In the pursuit of bridging the knowledge gap existing in the functionality of Wag31 (Fig. 1a), we generated a Wag31 conditional mutant in Msm, by integrating a copy of wag31 under tetracycline (ATc- tet off) regulation at the L5 locus and replacing the native copy with a hygromycin cassette (Fig. S1a). The mutant, Δwag31, thus generated, was confirmed by PCRs (Fig. S1b) and Western blotting (Fig. 1b). The lack of Wag31 led to a drop in the turbidity of culture (Fig. 1c). Analysis of bacillary survival in vitro by enumerating CFUs 12h post-ATc addition, revealed a 2 log fold difference in the survival of Δwag31 (Fig. 1d). Given the essential role of Wag31 in causing polar growth of the cell, we investigated the effect of depletion of Wag31 on bacterial cellular morphology using electron microscopy (EM). Scanning EM (SEM) suggested that the absence of Wag31 caused a transition in the morphology of the cells from rod to round, as previously documented (10, 15, 38). Apart from round morphology (∼36 %), we also observed a rod-to-round transformation stage in which cells were bulged at one pole with the other pole intact (Fig. 1e-g). To obtain detailed insights, we performed transmission EM (TEM), which divulges information on the internal structure of cells. We discovered spherical, electron-lucent bodies (indicated by an arrowhead in Fig.1h) in the cytosol of all the samples. While Msm and Δwag31- ATc harbored a few (∼2-3) electron-lucent bodies, their numbers were drastically higher in the case of Δwag31+ATc (Fig. 1h). The cytosol of Δwag31+ATc was filled with multiple variable-sized bodies which resembled Lipid bodies that Mycobacterium tuberculosis (Mtb) accumulates inside the host (39, 40). As the term ‘lipid bodies/lipid droplets’ is used specifically for the lipids acquired by Mtb from the host cell, we referred to them as intracellular lipid inclusions (ILIs). We categorized the cells based on the number of ILIs they contained. Their distribution remained reasonably similar within Msm and Δwag31-ATc cells for all the classes (Fig. 1i). Whereas Δwag31 treated with ATc had as high as 85% cells harboring more than 5 ILIs and very few cells with less than 5 ILIs (Fig. 1i). Our data, therefore, reports a novel observation of lipid accumulation when Wag31 is depleted from mycobacteria.

Next, we set out to analyze if the ILIs are indeed lipidic in nature by staining cells with BODIPY (493/503), a dye that preferentially partitions into neutral lipid droplets (41, 42). We stained either untreated or ATc-treated Msm and Δwag31 with BODIPY and visualized them on a microscope. ATc- treated Δwag31 cells displayed the highest level of BODIPY fluorescence, indicating the maximum abundance of lipid bodies among the three strains (Fig. 2a, c). Further, we examined whether the appearance of ILIs is associated just with the loss of Wag31 or is a consequence of deviations from the native expression level of Wag31. Towards this, we overexpressed Wag31 episomally using an isovaleronitrile (IVN) inducible system (Msm::wag31) (Fig. S2a) and stained the cells with BODIPY. Unlike Wag31 depletion, which resulted in spherical cells (Fig. 1e), Wag31 overexpression resulted in the bulging of cells from one pole (Fig. 2b). Additionally, the overexpression of Wag31 also led to lipid aggregates in the cell with a few larger ‘donut-like’ aggregates in the bulged pole (Fig 2b,d).

Lipid accumulation in both cases led us to inspect whether lipid metabolism is closely tied to Wag31 expression levels in mycobacteria. To investigate that possibility, total cellular lipids were extracted from Msm, Wag31 depletion, and overexpression conditions and subjected to semi-quantitative LC-MS analysis (illustrated in Fig. 2e). Lipidomic analysis of Δwag31+ATc revealed an imbalance in the levels of neutral lipids, phospholipids, and free fatty acids in comparison to Msm-ATc (Fig. 2f-g). Δwag31+ATc cells had a significant accumulation of unsaturated diacylglycerols (∼3-5 fold) and a very long chain containing (>C36) phosphatidylethanolamines (∼5-32 fold). This was accompanied by increased levels of different chain lengths (C18-C24) of free fatty acids (∼1.7-4 fold) and reduced levels of cardiolipins C70:0 & C70:1 (∼3 fold). Wag31 overexpression, on the other hand, led to a 2-fold increase in the level of different species of alpha-mycolic acids (C74-C78) as compared to Msm+IVN (Fig. 2h), while other tested lipids remained unchanged (Fig. S2). Altogether, the results presented above demonstrate that Wag31 levels are crucial for maintaining lipid homeostasis in mycobacteria, the depletion and overexpression of which impact lipid metabolism differently.

