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Functional Analyses of Mycobacterial Lipoprotein DiacylglycerylTransferase and Comparative Secretome Analysis of a Mycobacteriallgt Mutant

Andreas Tschumi,a Thomas Grau,a* Dirk Albrecht,b Mandana Rezwan,a* Haike Antelmann,b and Peter Sandera,c

Institute of Medical Microbiology, University of Zurich, Zurich, Switzerlanda; Institute for Microbiology, Ernst-Moritz-Arndt-University of Greifswald, Greifswald, Germanyb;and Swiss National Centre for Mycobacteria, Zurich, Switzerlandc

Preprolipopoprotein diacylglyceryl transferase (Lgt) is the gating enzyme of lipoprotein biosynthesis, and it attaches a lipidstructure to the N-terminal part of preprolipoproteins. Using Lgt from Escherichia coli in a BLASTp search, we identified thecorresponding Lgt homologue in Mycobacterium tuberculosis and two homologous (MSMEG_3222 and MSMEG_5408) Lgt inMycobacterium smegmatis. M. tuberculosis lgt was shown to be essential, but an M. smegmatis �MSMEG_3222 mutant could begenerated. Using Triton X-114 phase separation and [14C]palmitic acid incorporation, we demonstrate that MSMEG_3222 is themajor Lgt in M. smegmatis. Recombinant M. tuberculosis lipoproteins Mpt83 and LppX are shown to be localized in the cell en-velope of parental M. smegmatis but were absent from the cell membrane and cell wall in the M. smegmatis �MSMEG_3222strain. In a proteomic study, 106 proteins were identified and quantified in the secretome of wild-type M. smegmatis, including20 lipoproteins. All lipoproteins were secreted at higher levels in the �MSMEG_3222 mutant. We identify the major Lgt in M.smegmatis, show that lipoproteins lacking the lipid anchor are secreted into the culture filtrate, and demonstrate that M. tuber-culosis lgt is essential and thus a validated drug target.

Mycobacteria belong to the group of GC-rich actinobacteriaamong the Gram-positive bacteria. Comprising more than130 species, the genus Mycobacterium is rather diverse. Membersof this genus are, among others, the slow-growing, pathogenicMycobacterium tuberculosis, the causative agent of tuberculosis,Mycobacterium bovis bacillus Calmette-Guérin, the live attenu-ated vaccine applied to protect against tuberculosis, Mycobacte-rium leprae, the causative agent of leprosy, and the fast-growingMycobacterium smegmatis, a nonpathogenic, saprophytic myco-bacterial model organism. Although classified as Gram-positivebacteria, the cellular envelope of mycobacteria resembles the cellenvelope of Gram-negative bacteria, having an outer membrane-like structure (15). Mycobacteria interact with their environmentby secreted and surface-localized proteins. Lipoproteins are a het-erogeneous subgroup of membrane-associated proteins univer-sally present in bacteria. One to 3% of bacterial genomes encodelipoproteins (2, 16). The common feature of lipoproteins is a uni-versally conserved N-terminal cysteine modified with a lipidstructure functioning as a membrane anchor. Synthesized as pre-cursors in the cytoplasm, lipoproteins are translocated across thecytoplasmic membrane by either the Sec translocation machineryor the twin-arginine translocation (Tat) system (21, 28, 42, 48).Lipoprotein maturation subsequently occurs on the periplasmicside of the cytoplasmic membrane by the consecutive action of thethree enzymes Lgt (preprolipoprotein diacylglyceryl transferase),LspA (prolipoprotein signal peptidase), and Lnt (apolipoproteinN-acyltransferase). These posttranslational modifications are di-rected by a lipobox motif comprising four amino acids, includingthe invariant cysteine (LVI)(ASTVI)(GAS)C (2). As a first step,Lgt attaches a diacylglycerol residue to the thiol group of the uni-versally conserved cysteine within the lipobox. Second, LspAcleaves off the signal peptide N-terminal of the modified cysteine,followed by the attachment of a third acyl residue to the freeamino group of the cysteine mediated by Lnt (22). The membrane

anchor of mycobacterial lipoproteins has been resolved at the mo-lecular level recently (45). Mycobacterial lipoproteins are modi-fied with a thioether-linked diacylglyceryl residue composed of anester-linked tuberculostearic and an ester-linked palmitic acid aswell as an additional palmitic acid amide linked to the N-terminal�-amino group. Diacylglycerol modification and the signal se-quence cleavage are prerequisites for N-acylation (5, 45, 48).

The functional diversity of lipoproteins is manifold; amongothers, they have direct virulence-related functions, such as inva-sion of host cells, evasion from host defense, and immunomodu-lation in Gram-positive and Gram-negative bacterial pathogens(18). All three enzymes of the lipoprotein biosynthesis pathwayare essential in Gram-negative but not in Gram-positive bacteria.In M. tuberculosis, targeted deletion of lspA demonstrated a role ofthe lipoprotein biosynthesis pathway in pathogenesis. An M. tu-berculosis lspA mutant is unable to cleave the signal peptide oflipoproteins, and this was associated with a 3- to 4-log-reducednumber of CFU in an animal model of tuberculosis. Additionally,this strain induced hardly any lung pathology and did not spreadto the secondary organs spleen and liver (27, 33).

Lipoproteins (from different bacteria, including mycobacte-ria) are potent agonists of Toll-like receptor 2 (TLR2). TLR2 ago-

Received 30 January 2012 Accepted 13 May 2012

Published ahead of print 18 May 2012

Address correspondence to Peter Sander, [email protected].

* Present address: Thomas Grau, Roche Diagnostics Ltd., Rotkreuz, Switzerland;Mandana Rezwan, Dualsystems Biotech AG, Schlieren, Switzerland.

A.T. and T.G. contributed equally to this work.

Supplemental material for this article may be found at http://jb.asm.org/.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JB.00127-12

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nist activity has been shown for several M. tuberculosis lipopro-teins, including LpqH, LprA, LprG, and PstS1 (10). Successfulimmune evasion of M. tuberculosis has been partly attributed toTLR2-dependent inhibition of antigen processing and presenta-tion (10, 12). Although TLR signaling enhances both innate andadaptive immune responses, it can also downregulate some im-mune functions.

Virulence assays indicated an important role of the second en-zyme (LspA) of the lipoprotein biogenesis in the pathogenesis oftuberculosis, and functional investigations elucidated that the my-cobacterial lipoprotein anchor carries three fatty acids and thus issimilar to the membrane anchor of Gram-negative bacteria. How-ever, the physiological role of mycobacterial Lgt, the gating en-zyme of lipoprotein biosynthesis, remains to be demonstrated. Ofnote, a high-density mutagenesis study suggested that M. tubercu-losis Lgt is essential (36). There is an urgent need for novel drugsand verification of drug targets, since the antituberculosis drugpipeline is not sufficiently filled and more and more drug-resistant M. tuberculosis strains emerge (31). Essential genes,particularly those which are restricted to bacteria, encode drugtargets that have great potential. Therefore, we here investi-gated the prolipoprotein diacylglyceryl transferase in myco-bacteria.

