## 3 Results and Discussion
### Loss of MSMEG_3070 3069 or mmpL11 Leads to Similar Defects in Biofilm Formation
TLC plates were immersed in 10% phosphomolybdic acid and charred to visualize lipids. The line near the top of the plates indicates the solvent front. Data shown represents one biological replicate. (Figure 4). In summary we found no shared differences in surface nor total levels of lipids previously associated with biofilm formation that could explain the biofilm phenotypes we observed upon loss of _MSMEG_3070\(-\)3069_ and _mmpL11_. Loss of MSMEG_3070\(-\)3069 or mmpL11 Does Not Correlate with Changes in Expression of Genes Associated with Biofilm Formation  
Since we found no shared differences in lipids previously associated with biofilm formation, we hypothesized that the biofilm defect in the \(\Delta\)_MSMEG_3070\(-\)3069_, \(\Delta\)_mmpL11_, and _mmpL11::Tn_ strains could be due to changes in the expression of biofilm-related genes. To test this hypothesis, we measured the expression of _mmpL11_, the co-cistronic _MSMEG_0240_, and genes associated with biofilm maturation (_groE11_, _kasA_) [12] or required for TDM synthesis (_mmpL3_, _ag85A_, _ag85B_, _ag85C_) [11]. Compared to the wild-type, no significant changes in gene expression were detected for any of these genes (Figure 5). The only exception was _mmpL11_. As expected, expression was not detected in the targeted gene deletion _mmpL11_ (Figure 5A). Low levels were found in _mmpL11::Tn_, as assessed using a primer pair upstream of the mapped transposon insertion site, suggesting that the insertion suppressed but did not prevent transcription (Figure 5B). However, in _mmpL11::Tn_ complemented with _M. smegmatis mmpL11_, the complement was highly expressed (\(\sim\)64-fold increase compared to the wild-type, an average of three independent experiments) (Figure 5B). This suggests that _M. smegmatis mmpL11_ overexpression underlies the lack of biofilm complementation (Figures 1 and S2) and the unexpected growth defect (Figure 2) in this strain. In contrast, _Mtb mmpL11_ expression was not detected even though this strain showed complementation of the _mmpL11::Tn_ biofilm defect (Figures 1, 5B and S2). It is possible that this message is unstable when isolated, or that even very low expression is sufficient for complementation. In summary, differences in _mmpL11_ gene expression may contribute to the distinct biofilm phenotypes seen between \(\Delta\)_mmpL11_ and _mmpL11::Tn_, but cannot explain the biofilm defect shared between \(\Delta\)_mmpL11_ and \(\Delta\)_MSMEG_3070\(-\)3069_. Figure 4: The free mycolic acid lipid profile in the total and surface lipid extracts, and the trehalose dimycolate (TDM) and trehalose monomycolate (TMM) lipid profiles in the total lipid extracts are not altered in any strain. (**A**) 50 \(\upmu\)g total lipid and (**B**) 50 \(\upmu\)g surface lipid extracts were resolved via TLC in a chloroform: methanol (96:4, \(\
u/\
u\)) solvent system. (**C**) 50 \(\upmu\)g total lipid extracts were resolved via TLC in chloroform: methanol: water (30:8:1, \(\
u/\
u\)). TLC plates were immersed in 10% phosphombolybdic acid and charred to visualize lipids. The line near the top of the plate indicates the solvent front. TDM: trehalose dimycolate, TMM: trehalose monomycolate, PE: phosphatidylethanolamine, CL: cardiolipin, PIMs: phosphatidylinositolmannosides. Data shown represent one replicate. Figure 5: RT-qPCR analysis of genes connected to biofilm formation in M. smegmatis. RT-qPCR analysis of (**A**,**B**) _MSMEG\({}_{-}\)0240_ and _mmpL11_, (**C**,**D**) _groEL1_, _kasA_, and _inhA_ and (**E**,**F**) _qg85A_, _qg85B_, _qg85C_, and mmpL3 in the wild-type, mutant, and complement strains. Changes in gene expression are relative to the appropriate wild-type parent strain control and were calculated using the Pfaff method [45]. The housekeeping gene _sigA_ was used as the endogenous reference for normalization between samples. Data represent mean \(\pm\) SEM of three independent biological replicates. Statistical significance was determined using one-way ANOVA (Dunnett’s test) in GraphPad Prism version 10. (*, \(p\) = 0.03; ns: not significant).