{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T13:52:54Z","timestamp":1791294774572,"version":"4.1.0"},"reference-count":78,"publisher":"American Society for Microbiology","license":[{"start":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T00:00:00Z","timestamp":1791244800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T00:00:00Z","timestamp":1791244800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/100000060","name":"National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["R01AI188467, R01AI155492"],"award-info":[{"award-number":["R01AI188467, R01AI155492"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000057","name":"National Institute of General Medical Sciences","doi-asserted-by":"publisher","award":["R01GM138630"],"award-info":[{"award-number":["R01GM138630"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["mBio"],"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      In most bacteria, peptidoglycan contains mainly 4-3 crosslinks formed by penicillin-binding proteins (PBPs). However, in the opportunistic pathogen\n                      <jats:italic toggle=\"yes\">Clostridioides difficile<\/jats:italic>\n                      , 70% of the crosslinks are 3-3 crosslinks formed by L,D-transpeptidases (LDTs), and LDTs are essential for viability. PBPs and LDTs use different acyl donors for crosslinking; PBPs require a pentapeptide, while LDTs require a tetrapeptide. Here, we determined the source of the tetrapeptides in\n                      <jats:italic toggle=\"yes\">C. difficile<\/jats:italic>\n                      and investigated the consequences of reengineering PG crosslinking from predominantly 3-3 to exclusively 4-3. We found that two D-alanyl-D-alanine carboxypeptidases (DD-CPase), DacA and DacC, supply LDTs with tetrapeptides during vegetative growth. Deleting these enzymes was sufficient to bypass the normal requirement for LDTs. The resulting mutant (\u0394\n                      <jats:italic toggle=\"yes\">dacAC<\/jats:italic>\n                      \u0394\n                      <jats:italic toggle=\"yes\">ldt<\/jats:italic>\n                      ) was remarkably healthy despite the absence of 3-3 crosslinks. Its only major phenotypic defect was a 3- to 4-log decrease in sporulation, which could, however, be overcome by deleting a third DD-CPase,\n                      <jats:italic toggle=\"yes\">dacB<\/jats:italic>\n                      . These findings fill gaps in our understanding of the pathway for LD-transpeptidation in\n                      <jats:italic toggle=\"yes\">C. difficile<\/jats:italic>\n                      and imply that LDTs are not essential components of the elongasome or divisome, both of which function well in the complete absence of LDTs, provided there is sufficient pentapeptide to sustain crosslinking by PBPs. Thus, LDTs are essential for viability because\n                      <jats:italic toggle=\"yes\">C. difficile<\/jats:italic>\n                      has intrinsically high levels of DD-CPase activity. Finally, we propose a model for how PBPs and LDTs work together during PG synthesis. In this model, PBPs construct a sparsely crosslinked PG sacculus that is subsequently strengthened with crosslinks introduced by LDTs.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        Synthesis of peptidoglycan (PG) in the opportunistic gut pathogen\n                        <jats:italic toggle=\"yes\">Clostridioides difficile<\/jats:italic>\n                        depends mostly on 3-3 crosslinks formed by L,D-transpeptidases (LDTs). Here, we converted\n                        <jats:italic toggle=\"yes\">C. difficile<\/jats:italic>\n                        from an organism that relies primarily on 3-3 crosslinks made by LDTs to one that relies exclusively on 4-3 crosslinks made by an alternative family of crosslinking enzymes, the penicillin-binding proteins (PBPs). Our findings explain why 3-3 crosslinking predominates over 4-3 crosslinking, clarify why\n                        <jats:italic toggle=\"yes\">C. difficile<\/jats:italic>\n                        does not compensate for the loss of LDTs by increasing PBP activity, and suggest a model for how PBPs and LDTs work together in this problematic pathogen.