MATERIALS AND METHODSBacterial strains and growth conditions. M. smegmatis was grown on LB(Luria-Bertani) agar or on Middlebrook 7H10 agar supplemented witholeic acid albumin dextrose (OADC; Difco). M. tuberculosis was grown onMiddlebrook 7H10 agar supplemented with OADC. Tween 80 (0.05%,vol/vol) was added to liquid broth LB, 7H9, and 7H9-OADC to avoidclumping. When appropriate, antibiotics were added at the followingconcentrations: kanamycin, 50 �g ml�1; streptomycin, 100 �g ml�1; hy-gromycin, 25 �g ml�1; and gentamicin, 10 �g ml�1. Strain designationswere the following: �lgt, lgt knockout mutant; �lgt-lgt, M. smegmatis �lgttransformed with complementing vector pMV361-hyg-lgt expressing M.tuberculosis lgt; �lgt-MSMEG_3222, M. smegmatis �lgt transformed withcomplementing vector pMV361-hyg-MSMEG_3222 expressing M. smeg-matis lgt.

Disruption of lgt in M. smegmatis. For disruption of M. smegmatis lgt(MSMEG_3222), a 1,330-bp fragment upstream and a 1,415-bp fragmentdownstream of the predicted ORF were amplified by PCR. XbaI/EcoRIlinker sequences were added to the upstream fragment and EcoRI/MluIlinker sequences were added to the downstream fragment to facilitateoriented cloning. The resulting fragments were cloned into pMCS5-rpsL,resulting in pMCS5-rpsL-lgt. A fragment containing the aph cassette wascloned into the EcoRI site between the upstream and downstream frag-ments, resulting in plasmid pMCS5-rpsL-lgt::aph. Using the rpsL counter-selection strategy (32), the �lgt allele was substituted for lgt in M. smeg-matis, deleting 959 bp from the open reading frame (ORF) coding for Lgt.Substitution was confirmed by Southern blot analysis by probing with a967-bp ApaI lgt gene fragment. For complementation, an 8,024-bp SfiI/PvuII fragment of the M. tuberculosis chromosome, encompassing thecomplete lgt (Rv1614) under the control of its own promoter, was clonedin pMV361-hyg, and the resulting plasmid was transformed into �lgt. Acorresponding complementation vector, carrying a 2.5-kbp M. smegmatislgt fragment, was also constructed and transformed. A strategy similar tothat for generating an M. smegmatis deletion mutant was applied to gen-erate corresponding M. tuberculosis mutants.

Whole-genome sequencing, data analysis, and single-nucleotidepolymorphism (SNP) confirmation. Genomic DNA of M. smegmatisSmr5, a streptomycin-resistant derivative of M. smegmatis mc2155 (37)whose sequence has been published, and M. smegmatis �lgt was preparedas follows. Bacteria were grown for 2 to 3 days on plates. Bacteria were

resuspended in 340 �l Tris-EDTA (TE) buffer and heat inactivated for 20min at 80°C. After cooling down to room temperature, 2 �l 20% Tween 80and 10 �l lysozyme (80 mg ml�1; Roche) were added, followed by incu-bation for 2 h at 37°C. After addition of 20 �l 20% SDS and 20 �l protei-nase K (2 mg ml�1; Roche), samples were incubated for 1 h at 50°C. Fourhundred �l phenol-chloroform-isoamylalcohol (25:24:1, vol/vol) wasadded, and samples were shaken for 1 h. Subsequently, samples werecentrifuged (16,000 � g for 20 min at 4°C), and the supernatant wastransferred into a fresh 1.5-ml tube. Eight �l 5 M NaCl and 2.5 volumes (1ml) of ethanol were added, and the mixtures were incubated overnight at�20°C. After centrifugation of the samples at 16,000 � g for 20 min at4°C, the pellet was washed twice with 70% ethanol, dried under vacuum,and resuspended in 100 to 300 �l water.

The strains were sequenced using the Illumina Genetic Analyzer (Illu-mina, Saffron Walden, United Kingdom) to produce paired-end frag-ment reads of 35 bp. Sequencing was performed at GATC Biotech Ltd.(Constance, Germany). Reads of both strains were mapped against M.smegmatis mc2155 (37) (NC_008596) using CLC Genomics Workbench4.8 (CLCbio). SNP detection tool (CLCbio) parameters were set as thefollowing: window length, 11; maximum number of gaps and mis-matches, 2; minimum average quality of surrounding bases, 15; minimumquality of central base, 20; minimum coverage, 4; and minimum variantfrequency, 35%. SNP confirmation was performed by Sanger sequencingwith an ABI Prism 310 Genetic Analyzer (Applied Biosystems).

Microscopy. For electron microscopy, bacteria were centrifuged andfixed for 30 min at room temperature in 3% paraformaldehyde— 0.1%glutaraldehyde. The cells were washed in phosphate-buffered saline (PBS)and postfixed for 30 min in 2% OsO4 before dehydration in ethanol andembedding in Epon. Thin sections were stained with uranyl acetate andlead citrate and examined in a Phillips CM12 electron microscope. Stan-dard laboratory techniques were used for Ziehl-Neelsen and auramineO/rhodamine staining. For the study of microcolonies, bacteria were grownfor 3 days on 7H10 agar plates.

Cloning of Mpt83 and LppX. Plasmid pMV261-Gm, a derivative ofpMV261, is a shuttle vector replicating in E. coli as well as in mycobacteria(39). M. tuberculosis LppX and Mpt83 were amplified by PCR fromgenomic DNA and fused to the M. tuberculosis 19-kDa (lpqH) promoter.Two sequences encoding a hemagglutinin and a hexa-His epitope werefused to the 3= part of the genes to facilitate subsequent detection byWestern blotting. The insert was cloned into the EcoRI site, resulting inpMV261-Gm-FusLppX and pMV261-Gm-FusMpt83, respectively.

Western blotting. Bacteria from liquid cultures were harvested, resus-pended in PBS containing Complete EDTA-free tablets (Roche) to inhibitprotein degradation, and subjected to 15 to 30 min of ultrasonication inan ice bath. Soluble and insoluble fractions were separated by centrifuga-tion at 15,000 � g for 20 min at 4°C. Extracts corresponding to 1 to 5 �gof total protein were separated by SDS-PAGE (12%) and analyzed byWestern blotting. Antiserum against hemagglutinin (HA) epitope(Roche) and LprG (provided by H. Bercovier) were used in dilutions of1:300 or 1:10,000, respectively. Appropriate secondary antibodies conju-gated with horseradish peroxidase were used at dilutions of 1:10,000. Theblots were developed using enhanced chemiluminescence (Bio-Rad).

[14C]palmitic acid incorporation. Lipoprotein labeling was per-formed as described previously (38, 50).

Subcellular fractionation of cells. Subcellular fractionation was per-formed as described in reference 29. Briefly, cells of 1-liter cultures wereharvested, washed in 0.16 M NaCl, and resuspended in lysis buffer (0.05 Mpotassium phosphate, 0.022% [vol/vol] �-mercaptoethanol, pH 6.5). Celllysis was performed by a French press (American Instrument Company)followed by low-speed centrifugation at 1,000 � g to remove unbrokencells. Centrifugation was repeated 3 to 5 times for 40 min at 27,000 � g topellet outer lipid layer material. The supernatant was called SN27.1. Thepellet was resuspended in lysis buffer and disrupted by a French press.Subsequent centrifugation at 27,000 � g resulted in a pellet enriched forcell wall components (mycolic acids) and supernatant SN27.2. The super-

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natants SN27.1 and SN27.2 were pooled and centrifuged at 100,000 � gfor 1 h. The resulting pellet was enriched in cytoplasmic membrane andthe supernatant in cytosolic components.