We reasoned that Wag31, an adaptor protein, might maintain lipid homeostasis by interacting with proteins involved in these pathways. To examine the protein interaction network of Wag31, we performed an interactome analysis by utilizing Δwag31, which expresses FLAG-Wag31 integrated at the L5 locus, and used FLAG-GFP as the control to differentiate from non-specific interactions. FLAG- Wag31 and FLAG-GFP from biological triplicates were immunoprecipitated using whole cell lysates from the log phase cells and subjected to mass spectrometry analysis (Fig. S3a & Table S2). This study identified both direct and indirect protein-partners of Wag31. The proteins identified were categorized based on their functions. It was found that Wag31 interacts with proteins involved in cell-wall elongation and division, lipid metabolism, intermediary metabolism and respiration and information pathways (translation, replication) (Fig. 3a, Fig. S4). Interactions of Wag31 with AccA3 and AccD5, subunits of the ACCase complex, and Rne or RNaseE (Msm4626), a membrane-associated protein, have been reported previously (28, 43). Subunits of ACCase complex and Rne were among the top 5 hits in our MS/MS data, validating our approach (Fig. 3a, marked with asterisk). The classification of the top 20 hits is represented in Fig. 3a. The highest number of protein interactors, expectedly, fell in the cell wall and cell processes category (Fig. 3a). For the present study, we focussed on the interactors found in the cell wall elongation and cell division and lipid metabolism functional categories to probe the cause behind the lipid dysbiosis phenotype.

MurG, involved in PG metabolism (36, 44–46), and SepIVA, involved in regulating septation and the spatial regulator of MurG (47) also surfaced in the interactome of Wag31. Msm1813, or acyl CoA carboxylase (ACAD), crucial for nascent fatty acid precursor synthesis, was also enriched in the interactome. Other lipid metabolism interactors of Wag31 included acyl-CoA dehydrogenase (Msm2080), involved in lipid catabolism; GpsA, involved in phospholipid biosynthesis; and Msm0372, involved in fatty acid biosynthesis. To validate the high-throughput data, we chose MurG, SepIVA and Msm2092 and performed pull-down assays with His-tagged Wag31. MmpL4 and MmpS5 which didn’t appear in the interactome database were used as negative controls in this experiment. His-tagged Wag31 (Fig. 3b) was incubated with Msm lysates expressing 3X-FLAG tagged versions of the interactors and non-interactors mentioned above (Fig. 3c) and pulled-down with the help of cobalt beads followed by a western blot analysis. Results obtained confirmed the interaction of Wag31 with MurG, SepIVA and Msm2092, but not with the MmpL4 and MmpS5, thus validating our data (Fig. 3d).

Since Wag31 is a spatiotemporal regulator of other proteins, we were intrigued to discover the localisation of its interacting partners. We used the data curated by Zhu et al. (48) and determined that Wag31 majorly interacts with membrane-associated proteins with a few exceptions (Fig. 3e, Fig. S3b). The mycobacterial plasma membrane has been recently shown to have a cell wall-associated compartment called PM-CW and a cell wall-free compartment called PM_f or IMD, which is the site for lipid synthesis. Based on the available data (36, 48, 49), we found that Wag31 interacts more with IMD- proteins than with PM-CW proteins (Fig. 3f.) In accordance with assigned roles (36), PM-CW residing proteins in the interactome are involved in cell wall and cell processes and intermediary metabolism and respiration. Similarly, IMD-homing proteins clustered in the lipid metabolism and cell wall synthesis category. These results suggest that Wag31 maintains lipid homeostasis and cellular morphology primarily through its interactions with the membrane proteins participating in such pathways.

Wag31 is a cytoplasmic protein that recognizes negative curvature regions in the cell, i.e., poles and septum (28). It also forms higher-order structural assemblies at the poles (21, 50). However, the precise mechanism by which Wag31 assemblies localize to the poles remains elusive. The predominant interactions of Wag31 with membrane-associated proteins (Fig. 3) were intriguing and led us to investigate if it can directly bind to membranes. We reasoned that such membrane binding activity might render it capable of acting as a scaffold that anchors and organizes other proteins, thereby modulating their functions. To test this, we performed a lipid crosslinking-based PLiMAP assay (Fig. 4a). Purified Wag31 was incubated with ∼100 nm extruded liposomes containing each of the abundant classes of bacterial lipids phosphatidylethanolamine (PE), phosphatidylglycerol (PG), or cardiolipin (CL). Liposomes contained trace amounts of a reactive fluorescent lipid (RFL), which crosslinks with membrane-bound proteins upon photoactivation. These assays revealed that Wag31 preferentially binds CL- but not PG- or PE-containing liposomes (Fig. 4b). Both CL and PG are anionic lipids, and the preferential binding of Wag31 to CL indicates that the binding cannot be attributed to a charge-based interaction. Remarkably, the sensitive fluorescence-based detection revealed the presence of SDS- resistant higher-order oligomers of membrane-bound Wag31 (Fig. 4b).

Wag31 is a homolog of the DivIVA protein from B. subtilis. The latter has been shown to bind to the membrane via the N-terminal DivIVA domain (N-DivIVA_BS) (51). However, the functional significance of the N- & C- terminal domains of mycobacterial Wag31 has not been dissected. Considering that the Wag31 N-terminal region shares 42% sequence identity with N-DivIVA_BS (50), we sought to inspect the effect of disruption of a particular residue (K20) in the N-terminus and the deletion of the entire N-terminus, encompassing the DivIVA domain (1-60 amino acids) on membrane binding (Fig. S5a). The K20 residue lies within a positively charged patch, which likely constitutes a membrane-associating surface in the intertwined loop region of Wag31(50). Moreover, mutating the K20 residue has been shown to decrease the cell length and elongation rate by 18% and 22%, respectively (34). The K20A mutation showed a marginal defect in binding to CL-liposomes (Fig. 4c-d), indicating that K20 alone is insufficient for membrane binding. However, Wag31_K20A showed a marked defect in forming SDS-resistant higher-order oligomers, as seen in the binding assays. Interestingly, deletion of the entire N-terminal domain caused a significant defect in both membrane binding and forming SDS- resistant higher-order oligomers (Fig. 4c-d). Surprisingly, binding was not entirely abolished, suggesting that the C-terminus of Wag31 may also contribute to membrane binding. Taken together, our results indicate that Wag31 preferentially binds CL-containing membranes and that regions other than the N- terminal domain contribute to membrane binding. The latter feature distinguishes Wag31 from DivIVA_BS, and Wag31 likely contains membrane binding sites located at both the N- and C-terminal regions, which might lead to differences in the manner in which it interacts with the membrane and partner proteins compared to DivIVA_BS.

