## 3 Results and Discussion  
### Loss of MSMEG_3070 3069 or mmpL11 Leads to Similar Defects in Biofilm Formation  
We hypothesized that if LprG\(-\)Rv1410c functions parallel to or downstream of MmpL11, loss of function would lead to a similar defect in biofilm formation. To test this, we used a previously reported _M. smegmatis_\(\Delta\)_MSMEG_3070\(-\)3069_ deletion strain [39; 41]. To enable direct comparison, we constructed a targeted deletion of _mmpL11_ in the same parent wild-type strain. Following established protocols [7], we compared the development of pellicle biofilms over a total of 5 days for these and corresponding complement strains (Figures 1A and S2). The wild-type strain formed an immature pellicle biofilm at the air-liquid interface after 2 days; a monolayer-like pellicle biofilm after 3 days; and a mature biofilm with reticulations after 4 days. In contrast, both \(\Delta\)_MSMEG_3070\(-\)3069_ and \(\Delta\)_mmpL11_ showed a delay in pellicle biofilm formation and failed to form mature reticulated biofilms by day 4 (Figure 1A) or even day 5 (Figure S2). Importantly, both the kinetics and full maturation of biofilm formation were restored via complementation (Figures 1A and S2). Since an in vitro interaction was previously detected between MmpL11 and LprG, but not with Rv1410c [40], we also assayed biofilm formation in \(\Delta\)_MSMEG_3070\(-\)3069_, complemented with either _Mtb lprG_ or _rv1410c_, rather than the full operon (Figure S3). Both singly complemented strains showed an identical phenotype to the wild-type strain, suggesting that either gene is sufficient for wild-type biofilm formation but only removal of both genes leads to a biofilm phenotype that is similar to that of \(\Delta\)_mmpL11_. Figure 1: _MSMEG_3070\(-\)3069 and _mmpL11_ mutants displayed impaired biofilm formation. (**A**,**B**) Pellicle biofilm formation at the air–liquid interface at 2 and 4 days after inoculation for (**A**) parent wild-type, \(\Delta\)_MSMEG_3070\(-\)3069_, _mmpL11_, and associated complement strains and (**B**) parent wild-type, mmpL:Tn, and associated complement strains. Equal numbers of bacteria were inoculated in [PERSON]’s medium, without Tween 80, in polystyrene dishes and the plates were incubated at 30 \({}^{\circ}\)C without disturbance for 5 days. Data shown are representative of three biological replicates. The delayed maturation biofilm phenotype prompted us to ask whether the gene deletion strains were attenuated for growth under biofilm conditions. Indeed, we found that both \(\Delta\)_MSMEG_3070\(-\)3069_ and \(\Delta\)_mmpL11_ strains grew more slowly, with approximately 1-log fewer colony forming units (CFU) compared to the wild-type strain 2 days after inoculation (Figure 2A,B). Although this difference was not statistically significant 4 days after inoculation, the trend and magnitude were similar (Figure 2C,D). For both mutants, growth at both timepoints was restored via complementation (Figure 2A,C). Thus, both \(\Delta\)_MSMEG_3070\(-\)3069_ and \(\Delta\)_mmpL11_ display a moderate growth defect that may contribute to the observed biofilm defect. This contrasts with _M. smegmatis mmpL11:Tn_, which was reported to have a biofilm maturation defect, but wild-type-like growth under similar conditions [7]. Therefore, we also assayed the biofilm and growth characteristics of the transposon insertion and corresponding complement strains. For _mmpL11:Tn_, biofilm formation resembled that of the parent wild-type strain until day 3 (Figures 1B and S2), after which it remained an easily disturbed monolayer without reticulations (Figures 1B and S2), and its growth was similar to that of the wild-type at both 2 days and 4 days post inoculation (Figure 2). While this characterization is similar to previous reports [7], it is notably distinct from the phenotype of our \(\Delta\)_mmpL11_-targeted deletion strain, suggesting that the different methods of gene  
Figure 2: _MSMEG_3070\(-\)3069_ and \(\Delta\)_mmpL11_ show delayed growth compared to the wild-type during biofilm formation.