The Conserved Translation Factor LepA Is Required for Optimal Synthesis of a Porin Family in Mycobacterium smegmatis

In a previous screen, we found that strains carrying transposon insertions in lepA were predicted to be associated with decreased accumulation of a fluorescent dye, calcein acetoxymethyl ester (AM), and decreased killing by rifampin, a first-line tuberculosis drug (18, 34). To validate that loss of LepA was responsible for the observed phenotype, we used the lepA deletion strain (ΔlepA) (18) to generate a complemented strain in which we reintroduced lepA in single copy at a phage integration site (ΔlepA L5::lepA). We found the ΔlepA strain exhibited an approximately 2-fold decrease in calcein signal (Fig. 1A) relative to the wild-type (WT) and complemented strains. Loss of LepA also resulted in increased tolerance to rifampin and vancomycin (see Fig. 1B and C) (18), but had no effect on tolerance to isoniazid or linezolid (see Fig. S1A and B in the supplemental material) or on susceptibility to a variety of translation inhibitors (Table S1).

To define the link between a ribosomal factor and the phenotypes we observed, we first examined LepA’s influence on mycobacterial ribosomes and ribosome activity in vitro, given its well-studied role as a ribosome-associated GTPase (27, 31, 32). To test whether the GTPase activity of LepA was critical to the phenotypes observed in ΔlepA cells, we assayed calcein staining and drug killing with a ΔlepA strain complemented with a putative GTPase-null mutant of LepA (ΔlepA L5::lepA H109A). Mutation of this conserved catalytic histidine was previously shown to abolish LepA GTPase activity in E. coli (28). We found that both calcein levels and drug tolerance in the ΔlepA L5::lepA H109A strain were equivalent to those in the ΔlepA strain (Fig. 1A to C), indicating that, in mycobacteria, the GTPase activity of LepA is necessary for both calcein staining and antibiotic tolerance in WT.

We reasoned that LepA could be interacting with the ribosome during ribosome biogenesis, translation initiation, or elongation (23, 24, 28). To determine if mycobacterial LepA altered ribosome biogenesis or stability, we profiled ribosome populations in ΔlepA and the complemented strain via sucrose density centrifugation. Unlike in E. coli, loss of LepA in M. smegmatis did not alter levels of ribosome subunits, assembled 70S, or polysome formation, markers of active translation (Fig. 1D). To directly test mycobacterial LepA activity at the ribosome in vitro, we purified M. smegmatis LepA and M. smegmatis LepA H109A. As previously observed with E. coli LepA (19, 28), addition of mycobacterial LepA to an in vitro cell-free translation reaction mixture containing Venus mRNA increased Venus signal (Fig. S1C) relative to the catalytic mutant and control reactions. Our in vitro experiments support a role for LepA in active translation rather than in ribosome biogenesis.

Based on the altered drug tolerance of the lepA mutant, we hypothesized that LepA might affect the translation of proteins that mediate drug susceptibility. To find candidate proteins whose translation was affected by the loss of LepA, we measured simultaneous steady-state levels of proteins and transcripts from wild type, the deletion mutant, and the complemented strain. Given multiple phenotypes demonstrating that the wild type and the complemented strain are physiologically comparable relative to ΔlepA, we compared protein and RNA levels between strains with LepA (ΔlepA L5::lepA and WT) and without LepA (ΔlepA).

To quantify the relative abundance of proteins, we used tandem-mass-tag (TMT) labeling of peptides, after tryptic digestion of cell lysates, coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). We identified a total of 4,646 proteins with 4,549 of them quantified by 2 or more peptides (Data Set S1). Among these 4,549 proteins, 78 were significantly altered by the loss of LepA (Fig. 2A). Interestingly, a number of membrane processes were enriched in the subset of proteins altered by LepA (Fig. S2A) (35). One of the most significant changes in the ΔlepA strain was the decreased abundance of peptides corresponding to a highly similar set of four porins: MspA, -B, -C, and -D (Fig. S2B). Mycobacterial porins are octameric channels built into the mycomembrane and are responsible for the uptake of nutrients critical for mycobacterial growth (36–39). The four porins in M. smegmatis, encoded by mspA to mspD, are paralogs distributed across the genome. Each porin transcript encodes a Sec signal peptide that enables cotranslational targeting of these proteins into the mycobacterial membrane (40, 41).

