## 1 Introduction
Indeed, other studies have identified correlations with assorted genes and lipids: Loss of function in enzymes that make the second messengers (pp)Gpp (_rel_) or c-di-GMP (_dcpA_) are defective for biofilm formation and show reductions in total levels of not only GPLs, but also other polar lipids [30]. Notably, the involvement of _lsr2_ in biofilms was uncovered via a transposon insertion library screen in _M. smegmatis_[26]. Additional prospective studies have sought to identify other factors that influence mycobacterial biofilm formation through not only transposon insertion library screens [10; 31; 32] in other species, but also gene expression profiling [10; 33; 34], and comparative proteomics [35; 36]. These have further underscored the association of lipids with the identification of additional genes and proteins related to lipid biosynthesis. Overall, the correlations between membrane lipid composition and biofilm formation highlight lipid biosynthesis and transport as targets for disrupting these antibiotic-resistant cell aggregates. Many of the enzymes that produce mycobacterial lipids have been identified, but the mechanisms by which these products reach the cell wall and mycomembrane remain poorly understood. Of the few pathways that have been characterized, the lipoprotein LprG and the co-cistronic integral membrane transporter Rv1410c are conserved across mycobacteria and are associated with the transport of triacyl lipids such as triacylglycerolide and lipoarabinomannan [37; 38; 39]. Intriguingly, LprG has been shown to interact in vitro with the periplasmic domains of MmpL11 [40], which is involved in biofilm maturation and the surface localization of mycolic acid-containing lipids in biofilms such as MWE and MMDAG [7; 27]. This evidence for a physical interaction led us to investigate the possible functional interaction between these pathways. In this study, our aim was to investigate the potential overlapping roles of LprG\(-\)Rv1410c and MmpL11 in lipid transport and biofilm formation. Based on previous work, we focused these studies on _M. smegmatis_ and the effects of disrupting the homologues_MSMEG_3070\(-\)3069 (lprG\(-\)rv1410c)_ and _mmpL11_ (_MSMEG_0241_; based on prevailing nomenclature, we use _mmpL11_ to designate both the _M. smegmatis_ and _Mtb_ homologues throughout). We found that the targeted deletion of _mmpL11_ or _MSMEG_3070\(-\)3069_ led to similar biofilm defects that were distinct from that of the previously reported _mmpL11_ transposon insertion mutant [7] and were not correlated with shared changes in the total or cell-surface levels of biofilm-related lipids. Instead, we found that differences in _mmpL11_ gene expression could underlie the distinct biofilm phenotype between _\(\Delta\)mmpL11_ and _mmpL11:Tn_, and a modest growth defect could explain the biofilm defect shared between _\(\Delta\)MSMEG_3070\(-\)3069_ and _\(\Delta\)mmpL11_. Our findings thus suggest that although LprG and MmpL11 may physically interact in vitro [40], the biofilm defects in the _\(\Delta\)MSMEG_3070\(-\)3069, _\(\Delta\)mmpL11_, and _mmpL11:Tn_ strains are not due to overlapping functions of these loci in lipid transport.