## 3 Results and Discussion
### Loss of MSMEG_3070 3069 or mmpL11 Leads to Similar Defects in Biofilm Formation
This result contradicts the prevailing model from _Mtb_, in which LprG/Rv1410c transport TAG to the mycomembrane, but is consistent with the accumulation of select TAG isoforms in the total lipid extracts of _Mtb_\(\Delta\)lprG\(-\)rv1410c. One possible explanation is that the extraction method is not surface-selective: sonication was performed at 50 \({}^{\circ}\)C [27], which, may have led to the solubilization of lipids deeper within the mycobacterial cell envelope. We thus compared this to a gentler extraction in hexanes without sonication or heating, an approach that has been previously used to characterize lipid transport in _Mtb_[46; 47; 48]. Although TAG is considered a mycomembrane lipid based on surface-selective lipid extraction with reverse micelles, hexanes extracts obtained without sonication or heating showed markedly reduced extraction of TAGs, based on the comparison between treatment conditions and using a commercial tripalmitate standard (Figure 3B). However, when applied to _mmpL11::Tn_, this gentler extraction of hexanes still consistently recapitulated the previously reported loss of MWE, MMDAG, and lipid A (Figure 3B). We concluded that while this method may not exhaustively extract all surface lipids, it was more selective and therefore preferred for further analyses since it was less likely to lead to a false negative lipid transport phenotype. We therefore proceeded to analyze selective \"gentle\" hexanes and total chloroform-methanol extracts for all _MSMEG_3070\(-\)3069_- and _mmpL11_-associated strains (Figure 3). For all strains, no major differences were detected in the total cellular levels of MWE, MMDAG, or lipid A (Figure 3D). In contrast, hexanes extracts revealed distinctions between the strains that did not, however, correlate with biofilm phenotypes (Figures 1, 3B,C and S2). First, _M. smegmatis mmpL11_ complementation restored MWE, MMDAG, and lipid A levels in _mmpL11:Tn_. Second, _mmmpL11_ only partially recapitulated _mmpL11:Tn_: While a reduction in MWE was evident, changes in MMDAG or lipid A were not, and this phenotype was restored via complementation with _Msm mmpL11_. (Because MMDAG is not well resolved in the replicate shown in Figure 3B, a replicate with a subset of strains more clearly illustrating the reduction of MMDAG for _mmmpL11_ is provided in Figure 3C.) Finally, the loss of _MSMEG_3070 3069_ function did not strongly affect any of the three lipid classes. These results suggest that changes in the surface localization of MWE, MMDAG, and lipid A can broadly be correlated with the loss of _mmpL11_ function, but not the loss of _MSMEG_3070\(-\)3069_ function. Consequently, the shared defect in biofilm formation cannot be explained by the changes in the surface localization of these three classes of lipids in these strains. As noted in the introduction, free mycolic acids (FMA) have been implicated in biofilm formation through changes in the mycolic acid synthesis (specifically, the modulation of KasA activity by interaction with the chaperone GroEL1 [12]) and incorporation into the extracellular matrix (through the activity of a trehalose dimycolate hydrolase [11]). To test whether changes in FMA underlie the shared biofilm phenotype, we also analyzed free mycolic acid and trehalose (di)mycolate levels in all _MSMEG_3070\(-\)3069_ and _mmpL11_-related strains. However, no major changes in any of these lipid classes were detected  
Figure 3: _MSMEG_3070\(-\)3069_ mutant has an altered “harsh” hexane extract surface- exposed lipid profile of Lipid A, MWE, MMDAG and TAG but does not have an altered “gentle” hexane extract surface-exposed lipid profile. (**A**) 195 \(\upmu\)g of surface lipid extracts, (**B**,**C**) 200 \(\upmu\)g of surface lipid extracts and (**D**) 200 \(\upmu\)g of total lipid extracts were resolved by TLC in tolueneacetone (99:1, \(v/v\)). Tripalmitate (labeled as TAG) was used as a migration standard. Lipid A, MWE and MMDAG were assigned by comparison to previously published TLCs performed under the same conditions [7; 27].