We used RNA sequencing (RNA-seq) to estimate the transcriptional contribution to the set of regulated proteins from our proteomics (Data Set S2). Despite finding a number of mRNAs encoding membrane proteins to be increased in the ΔlepA strain, transcript levels across the four porin transcripts were altered in both directions, which was not consistent with our observation of the bulk decrease in protein levels of the porin family (Fig. 2B). To precisely measure the levels of each porin transcript, we used quantitative RT-PCR (RT-qPCR) and, indeed, found that none were significantly altered by loss of lepA (Fig. S2C). Together, these data show that loss of lepA leads to a disproportionate decrease in porin-derived peptides compared to their encoding transcripts.

As our whole-cell proteomics data identified peptides that mapped to all four porins, we sought to identify the porin whose translation was most affected by LepA. We fused each protein to a C-terminal luciferase reporter and expressed the fusions in single copy in a merodiploid, a strain that continues to produce the wild-type copies of each protein. Using luminescence levels as a proxy for protein abundance, we examined levels of each porin in the presence of functional LepA, the GTPase mutant, or the knockout strain. The presence of functional LepA, but not the GTPase mutant, increased luminescence 2- to 3-fold for fusions with coding sequences of MspA, MspB, and MspC but not for MspD or luciferase alone (Fig. 2C). Additionally, we used the luciferase fusion approach to test other candidates that were significantly altered at the protein level and found that a cell wall amidase (AmiB) and an ABC transporter-associated protein (MSMEG_0114) were significantly increased by the presence of lepA, as indicated by reporter fusion experiments (Fig. S2D).

As the Msp porins are known to mediate drug susceptibility, likely through intracellular drug accumulation (42, 43), we hypothesized that LepA’s influence on porin abundance could explain the lepA phenotypes. To verify the function of our porin reporter fusions, we used fluorescence microscopy to examine the location of an MspA-monomeric red fluorescent protein (mRFP) fusion relative to proteins with known or predicted localization patterns. For this comparison, we used fusions of the fluorescent protein Dendra2 to GroEL, a cytoplasmic protein, and MmpL3, a membrane protein (Fig. S3A). We observed that MspA-mRFP had localization strongly suggestive of membrane association, characterized by a halo of fluorescence around the cell body and an absence of signal along the medial axis. In addition, we found that an MspA-mRFP fusion is less abundant at the membrane in the absence of LepA (Fig. 2D), an observation that supports the findings from the luciferase fusion studies. These data support our hypothesis that LepA acts as a ribosomal GTPase to improve synthesis of a number of mycobacterial porins into the mycobacterial membrane.

What is the physiological cost of decreased synthesis of each porin in the absence of LepA? To understand this, we employed an inducible CRISPRi strategy to transcriptionally deplete each porin individually (Fig. S3B) (44). To assess permeability, we compared calcein fluorescence of each porin knockdown in the presence or absence of LepA. Of the four porins tested, only depletion of MspA eliminated the LepA-dependent increase in calcein signal (Fig. 3A). Correspondingly, expressing higher levels of MspA, but not MspD, in the presence of lepA increased calcein staining (Fig. S3C). In contrast, MspD did not increase the permeability in either genetic background. These data suggest that LepA-mediated regulation of MspA abundance during translation is primarily responsible for the observed lepA deletion phenotypes.

We reasoned that if mspA and lepA were functioning in the same pathway, we should be able to detect this by epistasis experiments using a number of reporters of permeability (45). Accordingly, we deleted mspA in the original lepA deletion strain and measured fluorescence of both calcein and ethidium bromide (EtBr) in our mutants. We found that the loss of both genes does not lead to a more severe reduction in calcein or EtBr accumulation than in the single mspA mutant (Fig. 3B and C). Thus, lepA is epistatic to mspA in M. smegmatis, suggesting that they function in the same pathway. Together, these data indicate that while LepA is sufficient to affect the translation of multiple porins, it mainly controls the synthesis of MspA, which in turn mediates permeability to multiple compounds, including antibiotics.

