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Rv1410 was originally postulated to function as a drug efflux pump, but recent studies suggested that Rv1410 and LprG work in concert to insert triacylglycerides and lipoarabinomannans into the outer membrane. Here, we conducted microscopic analyses of <jats:italic>Mycobacterium smegmatis <\/jats:italic>lacking the operon and observed a cell separation defect, while surface rigidity measured by atomic force microscopy was found to be increased. Whereas Rv1410 expressed in <jats:italic>Lactococcus lactis<\/jats:italic> did not confer drug resistance, deletion of the operon in <jats:italic>Mycobacterium abscessus <\/jats:italic>and <jats:italic>M. smegmatis <\/jats:italic>resulted in increased susceptibility toward vancomycin, novobiocin and rifampicin. A homology model of Rv1410 revealed a periplasmic loop as well as a highly conserved aspartate, which were found to be essential for the operon\u2019s function. Interestingly, influx of the fluorescent dyes BCECF\u2010AM and calcein\u2010AM in de\u2010energized <jats:italic>M. smegmatis <\/jats:italic>cells was faster in the deletion mutant. Our results unambiguously show that elevated drug susceptibility in the deletion mutant is caused by increased drug influx through a defective mycobacterial cell envelope and not by drug efflux mediated by Rv1410.<\/jats:p>","DOI":"10.1111\/mmi.14220","type":"journal-article","created":{"date-parts":[[2019,2,11]],"date-time":"2019-02-11T17:02:35Z","timestamp":1549904555000},"page":"1263-1282","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":35,"title":["Increased drug permeability of a stiffened mycobacterial outer membrane in cells lacking MFS transporter Rv1410 and lipoprotein LprG"],"prefix":"10.1111","volume":"111","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6046-4865","authenticated-orcid":false,"given":"Michael","family":"Hohl","sequence":"first","affiliation":[{"name":"Institute of Medical Microbiology University of Zurich Z\u00fcrich 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