimination by PrkC, a key kinase involved in bacterial resuscitation from dormancy. J Am Chem Soc 133:20676\u201320679. https:\/\/doi.org\/10.1021\/ja208080r","journal-title":"J Am Chem Soc"},{"key":"14013_CR56","doi-asserted-by":"publisher","first-page":"279","DOI":"10.7164\/antibiotics.55.279","volume":"55","author":"D Sun","year":"2002","unstructured":"Sun D, Cohen S, Mani N, Murphy C, Rothstein DM (2002) A pathway-specific cell based screening system to detect bacterial cell wall inhibitors. J Antibiot (Tokyo) 55:279\u2013287. https:\/\/doi.org\/10.7164\/antibiotics.55.279","journal-title":"J Antibiot (Tokyo)"},{"key":"14013_CR57","doi-asserted-by":"publisher","first-page":"2973","DOI":"10.1128\/jb.01563-08","volume":"191","author":"P Suntharalingam","year":"2009","unstructured":"Suntharalingam P, Senadheera MD, Mair RW, L\u00e9vesque CM, Cvitkovitch DG (2009) The LiaFSR system regulates the cell envelope stress response in Streptococcus mutans. J Bacteriol 191:2973\u20132984. https:\/\/doi.org\/10.1128\/jb.01563-08","journal-title":"J Bacteriol"},{"key":"14013_CR58","doi-asserted-by":"publisher","first-page":"5419","DOI":"10.1128\/jb.187.15.5419-5426.2005","volume":"187","author":"H Szurmant","year":"2005","unstructured":"Szurmant H, Nelson K, Kim E-J, Perego M, Hoch JA (2005) YycH regulates the activity of the essential YycFG two-component system in Bacillus subtilis. J Bacteriol 187:5419\u20135426. https:\/\/doi.org\/10.1128\/jb.187.15.5419-5426.2005","journal-title":"J Bacteriol"},{"key":"14013_CR59","doi-asserted-by":"publisher","first-page":"3280","DOI":"10.1128\/jb.01936-06","volume":"189","author":"H Szurmant","year":"2007","unstructured":"Szurmant H, Mohan MA, Imus PM, Hoch JA (2007) YycH and YycI interact to regulate the essential YycFG two-component system in Bacillus subtilis. J Bacteriol 189:3280\u20133289. https:\/\/doi.org\/10.1128\/jb.01936-06","journal-title":"J Bacteriol"},{"key":"14013_CR60","doi-asserted-by":"publisher","DOI":"10.1016\/j.jbc.2022.102473","author":"S Tan","year":"2022","unstructured":"Tan S, Cho K, Nodwell JR (2022) A defect in cell wall recycling confers antibiotic resistance and sensitivity in Staphylococcus aureus. J Biol Chem. https:\/\/doi.org\/10.1016\/j.jbc.2022.102473","journal-title":"J Biol Chem"},{"key":"14013_CR61","doi-asserted-by":"publisher","unstructured":"Tol MB, Morales Angeles D, Scheffers D-J (2015) In vivo cluster formation of nisin and lipid II is correlated with membrane depolarization. Antimicrob Agents Chemother 59:3683\u20133686. https:\/\/doi.org\/10.1128\/aac.04781-14","DOI":"10.1128\/aac.04781-14"},{"key":"14013_CR62","doi-asserted-by":"publisher","first-page":"677","DOI":"10.1007\/s00203-011-0710-0","volume":"193","author":"C-L Tseng","year":"2011","unstructured":"Tseng C-L, Chen J-T, Lin J-H, Huang W-Z, Shaw G-C (2011) Genetic evidence for involvement of the alternative sigma factor SigI in controlling expression of the cell wall hydrolase gene lytE and contribution of LytE to heat survival of Bacillus subtilis. Arch Microbiol 193:677\u2013685. https:\/\/doi.org\/10.1007\/s00203-011-0710-0","journal-title":"Arch Microbiol"},{"key":"14013_CR63","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0030403","volume":"7","author":"M T\u00fcrck","year":"2012","unstructured":"T\u00fcrck M, Bierbaum G (2012) Purification and activity testing of the full-length YycFGHI proteins of Staphylococcus aureus. PLoS ONE 7:e30403. https:\/\/doi.org\/10.1371\/journal.pone.0030403","journal-title":"PLoS ONE"},{"key":"14013_CR64","doi-asserted-by":"publisher","first-page":"6436","DOI":"10.1128\/AEM.00586-07","volume":"73","author":"A Urban","year":"2007","unstructured":"Urban A, Eckermann S, Fast B, Metzger S, Gehling M, Ziegelbauer K, R\u00fcbsamen-Waigmann H, Freiberg C (2007) Novel whole-cell antibiotic biosensors for compound discovery. Appl Environ Microbiol 73:6436\u20136443. https:\/\/doi.org\/10.1128\/AEM.00586-07","journal-title":"Appl Environ Microbiol"},{"key":"14013_CR65","doi-asserted-by":"publisher","first-page":"6121","DOI":"10.1128\/iai.70.11.6121-6128.2002","volume":"70","author":"C Wagner","year":"2002","unstructured":"Wagner C, de Saizieu AA, Sch\u00f6nfeld H-J, Kamber M, Lange R, Thompson CJ, Page MG (2002) Genetic analysis and functional characterization of the Streptococcus pneumoniae vic operon. Infect Immun 70:6121\u20136128. https:\/\/doi.org\/10.1128\/iai.70.11.6121-6128.2002","journal-title":"Infect Immun"},{"key":"14013_CR66","doi-asserted-by":"publisher","first-page":"1619","DOI":"10.1128\/aac.01046-08","volume":"53","author":"T Wecke","year":"2009","unstructured":"Wecke T, Z\u00fchlke D, M\u00e4der U, Jordan S, Voigt B, Pelzer S, Labischinski H, Homuth G, Hecker M, Mascher T (2009) Daptomycin versus Friulimicin B: in-depth profiling of Bacillus subtilis cell envelope stress responses. Antimicrob Agents