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/mbio.01580-26","type":"journal-article","created":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T13:00:46Z","timestamp":1791291646000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Carboxypeptidase activity drives L,D-transpeptidase essentiality during vegetative growth and sporulation in\n                    <i>Clostridioides difficile<\/i>"],"prefix":"10.1128","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7694-2200","authenticated-orcid":true,"given":"Kevin W.","family":"Bollinger","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/036jqmy94","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Microbiology and Immunology, Carver College of Medicine, University of Iowa","place":["Iowa City, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-7114-019X","authenticated-orcid":true,"given":"Ute","family":"M\u00fch","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/036jqmy94","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Microbiology and Immunology, Carver College of Medicine, University of Iowa","place":["Iowa City, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paige B.","family":"Brannen","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/036jqmy94","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Microbiology and Immunology, Carver College of Medicine, University of Iowa","place":["Iowa City, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2614-143X","authenticated-orcid":true,"given":"David L.","family":"Popham","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02smfhw86","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biological Sciences, Virginia Tech","place":["Blacksburg, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2217-0070","authenticated-orcid":true,"given":"David S.","family":"Weiss","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/036jqmy94","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Microbiology and Immunology, Carver College of Medicine, University of Iowa","place":["Iowa City, USA"]},{"id":[{"id":"https:\/\/ror.org\/036jqmy94","id-type":"ROR","asserted-by":"publisher"}],"name":"Graduate Program in Genetics, University of Iowa","place":["Iowa City, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8953-5081","authenticated-orcid":true,"given":"Craig D.","family":"Ellermeier","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/036jqmy94","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Microbiology and Immunology, Carver College of Medicine, University of Iowa","place":["Iowa City, USA"]},{"id":[{"id":"https:\/\/ror.org\/036jqmy94","id-type":"ROR","asserted-by":"publisher"}],"name":"Graduate Program in Genetics, University of Iowa","place":["Iowa City, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","published-online":{"date-parts":[[2026,10,6]]},"reference":[{"key":"e_1_3_4_2_2","doi-asserted-by":"publisher","DOI":"10.1111\/nyas.12006"},{"key":"e_1_3_4_3_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1574-6976.2007.00094.x"},{"key":"e_1_3_4_4_2","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-micro-020518-120056"},{"key":"e_1_3_4_5_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbamem.2007.06.007"},{"key":"e_1_3_4_6_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1574-6976.2008.00105.x"},{"key":"e_1_3_4_7_2","doi-asserted-by":"publisher","DOI":"10.3390\/antibiotics14121210"},{"key":"e_1_3_4_8_2","doi-asserted-by":"publisher","DOI":"10.1128\/mmbr.00090-24"},{"key":"e_1_3_4_9_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M111.259150"},{"key":"e_1_3_4_10_2","doi-asserted-by":"publisher","DOI":"10.1128\/mBio.02383-14"},{"key":"e_1_3_4_11_2","doi-asserted-by":"publisher","DOI":"10.1128\/JB.00711-18"},{"key":"e_1_3_4_12_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-023-43595-3"},{"key":"e_1_3_4_13_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2408540121"},{"key":"e_1_3_4_14_2","doi-asserted-by":"publisher","DOI":"10.1128\/jb.00220-25"},{"key":"e_1_3_4_15_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pgen.1011449"},{"key":"e_1_3_4_16_2","doi-asserted-by":"publisher","DOI":"10.1128\/iai.00047-25"},{"key":"e_1_3_4_17_2","doi-asserted-by":"publisher","DOI":"10.1111\/febs.16066"},{"key":"e_1_3_4_18_2","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.19469"},{"key":"e_1_3_4_19_2","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.37516"},{"key":"e_1_3_4_20_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.isci.2025.112227"},{"key":"e_1_3_4_21_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.01607-17"},{"key":"e_1_3_4_22_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jbc.2023.105529"},{"key":"e_1_3_4_23_2","doi-asserted-by":"publisher","DOI":"10.1093\/femsre\/fux048"},{"key":"e_1_3_4_24_2","doi-asserted-by":"publisher","DOI":"10.1101\/2025.09.22.677799"},{"key":"e_1_3_4_25_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.isci.2025.112586"},{"key":"e_1_3_4_26_2","doi-asserted-by":"publisher","DOI":"10.1038\/embor.2012.186"},{"key":"e_1_3_4_27_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41564-018-0248-x"},{"key":"e_1_3_4_28_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1013155"},{"key":"e_1_3_4_29_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.RA118.004273"},{"key":"e_1_3_4_30_2","doi-asserted-by":"publisher","DOI":"10.1128\/jb.178.22.6451-6458.1996"},{"key":"e_1_3_4_31_2","doi-asserted-by":"publisher","DOI":"10.1093\/jac\/dkac404"},{"key":"e_1_3_4_32_2","doi-asserted-by":"publisher","DOI":"10.1007\/s10096-019-03539-6"},{"key":"e_1_3_4_33_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M507384200"},{"key":"e_1_3_4_34_