M. smegmatis strains were inoculated from frozen stocks into Middlebrook 7H9 medium supplemented with 0.2% glycerol, 0.05% Tween 80, and ADC (5 g/liter bovine serum albumin, 2 g/liter dextrose, 3 µg/ml catalase) and grown at 37°C. Appropriate antibiotics or inducing agents were used at the following concentrations in M. smegmatis: nourseothricin (Nat; 20 μg/ml), zeocin (Zeo; 20 μg/ml), kanamycin (Kan; 25 μg/ml), hygromycin B (Hyg; 50 μg/ml), and anhydrous tetracycline (aTc; 100 ng/ml). Transformations, performed for the construction of M. smegmatis strains, were plated onto LB agar plates supplemented with the appropriate antibiotic. Unless otherwise specified for an experiment, strains were grown to log phase (optical density at 600 nm [OD₆₀₀] of 0.3 to 0.8) without antibiotics. For cloning purposes, E. coli strains were grown in LB broth or on LB agar with antibiotics as follows: Nat (40 μg/ml), Zeo (50 μg/ml), Kan (25 μg/ml), and Hyg (100 μg/ml).

All bacterial strains constructed in this study can be found in Table S2 in the supplemental material. Description of the plasmids, primers, and recombinant DNA used to construct the strains can be found in Tables S3, S4, and S5, respectively. Generally, all plasmids were constructed by restriction digestion of the parental vector (with the desired antibiotic resistance gene and phage integration gene for M. smegmatis propagation) and all inserts were prepared by amplifying gene fragments with 18- to 25-bp Gibson assembly overhangs. Vector and insert combinations were ligated together by Gibson isothermal assembly (63). Plasmids were isolated from E. coli and insert orientation was sequenced via Sanger sequencing. Sequencing reactions were carried out with an ABI3730xl DNA analyzer at the DNA Resource Core of Dana-Farber/Harvard Cancer Center.

Deletion mutants. The lepA mutant, the ΔlepA::zeo strain (HR334), and the mspA mutant, the ΔmspA::zeo strain (HR329), were previously constructed (18). The ΔlepA::zeo ΔmspA::hyg strain (SF789) was constructed with HR334 as the parental strain, using double-stranded recombineering. For recombination, a linear double-stranded DNA (dsDNA) fragment was generated by amplifying the following fragments: the 500-bp upstream region of mspA, the 500-bp downstream region of mspA, and a lox-hyg-lox fragment. The three fragments were ligated together using Gibson assembly. The deletion cassette was transformed into a ΔlepA recombineering strain as previously described (64, 65) and plated on Hyg to select for double mutants.

lepA and mspA alleles. Plasmid pSF121, used for lepA complementation, was generated using a parental vector (pCT94) that integrates into the L5 phage site and is marked with a Kan resistance (kan) gene. The vector was digested with XbaI and HindIII (New England BioLabs, Ipswitch, MA). lepA and its 5′ untranslated region (UTR; 300 bp upstream) were amplified via PCR. The vector and insert were gel extracted and ligated using Gibson assembly. The complemented strain (SF178 ΔlepA::zeo L5::lepA-kan) and the marked mutant ΔlepA strain (SF181 ΔlepA::zeo L5::empty-kan) were derivatives of HR334. Plasmid pSF417, used for lepA complementation in the CRISPRi experiments, was generated using a parental vector (pCT204) that integrates at the Tweety (Tw) phage integration site and is marked with a Hyg resistance (hyg) gene. The lepA insert was amplified from the M. smegmatis genome as above, and the parental vector was linearized by digesting with SspI and NdeI. The resulting vector was assembled as above.