Preparation of the extracellular protein fraction. M. smegmatis cellswere grown in 1 liter of LB broth (Difco), and 500 ml cells was harvestedduring exponential growth (optical density at 600 nm of 0.4 to 0.7) at thetransient phase (t0), as well as after the entry into the stationary phase (t1),by centrifugation for 10 min at 4°C (1 mM phenylmethylsulfonyl fluoride[PMSF] was added immediately after harvesting). The extracellular pro-teins of the supernatant were precipitated with ice-cold 10% (wt/vol)trichloroacetic acid (TCA) overnight on ice and centrifuged for 45 min at13,500 � g and 4°C. The resulting protein pellet was scraped with a spatulafrom the wall of the centrifuge tube, washed with 96% (vol/vol) ethanol 5times, and dried.

Extracellular proteome analysis and image analysis. The TCA-pre-cipitated extracellular proteins of the M. smegmatis wild type and lgt mu-tant were washed extensively with ethanol, dried in a speed vacuum, andresolved in a solution containing 2 M thiourea and 8 M urea. Insolublematerial was removed by centrifugation. The protein content was deter-mined using the Bradford assay (4). For two-dimensional polyacrylamidegel electrophoresis (2D PAGE), 200 �g of the protein extracts was sepa-rated using the nonlinear immobilized pH gradients (IPG; pH range, 4 to7; Amersham Biosciences) and a Multiphor II apparatus (AmershamPharmacia Biotech) as described previously (1). The resulting 2D gelswere fixed in 40% (vol/vol) ethanol, 10% (vol/vol) acetic acid and stainedwith colloidal Coomassie brilliant blue (Amersham Biosciences). The im-age analysis and quantification were performed with Decodon Delta 2Dsoftware.

Identification of proteins in the secretome using MALDI-TOF-TOFtandem mass spectrometry (MS/MS). Proteins were cut manually fromthe Coomassie-stained proteome and tryptically in-gel digested using theEttan spot handling platform as described previously (7). The matrix-assisted laser desorption ionization—tandem time-of-flight (MALDI-TOF-TOF) measurement of spotted peptide solutions was carried out ona Proteome-Analyzer 4800 (Applied Biosystems, Foster City, CA) as de-scribed previously (7). The spectra were recorded in reflector mode in amass range from 900 to 3,700 Da with a focus mass of 2,000 Da. For onemain spectrum, 25 subspectra with 100 shots per subspectrum were accu-mulated using a random search pattern. If the autolytic fragment of tryp-sin with the monoisotopic (M�H)� m/z at 2,211.104 reached a signal-to-noise ratio (S/N) of at least 10, an internal calibration was automaticallyperformed using this peak for one-point calibration. The peptide searchtolerance was 50 ppm, but the actual RMS value was between 10 and 20ppm. After calibration, the peak lists were created by using the “peak tomascot” script of the GPS Explorer software, v.3.6, with the followingsettings: mass range from 900 to 3,700 Da, peak density of 50 peaks perrange of 200 Da, minimal area of 100, maximal 200 peaks per protein spot,and minimal S/N ratio of 6. The peak lists were searched against an M.smegmatis database extracted from UniprotKB release 12.7 (46) using theMascot search engine, v.2.1.04 (Matrix Science Ltd., London, UnitedKingdom).

MALDI-TOF-TOF MS/MS analysis was performed for the threestrongest peaks of the TOF spectrum. For one main spectrum, 20 subspec-tra with 125 shots per subspectrum were accumulated using a randomsearch pattern. The internal calibration was automatically performed asone-point calibration if the monoisotopic arginine (M�H)� m/z at175.119 or lysine (M�H)� m/z at 147.107 reached an S/N ratio of at least5. The peak lists were created by using the “peak to mascot” script of theGPS Explorer software, v.3.6, with the following settings: mass range from60 Da to a mass that was 20 Da lower than the precursor mass, peakdensity of 5 peaks per 200 Da, minimal area of 100, maximal 20 peaks perprecursor, and a minimal S/N ratio of 5. Peptide mixtures that yielded amolecular weight search (MOWSE) score of at least 50 in the reflectormode and a sequence coverage of at least 30% that were confirmed bysubsequent MS/MS analysis were regarded as positive identification.

RESULTSGeneration of mycobacterial lgt deletion mutants. Using E. coliLgt as a query in a BLASTp search, we identified Rv1614, anno-tated as Lgt, in M. tuberculosis as a preprolipoprotein diacylglyc-eryl transferase. We applied the rpsL counterselection strategy togenerate an M. tuberculosis lgt deletion mutant. rpsL has beenshown to be a powerful tool to generate deletion mutants (32, 33).The vector-carried wild-type rpsL confers a streptomycin-sensi-tive phenotype in a streptomycin-resistant rpsL mutant strain dueto the dominance of the wild-type allele. Transformation of astreptomycin-resistant M. tuberculosis H37Rv derivative with asuicide plasmid for targeted deletion of lgt resulted in single-cross-over recombinants which had integrated the suicide plasmid at thelgt locus. Upon counterselection, an lgt deletion mutant could notbe generated. Only spontaneously streptomycin-resistant single-crossover mutants, which had not undergone second recombina-tion, were obtained. However, the construction of an M. tubercu-losis lgt deletion mutant was successful in an lgt-complementedstrain, indicating that replacement of lgt is feasible in principle butthat a functional copy elsewhere is required. The data from ourtargeted gene deletion confirm the findings of Sassetti et al. pre-dicting lgt to be essential for growth based on the high-densitymutagenesis of M. tuberculosis (36). We previously succeeded ingenerating an lspA as well as an lnt deletion mutant in M. smeg-matis (45). Therefore, we used this nonpathogenic mycobacterialmodel organism, which is fast growing and amenable to geneticmanipulation, to investigate the function of lgt.

Using E. coli Lgt as a query in a BLASTp search, we identifiedtwo putative paralogous open reading frames, i.e., MSMEG_5408and MSMEG_3222. We used pairwise sequence alignment with aNeedleman-Wunsch algorithm (http://www.ebi.ac.uk/Tools/psa/emboss_needle/) with default settings to compare both M. smeg-matis ORFs to M. tuberculosis, E. coli, and Bacillus subtilis Lgt se-quences (see Table S1 in the supplemental material).MSMEG_5408 shows higher percentages of identities/similaritiesto E. coli or B. subtilis Lgt than does MSMEG_3222. Of note,MSMEG_3222 has an extended C-terminal sequence which ismissing from E. coli or B. subtilis Lgt. When both M. smegmatisORFs were compared to M. tuberculosis Lgt, MSMEG_3222 showsthe highest identities/similarities (see Table S1). A recent studyfrom Pailler et al. identified residues essential and important forthe function of E. coli Lgt (24). We also used pairwise sequencealignment with the Needleman-Wunsch algorithm (http://www.ebi.ac.uk/Tools/psa/emboss_needle/) with default settings to an-alyze the conservation of these residues in MSMEG_3222 andMSMEG_5408 (see Table S2). MSMEG_3222 showed conserva-tion of all four essential residues, the Lgt signature motif, and 10 of17 important residues. Seven of 17 important residues were dif-ferent from E. coli Lgt, but only four residues were different fromM. tuberculosis Lgt. In contrast, comparison of MSMEG_5408 andE. coli Lgt revealed two essential residues which were not con-served in MSMEG_5408, i.e., Y26 and N146 (E. coli numbering).At position Y26 a histidine was found, and at position N146, lo-cated in the Lgt signature motif, a cysteine was found in MS-MEG_5408. Moreover, six important residues were not conservedin MSMEG_5408 in addition to alteration in two essential resi-dues. Because of the conservation of all residues essential for Lgtfunction and higher homology to M. tuberculosis Lgt, we focusedon MSMEG_3222 for further characterization. MSMEG_3222 lo-

