## 4 Conclusions  
Taken together, our findings suggest that the loss of function of _MSMEG_3070\(-\)3069_ and _mmpL11_ leads to a defect in biofilm formation that is distinct from that of a previously characterized _mmpL11_ transposon insertion mutant in _M. smegmatis_. This work also revealed that neither the difference in the biofilm phenotype between \(\Delta\)_mmpL11_ and _mmpL11::Tn_, nor the similar biofilm defect in \(\Delta\)_MSMEG_3070\(-\)3069_ and \(\Delta\)_mmpL11_, can be directly explained by changes in the total or cell-surface levels of lipid classes that have been correlated with biofilm formation, or by changes in the expression of genes associated with biofilm formation or the synthesis of biofilm-related lipids. Instead, both null strains show a moderate growth defect that may underlie the observed delay in biofilm formation. Given that the common growth defect is not correlated with shared changes in the lipid profile, it is possible that the disruption of lipid transport in these strains dysregulates lipid metabolism in ways that similarly compromise growth. This model has been proposed for _lprG_\(-\)_rv1410c in Mtb_[39], but remains to be experimentally explored. Overall, we found that while LprG and MmpL11 may physically interact [40], these proteins do not function in overlapping lipid transport pathways that correlate with biofilm formation. Finally, the growth and the biofilm defects in \(\Delta\)_mmpL11_ were not shared by the _mmpL11::Tn_ transposon mutant, but the two strains were otherwise similar in all other assays. The only clear difference was that the chemical undefined species lipid A was retained in hexanes extracts of \(\Delta\)_mmpL11_ (Figure 3B). While we showed that this change was not correlated with differences in expression of the downstream _MSMEG_0240_, mutations in unlinked genes elsewhere in the chromosome or strain-specific responses to genetic modification could be responsible. An alternative, but less likely, explanation is that the low-level transcription of _mmpL11_ in _mmpL11::Tn_ permits the expression of an N-terminal fragment that complements growth without restoring lipid phenotypes. Nevertheless, comparison of the two strains confirms that _mmpL11_ is required in biofilms for the surface localization of MMDAG and MWE. Our data also provide a note of caution concerning complementation, as the multi-copy expression of _mmpL11_ from a constitutive promoter led to significant overexpression and could underlie the unexpected phenotypes seen when compared to the parent strain. The following supporting information can be downloaded at [[https://www.mdpi.com/article/10.3390/pathogens12121375/s1](https://www.mdpi.com/article/10.3390/pathogens12121375/s1)]([https://www.mdpi.com/article/10.3390/pathogens12121375/s1](https://www.mdpi.com/article/10.3390/pathogens12121375/s1)), Figure S1: schematic of plasmids used in the study and recombineering strategy used to generate \(\Delta\)_mmpL11_ strain; Figure S2: 5-day time course of pellicle biofilm formation at the air-liquid interface; Figure S3: 5-day time course of _MSMEG_3070\(-\)3069_ mutant and complement strains' pellicle biofilm formation at the air-liquid interface; Table S1: strains used in this study; Table S2: plasmids and primers used in this study. Conceptualization, L.-M.N. and J.C.S. ; resources, M.L.P. ; validation, L.-M.N., M.L.P. and J.C.S. ; methodology, L.-M.N., M.L.P. and J.C.S. ; formal analysis, L.-M.N. and J.C.S. ; investigation, L.-M.N. and J.C.S. ; writing--original draft preparation, L.-M.N., M.L.P. and J.C.S. ; writing--review and editing, L.-M.N. and J.C.S. ; visualization, L.-M.N. and J.C.S. ; supervision, J.C.S. ; project administration, J.C.S. ; funding acquisition, J.C.S. All authors have read and agreed to the published version of the manuscript. This research was supported by funding from the National Institutes of Health (R01 AI141513 to J.C.S. and IRACDA NY-CAPS K12 GM102778 to L.-M.N.). Not applicable.