To further confirm membrane binding, we imaged 100 nm-extruded CL-containing liposomes with a membrane dye DiI in the presence of Wag31. We used a GFP-tagged Wag31 mixed with an untagged Wag31 at a 1:10 molar ratio for these experiments. In the absence of Wag31-GFP, liposomes appeared as faint but distinct fluorescent puncta (Fig. 4e, first panel). Surprisingly, in reactions containing Wag31-GFP, we observed liposomes clustered into bright fluorescent puncta that were positive for Wag31-GFP (Fig. 4e, remaining panels). The Wag31 tetramer has been modelled as a long rod-shaped protein filament (21, 50) and such clustering of liposomes falls in congruence with the model. It indicates that these long filaments of Wag31 act to tether liposomes. We then systematically evaluated this effect by testing liposomes of different compositions and Wag31 mutants. Again, in the absence of Wag31, both PE- and CL-containing liposomes appeared as faint but distinct fluorescent puncta (Fig. 4f). The addition of Wag31 caused clustering of CL-containing liposomes into bright foci (Fig. 4f), which is quantified by a particle intensity analysis (Fig. 4g). This clustering was caused by membrane binding of Wag31 because PE-containing liposomes that do not bind Wag31 (Fig. 4b) showed no clustering (Fig. 4f,g). As expected, Wag31_ΔN was defective in membrane binding (Fig. 4c,d) and showed a severe tethering capacity defect (Fig. 4f,g). Surprisingly, Wag31_K20A, which showed a marginal defect in membrane binding (Fig. 4c,d), showed a marked defect in tethering capacity (Fig. 4f,g). Together, these results suggest that membrane-binding and membrane-tethering are non-overlapping attributes in the structure of Wag31, and the N-terminal DivIVA-domain is indispensable for membrane-tethering activity.

Results presented in Fig.4c indicate that Wag31 binds strongly to CL-containing membranes. CL is a negatively charged inverted cone-shaped lipid that has been shown to be concentrated at the sites of negative curvature in the bacterial cell, i.e., pole and septum (52, 53). Wag31 has been demonstrated previously to localize at sites of negative curvature (28). Their similar localization pattern, coupled with Wag31’s affinity to bind to CL, led us to examine the effect of this binding in vivo. We hypothesized that modulations in Wag31 levels might alter CL distribution in the cell.

To examine this, we utilized a fluorophore 10-N-Nonyl-acridine orange (NAO) that is widely used to examine the localization of CL in prokaryotes and in the mitochondria of eukaryotes (52, 54, 55). NAO binds to anionic phospholipids with an affinity constant of 7 × 10⁴ M⁻¹ and to CL with an affinity constant of 2 × 10⁶ M⁻¹ (56). We stained Msm and Wag31 depleted and overexpressing cells with NAO and imaged them using a fluorescence microscope. While the localization pattern of NAO was polar in Msm+ATc and Δwag31-ATc, there was a redistribution of red fluorescence to the cytosol and along the perimeter of the cell in Δwag31+ATc (Fig.5a-b). We quantified the distribution of fluorescence using a line profile which yielded peaks of intensity at the poles and a basal level of fluorescence along the lateral cell body for both Msm+ATc and Δwag31-ATc (Fig.5b). Wag31 overexpression also led to a change in the distribution of NAO from the poles and septum to ‘donut-like’ fluorescent foci in the cytosol (Fig.5c-d). As the affinity of NAO for CL is more than other anionic phospholipids and our result (shown in Fig. 4b) indicate that Wag31 prefers to bind CL over PG (another negatively charged phospholipid), the change in the distribution of NAO fluorescence implies that the loss or overexpression of Wag31 causes the mislocalization of CL in mycobacterium.

Subsequently, we investigated the impact of tethering-deficient Wag31 mutants on cell survival. Wag31 has an N-terminal domain joined to the C-terminal via a linker region. The N-terminal domain contains a positively charged patch in the intertwined region (the region between two helices in the N- terminus) and membrane binding has been attributed to this region (50). This patch is lined with conserved arginine and lysine residues, K15, K20, and R21 (50) (Fig. 5e). As our in vitro results suggest the importance of K20 and the DivIVA-domain in tethering, we performed complementation assays with the tethering-defective mutants to examine cell survival and morphology. Δwag31 was complemented with an integrative copy of either Wag31 or Wag31_K20A or Wag31_ΔN. Δwag31+ATc showed a two log-fold difference in survival and a 40 % decrease in the percentage of rod-shaped cells compared with Δwag31-ATc (Fig. 5f-g). Complementation with Wag31 and Wag31_K20A rescued the loss of cell viability and restored the rod shape of the cells (Fig. 5f-g), whereas Wag31_ΔN behaved similar to Δwag31+ATc. It neither restored the rod shape of the cells nor reversed the survival defect (Fig. 5f-g). These results highlight the importance of the N-terminal DivIVA-domain for sustaining mycobacterial morphology and survival.