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calizes at positions 3,299,959 to 3,301,809 in the genome sequenceof M. smegmatis that is available from NCBI (http://www.ncbi.nlm.nih.gov/genome/?term�NC_008596). The ORF encodes aprotein of 616 amino acids. The six conserved domains observedin Lgt (26) are all present in MSMEG_3222. Following transfor-mation with the suicide plasmid pMCS5-rpsL-lgt::aph, single-crossover recombinants were isolated and subjected to counterse-lection. Intramolecular homologous recombination occurs with afrequency of about 10�4 in mycobacteria (25). Counterselectionof the MSMEG_3222 single-crossover recombinants was muchless frequent (10�8). However, a single-mutant strain resultingfrom allelic exchange was found after counterselection and is re-ferred to as M. smegmatis �lgt. Replacement of lgt with a kanamy-cin resistance marker was verified by Southern blot analysis andPCR (Fig. 1). The mutant strain was complemented by introduc-ing plasmid pMV361-hyg-lgt expressing M. tuberculosis lgt, result-ing in strain M. smegmatis �lgt-lgt. In addition, the mutant strainwas complemented with a corresponding vector carrying M.smegmatis lgt (MSMEG_3222) (data not shown).

Whole-genome comparison of M. smegmatis Smr5 and �lgt.Isolation of a single mutant only and the apparent discrepancy

with respect to the essentiality of lgt in M. tuberculosis H37Rv andM. smegmatis Smr5 prompted us to compare the whole genome ofparental M. smegmatis Smr5 and M. smegmatis �lgt by using Illu-mina sequencing technology. We reasoned that suppressor muta-tions in the knockout strain might explain the success of lgt dele-tion in M. smegmatis. The genome of M. smegmatis mc2155 (37)contains 6,988,209 nucleotides (90% coding) with a GC content of67%. It comprises 6,938 genes, with 6,717 encoding proteins(http://www.ncbi.nlm.nih.gov/genome?term�nc_008596). M.smegmatis Smr5, the parental strain of the �lgt mutant, is a directderivative of strain mc2155 and is therefore assumed to differ fromstrain mc2155 at least by an rpsL mutation that renders the strainstreptomycin resistant (32). The average sequence coverage was122.90-fold (12.8 million paired-end reads) for M. smegmatisSmr5 and 71.84-fold (7.4 million paired-end reads) for M. smeg-matis �lgt. Using M. smegmatis mc2155 (NC_008596) as a refer-ence, the genomes were mapped with the CLC Genomics Work-bench. Results indicated that 99.9266% (Smr5) and 99.3704%(�lgt) of the genomes were covered with at least one read. Neitherinsertions nor deletions were detected in the Smr5 wild type andthe �lgt mutant, except for the genetically engineered lgt deletion

FIG 1 Disruption of M. smegmatis lgt (MSMEG_3222). (Left) Genomic DNAs from M. smegmatis (lane 1), lgt single-crossover (5=sco) mutant (lane 2), �lgtmutant (lane 3), and �lgt-lgtMtb mutant (lane 4; lgtMtb indicates lgt from M. tuberculosis) were digested with XcmI and probed with a 967-bp ApaI lgt genefragment. The presence of a single 5.6-kbp fragment in the �lgt knockout strain compared to the single 5.4-kbp fragment in the parental strain demonstratesinactivation of lgt. The shift in fragment size in the �lgt knockout strain results from replacement of a 959-bp lgt fragment with a 1.2-kbp kanamycin resistancecassette. (Right) Genomic DNAs of M. smegmatis (lane 5), lgt-5= single-crossover mutant (lane 6), �lgt mutant (lane 7), and �lgt-lgtMtb mutant (lane 8) weredigested with BamHI and probed with a 491-bp SacI M. tuberculosis lgt gene fragment to demonstrate complementation. Complementation is indicated by ahybridization signal with genomic DNA derived from strain �lgt-lgtMtb (the M. tuberculosis lgt probe does not hybridize with M. smegmatis lgt).




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in the �lgt mutant. Parental Smr5 and �lgt had 35 single-nucle-otide polymorphisms in common that were not found in the ref-erence strain mc2155. One of these SNPs was the rpsL mutationconferring streptomycin resistance. The other putative SNP foundin both strains located to GC-rich regions with low coverage. OneSNP, a missense mutation, was unique to the �lgt mutant. TheSNP was identified in the ORF MSMEG_3278, a gene with un-known function. An ORF (MSMEG_3280) encoding a lipopro-tein is located in that region. Sanger sequencing confirmed thisSNP.

In vitro growth characteristics of M. smegmatis �lgt. The invitro growth characteristics of parental M. smegmatis, �lgt mutant,and complemented strain expressing M. tuberculosis lgt were in-vestigated. Growth retardation of the �lgt mutant strain was ob-served in liquid Middlebrook-Tween broth (7H9) supplementedwith OADC. Generation times of the strains at maximum growthrate were the following: wild-type, 3 h 20 min; �lgt, 5 h 10 min;

and �lgt-lgt, 3 h 50 min. A 1.5-fold growth retardation of the �lgtmutant was also observed in nutrient-poor medium, i.e., in liquidMiddlebrook-7H9-Tween broth without OADC (Fig. 2A). Gen-eration times were 4 h 17 min for the wild type, 6 h 30 min for �lgt,and 4 h 40 min for �lgt-lgt.

On 7H10 agar, microcolonies of M. smegmatis typicallyshowed rough, dry, and irregular surfaces. In contrast, colonies ofthe �lgt mutant were smaller and less ruffled (Fig. 2B). In contrastto the parental strain, M. smegmatis �lgt was barely stained byauramine O/rhodamine and Ziehl-Neelsen, respectively (Fig. 2B).These morphological alterations had no correlation at the ultra-structural level as revealed by electron microscopy. Both the wild-type and �lgt strains showed a multilayered cell envelope typicalfor mycobacteria, i.e., the plasma membrane and the electron-dense, presumably peptidoglycan layer are covered by an electron-transparent layer and an irregular electron-dense outer layer (Fig.2B). Wild-type-like growth on solid agar and staining with aura-

FIG 2 Growth characteristics of M. smegmatis �lgt. (A) M. smegmatis wild type, M. smegmatis �lgt, and M. smegmatis �lgt-lgtMtb were grown in rich medium (a)or in nutrient-poor medium (b). (B) Inactivation of lgt affects cell and colony morphology. (a) Microcolonies of M. smegmatis grown on 7H10 agar supplementedwith OADC for 3 days (total magnification, �10); (b) auramine-stained M. smegmatis grown in 7H9-Tween (total magnification � 2000); (c) Ziehl-Neelsen-stained M. smegmatis grown in 7H9-Tween (total magnification, �1,000); (d) electron microscopic photographs of M. smegmatis (total magnification,�200,000). Column 1, parental M. smegmatis; column 2, �lgt mutant; column 3, �lgt-lgtMtb complemented strain; arrow, cytoplasmic membrane; square,electron-translucent layer; circle, electron-dense outer layer.