For microscopy, mspA was fused to monomeric red fluorescent protein (mRFP), with no linker, and constitutively expressed from P_rpsA, in a Tw-integrating vector with a nourseothricin resistance (nat) gene. The mspA-mRFP vector was built using Gibson assembly and transformed into ΔlepA and the complemented strain for localization of MspA-mRFP.

Candidate-luciferase fusions. All luciferase reporters were generated from the parental vector CT250, a Tw-integrating vector. The vector was linearized using NdeI and HindIII, to preserve the upstream promoter (P_rpsA). For each reporter, the candidate gene was amplified from the M. smegmatis genome and the luciferase gene was amplified from a plasmid (pJR976) (44). Using Gibson overhangs that contained glycine-serine-glycine (GSG) linkers, each reporter vector was constructed using Gibson assembly. Each reporter vector was transformed into ΔlepA and the complemented strain.

Porin knockdown constructs. Knockdown of each porin was accomplished using mycobacterial CRISPRi, with knockdown systems constructed as previously described (44). Porin knockdown vectors were created by annealing oligonucleotides for each porin and ligating these fragments into a linearized vector (pJR965, digested by BsmBI), containing the mycobacterial CRISPRi system. Knockdown vectors were cotransformed into HR334 with pSF417 or pSF418.

Strains were grown to log phase and stained with 0.5 µg/ml of calcein AM (Invitrogen, Carlsbad, CA) for 1 h. Strains were analyzed by flow cytometry on a MACSQuant (VYB excitation: 488 nm; emission filter: 525/50) in the same manner as previously described (18). Median fluorescence was used from each replicate to compute an overall mean fluorescence intensity.

Strains were grown to mid-log phase, diluted to an OD₆₀₀ of 0.05 and treated with 10× MICs of the following drugs: rifampin (20 µg/ml), isoniazid (40 µg/ml), vancomycin (4 µg/ml), and linezolid (500 ng/ml). Survival was assessed over time as described previously (18).

Drug susceptibility was determined using a MIC assay, as described previously (66). In 96-well plates, strains were diluted to 0.005 and tested in biological triplicate in serial dilutions of tetracycline (Sigma-Aldrich, St. Louis, MO), clarithromycin (Sigma-Aldrich, St. Louis, MO), chloramphenicol (Sigma-Aldrich, St. Louis, MO), amikacin (Sigma-Aldrich, St. Louis, MO), and erythromycin (Santa Cruz Biotechnology, Santa Cruz, CA). The highest concentrations of each drug tested were 4 µg/ml for tetracycline, 4 µg/ml for clarithromycin, 320 µg/ml for chloramphenicol, 3.2 µg/ml for amikacin, and 16 µg/ml for erythromycin. Plates were agitated at 37°C for 21 h. To determine MICs for each condition, 0.0002% resazurin was added to each well and plates were agitated at 37°C for 3 h. The first well with no growth (blue) in each concentration gradient was considered the MIC. A biological replicate, in this case, is considered a single row in a 96-well plate of drug and bacterial incubation, using bacteria from the same culture.

M. smegmatis LepA and LepA H109A were each cloned with an N-terminal 6× His tag using Gibson assembly and expressed from pET28a in BL21 E. coli, as previously described for E. coli LepA (28). Briefly, 200 ml of log-phase culture was induced with 1 mM isopropyl-β-d-1-thiogalactopyranoside (IPTG) for 4 h at room temperature. Cells were harvested at 5,000 × g for 10 min, and pellets were frozen at −80°C overnight. The pellet was lysed at room temperature (RT) for 30 min in BugBuster 10× protein extraction reagent (Millipore Sigma, St. Louis, MO). Cell lysates were clarified by centrifugation at 15,000 × g for 30 min at 4°C. Lysate was brought up to 30 mM imidazole, pH 7.6, and His-tagged LepA was extracted via an Ni-nitrilotriacetic acid (Ni-NTA) column (New England BioLabs, Ipswitch, MA) purification. Beads were collected in plastic columns with a 10-ml bed volume and washed with 4 × 10 ml wash buffer (50 mM Tris-HCl [pH 7.6], 300 mM KCl, 5% glycerol, 6 mM β-mercaptoethanol [BME], 30 mM imidazole). One-milliliter elution fractions were collected using elution buffer (50 mM Tris-HCl [pH 7.6], 40 mM KCl, 5% glycerol, 6 mM BME, 200 mM imidazole) and analyzed via SDS-PAGE. The cleanest elution fractions were pooled and dialyzed into 6 liters (3 × 2 liters) of storage buffer (50 mM Tris-HCl [pH 7.6], 50 mM KCl, 5% glycerol, 6 mM BME) using dialysis cassettes with a 10-kDa molecular weight cutoff (MWCO). Aliquots (10 μl) were flash-frozen with liquid nitrogen and stored at –80°C. LepA protein concentration was calculated using the Qubit protein assay kit (Thermo Fisher, Berkeley, MO).

