{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,9,17]],"date-time":"2026-09-17T06:04:29Z","timestamp":1789625069909,"version":"build-2803163510"},"posted":{"date-parts":[[2026,9,11]]},"group-title":"Microbiology","reference-count":44,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T00:00:00Z","timestamp":1789084800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,9,12]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  The cell envelope of many bacteria possesses components that play central roles in bacterial pathogenesis. Unique to mycobacteria is the presence of a lipid-rich cell wall which is critical for the virulence of pathogenic mycobacteria such as\n                  <jats:italic>Mycobacterium tuberculosis<\/jats:italic>\n                  (\n                  <jats:italic>Mtb<\/jats:italic>\n                  ). The biosynthesis of mycobacterial cell wall lipids has been well characterized, but the mechanisms of lipid transport still remain largely unknown. MmpL (mycobacterial membrane protein large) proteins have been implicated in the biosynthesis and\/or transport of mycobacterial cell wall lipids, but due to their cellular location, it is still unclear how cell wall lipids are transported beyond the inner membrane. Here, we further investigate the role of two conserved lipid transport pathways LprG-Rv1410c and MmpL11 in cell envelope biogenesis during biofilm formation in\n                  <jats:italic>Mycobacterium smegmatis<\/jats:italic>\n                  . We found that deletion of the\n                  <jats:italic>lprG<\/jats:italic>\n                  -\n                  <jats:italic>rv1410c<\/jats:italic>\n                  operon homologues\n                  <jats:italic>MSMEG_3070-3069<\/jats:italic>\n                  and\n                  <jats:italic>mmpL11<\/jats:italic>\n                  (\n                  <jats:italic>MSMEG_0241<\/jats:italic>\n                  ) simultaneously led to similar biofilm defects as observed in the\n                  <jats:italic>MSMEG_3070-3069<\/jats:italic>\n                  and\n                  <jats:italic>mmpL11<\/jats:italic>\n                  mutants. Analysis of pellicle biofilms, total lipid extracts, gene expression and cellular c-di-GMP levels revealed that the shared biofilm defect is directly correlated with significant decreases in cellular c-di-GMP levels, but may also be due to changes in the synthesis and\/or localization of glycopeptidolipids (GPLs), and changes in the expression of GPL biosynthesis and transport genes. Our findings therefore suggest that while both LprG-Rv1410c and MmpL11 pathways are involved in modulating cellular c-di-GMP levels during biofilm formation, only LprG-Rv1410c are important for regulating GPL synthesis and\/or surface localization, and MmpL11 may play a broad role in fine-tuning GPL levels.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>IMPORTANCE<\/jats:title>\n                  <jats:p>\n                    The lipid-rich cell wall has been demonstrated to be essential for the virulence of pathogenic mycobacteria such as\n                    <jats:italic>Mycobacterium tuberculosis<\/jats:italic>\n                    (\n                    <jats:italic>Mtb<\/jats:italic>\n                    ). The biosynthesis of mycobacterial cell wall lipids has been well characterized, but the mechanisms of lipid transport still remain largely unknown. Here, we demonstrate that LprG-Rv1410c and MmpL11 impact biofilm formation in\n                    <jats:italic>M. smegmatis<\/jats:italic>\n                    via modulation of GPL biosynthesis and\/or cell wall localization and cellular c-di-GMP levels. This work further underscores the importance of lipid transport pathways in cell wall biogenesis and provides additional insights into how GPL synthesis and\/or cell wall transport may be regulated during mycobacterial biofilm formation, with implications for better understanding the connection between mycobacterial cell wall biogenesis, biofilm formation and mycobacterial physiology overall.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.09.10.750705","type":"posted-content","created":{"date-parts":[[2026,9,12]],"date-time":"2026-09-12T03:20:23Z","timestamp":1789183223000},"source":"Crossref","is-referenced-by-count":0,"title":["Loss of function of LprG-Rv1410c and MmpL11 homologues in\n                  <i>Mycobacterium smegmatis<\/i>\n                  leads to altered glycopeptidolipid profile and decreased cellular c-di-GMP levels during biofilm formation"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-5746-1625","authenticated-orcid":false,"given":"Mariah","family":"Desroches","sequence":"first","affiliation":[{"name":"Department of Microbiology, Cornell University, Ithaca, New York, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-7183-166X","authenticated-orcid":false,"given":"Miranda","family":"Coldren","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Cornell University, Ithaca, New York, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9252-5044","authenticated-orcid":false,"given":"Lisa-Marie","family":"Nisbett","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Cornell University, Ithaca, New York, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026091622451467000_2026.09.10.750705v2.1","unstructured":"World Health Organization. 2025. 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