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mine O/rhodamine and Ziehl-Neelsen was restored by introduc-ing a wild-type copy of M. tuberculosis lgt, demonstrating thatmorphological and staining alterations in the mutant are due tothe deletion of lgt and that M. tuberculosis lgt is functional in M.smegmatis. Growth rate and colony morphology of the �lgt mu-tant were also restored when complemented with MSMEG_3222(see Fig. S2 in the supplemental material).

Impact of Lgt depletion on mycobacterial lipoproteins. Toverify that MSMEG_3222 is the gating enzyme of lipoprotein bio-synthesis, we performed Triton-X114 extraction using Mpt83 andLppX, two well-characterized M. tuberculosis lipoproteins, as re-porters to monitor the fate of representative lipoproteins. Phasepartition of protein extracts with the detergent Triton-X114 leadsto the accumulation of lipophilic proteins in the detergent phase,whereas hydrophilic proteins accumulate in the aqueous phase.The diacylglyceryl residue, composed of a glyceryl and two ester-bound fatty acids, renders hydrophilic proteins lipophilic. Mpt83and LppX, extracted from the parental M. smegmatis, accumu-lated in the detergent phase, whereas both lipoproteins extractedfrom the �lgt mutant accumulated in the aqueous phase, suggest-ing the absence of the lipid anchor from the �lgt mutant. Com-plementation with M. tuberculosis lgt reverted the phenotype ofthe �lgt mutant (Fig. 3A and B). To corroborate these findings

obtained with representative lipoproteins, metabolic labelingexperiments with [14C]palmitic acid were performed for abroader set of proteins. The same amount of total protein fromthe parental strain and �lgt mutant was loaded on SDS gels.Autoradiography of protein extracts separated by SDS-PAGErevealed multiple 14C-labeled proteins in the wild-type extract.In contrast, the �lgt mutant showed almost no incorporationof [14C]palmitic acid (Fig. 3C). The accumulation of Mpt83and LppX from the �lgt mutant in the aqueous phase indicatesa lack of the diacylglyceryl anchor. The almost-abolished[14C]palmitic acid incorporation supports this indication. Thisdemonstrates that MSMEG_3222 encodes an M. smegmatisprolipoprotein diacylglyceryl transferase Lgt.

Lgt is the gating enzyme for lipoprotein biosynthesis. It cata-lyzes the first step in the biosynthesis of lipoproteins by forming athioether linkage between a diacylglyceryl residue and the sulfhy-dryl group of the �1 cysteine, thereby attaching the first buildingblock of the membrane anchor. It is assumed that afterwards, in asequential manner, lipoprotein-specific signal peptidase A (LspA)then cleaves off the signal sequence, which in turn is a prerequisitefor N-acylation. The lack of the diacylglyceryl modification shouldabolish recognition of immature lipoproteins by LspA and subse-quent modifications. Western blot analyses with protein extractsfrom the M. smegmatis parental strain, lgt mutant, and lgt mutantcomplemented with the M. tuberculosis lgt (Rv1614) homologuewere performed to investigate the processing of lipoproteins in theM. smegmatis �lgt mutant. An M. smegmatis lspA mutant served asa control (45). Temperature-sensitive lspA mutants of E. coli andlspA knockout mutants of Gram-positive bacteria accumulatediglyceride prolipoprotein, since conversion of prolipoprotein toapolipoprotein is inhibited due to the inability to cleave the signalsequence from the diglyceride prolipoprotein (35, 47). Mpt83 ex-pressed in the lgt mutant showed the same apparent molecularmass as Mpt83 from the parental strain, as demonstrated by West-ern blot analysis (Fig. 4A). This indicates Lgt-independent cleav-age of the signal sequence of Mpt83. In contrast, an increasedmolecular mass of Mpt83 derived from the lspA mutant was ob-served. The difference in size of about 2 to 3 kDa corresponds tothe mass of the signal sequence. The absence of smaller forms ofMpt83 in the lspA mutant indicates that Mpt83 is not processed byalternative signal peptidases when lgt is functional. Lgt-indepen-dent LspA cleavage has been shown for a limited number of lipo-

FIG 3 M. smegmatis �lgt mutant fails to attach the lipid anchor. (A) TritonX-114 phase partition of M. smegmatis strains expressing recombinantlipoprotein Mpt83. (B) Triton X-114 phase partition of M. smegmatis pa-rental strain, �lgt mutant, and �lgt-lgtMtb complemented strain expressingrecombinant LppX using anti-LprG antibody. Of note, anti-LprG antibody(provided by H. Bercovier) cross-reacts with LppX (see Fig. S1 in the sup-plemental material). (C) [14C]palmitic acid incorporation in the wild typeand �lgt mutant.

FIG 4 Lipoproteins without a lipid anchor are not cleaved by LspA. Westernblot analysis of whole-cell extracts of M. smegmatis strains expressing Mpt83(A) or LppX (B). The signal peptide of lipoproteins Mpt83 and LppX accountsfor approximately 2 kDa. The signal peptide is not cleaved in the lgt mutant inthe case of LppX. The signal peptide of Mpt83 is cleaved in the �lgt mutant,indicating Lgt-independent signal sequence cleavage. Western blot analyseswere performed using anti-HA antibody and corresponding secondary anti-body conjugated with horseradish peroxidase.




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proteins in Listeria monocytogenes (3). Therefore, we also analyzedthe M. smegmatis parental strain, �lgt mutant, and complementedstrain expressing a second heterologous lipoprotein, namely, M.tuberculosis LppX. Western blot analysis of whole-cell extracts re-vealed an increased molecular mass of recombinant LppX ofabout 2 kDa in the lgt mutant compared to wild-type cells (Fig.4B). The increased molecular size corresponds to the size of theN-terminal signal sequence of LppX. The same increase in size wasalso observed in the lspA mutant. LppX from the complementedlgt mutant showed the same molecular mass as that from the wildtype. The LppX signal peptide was also cleaved when �lgt mutantwas complemented with MSMEG_3222 (see Fig. S2 in the supple-mental material). This demonstrates that accumulation of pro-LppX in the �lgt mutant is related to Lgt depletion. The accumu-lation of prolipoproteins in the lspA mutant shows that the clonedproteins are recognized as lipoproteins. The accumulation of theprolipoprotein form of LppX in the lgt mutant clearly demon-strates that the lack of Lgt abolishes the signal sequence cleavagefor LppX but not for Mpt83.

Taken together, Triton X-114, [14C]palmitic acid labeling, andWestern blot analyses strongly indicate that lipoproteins derivedfrom the �lgt mutant lack the diacylglyceryl residue. The lack ofthe lipid anchor results in a failure of LspA-mediated signal se-quence cleavage for some lipoproteins. In contrast, the signal se-quence of Mpt83 derived from the lgt mutant is cleaved off. Lipo-proteins can be released into the culture filtrate by shedding orsignal peptide processing (shaving). In a secretome analysis of B.subtilis, seven lipoproteins were found to be shed into the extra-cellular medium in the wild type, and more than 20 lipoproteinswere released into the medium in large amounts in the B. subtilislgt mutant (1). Lipoproteins lacking the lipid anchor were re-ported to be released into the extracellular medium after signalsequence cleavage by Lsp, SpaseI, or alternative proteases or with-out further processing in Streptococcus species, L. monocytogenes,and B. subtilis, respectively (1, 3, 8, 9, 43).

