{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,8,3]],"date-time":"2026-08-03T17:53:24Z","timestamp":1785779604219,"version":"3.56.0"},"posted":{"date-parts":[[2026,7,31]]},"group-title":"Microbiology","reference-count":77,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,7,31]],"date-time":"2026-07-31T00:00:00Z","timestamp":1785456000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,7,31]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  Bacteria in the Mycobacteriales order like mycobacteria and corynebacteria surround themselves with a multilayered cell envelope. Their cytoplasmic membrane is fortified by a peptidoglycan cell wall that is decorated with branched arabinogalactan (AG) polymers. The AG glycans are further modified with mycolic acids to form an outer membrane. Biogenesis of this mycomembrane requires the transport of mycolates from their site of synthesis in the cytoplasmic membrane to the cell surface. How mycolate transport is controlled and coordinated with AG synthesis has remained unclear. Mycolate transport is mediated by the essential RND-family transporter MmpL3 in mycobacteria and a pair of related, partially redundant transporters called CmpL1 and CmpL4 in corynebacteria. The acetyltransferase TmaT has also been implicated in mycolate transport in both types of bacteria. In corynebacteria, it is required for the production of acetylated mycolates, and this modification has been proposed to promote mycolate transport via both CmpL transporters in the model organism\n                  <jats:italic>Corynebacterium glutamicum<\/jats:italic>\n                  (\n                  <jats:italic>Cglu<\/jats:italic>\n                  ). Here, we reinvestigated the function of TmaT in\n                  <jats:italic>Cglu<\/jats:italic>\n                  and found that it and several factors encoded in the\n                  <jats:italic>tmaT<\/jats:italic>\n                  locus are specifically required for mycolate transport via the CmpL4 transporter pathway. Notably, one of these additional genes encodes the arabinosyltransferase AftD involved in AG biogenesis. TmaT and AftD were found to interact, and our results indicate that this interaction is required for acetylated mycolate production, mycolate transport via the CmpL4 pathway, and normal arabinan synthesis. Thus, the TmaT-AftD interaction may serve as a regulatory link connecting mycolate transport with AG biogenesis.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>SIGNIFICANCE<\/jats:title>\n                  <jats:p>\n                    Mycobacteriales bacteria, including pathogens like\n                    <jats:italic>Mycobacterium tuberculosis<\/jats:italic>\n                    (\n                    <jats:italic>Mtb<\/jats:italic>\n                    ), have a complex cell surface comprising an inner membrane, a cell wall modified with arabinogalactan (AG), and an outer mycomembrane made of mycolic acids linked to AG polymers. Because these surface biogenesis pathways are targeted by frontline anti-\n                    <jats:italic>Mtb<\/jats:italic>\n                    drugs, there is great interest in elucidating their underlying mechanisms. Here, we identify an interaction between factors involved in mycomembrane (TmaT) and AG biosynthesis (AftD) in the model organism\n                    <jats:italic>Corynebacterium glutamicum.<\/jats:italic>\n                    We show that this interaction is important for proper surface biogenesis and may therefore function to coordinate mycomembrane assembly with AG synthesis. This and other potential regulatory connections controlling envelope biogenesis represent attractive targets for future antibiotic development.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.07.31.741767","type":"posted-content","created":{"date-parts":[[2026,8,1]],"date-time":"2026-08-01T04:25:14Z","timestamp":1785558314000},"source":"Crossref","is-referenced-by-count":0,"title":["A TmaT-AftD interaction is required for the CmpL4 mycomembrane biogenesis pathway in\n                  <i>Corynebacterium glutamicum<\/i>"],"prefix":"10.64898","author":[{"given":"Anastacia R.","family":"Parks","sequence":"first","affiliation":[{"name":"Department of Microbiology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA"},{"name":"Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, 02115, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eric D.","family":"Snow","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Victoria M.","family":"Marando","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael J.","family":"James","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1019-3558","authenticated-orcid":false,"given":"Vincent","family":"de