As described above, Wag31-mediated membrane tethering is an absolute for the survival of mycobacteria. Perturbations in lipid homeostasis and mislocalization of CL due to altered Wag31 levels hint at a correlation between its ability to function as a homotypic tether and maintain lipid composition and localization. To derive the mechanism behind it, we performed fluorescence imaging experiments with native and tethering defective mutants of Wag31. To investigate a role of tethering in lipid homeostasis in mycobacteria, we stained wildtype and complementation strains with BODIPY and quantified the fluorescence intensities. We observed comparable staining intensity in Msm +ATc and, Δwag31 -ATc (Fig. 6a-b). Δwag31+ATc expectedly had the highest BODIPY staining intensity, indicating higher than normal lipid levels (Fig. 6a-b). Complementation with Wag31 fully restored the lipid levels to normal, whereas complementation with Wag31_K20A couldn’t restore the lipid levels completely (Fig. 6a-b). Wag31_ΔN exhibited higher lipid levels than Δwag31-ATc (Fig. 6a-b). These results highlight that the maintenance of lipid homeostasis by Wag31 is a consequence of its tethering activity.

Next, we examined the dependence of CL localisation on tethering by staining Msm +ATc, Δwag31 -ATc, Δwag31+ATc, Δwag31::wag31+ATc, Δwag31::wag31_K20A+ATc, and Δwag31:: wag31_ΔN +ATc with NAO. NAO staining showed a polar and septal (in dividing cells) localization pattern in the case of Msm and Δwag31-ATc, whereas it stained the cytosol and perimeter of the Wag31 depleted cells (Fig. 6c-d). Complementation with Wag31 restored NAO localization completely, whereas Wag31_K20A restored it partially, which could be attributed to its limited tethering activity (Fig. 6c-d). Importantly, we observed a mis-localized pattern of NAO with Wag31_ΔN similar to Wag31 depletion (Fig. 6c-d). These results inform of the dependence of CL localization on membrane tethering activity of Wag31. Taken together, the data suggests the importance of the N-terminal region of Wag31 in regulating lipid homeostasis and localization.

Wag31 is a 272 amino-acids long protein that contains an N- and a C-terminal domain that are joined by a linker region (Fig. 7a). In this report, we established a role for the N-terminal region of Wag31 that comprises DivIVA-domain in membrane-tethering (Fig. 4). We also identified the protein-interaction network of Wag31 with the help of MS/MS analysis (Fig. 3 & Fig. S4). To investigate which domain of Wag31 promotes its interactions with other proteins, we generated Msm strains expressing either N- terminal (Wag31_ΔN) or C-terminal truncated (Wag31_ΔC) Wag31 (Fig. 7a) and performed a series of His pull-down experiments with both the mutants. To investigate the protein-binding ability of the domain truncation mutants, we tested them against novel interactors of Wag31 viz. MurG, SepIVA, and Msm2092 from our interactome database (Fig.3a) and AccA3, a known interactor of Wag31. MmpS5 served as a non-interactor negative control in the experiment.

We performed His pull-down assays utilizing E. coli lysates expressing either Wag31 or Wag31_ΔC or Wag31_ΔN (Fig. 7b, e). These lysates were mixed and incubated with Msm lysates expressing 3x-FLAG tagged versions of the above-mentioned proteins (Fig.7c, f) in separate reactions and subsequently pulled-down with Cobalt beads. The western blot analysis revealed that all the tested interactors bound to Wag31 (Fig. 7d, g). Expectedly, MmpS5, being a non-interactor of Wag31 did not bind to it (Fig. 7d). As shown in Fig. 7d, none of the interactors bound to Wag31_ΔC, indicating that the presence of N-terminal DivIVA-domain is not sufficient to enable Wag31 to interact with other proteins. Interestingly, interactions with all the proteins were retained in the case of Wag31_ΔN (Fig. 7g), thus suggesting that Wag31 interacts with other proteins via its C-terminal. Taken together, the results suggest that the N-terminal region of Wag31 is involved in membrane-tethering through interactions with CL, and the C-terminal region is involved in modulating interactions with other membrane-associated proteins. Thus N-terminal and C-terminal of Wag31 have distinct molecular functions that together are crucial for maintaining cell shape, lipid homeostasis and survival of mycobacteria.

Middlebrook 7H9 medium (BD Biosciences) or 7H11 agar (BD Biosciences) was used to cultivate mycobacterial strains. 7H9 medium was supplemented with 10% ADC (albumin, dextrose, NaCl, and catalase) and 0.2% glycerol (Sigma) and, 0.05% Tween-80 (Sigma) or 7H11 agar (BD Biosciences) with 10% OADC (oleic acid added to ADC) and 0.2% glycerol were added to the media. Antibiotic concentrations used: Hygromycin (Hyg) was used at 100 μg/mL, Kanamycin (Kan) at 25 μg/mL, and Apramycin (Apra) at 30 μg/mL for Msm. E. coli was cultured in Miller Luria Bertani Broth (Himedia) or Miller Luria Bertani Agar. Antibiotic concentrations used: Hyg was used at 150 μg/mL, Kan at 50 μg/mL, Apra at 30 μg/mL, Ampicillin (Amp) at 100 μg/mL for E. coli.