Lipoproteins devoid of the lipid anchor are released into cul-ture filtrate. We performed subcellular fractionations (29) of theparental strain and lgt mutant expressing LppX and Mpt83, re-spectively, to localize mature lipoproteins and lipoproteins lack-ing the lipid anchor. In the parental strain, both lipoproteins,LppX and Mpt83, were present in the cell envelope fractions (Fig.5). In the �lgt mutant, both lipoproteins were absent from the cellwall fraction and from the cytoplasmic membrane fraction. Takentogether, this demonstrates that the lack of lipid modification af-fects subsequent processing and localization. For some lipopro-teins, e.g., Mpt83, signal sequence cleavage may occur withoutprior Lgt-mediated lipid modification, but the absence of a mem-brane anchor leads to the failure to transport and retain lipopro-teins in the mycobacterial cell envelope (Fig. 5).

Lipoproteins without the lipid anchor either may be se-creted into the culture supernatant because they lack a hydro-phobic structure which incorporates them into the membrane,or they may be degraded by proteases. So far, we have focusedon the two recombinant M. tuberculosis lipoproteins, Mpt83and LppX. To get a more complete view of the fate of endoge-nous lipoproteins, we used a proteomic approach. We analyzedthe composition of the secretome of the parental strain and thelgt mutant by two-dimensional gel electrophoresis (2D PAGE)and subsequent protein identification by MALDI-TOF massspectrometry as described previously (1) (see Fig. S3 in the

supplemental material). Experiments were performed in dupli-cate, and samples were taken during exponential growth (t0)and after entry into the stationary phase (t1). We identified 129protein spots in the culture supernatant (see Table S1). Theidentified extracellular proteins are involved in protein trans-port and solute binding, protein folding and degradation, cellenvelope maintenance, intermediary and energy metabolism,transcription and translation, and defense mechanisms. Thedifferences in the levels of 106 secreted proteins between wild-type and lgt mutant cells are quantified in Table S2. In total, 54proteins were found at 2- to 10-fold larger amounts in the lgtmutant secretome than in the wild-type secretome (Table 1). Inthe stationary phase, the cytoplasmic proteins Tkt and EF-Tuwere more abundant in the lgt mutant secretome than in thesecretome of the parental strain, which is probably due to in-creased cell lysis in t1. We then analyzed all identified proteinsfor the presence of signal peptides and signal peptidase I and IIcleavage sites using the LipoP 1.0 server (http://www.cbs.dtu.dk/services/LipoP/). In total, 45 extracellular proteins werepredicted to be synthesized with N-terminal signal peptides,including 25 proteins with type I signal peptides and 20 pro-teins with lipoprotein-specific type II signal peptides (Table 1).These 45 predicted secretory proteins include 35 proteins thatare more abundant in the lgt mutant secretome. In particular,all 20 lipoproteins identified in the culture supernatant werepresent in larger amounts in the �lgt mutant secretome (Fig.6). Tryptic peptide fragments corresponding to signal peptidesof lipoproteins were not detected (data not shown). These li-poproteins function mainly as ABC transporter substrate-binding proteins. Other lipoproteins are involved in energyand intermediary metabolism, protein folding and stabiliza-tion, transcriptional regulation, and unknown functions. Someproteins with predicted signal peptides were found to be pres-ent in the secretome in smaller amounts in the �lgt mutant.Among these are two secreted proteins (MSMEG_0066 and

FIG 5 Subcellular localization of Mpt83 and LppX in M. smegmatis �lgtmutant and parental strains. Western blot analyses of fractionated M.smegmatis extracts are shown. Lipoproteins Mpt83 and LppX localize in thecell wall fraction in the parental strain but are absent from the cytoplasmicmembrane and the cell wall fraction in the �lgt mutant. Absence of lipo-proteins in the cell envelope fractions suggests that lipoproteins in the �lgtmutant are released into the supernatant (see Fig. 6 in the supplementalmaterial).

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TABLE 1 Quantification of proteins that are present in larger amountsa in the secretome of the M. smegmatis �lgt mutant than in the parental strain

Protein group and name Function or description

�lgt/wild-type ratio at:

t0-1 t0-2 t1-1 t1-2

Transport and solute bindingMSMEG_3247d Branched-chain amino acid ABC transporter substrate-binding protein 4.74 7.57 4.68 4.51MSMEG_3280 Lipc Polyamine-binding lipoprotein 9.93 10.64 9.15 12.00MSMEG_3598 Lip Periplasmic sugar-binding proteins 7.06 8.68 7.14 6.21MSMEG_3235 Lip ABC-type amino acid transport system, secreted component 3.87 6.90 4.91 6.31MSMEG_2727 Lip Glutamate-binding protein 5.68 4.80 2.53 6.06MSMEG_6804 Lip Sugar ABC transporter substrate-binding protein 5.85 6.78 5.02 6.31MSMEG_1704 Lip ABC transporter 5.27 5.71 5.12 7.23MSMEG_3636 Lip Ferric iron-binding periplasmic protein of ABC transporter 9.51 7.45 5.57 5.13MSMEG_0643 Lip Extracellular solute-binding protein, family protein 5, putative 4.29 5.56 3.66 3.50MSMEG_6524 SPb ABC polyamine/opine/phosphonate transporter, periplasmic ligand binding protein 3.17 1.84 5.80 4.35MSMEG_6047 Lip Cation ABC transporter, periplasmic cation-binding protein, putative 5.10 4.62 3.18 2.51PstS (MSMEG_5782) Lip Periplasmic phosphate-binding protein 4.68 3.88 5.53 4.21EhuB (MSMEG_5368) Lip Ectoine/hydroxyectoine ABC transporter solute-binding protein 3.88 3.25 2.36 3.46MSMEG_4533 Lip Sulfate-binding protein 2.21 2.93 0.72 1.74MSMEG_1712 Lip ABC transporter periplasmic-binding protein YtfQ 4.85 4.90 3.62 3.26MSMEG_5574 Lip Substrate-binding protein 3.06 4.44 3.38 6.85MSMEG_4999 Lip Bacterial extracellular solute-binding protein, family protein 5 1.06 1.24 3.95 4.21

Protein fate: folding, stabilization and degradationMSMEG_3070 Lip LprG protein 4.31 2.88 2.20 1.06MSMEG_3903 SP Low-molecular-wt antigen MTB12 5.67 1.98 6.11 3.10tig (MSMEG_4674) TF 1.09 1.30 0.88 0.91tig-2 (MSMEG_4674)e TF 5.35 3.42 2.62 2.97MSMEG_5664 Peptidyl-prolyl cis-trans isomerase 1.30 0.70 1.25 1.28MSMEG_5015 SP Secreted protein 2.98 3.46 2.06 3.04

Defense mechanismsMSMEG_6567 SP Iron-dependent peroxidase 1.46 1.05 2.33 9.89MSMEG_2658 SP Beta-lactamase 1.76 1.47 1.02 1.18MSMEG_3811 Universal stress protein family protein, putative 1.79 1.44 0.68 0.51