To assess the effect of LepA on translation, in vitro translation reactions were prepared with purified mRNA. Plasmid pSF741 was used in a HiScribe T7 in vitro transcription kit (New England BioLabs, Ipswich, MA) to generate Venus mRNA. A master mix of purified Venus mRNA (500 ng per reaction) and PURExpress (New England BioLabs, Ipswich, MA) components was prepared in duplicate reactions with purified M. smegmatis LepA or LepA H109A (300 ng per reaction). When no LepA was added to the reaction, an equal volume of storage buffer was added in place of protein. Reactions were carried out in 20 μl in a 384-well plate for 4 h at 37°C, and fluorescence (measured at an excitation of 505 nm and an emission of 540 nm) was collected on a SpectraMax M2 microplate reader.

Preparation of mycobacterial ribosomes was performed as previously described for E. coli (67), yet optimized for M. smegmatis. A culture of 500 ml of cells was grown to mid-log phase, filtered over 0.22-μm, 90-mm membranes (Millipore Sigma, St. Louis, MO) on a fritted glass microfiltration apparatus (Kimball-Chase, Rockwood, TN), and scraped into liquid nitrogen. A 500-μl aliquot of lysis buffer (20 mM Tris [pH 8], 10 mM MgCl₂, 100 mM NH₄Cl, 5 mM CaCl₂, 0.4% Triton X-100, 0.1% NP-40, 34 mg/ml chloramphenicol, 100 U/ml RNase-free DNase I) was added to the cell scrapes. Frozen cells and lysis buffer were ground in a Retsch 400 mixer mill using 10-ml grinding jars and 12-mm grinding balls at 15 Hz for 5 × 3 min. Cell lysates were thawed and clarified at 15,000 × g for 15 min at 4°C. Aliquots of 250 μl of lysate were layered onto a 10 to 40% linear sucrose gradient. The sucrose gradients were spun in a Beckman ultracentrifuge at 150,000 × g for 2.5 h at 4°C. The gradients were fractionated and analyzed using a gradient fractionator (BioComp Instruments, Inc., NB, Canada).

Cultures of 60 ml of each strain were grown to log phase (OD₆₀₀ ∼0.4) and split into two parts to extract protein and RNA separately. Both aliquots were spun at 5,000 × g for 10 min. For proteomics, cells were resuspended in 500 μl of urea lysis buffer (8 M urea in 50 mM Tris [pH 8.2], 75 mM NaCl, Roche complete EDTA-free protease inhibitor cocktail tablet) and subjected to bead beating for 4 × 45 s with 3 min on ice in between. Cell lysates were spun down at 20,000 × g for 10 min at 4°C and the supernatant was isolated for proteomics sample preparation. For RNA sequencing, RNA was isolated as described previously (68), depleted for rRNA using RiboZero (Epicenter, Madison, WI), and prepared for sequencing using KAPA stranded RNA-Seq library preparation kit (Millipore Sigma, St. Louis, MO).