Recombineering method, as described in (65), was used to generate a Wag31 conditional gene replacement mutant. First, wag31 was cloned under a tet regulatable promoter in pST-KiT_off (kan res; (66)), which turns off transcription upon the addition of anhydrotetracycline. pST-wag31 construct thus generated was electroporated into a recombineering proficient strain, Msm::pJV53. An allelic exchange substrate was generated by cloning 675 bp from upstream (Left hand sequence, LHS) and 700 bp from downstream (Right hand sequence, RHS) of wag31 (including ∼150 bp at both ends) with hyg^res cassette and oriE+cosλ and digested with SnaBI to release the donor LHS-hyg^res-RHS. The linear donor was electroporated into Msm::wag31 expressing gp60 and gp61 recombinases. The colonies were selected on Hyg and ten colonies were screened with multiple primer pairs to confirm recombination at the native locus. Confirmed recombinants were passaged multiple times to cure them of pJV53 to generate conditional mutant Δwag31.To perform in vitro growth kinetics, secondary cultures of Msm and Δwag31 were seeded at a density of A₆₀₀∼ 0.05 and either left untreated or treated with 100 ng/mL ATc for 12 h. At 0 and 12 h, appropriate serial dilutions were plated on 7H11 agar plate and incubated at 37^°C for 2-3 days, and CFUs were enumerated. CFUs were plotted using GraphPad Prism 9 and analysed by performing Two-way ANOVA (0.0021 (**), 0.0002 (***).

Msm, Msm::wag31 and Δwag31 transformants grown in the presence or absence of IVN/ ATc for 12 h and harvested by centrifugation at 4000 rpm for 10 min at 4°C. The pellets were resuspended in 1:3 (pellet weight: buffer volume) Phosphate Buffer Saline containing 5% glycerol and 1 mM protease inhibitor-PMSF. The resuspension was transferred into pre-equilibrated bead-beating tubes filled to one-fourth with 0.1mm Zirconia beads. Pre-equilibration was performed by washing the beads with autoclaved MilliQ followed by lysis buffer. The tubes were placed in a bead-beater and subjected to 6-8 cycles of bead-beating -1 min on and 2 min rest on ice. To obtain WCL, the tubes were centrifuged at 13000 rpm for 10 min at 4^°C, and the supernatant was collected in a microcentrifuge tube. The concentration of protein in each sample was estimated by Pierce BCA protein Assay kit (Thermo Fischer Scientific), and 40 μg WCLs were resolved on 10-12% SDS-PAGE gels at constant current and transferred onto Nitrocellulose (NC) membrane. This was followed by blocking the membrane with 5% Bovine Serum Albumin (BSA) for an hour at RT and subsequently incubating it with the desired primary antibody for 2 h at RT or overnight at 4^°C. The primary antibodies used were either anti-RpoA (raised in rabbit), anti-Wag31 (raised in rabbit), anti-FLAG (raised in mice, Sigma), anti-His (raised in rabbit, CST). The membrane was washed thrice for 10 min each with PBST (PBS + 0.5% Tween-20) to remove any non-specific binding. Next, the membrane was probed with appropriate secondary antibody (anti-rabbit, Invitrogen or anti-mice, Invitrogen) for an hour at RT, washed thrice for 10 min each with PBST, developed using ECL reagent (Millipore) and visualized on BioRad ChemiDoc Touch Gel imaging system.

Msm and Δwag31 were set up at A₆₀₀∼0.05 and grown overnight in either the presence of absence of 100 ng/ml ATc. Post 12 h, the cells were harvested by centrifugation at 4000 rpm for 10 min at RT. Cells equivalent to A₆₀₀ ∼2 were taken further for sample preparation and washed thrice with 5 mL sterilized PBS. The cells were fixed with 5 mL of fixative (4% Paraformaldehyde (Sigma), 2.5% Glutaraldehyde (EMS), 0.1M Na-Cacodylate) for 4 h at RT protected from light. Cells were tap-mixed every 30 min to ensure that they remained in suspension. This was followed by washing the cells twice for 10 min with 0.1 M Na-Cacodylate buffer. Subsequently, cells were stained with 3 mL of 1% Osmium Tetraoxide (Os₄O₂) protected from light for a maximum of 1.5 h with tap-mixing every 30 min. Os₄O₂ was removed from the samples by centrifugation at 4000 rpm at 10 min at RT and discarded as per safety guidelines. The cells were then subjected to serial dehydration at RT with increasing concentrations of ethanol from 25% to 100%. Briefly, the cells were incubated for 5 min with 25% ethanol, harvested, and incubated for 7 min with 50% ethanol. This was followed by serially incubating the cells for 15, 20, and 30 min for 75%, 95%, and 100% (thrice) ethanol, respectively. Cells were then taken forward for critical-point drying with the help of 100 µl Hexamethyldisilazine (HMDS). Meanwhile, stubs were prepared by placing carbon tape and coverslip on them. 10 µl of the dried sample was spotted on the coverslip and evenly spread. Next, the stub was coated with 0.8 mm Gold and stored in a desiccator or imaged at 20,000X using FEI Nova NanoSEM 450. For TEM, cells were grown and harvested as described above for SEM. For TEM sample preparation, 50 OD cells were taken, fixed, stained, and gradually dehydrated with ethanol, as explained above. The cells were then infiltrated with Epon 812 resin. This was followed by cutting the resin into several 62 nm thick ultra-thin sections using an ultramicrotome (Leica EM UC7). The sections were then stained with Uranyl acetate and lead citrate and examined on a 120 kV transmission electron microscope (L120C, Talos, Thermo Scientific) at 17,500 magnification.