Bakker","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Laura L.","family":"Kiessling","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Suzanne","family":"Walker","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3566-7756","authenticated-orcid":false,"given":"Thomas G.","family":"Bernhardt","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA"},{"name":"Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, 02115, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026080310100666000_2026.07.31.741767v1.1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.bi.64.070195.000333"},{"key":"2026080310100666000_2026.07.31.741767v1.2","first-page":"116","article-title":"Mycobacterial cell wall biosynthesis: a multifaceted antibiotic target","volume":"145","year":"2016","journal-title":"Parasitology"},{"key":"2026080310100666000_2026.07.31.741767v1.3","doi-asserted-by":"crossref","first-page":"1023","DOI":"10.1586\/eri.12.91","article-title":"Targeting the mycobacterial envelope for tuberculosis drug development","volume":"10","year":"2012","journal-title":"Expert Rev. Anti-Infect. Ther"},{"key":"2026080310100666000_2026.07.31.741767v1.4","doi-asserted-by":"publisher","DOI":"10.1021\/cb5007689"},{"key":"2026080310100666000_2026.07.31.741767v1.5","doi-asserted-by":"crossref","unstructured":"Rens, C. , Chao, J. D. , Sexton, D. L. , Tocheva, E. I. & Av-Gay, Y. Roles for phthiocerol dimycocerosate lipids in Mycobacterium tuberculosis pathogenesis. Microbiology 167, (2021).","DOI":"10.1099\/mic.0.001042"},{"key":"2026080310100666000_2026.07.31.741767v1.6","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0305439101"},{"key":"2026080310100666000_2026.07.31.741767v1.7","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M408578200"},{"key":"2026080310100666000_2026.07.31.741767v1.8","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1700062114"},{"key":"2026080310100666000_2026.07.31.741767v1.9","doi-asserted-by":"publisher","DOI":"10.1038\/nchembio.794"},{"key":"2026080310100666000_2026.07.31.741767v1.10","doi-asserted-by":"publisher","DOI":"10.1016\/j.chembiol.2012.03.006"},{"key":"2026080310100666000_2026.07.31.741767v1.11","first-page":"1340","article-title":"Transport of outer membrane lipids in mycobacteria","volume":"1862","year":"2017","journal-title":"Biochim. Biophys. Acta (BBA) - Mol. Cell Biol. Lipids"},{"key":"2026080310100666000_2026.07.31.741767v1.12","doi-asserted-by":"publisher","DOI":"10.1126\/science.276.5317.1420"},{"key":"2026080310100666000_2026.07.31.741767v1.13","doi-asserted-by":"publisher","DOI":"10.1128\/JB.00285-13"},{"key":"2026080310100666000_2026.07.31.741767v1.14","doi-asserted-by":"publisher","DOI":"10.1016\/J.CELL.2021.06.033"},{"key":"2026080310100666000_2026.07.31.741767v1.15","first-page":"10.1128\/msystems.00070-19","article-title":"Genomewide Assessment of Mycobacterium tuberculosis Conditionally Essential Metabolic Pathways","volume":"4","year":"2019","journal-title":"mSystems"},{"key":"2026080310100666000_2026.07.31.741767v1.16","first-page":"10.1128\/mbio.02133-16","article-title":"Comprehensive Essentiality Analysis of the Mycobacterium tuberculosis Genome via Saturating Transposon Mutagenesis","volume":"8","year":"2017","journal-title":"mBio"},{"key":"2026080310100666000_2026.07.31.741767v1.17","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1002251"},{"key":"2026080310100666000_2026.07.31.741767v1.18","doi-asserted-by":"publisher","DOI":"10.1046\/j.1365-2958.2003.03425.x"},{"key":"2026080310100666000_2026.07.31.741767v1.19","doi-asserted-by":"crossref","first-page":"702","DOI":"10.1021\/acsinfecdis.6b00095","article-title":"Structure\u2013Function Profile of MmpL3, the Essential Mycolic Acid Transporter from Mycobacterium tuberculosis","volume":"2","year":"2016","journal-title":"ACS Infect. Dis"},{"key":"2026080310100666000_2026.07.31.741767v1.20","doi-asserted-by":"publisher","DOI":"10.3390\/biom11121760"},{"key":"2026080310100666000_2026.07.31.741767v1.21","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.RA119.011688"},{"key":"2026080310100666000_2026.07.31.741767v1.22","doi-asserted-by":"crossref","first-page":"2","DOI":"10.1016\/j.tcsw.2017.10.001","article-title":"AftD functions as an \u03b11 \u2192 5 arabinofuranosyltransferase involved in the biosynthesis of the mycobacterial cell wall core","volume":"1","year":"2018","journal-title":"Cell Surf"},{"key":"2026080310100666000_2026.07.31.741767v1.23","doi-asserted-by":"publisher","DOI":"10.1093\/glycob\/cwp116"},{"key":"2026080310100666000_2026.07.31.741767v1.24","unstructured":"Sin, R. H. S. Y. C. S. , Chen, Y. & Chng, S.