Quantitative proteomics. Samples for quantitative proteomics experiments were processed as described previously (69). Briefly, three biological replicates of each strain’s lysates were reduced with 5 mM dithiothreitol (DTT), alkylated with 10 mM iodoacetamide (IAA), and digested with endoproteinase Lys-C (Wako Laboratories) for 2 h at a 1:50 enzyme to substrate ratio at 30°C, followed by an overnight digestion with trypsin (Promega, Madison, WI) at a 1:50 enzyme to substrate ratio at 37°C. Reactions were quenched with neat formic acid (FA) to a final concentration of 1%. Digests were desalted using tC18 SepPak reversed phase cartridges (Waters, Milford, MA) following the manufacturer’s protocol. A tandem mass tag (TMT) isobaric labeling strategy was used for this experiment. An aliquot (50 μg) of each of the 9 samples were labeled by TMT10plex reagent (Thermo Fisher Scientific, Waltham, MA) following the manufacturer’s protocol. A pooled reference standard was generated by mixing equal amounts of each of the nine samples and included in the tenth channel of the TMT10plex. Labeling efficiency was assessed prior to quenching the reactions. Once sufficient (>99%) labeling efficiency was achieved, reactions were quenched and samples were mixed together. Combined sample was desalted using tC18 Sep-Pac reversed-phase cartridges, and the eluate was dried down completely. Sample was reconstituted and fractionated on a Zorbax 300 Extend-C₁₈ 4.6- by 250-mm column (Agilent Technologies, Santa Clara, CA), as described previously (69). Fractions were collected every minute during the gradient and further concatenated into a total of 24 fractions that were analyzed on a Q Exactive Plus mass spectrometer (MS) coupled to an EASY-nLC 1200 ultra-high-performance liquid chromatography (UHPLC) system (Thermo Fisher Scientific, Waltham, MA). One microgram of each of the fractions was injected on a 75-μm ID Picofrit column (New Objective, Woburn, MA) packed with Reprosil-Pur C₁₈-AQ 1.9 μm beads (Maisch, GmbH) in-house to a length of 22 cm. Sample was eluted at a 200-nl/min flow rate with solvent A of 0.1% FA–3% acetonitrile (ACN), solvent B of 0.1% FA–90% ACN and a gradient of 2 to 6% B in 1 min, 6 to 30% B in 84 min, 30 to 60% B in 9 min, 60 to 90% B in 1 min, and a hold at 90% B for 5 min. MS data were acquired in data-dependent mode with MS1 resolution of 70,000 and automatic gain control (AGC) of 3e6. MS/MS was performed on the most intense 12 ions with a resolution of 35,000, AGC of 5e4, isolation width of 1.6 amu, and normalized collision energy of 29. Data were extracted and searched against an M. smegmatis database using Spectrum Mill MS proteomics workbench (Agilent Technologies, Santa Clara, CA). Extracted spectra were searched using carbamidomethylation of cysteines and TMT labeling of N termini and lysine residues as fixed modifications and methionine oxidation, asparagine deamidation, and protein N-terminal acetylation as variable modifications. Spectrum to database matching was controlled with peptide level false discovery rate (FDR) of less than 1%. Peptides were rolled into protein groups and subgroups in Spectrum Mill with a protein level FDR of 0%. Protein summary export consisting of a list of quantified proteins with a reporter ion ratio of every TMT channel to the pooled reference channel was generated for quantitation of proteins. TMT10 reporter ion intensities were corrected for isotopic impurities in the Spectrum Mill protein/peptide summary module using the afRICA correction method, which implements determinant calculations according to Cramer's rule and correction factors obtained from the reagent manufacturer’s certificate of analysis (https://www.thermofisher.com/order/catalog/product/90406) for lot number SE240163 (70). Proteins identified with 2 or more peptides were used for further statistical analysis. Comparisons of protein levels in each strain were assessed for significance using a two-sample moderated t test with an adjusted P value threshold of less than 0.05 for assessing significantly altered proteins. For visualization purposes in Fig. 2, the protein and RNA ratios associated with LepA were excluded from the volcano plot.