1 mL cells of Msm and Δwag31 either untreated or treated with 100 ng/mL ATc for 12 h were stained with 20 μg/mL BODIPY (Invitrogen). For examining lipids in Wag31 overexpression strain, Msm and Msm::wag31 were treated with 5 μM IVN for 12 h and 1 mL cells of both strains were stained with 2.5 μg BODIPY. Staining was performed for 30 min protected from light in an incubator shaker followed by washing with sterilized Phosphate buffer saline (PBS). For Msm and Δwag31, ∼10 μl of cells were spotted on a 1% agar pad (prepared in 7H9) and visualized using a 100X/1.4 NA oil immersion objective on a Zeiss, LSM880 at 488 nm/510 nm (Excitation: Emission). Background correction in the collected images followed by Corrected Total Cell Fluorescence analysis was performed using Fiji (2) and plotted using Graph Pad Prism 9. For examining lipids in BODIPY-stained Msm and Msm::wag31, 2 μl of cells were spotted on a 1% agar pad (prepared in 7H9) and visualized using a 100X/1.32 NA oil immersion objective on a Leica THUNDER Imaging systems at 475nm:519nm (Excitation: Emission). About 20 Z-stack images, each spaced 0.1 μM apart, were acquired using a scientific CMOS K8 camera (Leica microsystems). Background correction and Thunder image processing was performed on the images using the Las-X software module (Leica microsystems). Processed images were then analysed for CTCF using Fiji (2) and plotted using Graph Pad prism 9.

Secondary cultures of Msm and Δwag31 were seeded at a density of A600∼ 0.05 and either left untreated or treated with 100 ng/mL ATc for 12 h until A600∼ 0.8-1.0. Post 12 h incubation with ATc, Cells corresponding to A₆₀₀∼20 were harvested and processed for lipid extraction. Bacterial pellets were resuspended in 1 ml sterilized PBS and transferred to a 3 ml glass vial. 2:1 Chloroform::Methanol mixture was added to it and vortexed vigorously for 2 min. Chloroform contained 1 nm 15:0 FFA to be used as the internal standard for normalization. The samples were centrifuged at 1500 rpm for 5 min at RT to separate the organic layer. The latter was transferred to a new glass vial, and 2 ml Chloroform (containing 2% Formic acid) was added to the old vial to extract phospholipids from the sample. The samples were centrifuged at 1500 rpm for 5 min at RT to extract the phospholipid-containing organic layer. Both organic layers were pooled and dried under a stream of Nitrogen gas at RT. The dried lipid extracts were re-solubilized in 200 μL of 2:1 CHCl3: CH₃OH, and 10 μL was injected into an Agilent 6545 QTOF (quadrupole-time-of-flight) instrument for semi-quantitative analysis using high-resolution auto MS/MS methods and chromatography techniques. A Gemini 5U C-18 column (Phenomenex) coupled with a Gemini guard column (Phenomenex, 4×3 mm, Phenomenex security cartridge) was used for LC separation. The solvents used for negative ion mode were buffer A: 95:5 H2O:CH₃OH + 0.1% ammonium hydroxide and buffer B: 60:35:5 Isopropanol: CH₃OH:H2O + 0.1% ammonium hydroxide. The 0.1% ammonium hydroxide in each buffer was replaced by 0.1% Formic acid + 10 mM Ammonium Formate for positive ion mode runs. All LC/MS runs were for 60 min, starting with 0.3 mL/min 100% buffer A for 5 minutes, 0.5 mL/min linear gradient to 100% buffer B over 40 minutes, 0.5 mL/min 100% buffer B for 10 minutes, and equilibration with 0.5 mL/min 100% buffer A for 5 minutes. The following settings were used for the ESI-MS analysis: drying gas and sheath gas temperature: 320°C, drying gas and sheath gas flow rate: 10 L/min, fragment or voltage: 150 V, capillary voltage: 4000 V, nebulizer (ion source gas) pressure: 45 psi and nozzle voltage: 1000 V. For analysis, a lipid library was employed in the form of a Personal Compound Database Library (PCDL), and the peaks were validated based on relative retention times and fragments obtained. This library was selectively curated from the MycoMass database and the LIPID MAPS Structure Database (LMSD). All robustly detected lipid species were quantified by normalizing areas under the curve (AUC) to the AUC of the relevant internal standard added and by normalizing to the total cell number. Log2 fold changes were then plotted for each lipid compared to the Msm-ATc samples.

