## 3 Results and Discussion
### Loss of MSMEG_3070 3069 or mmpL11 Leads to Similar Defects in Biofilm Formation
Growth was assessed (**A,B**) 2 days and (**C,D**) 4 days after biofilm inoculation by (**A,C**) spotting serial dilutions on 7H10/ADC/glycerol agar or (**B,D**) enumerating CFU. For (**A,C**), data representative of three independent experiments are shown. For (**B,D**), the data shown are the mean \(\pm\) S.D. of three independent experiments. Statistical significance was determined using one-way ANOVA (Dunnett’s test). (\({}^{**}\), \(p\) = 0.0021; \({}^{***}\), \(p\) = 0.0002; ns: not significant). disruption have different consequences for biofilm formation and growth. Notably, _mmpL11_ is expressed in an operon upstream of _MSMEG_0240_, and transposon insertion could have different effects on _MSMEG_0240_ expression than gene deletion, although how expression changes would affect bacterial physiology is not obvious from the available information: _MSMEG_0240_ is annotated as a transcription factor, but is otherwise an uncharacterized hypothetical protein. Finally, in our hands and in contrast to previous reports [7], complementation of _mmpL11::Tn_ with _mmpL11_ from _M. smegmatis_ did not restore biofilm formation and growth trended lower compared to the wild-type (Figures 1, 2 and S2). In contrast, the _M. tuberculosis mmpL11_ complement yielded wild-type-like biofilms and growth. Differences in how strains were complemented complicate the interpretation of these conflicting results. Complementation of _mmpL11::Tn_ was achieved using a multi-copy plasmid with a strong constitutive promoter driving expression of _M. smegmatis mmpL11_, but in the _Mtb mmpL11_ complement, _mmpL11_ gene expression was driven by a native promoter from a single-copy integrating plasmid. Further, we complemented _\(\Delta\)mmpL11_ from a single-copy integrated plasmid with a native promoter driving expression of _Msm mmpL11_. Overall, the parallels in biofilm formation and growth between _\(\Delta\)MSMEG_3070\(-\)3069_ and _\(\Delta\)mmpL11_, but contrast with _mmpL11::Tn_, led us to examine other aspects of these strains that could underlie these similarities and differences. MSMEG_3070\(-\)3069 Is Not Required for the Surface Localization of Mycolate Wax Esters and Monomeromyclyl Diacylglyceroles  
The defect in biofilm formation in the _mmpL11::Tn_ strain has been correlated with a biofilm- and cell surface-specific loss of certain lipids: mycolate wax esters (MWE), monomeromyclyl diacylglyceroleeride (MMDAG), and a chromatographically resolved but chemically undefined species annotated as Lipid A [7]. This phenotype led to the assignment of MmpL11 as the transporter for these lipid classes. Changes in other mycomembrane lipids, such as disruptions in TDM synthesis and enzymatic hydrolysis by trehalose dimycolate hydrolase [11], have also been associated with biofilm defects. These results suggest a model in which protein interactions involving lipid biosynthesis enzymes modulate lipid composition and thereby, biofilm formation. Based on this model, the physical interaction between LprG and MmpL11 could modulate the transport of MmpL11-associated lipids like MWE and MMDAG and thereby affect biofilm formation. We would thus predict that _\(\Delta\)MSMEG_3070\(-\)3069_ and _\(\Delta\)mmpL11_ have similar changes in their surface lipids compared to the wild-type, although, based on biofilm formation and growth, possibly distinct from _mmpL11::Tn_. To test this hypothesis, we analyzed lipids extracted from _M. smegmatis_ biofilms 5 days post inoculation via thin layer chromatography, thus allowing for the comparison of lipid profiles with the biofilm phenotypes shown in Figures 1A and S2. Surface-selective loss of MWE, MMDAG, and lipid A in _mmpL11::Tn_ biofilms was previously shown by extracting lipids with hexanes and sonication with heating. To facilitate direct comparison, we applied this method to _\(\Delta\)MSMEG_3070\(-\)3069_ and the corresponding wild-type and complement strains. Surprisingly, _\(\Delta\)MSMEG_3070\(-\)3069_ extracts showed elevated triacylglycerides (Figure 3A).