-S. Discovery of a regulatory node that coordinates cell envelope assembly in mycobacteria. Biorxiv (2026)."},{"key":"2026080310100666000_2026.07.31.741767v1.25","doi-asserted-by":"publisher","DOI":"10.1016\/j.chembiol.2012.03.006"},{"key":"2026080310100666000_2026.07.31.741767v1.26","doi-asserted-by":"crossref","first-page":"484","DOI":"10.1002\/mbo3.182","article-title":"RND transporters protect Corynebacterium glutamicum from antibiotics by assembling the outer membrane","volume":"3","year":"2014","journal-title":"MicrobiologyOpen"},{"key":"2026080310100666000_2026.07.31.741767v1.27","doi-asserted-by":"publisher","DOI":"10.1002\/anie.201712020"},{"key":"2026080310100666000_2026.07.31.741767v1.28","doi-asserted-by":"publisher","DOI":"10.1194\/jlr.M082784"},{"key":"2026080310100666000_2026.07.31.741767v1.29","doi-asserted-by":"publisher","DOI":"10.3390\/biom11121760"},{"key":"2026080310100666000_2026.07.31.741767v1.30","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.RA119.011688"},{"key":"2026080310100666000_2026.07.31.741767v1.31","doi-asserted-by":"publisher","DOI":"10.1016\/J.CELL.2021.06.033"},{"key":"2026080310100666000_2026.07.31.741767v1.32","doi-asserted-by":"publisher","DOI":"10.1128\/msystems.00070-19"},{"key":"2026080310100666000_2026.07.31.741767v1.33","doi-asserted-by":"publisher","DOI":"10.1128\/mbio.02133-16"},{"key":"2026080310100666000_2026.07.31.741767v1.34","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1002251"},{"key":"2026080310100666000_2026.07.31.741767v1.35","doi-asserted-by":"publisher","DOI":"10.1046\/j.1365-2958.2003.03425.x"},{"key":"2026080310100666000_2026.07.31.741767v1.36","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.54761"},{"key":"2026080310100666000_2026.07.31.741767v1.37","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M116.772202"},{"key":"2026080310100666000_2026.07.31.741767v1.38","doi-asserted-by":"crossref","first-page":"494","DOI":"10.1107\/S2059798322001772","article-title":"Crystal structure of the putative cell-wall lipoglycan biosynthesis protein LmcA from Mycobacterium smegmatis","volume":"78","year":"2022","journal-title":"Acta Crystallogr. Sect. D"},{"key":"2026080310100666000_2026.07.31.741767v1.39","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M700271200"},{"key":"2026080310100666000_2026.07.31.741767v1.40","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M102272200"},{"key":"2026080310100666000_2026.07.31.741767v1.41","doi-asserted-by":"crossref","first-page":"38","DOI":"10.1016\/j.tcsw.2018.06.003","article-title":"The singular Corynebacterium glutamicum Emb arabinofuranosyltransferase polymerises the \u03b1(1 \u2192 5) arabinan backbone in the early stages of cell wall arabinan biosynthesis","volume":"2","year":"2018","journal-title":"Cell Surf"},{"key":"2026080310100666000_2026.07.31.741767v1.42","doi-asserted-by":"publisher","DOI":"10.1093\/glycob\/cwl030"},{"key":"2026080310100666000_2026.07.31.741767v1.43","doi-asserted-by":"publisher","DOI":"10.1111\/j.1365-2958.2008.06354.x"},{"key":"2026080310100666000_2026.07.31.741767v1.44","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.93.21.11919"},{"key":"2026080310100666000_2026.07.31.741767v1.45","doi-asserted-by":"crossref","first-page":"819","DOI":"10.1021\/cb200091m","article-title":"Reconstitution of functional mycobacterial arabinosyltransferase AftC proteoliposome and assessment of decaprenylphosphorylarabinose analogues as arabinofuranosyl donors","volume":"6","year":"2011","journal-title":"ACS Chem. Biol"},{"key":"2026080310100666000_2026.07.31.741767v1.46","doi-asserted-by":"crossref","first-page":"2","DOI":"10.1016\/j.tcsw.2017.10.001","article-title":"AftD functions as an \u03b11 \u2192 5 arabinofuranosyltransferase involved in the biosynthesis of the mycobacterial cell wall core","volume":"1","year":"2018","journal-title":"Cell Surf"},{"key":"2026080310100666000_2026.07.31.741767v1.47","doi-asserted-by":"publisher","DOI":"10.1093\/glycob\/cwp116"},{"key":"2026080310100666000_2026.07.31.741767v1.48","doi-asserted-by":"publisher","DOI":"10.1111\/j.1365-2958.2008.06354.x"},{"key":"2026080310100666000_2026.07.31.741767v1.49","doi-asserted-by":"crossref","first-page":"16337","DOI":"10.1021\/jacs.1c07430","article-title":"Biosynthetic Glycan Labeling","volume":"143","year":"2021","journal-title":"J. Am. Chem. Soc"},{"key":"2026080310100666000_2026.07.31.741767v1.50","doi-asserted-by":"publisher","DOI":"10.1038\/S41586-024-07487-W"},{"key":"2026080310100666000_2026.07.31.741767v1.51","doi-asserted-by":"crossref","first-page":"1728","DOI":"10.1021\/acs.biochem.4c00731","article-title":"Acyltransferases that Modify Cell Surface Polymers Across the