Energy, intermediary and fatty acid metabolismMSMEG_0806 Hydrolase 4.98 4.10 3.65 5.88MSMEG_0194 SP Serine esterase, cutinase family protein 2.64 2.64 2.39 4.67MSMEG_1403 SP Cutinase superfamily protein 1.85 0.94 0.70 1.38MSMEG_0361 SP Glycosyl hydrolase family protein 3 1.49 1.47 1.00 0.40MSMEG_0645 SP Putative beta-1,3-glucanase 1.82 1.89 1.36 1.56MSMEG_3962 Lactate 2-monooxygenase 1.07 1.27 0.51 0.73MSMEG_6398 SP Antigen 85-A 1.69 1.55 1.45 1.56MSMEG_5789 Putative thiosulfate sulfurtransferase (Rhodanese-like protein) 2.00 1.52 1.12 1.59MSMEG_3580 Antigen 85-C 1.20 1.61 1.01 1.19MSMEG_5345 Lip Glycosyl hydrolase family protein 16 1.29 2.26 0.89 1.26MSMEG_0216 3-Hydroxyacyl-coenzyme A dehydrogenase 1.62 1.38 0.25 0.31glcB (MSMEG_3640) Malate synthase G 0.98 1.90 1.06 0.94tkt (MSMEG_3103) Transketolase 0.43 0.62 0.99 1.31

Other and unknown functionMSMEG_5617 SP Immunogenic protein MPT63 2.19 1.78 0.42 5.55MSMEG_1051 SP Immunogenic protein MPB64/MPT64 1.59 1.57 1.27 0.82MSMEG_3599 Lip Sugar-binding transcriptional regulator, LacI family protein 6.31 8.48 6.52 6.28MSMEG_2408 Uncharacterized oxidoreductase MSMEG_2408 1.67 1.92 0.84 1.56MSMEG_1038 GTP cyclohydrolase II 1.37 1.10 0.33 0.78tuf (MSMEG_1401) Elongation factor Tu (EF-Tu) 0.56 0.49 0.14 5.28MSMEG_0233 SP Lipoprotein LppS 1.79 1.67 1.33 1.71MSMEG_3528 SP ErfK/YbiS/YcfS/YnhG family protein 6.25 7.05 6.02 9.87MSMEG_2381 Putative uncharacterized protein 4.53 2.34 3.15 1.45MSMEG_6078 Lip LpqE protein 2.31 4.04 2.22 0.29MSMEG_1322 SP ErfK/YbiS/YcfS/YnhG family protein 3.00 1.36 9.71 6.74MSMEG_0035 FHA domain protein 1.74 0.97 4.52 8.20MSMEG_6289 Trypsin 1.32 1.23 0.94 0.16MSMEG_0065 Putative uncharacterized protein 1.92 1.17 0.67 1.17MSMEG_4187 Lip Putative uncharacterized protein 1.65 1.79 4.92 5.70

a Larger amounts indicates that mean values of duplicates of �lgt/wt ratios are greater than 1.1 for at least one time point (t0 or t1). t0-1 and t0-2 indicate analysis of two biologicalreplicates at t0; t1-1 and t1-2 indicate analysis of two biological replicates at t1.b SP, signal peptidase I cleavage site.c Lip, lipobox motif according to LipoP 1.0.d According to LipoP 1.0, MSMEG_3247 is not a lipoprotein. MSMEG_3247 was identified as a lipoprotein manually based on the lipobox motif IAGC. This gene was not includedas a lipoprotein in the calculations.e Trigger factor (TF) was identified in two different protein spots (tig and tig-2). tig-2 is a putative processed form of tig.




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MSMEG_3493) and a 28-kDa antigen (MSMEG_6919). Thedecreased amount of these proteins could be due to overrepre-sentation of lipoproteins in the secretome of the �lgt mutant,since the secretome of the two strains was normalized withrespect to total protein concentration.

The increased amount of lipoproteins in the culture filtrate ofthe �lgt mutant clearly demonstrates that the diacylglyceryl resi-

due attached to the �1 cysteine is responsible for anchoring lipo-proteins in the cell envelope. Lipoproteins lacking this lipid struc-ture are not retained in the mycobacterial cell envelope; rather,they are secreted into the supernatant. The redistribution of alllipoproteins in the M. smegmatis �lgt mutant from the cell enve-lope to the medium most probably is responsible for the severegrowth and physiological phenotype.

FIG 6 Close-ups of the lipoproteins that are secreted into the medium in the �lgt mutant due to the missing lipid anchor. Shown are sections of the dual-channelimages of the secretome of the M. smegmatis �lgt mutant (red image) and the wild-type strain (green image) at the transition phase (t0; A) and 1 h after entry intothe stationary phase (t1; B). The secretome was precipitated with TCA and separated using 2D PAGE in the pH range of 4 to 7, as described in Materials andMethods. Quantification of the dual-channel image was performed using Decodon Delta 2D software. (C) The induction ratios of the identified lipoproteins inthe extracellular proteome of the lgt mutant to those of the wild type are shown in the corresponding diagram. Two biological replicates are used for quantificationin panel C. Proteins are annotated with their corresponding MSMEG_ number. Lipoproteins were identified using LipoP 1.0.

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DISCUSSION

Lipoproteins have been known since the discovery of the majorlipoprotein of E. coli by Braun and Rehn in 1969. Later, the threeenzymes involved in lipoprotein biosynthesis were identified firstin E. coli. Therefore, the biosynthesis pathway of E. coli lipopro-teins is well described. Lipoproteins are present in all bacterialspecies, but their structure and biosynthesis pathways differ, par-ticularly between Gram-negative and Gram-positive bacteria(22). Mycobacteria have features of both Gram-negative andGram-positive bacteria. Mycobacterial and E. coli lipoproteinshave three fatty acids in common, but mycobacterial lipoproteinsdiffer from E. coli lipoproteins with respect to the fatty acids of thediacylglyceryl residue linked to the sulfhydryl group of the �1cysteine. The mycobacterial diacylglyceryl contains the mycobac-terium-specific fatty acid 10-methyl octadecanoic acid (tubercu-lostaric acid) (45). Lipoproteins and the enzymes involved in theirsynthesis are virulence factors in bacterial pathogens (18). M. tu-berculosis lipoproteins in particular have been shown to suppressinnate immune mechanisms. LspA catalyzes the second step oflipoprotein synthesis, and its inactivation already attenuates M.tuberculosis severely (33). Characterization of the step precedingsignal peptide cleavage of mycobacterial lipoproteins is thereforeof major interest. The enzyme which catalyzes the diacylglycerolattachment and the function of the diacylglyceryl on lipoproteinsin mycobacteria is characterized here. Based on Himar-1 trans-poson mutagenesis, lgt in M. tuberculosis is an essential gene, andthis was confirmed by our targeted approach. Lgt and the otherlipoprotein biosynthesis enzymes (Lsp and Lnt) are essential inGram-negative bacteria (49). Interference with lipoprotein syn-thesis leads to mislocalization of lipoproteins involved in majorouter membrane biogenesis pathways (30, 44). In Gram-positivebacteria, lgt is not essential, although some lipoproteins are essen-tial, indicating that lipoproteins are active without their mem-brane anchor (20, 48). In mycobacteria, few lipoproteins are func-tionally characterized, but most of the approximately 140mycobacterial lipoproteins are of unknown function. Seven my-cobacterial lipoproteins have been shown to be essential (LppL,LppY, LpqB, LpqF, LpqK, LpqW, and LprB) (36). LpqW(MSMEG_5130, Rv1166) acts at the branching point of phospha-tidylinositol and lipoarabinomannan biosynthesis to control theabundance of the two species in the cell (17). Cell wall biogenesisis essential for mycobacteria and is a target of several antimyco-bacterial drugs. Mislocalization of LpqW and, in turn, failure tosynthesize the correct mannose-capped lipoarabinomannancould be a reason for essentiality of Lgt in M. tuberculosis.