250 mL secondary cultures of Msm::gfp and Δwag31 were inoculated at an A600∼ 0.05 and incubated in a shaker incubator until A600∼ 0.8-1.0. Cells were collected and lysed using bead-beating to make Whole cell lysate (WCL) and passed through a 0.2 μM filter. 3 mg lysate from each group was immunoprecipitated overnight with anti-FLAG^® M2 magnetic beads (Millipore), washed thrice, and heated in 4X Laemmli Buffer. One-fifth of the IP supernatant was resolved on a 10% gel, transferred onto a Nitrocellulose membrane, and probed with anti-FLAG Antibody (Sigma). Lysates from confirmed IPs (biological quadruplets) were taken further for mass spectrometry sample preparation and analysis. Sample preparation and, mass spectrometry analysis were outsourced to VProteomics, New Delhi. Briefly, the IP samples were reduced with 5 mM TCEP, followed by alkylation with 50 mM iodoacetamide. The samples were digested with 1μg Trypsin for 16 h at 37 °C. Digests were cleaned using a C18 silica cartridge and resuspended in a buffer containing 2% acetonitrile and 0.1% formic acid. 1 μg of peptide was used for analysis in an Easy-nlc-1000 system coupled to an Orbitrap Exploris mass spectrometer. The gradients were run for 110 min. MS1 spectra were acquired in the Orbitrap (Max IT = 60 ms, AGQ target = 300%; RF Lens = 70%; R=60K, mass range = 375−1500; Profile data). Dynamic exclusion was employed for 30 seconds, excluding all charge states for a given precursor. MS2 spectra were collected for the top 20 peptides. MS2 (Max IT=60ms, R= 15K, AGC target 100%). Proteome Discoverer (v2.5) was used to analyse the RAW data against the Uniprot Msm database. The precursor and fragment mass limitations for the dual Sequest and Amanda searches were established at 10 ppm and 0.02 Da, respectively. The false discovery rate for proteins and peptide spectrum matches was adjusted to 0.01 FDR. For identification of interactors unique to Wag31, we removed all the proteins obtained in the three replicates of Msm::gfp from the Δwag31 datasets. In the corrected datasets, proteins common to all the three replicates with minimum unique peptides #2 were considered interactors. To identify top hits, PSM cut-off was set to 18 and unique peptides to 5.

BL21(DE3) cells transformed with pET-wag31, or pET-wag31K20A or pET-wag31_ΔN were cultured at 37°C until the A600 ∼0.6. 0.1 mM IPTG was added to the culture to induce protein expression, followed by incubating the culture at 18°C for 12 h. Cells were pelleted and stored at −40°C. The bacterial pellet was resuspended in a lysis buffer (20 mM HEPES pH 7.4, 150 mM NaCl, and 20 mM imidazole with 1 mM phenylmethylsulphonyl fluoride (PMSF)) and lysed by sonication in an ice-water bath. Lysate was spun at 30,000 x g for 20 min, and the supernatant obtained was incubated with TALON® Metal Affinity Resin (Takara Bio) 4 °C for 30 min. The resin was extensively washed with lysis buffer, and the bound protein was eluted with 20 mM HEPES pH 7.4, 150 mM NaCl, and 250 mM imidazole. The elution was dialyzed against 20 mM HEPES pH 7.4 150 mM NaCl and stored at 4 °C for the duration of the experiments. Aggregates were removed by centrifugation at 100,000 g. Concentration of the proteins was quantified by measuring A280 using the molar extinction coefficient predicted by the Expasy ProtParam tool.

1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE) and1′,3′-bis[1,2-dioleoyl-sn-glycero-3-phospho]-glycerol (sodium salt), CL were obtained from Avanti Polar Lipids. 1,1’-dilinoleyl-3,3,3’,3’-tetramethylindocarbocyanine, 4- chlorobenzenesulfonate (FAST DiI™), and 3,3’-dilinoleyloxacarbocyanine perchlorate (FAST DiO™) were obtained from Invitrogen. The UV-activable, diazirine-containing reactive fluorescent lipid probe BODIPY-diazirine phosphatidylethanolamine (BDPE) was prepared as described earlier (1, 2). For liposome preparation, lipids were aliquoted from chloroform stocks at desired ratios in a clean glass tube and dried under a high vacuum for an hour to a thin film. The dried lipids were used at a final concentration of 1 mM (deionised water was added to them). After hydrating at 50 °C for 30 minutes, lipids were vigorously vortexed and then extruded through polycarbonate filters with pore sizes of 100 nm (Whatman). For PLiMAP assays, liposomes were CL, DOPG, or DOPE with 69 mol% DOPC and 1 mol% BDPE. Liposomes used in tethering assays were made similarly with 30 mol% CL or DOPE and 1 mol% of the fluorescent lipid DiI or DiO) in the DOPC background.

For carrying out the PLiMAP assay, Wag31 and its mutants were purified and desired liposomes were prepared as described in supplementary. PLiMAP was carried out and analysed as described (67). Briefly, 2 µM Wag31 or its mutants were incubated with liposomes containing 30 mol% CL, DOPG, or DOPE (200 µM total lipid) in a final volume of 30 μL. The reaction was incubated for 30 min in the dark at room temperature followed by exposure to a 1 min long pulse of 365 nm UV light (UVP crosslinker CL-1000L) at an intensity of 200mJ cm⁻². The reaction was mixed with Laemmli sample buffer, boiled, and equal volumes for all reactions were loaded onto a 10% polyacrylamide gel and resolved using SDS-PAGE. iBright1500 (Thermo Fischer Scientific) or Amersham Typhoon (GE) were used for imaging gels. Gels were first imaged for BODIPY fluorescence and later fixed and stained with Coomassie Brilliant Blue.