Membrane","volume":"64","year":"2025","journal-title":"Biochemistry"},{"key":"2026080310100666000_2026.07.31.741767v1.52","doi-asserted-by":"crossref","first-page":"110546","DOI":"10.1016\/j.jbc.2025.110546","article-title":"Structural unification of diverse transmembrane acyltransferases reveals a conserved fold for the transmembrane acyl transferase (TmAT) superfamily","volume":"301","year":"2025","journal-title":"J. Biol. Chem"},{"key":"2026080310100666000_2026.07.31.741767v1.53","doi-asserted-by":"publisher","DOI":"10.1128\/mbio.01364-20"},{"key":"2026080310100666000_2026.07.31.741767v1.54","doi-asserted-by":"crossref","first-page":"e81547","DOI":"10.7554\/eLife.81547","article-title":"A novel fold for acyltransferase-3 (AT3) proteins provides a framework for transmembrane acyl-group transfer","volume":"12","year":"2023","journal-title":"eLife"},{"key":"2026080310100666000_2026.07.31.741767v1.55","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-024-49614-1"},{"key":"2026080310100666000_2026.07.31.741767v1.56","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-024-53512-x"},{"key":"2026080310100666000_2026.07.31.741767v1.57","first-page":"243","article-title":"Fast and accurate protein structure search with Foldseek","volume":"42","year":"2023","journal-title":"Nat. Biotechnol"},{"key":"2026080310100666000_2026.07.31.741767v1.58","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.01634-13"},{"key":"2026080310100666000_2026.07.31.741767v1.59","doi-asserted-by":"crossref","first-page":"e1011127","DOI":"10.1371\/journal.pgen.1011127","article-title":"The conserved \u03c3D envelope stress response monitors multiple aspects of envelope integrity in corynebacteria","volume":"20","year":"2024","journal-title":"PLOS Genet"},{"key":"2026080310100666000_2026.07.31.741767v1.60","doi-asserted-by":"publisher","DOI":"10.1016\/j.gene.2006.12.003"},{"key":"2026080310100666000_2026.07.31.741767v1.61","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0029266"},{"key":"2026080310100666000_2026.07.31.741767v1.62","doi-asserted-by":"crossref","first-page":"147","DOI":"10.1186\/1475-2859-11-147","article-title":"Design and testing of a synthetic biology framework for genetic engineering of Corynebacterium glutamicum","volume":"11","year":"2012","journal-title":"Microb. Cell Factories"},{"key":"2026080310100666000_2026.07.31.741767v1.63","doi-asserted-by":"publisher","DOI":"10.1007\/s002530051557"},{"key":"2026080310100666000_2026.07.31.741767v1.64","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pgen.1008284"},{"key":"2026080310100666000_2026.07.31.741767v1.65","doi-asserted-by":"publisher","DOI":"10.1016\/j.mimet.2011.02.012"},{"key":"2026080310100666000_2026.07.31.741767v1.66","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.1923"},{"key":"2026080310100666000_2026.07.31.741767v1.67","doi-asserted-by":"publisher","DOI":"10.1093\/gigascience\/giab008"},{"key":"2026080310100666000_2026.07.31.741767v1.68","doi-asserted-by":"publisher","DOI":"10.1186\/s13059-014-0550-8"},{"key":"2026080310100666000_2026.07.31.741767v1.69","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkad350"},{"key":"2026080310100666000_2026.07.31.741767v1.70","doi-asserted-by":"crossref","first-page":"11189","DOI":"10.1021\/jacs.4c17913","article-title":"Intercepting a Mycobacterial Biosynthetic Pathway with Covalent Labeling","volume":"147","year":"2025","journal-title":"J. Am. Chem. Soc"},{"key":"2026080310100666000_2026.07.31.741767v1.71","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.54761"},{"key":"2026080310100666000_2026.07.31.741767v1.72","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkad350"},{"key":"2026080310100666000_2026.07.31.741767v1.73","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.54761"},{"key":"2026080310100666000_2026.07.31.741767v1.74","doi-asserted-by":"publisher","DOI":"10.1038\/S41586-024-07487-W"},{"key":"2026080310100666000_2026.07.31.741767v1.75","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkad350"},{"key":"2026080310100666000_2026.07.31.741767v1.76","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-024-49614-1"},{"key":"2026080310100666000_2026.07.31.741767v1.77","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-024-53512-x"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.64898\/2026.07.31.741767","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,3]],"date-time":"2026-08-03T17:10:15Z","timestamp":1785777015000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.64898\/2026.07.31.741767"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,31]]},"references-count":77,"URL":"https:\/\/doi.org\/10.64898\/2026.07.31.741767","relation":{},"subject":[],"published":{"date-parts":[[2026,7,31]]},"subtype":"preprint"}}