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M606920200"},{"key":"e_1_3_4_35_2","doi-asserted-by":"publisher","DOI":"10.1128\/JB.01396-13"},{"key":"e_1_3_4_36_2","doi-asserted-by":"publisher","DOI":"10.1055\/s-0035-1547333"},{"key":"e_1_3_4_37_2","doi-asserted-by":"publisher","DOI":"10.1128\/microbiolspec.gpp3-0017-2018"},{"key":"e_1_3_4_38_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.tim.2014.04.003"},{"key":"e_1_3_4_39_2","doi-asserted-by":"publisher","DOI":"10.1128\/microbiolspec.TBS-0005-2012"},{"key":"e_1_3_4_40_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.mib.2021.11.010"},{"key":"e_1_3_4_41_2","doi-asserted-by":"publisher","DOI":"10.1128\/mSphere.00205-18"},{"key":"e_1_3_4_42_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-026-75594-5"},{"key":"e_1_3_4_43_2","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-4939-6361-4_5"},{"key":"e_1_3_4_44_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pgen.1003660"},{"key":"e_1_3_4_45_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pgen.1003756"},{"key":"e_1_3_4_46_2","doi-asserted-by":"publisher","DOI":"10.1046\/j.1365-2958.1999.01255.x"},{"key":"e_1_3_4_47_2","doi-asserted-by":"publisher","DOI":"10.1128\/jb.00503-25"},{"key":"e_1_3_4_48_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.anaerobe.2021.102379"},{"key":"e_1_3_4_49_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41564-019-0519-1"},{"key":"e_1_3_4_50_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pgen.1011746"},{"key":"e_1_3_4_51_2","doi-asserted-by":"publisher","DOI":"10.1128\/jb.00032-26"},{"key":"e_1_3_4_52_2","doi-asserted-by":"publisher","DOI":"10.1038\/nm.2120"},{"key":"e_1_3_4_53_2","doi-asserted-by":"publisher","DOI":"10.1128\/JCM.39.6.2140-2145.2001"},{"key":"e_1_3_4_54_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.44.11.3224-3228.2000"},{"key":"e_1_3_4_55_2","doi-asserted-by":"publisher","DOI":"10.3390\/antibiotics11020258"},{"key":"e_1_3_4_56_2","doi-asserted-by":"publisher","DOI":"10.1093\/cid\/ciae087"},{"key":"e_1_3_4_57_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1365-2958.1994.tb00497.x"},{"key":"e_1_3_4_58_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.38.9.1899"},{"key":"e_1_3_4_59_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2008.01.031"},{"key":"e_1_3_4_60_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M204319200"},{"key":"e_1_3_4_61_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1365-2958.2009.07014.x"},{"key":"e_1_3_4_62_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-022-32086-6"},{"key":"e_1_3_4_63_2","doi-asserted-by":"publisher","DOI":"10.1186\/gb-2009-10-9-r102"},{"key":"e_1_3_4_64_2","doi-asserted-by":"publisher","DOI":"10.1186\/s12866-015-0614-2"},{"key":"e_1_3_4_65_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1365-2958.2007.05906.x"},{"key":"e_1_3_4_66_2","doi-asserted-by":"publisher","DOI":"10.1016\/0378-1119(91)90546-n"},{"key":"e_1_3_4_67_2","doi-asserted-by":"publisher","DOI":"10.1128\/jb.00121-22"},{"key":"e_1_3_4_68_2","doi-asserted-by":"publisher","DOI":"10.1128\/JB.00421-20"},{"key":"e_1_3_4_69_2","doi-asserted-by":"publisher","DOI":"10.1038\/nmicrobiol.2016.77"},{"key":"e_1_3_4_70_2","doi-asserted-by":"publisher","DOI":"10.3791\/61376"},{"key":"e_1_3_4_71_2","doi-asserted-by":"publisher","DOI":"10.21769\/BioProtoc.2125"},{"key":"e_1_3_4_72_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkac1003"},{"key":"e_1_3_4_73_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkw1017"},{"key":"e_1_3_4_74_2","doi-asserted-by":"publisher","DOI":"10.48550\/arXiv.1303.3997"},{"key":"e_1_3_4_75_2","doi-asserted-by":"publisher","DOI":"10.48550\/arXiv.1207.3907"},{"key":"e_1_3_4_76_2","doi-asserted-by":"publisher","DOI":"10.1128\/JB.185.4.1423-1431.2003"},{"key":"e_1_3_4_77_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.93.26.15405"},{"key":"e_1_3_4_78_2","doi-asserted-by":"publisher","DOI":"10.1016\/0003-2697(75)90324-3"},{"key":"e_1_3_4_79_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41564-021-01003-w"}],"container-title":["mBio"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/mbio.01580-26","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/mbio.01580-26","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T13:00:48Z","timestamp":1791291648000},"score":1,"resource":{"primary":{"URL":"https:\/\/journals.asm.org\/doi\/10.1128\/mbio.01580-26"}},"subtitle":[],"editor":[{"given":"Mariana G.","family":"Pinho","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2026,10,6]]},"references-count":78,"alternative-id":["10.1128\/mbio.01580-26"],"URL":"https:\/\/doi.org\/10.1128\/mbio.01580-26","relation":{},"ISSN":["2150-7511"],"issn-type":[{"value":"2150-7511","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,10,6]]},"assertion":[{"value":"2026-06-30","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-09-09","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-10-06","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e01580-26"}}