{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T20:27:22Z","timestamp":1792787242413,"version":"4.5.0"},"reference-count":73,"publisher":"American Society for Microbiology","issue":"9","license":[{"start":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T00:00:00Z","timestamp":1790640000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T00:00:00Z","timestamp":1790640000000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/501100008982","name":"National Science Foundation","doi-asserted-by":"publisher","award":["intramural IRD program"],"award-info":[{"award-number":["intramural IRD program"]}],"id":[{"id":"10.13039\/501100008982","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Yamagata Prefectural Government and Tsuruoka City","award":["n\/a"],"award-info":[{"award-number":["n\/a"]}],"id":[{"id":"https:\/\/ror.org\/04k6amx66","id-type":"ROR","asserted-by":"crossref"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["mSphere"],"published-print":{"date-parts":[[2026,9,29]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      In 2016, researchers at the J. Craig Venter Institute produced JCVI-Syn3.0, a derivative of\n                      <jats:italic toggle=\"yes\">Mycoplasma mycoides<\/jats:italic>\n                      subsp.\n                      <jats:italic toggle=\"yes\">capri<\/jats:italic>\n                      Large Colony strain GM12 with a re-engineered, minimized genome. The minimal synthetic genome of 532 kbp encodes 473 genes, selected from \u223c910 parent genes on the basis of their indispensability, that is, a lethal phenotype caused by a knockout of each such gene. Among these genes, 438 genes encode proteins, of which \u223c150 were not assigned a molecular function or predicted cellular role. A decade later, despite community annotation efforts, 50\u201360 of these putative proteins still lack any assigned function. We analyzed the intrinsic sequence characteristics, evolutionary conservation, and three-dimensional AlphaFold models of this least understood subset of the JCVI-Syn3.0 genes. We predicted conserved sequence motifs and structural features and inferred molecular functions of six proteins likely involved in membrane transport or membrane interactions, and six proteins that apparently belong to bacterial immunity systems, such as putative antitoxins, an essential toxin-like protein, and a member of a previously unnoticed clade of Cas4-like nucleases. The spatial models produced for this challenging data set by the AlphaFold pipeline help dissect proteins into discrete domains, which are often more convenient to analyze by sequence similarity than whole proteins. In several cases, AlphaFold models also enable functional inference more directly, based on spatial matching of the models to the databases of known protein structures.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        The smallest synthetic genome, JCVI-Syn3.0, encodes 438 proteins. JCVI-Syn3.0 was obtained by combining all genes whose deletion from a larger parental genome of\n                        <jats:italic toggle=\"yes\">M. mycoides<\/jats:italic>\n                        subsp.\n                        <jats:italic toggle=\"yes\">capri<\/jats:italic>\n                        Large Colony strain GM12 causes a complete stop or a significant delay in bacterial growth. Thus, nearly every protein in this minimal genome is essential; however, we do not know what \u223c15% of these genes do. Using computational methods, we predicted molecular functions for about a quarter of these enigmatic proteins, including several proteins that are related to the components of recently discovered bacterial immunity systems.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/msphere.00077-26","type":"journal-article","created":{"date-parts":[[2026,8,28]],"date-time":"2026-08-28T13:00:17Z","timestamp":1787922017000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Minimal organism JCVI-syn3.0, an engineered derivative of\n                    <i>Mycoplasma mycoides<\/i>\n                    , encodes an assortment of unrecognized membrane transporters and immunity factors"],"prefix":"10.1128","volume":"11","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6809-9225","authenticated-orcid":true,"given":"Arcady R.","family":"Mushegian","sequence":"first","affiliation":[{"name":"Division of Molecular and Cellular Biosciences, National Science Foundation","place":["Alexandria, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Phillip K.","family":"Yamamoto","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05kwbf598","id-type":"ROR","asserted-by":"publisher"}],"name":"Institute for Advanced Biosciences, Keio University","place":["Tsuruoka, Japan"]},{"id":[{"id":"https:\/\/ror.org\/02kn6nx58","id-type":"ROR","asserted-by":"publisher"}],"name":"Systems Biology Program, Graduate School of Media and Governance, Keio University","place":["Fujisawa, Japan"]},{"id":[{"id":"https:\/\/ror.org\/05rrcem69","id-type":"ROR","asserted-by":"publisher"}],"name":"Integrative Genetics and Genomics Program, University of California Davis","place":["Davis, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Naoki","family":"Torikai","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05kwbf598","id-type":"ROR","asserted-by":"publisher"}],"name":"Institute for Advanced Biosciences, Keio University","place":["Tsuruoka, Japan"]},{"id":[{"id":"https:\/\/ror.org\/02kn6nx58","id-type":"ROR","asserted-by":"publisher"}],"name":"Faculty of Environment and Information studies, Keio University","place":["Fujisawa, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2893-4919","authenticated-orcid":true,"given":"Kazuharu","family":"Arakawa","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05kwbf598","id-type":"ROR","asserted-by":"publisher"}],"name":"Institute for Advanced Biosciences, Keio University","place":["Tsuruoka, Japan"]},{"id":[{"id":"https:\/\/ror.org\/02kn6nx58","id-type":"ROR","asserted-by":"publisher"}],"name":"Systems Biology Program, Graduate School of Media and Governance, Keio University","place":["Fujisawa, Japan"]},{"id":[{"id":"https:\/\/ror.org\/02kn6nx58","id-type":"ROR","asserted-by":"publisher"}],"name":"Faculty of Environment and Information studies, Keio University","place":["Fujisawa, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_4_2_2","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.genom.1.1.99"},{"key":"e_1_3_4_3_2","doi-asserted-by":"publisher","DOI":"10.1128\/msphere.00139-24"},{"key":"e_1_3_4_4_2","doi-asserted-by":"publisher","DOI":"10.1101\/cshperspect.a023861"},{"key":"e_1_3_4_5_2","doi-asserted-by":"publisher","DOI":"10.1038\/scientificamerican0362-117"},{"key":"e_1_3_4_6_2","doi-asserted-by":"publisher","DOI":"10.1128\/br.36.3.263-290.1972"},{"key":"e_1_3_4_7_2","first-page":"750","article-title":"The completeness of molecular biology","volume":"20","author":"Morowitz HJ","year":"1984","unstructured":"Morowitz HJ. 1984. 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