RNA sequencing. Samples were sequenced on an Illumina HiSeq 2500 in paired-end mode with a read length of 125 bp. Approximately 4 million reads were collected for each sample. Reads were mapped to the genome sequence of M. smegmatis mc² 155 as a reference genome using Burrows-Wheeler aligner (BWA) (71). A Python script was used to separate reads in .sam files that mapped to the positive strand and negative strand of the chromosome. Then reads mapping to each open reading frame (ORF) (in a strand-specific manner) were tabulated. The raw read counts were converted to fragments per kilobase per million reads (FPKMs) by dividing by gene length (in base pairs) and total reads in the sample and scaling up by 10⁹. For analyses of differential gene expression, DESeq2 (72) was used to estimate log fold changes according to a hierarchical model based on the negative binomial distribution, and P values were calculated via a Wald test as a measure of significance. P values were adjusted for a false-discovery rate (FDR) of 5% over all genes by the Benjamini-Hochberg procedure.

Luciferase assays. Strains were grown to log phase and luciferase assays were conducted using Nanoglo luciferase assay system (Promega, Madison, WI). Briefly, 100 μl of cells was mixed with 100 μl of Nanoglo reagent (prepared as the kit protocol described). Within 2 min, luminescence measurements were taken in a TECAN Spark 10M plate reader with an integration time of 1,000 ms. The OD₆₀₀ was also measured in each well, and luminescent values were normalized by OD₆₀₀ to obtain relative luminescence values.

Strains were grown to log phase, diluted back into medium with or without aTc, and allowed to grow for 15 h to reach log phase. Cells were stained with calcein AM and analyzed by flow cytometry in biological triplicate, as described above.

Strains were grown to log phase, washed with phosphate-buffered saline (PBS)-0.4% glycerol (PBS-G) and prepared at an OD₆₀₀ of 0.8. An aliquot of 100 μl of each strain was mixed with 100 μl of 4 μg/ml of ethidium bromide (prepared in PBS-G) in a 96-well plate. Fluorescence was measured in a TECAN Spark 10M plate reader, using an excitation of 520 nm and emission of 600 nm.

GroEL-Dendra2 and MmpL3-Dendra2 strains were a gift from the Mycobacterial Systems Resource (principal investigator, K. Derbshire). Live still imaging of MspA-mRFP and Dendra2 fusion strains was performed using a Nikon TI-E inverted, wide-field microscope equipped with a Plan Apo 100×, 1.45-numerical-aperture (NA) objective, Spectra X LED light source, and an Andor Zyla sCMOS camera. M. smegmatis samples were spotted on 2% agarose pads. NIS-Elements version 4.5 software was used for data acquisition and ImageJ software was used for processing.

Unless otherwise noted all experiments were conducted at least twice, in biological triplicate.

Protein functional enrichment analysis was performed using “Functional Annotation” software within the DAVID platform (https://david.ncifcrf.gov/home.jsp). Specifically, InterPro terms considered biologically significant, and therefore visualized, refer to at least 6 proteins in the list of 80 proteins significantly altered by LepA. Statistical significance was determined using a modified Fisher’s exact test (35). All other statistical measurements and tests are specified in the figure legends.

mRNA was quantified as described previously (44). Briefly, purified RNA (DNase treated) was used as the template for cDNA synthesis, following the manufacturer’s instructions with Superscript V (Life Technologies, Carlsbad, CA). RNA was removed from the reaction using alkaline hydrolysis and the cDNA was cleaned using column purification (Zymo Research, Irvine, CA). Quantitative PCR (qPCR) was performed on purified cDNA using iTaq Universal SYBR green Supermix (Bio-Rad, Hercules, CA). mRNA fold change was calculated using the threshold cycle (ΔΔC_T) method, where porin transcript level was normalized by sigA level in each genetic background.

The original mass spectra and sequence database have been deposited in the public proteomics repository MassIVE and are accessible at ftp://[email protected] when providing the data set password “mycobacteria.”

The RNA-seq raw sequence files are deposited at BioProject accession number PRJNA518044, and the gene expression levels (FPKMs) are deposited in GEO under accession number GSE126130.

The further data that support these findings are available from the corresponding author upon reasonable request.