Chemother 53:1619\u20131623. https:\/\/doi.org\/10.1128\/aac.01046-08","journal-title":"Antimicrob Agents Chemother"},{"key":"14013_CR67","doi-asserted-by":"publisher","first-page":"1276","DOI":"10.1039\/C001260M","volume":"27","author":"KJ Weissman","year":"2010","unstructured":"Weissman KJ, M\u00fcller R (2010) Myxobacterial secondary metabolites: bioactivities and modes-of-action. Nat Prod Rep 27:1276\u20131295. https:\/\/doi.org\/10.1039\/C001260M","journal-title":"Nat Prod Rep"},{"key":"14013_CR68","doi-asserted-by":"publisher","first-page":"1242","DOI":"10.1016\/j.chembiol.2021.02.022","volume":"28","author":"KW Wex","year":"2021","unstructured":"Wex KW, Saur JS, Handel F, Ortlieb N, Mokeev V, Kulik A, Niedermeyer THJ, Mast Y, Grond S, Berscheid A, Br\u00f6tz-Oesterhelt H (2021) Bioreporters for direct mode of action-informed screening of antibiotic producer strains. Cell Chem Biol 28:1242-1252.e4. https:\/\/doi.org\/10.1016\/j.chembiol.2021.02.022","journal-title":"Cell Chem Biol"},{"key":"14013_CR69","doi-asserted-by":"publisher","first-page":"1772","DOI":"10.1074\/jbc.M006770200","volume":"276","author":"I Wiedemann","year":"2001","unstructured":"Wiedemann I, Breukink E, van Kraaij C, Kuipers OP, Bierbaum G, de Kruijff B, Sahl H-G (2001) Specific binding of nisin to the peptidoglycan precursor lipid II combines pore formation and inhibition of cell wall biosynthesis for potent antibiotic activity *. J Biol Chem 276:1772\u20131779. https:\/\/doi.org\/10.1074\/jbc.M006770200","journal-title":"J Biol Chem"},{"key":"14013_CR70","doi-asserted-by":"publisher","first-page":"4817","DOI":"10.1007\/s00253-016-7519-3","volume":"100","author":"D Wolf","year":"2016","unstructured":"Wolf D, Mascher T (2016) The applied side of antimicrobial peptide-inducible promoters from Firmicutes bacteria: expression systems and whole-cell biosensors. Appl Microbiol Biotechnol 100:4817\u20134829. https:\/\/doi.org\/10.1007\/s00253-016-7519-3","journal-title":"Appl Microbiol Biotechnol"},{"key":"14013_CR71","doi-asserted-by":"publisher","first-page":"789","DOI":"10.1007\/s00253-022-11762-z","volume":"106","author":"J Yin","year":"2022","unstructured":"Yin J, Cheng D, Zhu Y, Liang Y, Yu Z (2022) Development of a whole-cell biosensor for detection of antibiotics targeting bacterial cell envelope in Bacillus subtilis. Appl Microbiol Biotechnol 106:789\u2013798. https:\/\/doi.org\/10.1007\/s00253-022-11762-z","journal-title":"Appl Microbiol Biotechnol"},{"key":"14013_CR72","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0135198","volume":"10","author":"Z Yu","year":"2015","unstructured":"Yu Z, Cai Y, Qin W, Lin J, Qiu J (2015) Polymyxin E induces rapid Paenibacillus polymyxa death by damaging cell membrane while Ca2+ can protect cells from damage. PLoS ONE 10:e0135198. https:\/\/doi.org\/10.1371\/journal.pone.0135198","journal-title":"PLoS ONE"},{"key":"14013_CR73","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0203840","volume":"13","author":"Q-B Zhang","year":"2018","unstructured":"Zhang Q-B, Yu K, Liu Z, Wang D, Zhao Y, Yin S, Liu Z (2018) Prediction of prkC-mediated protein serine\/threonine phosphorylation sites for bacteria. PLoS ONE 13:e0203840. https:\/\/doi.org\/10.1371\/journal.pone.0203840","journal-title":"PLoS ONE"}],"container-title":["Applied Microbiology and Biotechnology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/link.springer.com\/article\/10.1007\/s00253-026-14013-7","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1007\/s00253-026-14013-7.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1007\/s00253-026-14013-7.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,28]],"date-time":"2026-08-28T07:56:31Z","timestamp":1787903791000},"score":1,"resource":{"primary":{"URL":"https:\/\/link.springer.com\/10.1007\/s00253-026-14013-7"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,28]]},"references-count":73,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2026,12]]}},"alternative-id":["14013"],"URL":"https:\/\/doi.org\/10.1007\/s00253-026-14013-7","relation":{},"ISSN":["1432-0614"],"issn-type":[{"value":"1432-0614","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,28]]},"assertion":[{"value":"13 May 2026","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"12 August 2026","order":2,"name":"revised","label":"Revised","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"14 August 2026","order":3,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"28 August 2026","order":4,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"Not applicable.","order":1,"name":"Ethics","label":"Ethical approval","group":{"name":"EthicsHeading","label":"Declarations"}},{"value":"The authors declare no competing interests.","order":2,"name":"Ethics","label":"Competing interests","group":{"name":"EthicsHeading","label":"Declarations"}}],"article-number":"260"}}