1 µM of Wag31 and its mutants were mixed with 10 µM of 100 nm extruded liposomes in Eppendorf. The mixture was then transferred to a LabTek chamber and imaged using 100x 1.4 NA oil-immersion objective on an Olympus IX73 inverted microscope connected to an LED light source (CoolLED) and an Evolve 512 EMCCD camera (Photometrics). Image acquisition was controlled by μManager, and images were analysed using Fiji. For single liposome intensity analysis, all images from the same condition from two independent experiments were combined into one montage. The montage was duplicated and thresholded using Fiji’s Otsu algorithm to generate a binary mask. The mask was overlaid onto the original montage to obtain the maximum intensity of liposomes.

Msm, Msm::wag31 were treated with 5 μM IVN for 12 h and Msm and Δwag31 were either left untreated or treated with 100 ng/mL ATc for 12 h. For complementation experiment, 1 mL of each strain was stained with 2 μM NAO for 1 h protected from light in an incubator shaker and washed thrice with sterilised 1X PBS. 2 μl of cells were spotted on a 1% agar pad (prepared in 7H9) and visualized using a 100X/1.32 NA oil immersion objective on a Leica THUNDER Imaging systems at 475nm:535nm (Excitation: Emission). About 20 Z-stack images, each spaced 0.1 μM apart, were acquired using a scientific CMOS K8 camera (Leica microsystems). For CL specific labelling, collection was also performed at 642nm.

Background correction and Thunder image processing was performed on the images using the Las-X software module (Leica microsystems). Processed images were then analysed using Fiji (68)and plotted using Graph Pad prism 9. Briefly, N=100 cells across two independent experiments were examined for their profile of CL distribution by normalizing the cell length and assigning pole with brighter NAO fluorescence intensity as 0 and the other pole as 100. The fluorescence intensity was normalised to the highest fluorescent value for each cell. Corresponding fluorescence intensity value for all cells for every point between 0 to 100 were calculated from the LOOKUP function of excel. The values representing CL fluorescence across 100 cells were averaged out and plotted against normalised cell length in GraphPad prism 9. 0th order smoothening with 4 neighbours on each size was performed on the curves obtained.

Secondary cultures of Msm, Δwag31, Δwag31::wag31, Δwag31::wag31_K20A, and Δwag31::wag31_ΔN were set up at A₆₀₀ ∼0.05. For each strain, two types of cultures were set up in triplicates-untreated and treated with 100ng/mL ATc and grown for 12 h. At 0 and 12 h, appropriate serial dilutions were plated on 7H11 agar plates and incubated at 37^°C for 2-3 days. CFUs were enumerated and plotted using GraphPad Prism 9. Statistical analysis was performed using two-way RM ANOVA followed by Tukey’s multiple comparison test, α=0.01, GP: 0.1234 (ns), 0.0332 (*), 0.0021 (**), 0.0002 (***), <0.0001 (****). For BODIPY- and NAO- labelling, the strains were grown as described above. At 12 h, 1 mL cultures of Msm+ATc, Δwag31, Δwag31 +ATc, Δwag31::wag31+ATc, Δwag31::wag31_K20A +ATc, and Δwag31::wag31_ΔN +ATc were taken and labelled with either BODIPY or NAO. The labelling, imaging and analysis for both fluorophores was performed as described in the respective sections above.

BL21 (DE3) cells transformed with no plasmid, or pET-wag31 or pET-wag31_ΔN or pET- wag31_ΔC were cultured and cell lysates were prepared as described for PLiMAP and tethering assays. Wag31 or Wag31_ΔN was assessed by resolving the samples on 10% SDS-PAGE followed by Coomassie staining whereas Wag31_ΔC was resolved on Tris-Tricine PAGE as described elsewhere (69). The interacting partners, namely murG, sepIVA, msm2092 and accA3 and the non-interactor mmpS5 were amplified from Msm genomic DNA using gene specific primers harbouring NdeI-HindIII sites and amplicons were digested and cloned into the corresponding sites in pNit-3X-FLAG shuttle vector. The constructs thus generated were electroporated in Msm to generate Msm::murG, Msm::SepIVA, Msm::msm2092, Msm::accA3 and Msm::mmpS5. The cultures grown till A600 ∼0.8 were used for fresh inoculation at A₆₀₀ of 0.05 in 7H9 media containing 0.5 μM IVN. Cultures were grown for 12 h and lysates were prepared by bead-beating and 40 μg were resolved on 10% gels and probed with α-FLAG antibody.

500 μg of E. coli lysates expressing Wag31 or mutant were mixed with 300 μg Msm lysates expressing Wag31 interactors in a total reaction volume of 700 μL and the samples were twirled at 4°C for 2 h. 50 μL cobalt beads/ sample (GoldBio) were washed twice with lysis buffer and incubated with 5% BSA solution for 2 h at 4°C. Subsequently, beads were washed with lysis buffer and incubated with samples for 2 h (twirled at 4°C). The pull downs were washed thrice in lysis buffer containing 1% Triton X-100 and finally resuspended in 2X-SDS sample buffer. Pulldown samples were resolved on 10-12% SDS-PAGE and probed with α-His and α-FLAG antibodies.