In contrast to M. tuberculosis, the generation of an lgt deletionmutant in M. smegmatis eventually was successful due to the pres-ence of a second lgt (MSMEG_5408). Our biochemical analysesdemonstrated that MSMEG_3222 is the major mycobacterial pro-lipoprotein diacylglyceryl transferase. M. smegmatis wild-type cul-tures pulsed with [14C]palmitic acid incorporated the labeled fattyacid into various proteins of different molecular masses, but in the�MSMEG_3222 mutant very little labeling was detected. UsingTriton phase partition, Mpt83 and LppX, rather hydrophilic pro-teins with a GRAVY (grand average of hydropathicity, calculatedas the sum of hydropathy values of all amino acids divided by thenumber of all residues) (19) score of 0.188 and 0.013, respectively,accumulate in the detergent phase when isolated from parentaland complemented strains. In contrast, these proteins accumulate

in the aqueous phase when isolated from the �lgt mutant. Thisclearly demonstrates that a hydrophobic structure is attached tothese proteins which is mediated by ORF MSMEG_3222. Third,the lipoprotein LppX was found in prolipoprotein form with asignal sequence in the M. smegmatis �lgt mutant. Fourth, lipopro-teins are abundant in the secretome of the �lgt mutant. The resid-ual labeling of proteins may be due to low Lgt activity ofMSMEG_5408. MSMEG_5408 could be an alternative Lgt withlower efficacy and activity than MSMEG_3222. MSMEG_5408has two amino acid substitutions in residues essential for Lgt func-tion, i.e., Y26H and N146C (see Table S2 in the supplementalmaterial). While a Y26F alteration does not affect the functionalityof Lgt, a Y26A alteration completely abolishes Lgt function in E.coli (24, 34). An N146A mutation also completely inactivates Lgt.N146 is located in the Lgt signature motif. Pailler et al. hypothe-sized that Y26, located in a putative acyltransferase motif togetherwith the Lgt signature motif, is involved in binding or recognitionof phosphatidylglycerol (24). Alterations Y26H and N146C there-fore could account for a decreased efficacy or activity ofMSMEG_5408. We currently cannot exclude that MSMEG_5408is a second Lgt. Generation of MSMEG_5408 alone or in combi-nation with MSMEG_3222 would be required to test this hypoth-esis. Alternatively, in vitro biochemical analysis with purifiedMSMEG_5408 could be performed (40). It may be worth notingthat the closely related actinobacterium Streptomyces coelicoloralso has two functional Lgt homologues. Both S. coelicolor lgt ho-mologues rescued a Streptomyces scabies �lgt mutant (48). Suc-cessful deletion of individual genes, but the inability to generate adouble mutant, likewise suggested an overlapping function (42).This may also be the case in M. smegmatis. MSMEG_5408 may actonly on a small subset of the putative 140 lipoproteins or be par-ticularly active under specific growth conditions. The residual la-beling may also be due to other enzymes, such as protein acetyl-transferase-modifying proteins in ε-amino groups of lysineresidues with degradation products of palmitic acid.

In Western blot analyses, Mpt83 was proteolytically processedin the �lgt mutant, presumably either by SpaseI or LspA. The twolipoproteins investigated here in more detail not only containLspA cleavage sites but also have confidently predicted SpaseIcleavage sites in their signal peptides in addition to the lipobox. Inthe �lspA mutant, both lipoproteins, Mpt83 and LppX, werefound in the prolipoprotein form. Unspecific proteolysis is notobserved for any of the tested lipoproteins in the lspA mutant.While pro-LppX is not cleaved by a signal peptidase, the signalsequence of Mpt83 eventually is cleaved independently of the lipi-dation by SpaseI or LspA. This phenomenon is well known in L.monocytogenes and Streptococcus spp. (3, 9).

The molecular nature of the diacylglycerol attached to lipopro-teins by Lgt was previously determined by MS analyses (5, 45). Thelipid structure is thought to anchor proteins to hydrophobicmembranes. In B. subtilis, E. coli, and Listeria, failure to attach thelipid anchor results in the loss of precursor lipoproteins (3, 8, 9,43). The lack of membrane retention results in the accumulationof these proteins in the culture filtrate. The molecular mechanismunderlying the release remains obscure, but it was shown in B.subtilis that proteolytic processing by alternative extracellular orcell wall-associated proteases contributes to the release of non-modified lipoproteins into the medium fraction. This has beenrevealed by N-terminal sequencing of the lipoproteins secreted inthe B. subtilis lgt mutant that all were alternatively processed and




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lacked the �1 cysteine residue (1). In the parental and the com-plemented M. smegmatis strain, LppX and Mpt83 were found toaccumulate in the cell wall fraction. When we subjected recombi-nant M. smegmatis �lgt mutant to subcellular fractionation, wefound neither LppX nor Mpt83 in the cytoplasmic membrane andthe cell wall fraction, respectively. Cell wall localization of Mpt83and LppX is consistent with the respective function and predictedlocalization of the two lipoproteins (41). However, it has also beennoted that MPB83, the corresponding M. bovis protein, is releasedfrom the cells as both a mature, lipidated, 25- to 26-kDa form andas a hydrophilic 22- to 23-kDa form (14).

The absence of Mpt83 and LppX from the cell wall of the �lgtmutant suggests that mutant bacteria are unable to retain lipopro-teins in their membranes. Therefore, we analyzed the secretome ofthe �lgt mutant and parental strain. Overall, 106 different proteinswere identified, including 54 proteins that are present in increasedamounts in the secretome of the �lgt mutant. The increased re-lease of some cytoplasmic proteins from M. smegmatis �lgt mu-tant such as Tkt and EF-Tu indicates that this strain is more sus-ceptible to cell lysis than the wild type. Furthermore, we identified20 lipoproteins that were present at higher levels in the �lgt mu-tant secretome than in the wild type, including 15 substrate-bind-ing or transport proteins. Substrate-binding proteins are abun-dant in the genomes of Gram-positive bacteria and mycobacteriaas binding components of ABC transport systems (41) and areanchored in the outer leaflet of the cytoplasmic membrane (6). InM. smegmatis, a total of approximately 140 putative lipoproteinsare annotated. This increased release of lipid anchor-lacking lipo-proteins in the lgt mutant verifies previous secretome results andconfirms the function of Lgt in M. smegmatis (1, 3, 8, 9, 43). Wealso searched the MALDI-TOF data set of the �lgt mutant secre-tome results for peptide masses corresponding to lipoprotein-spe-cific signal peptides to analyze whether lipoproteins are released inprocessed or nonprocessed forms. The MS results showed thatnone of these released lipoproteins harbored the N-terminal sig-nal peptide, indicating that the majority of released lipoproteinsare processed either by Lsp, SpaseI, or alternative proteases. Inter-estingly, release of nonmodified lipoproteins into the medium wasobserved in a daptomycin-resistant B. subtilis strain that acquireda point mutation of the pgsA gene encoding the phosphatidyl glyc-erol synthase (11). In the case of the pgsA mutant, the precursorfor the diacylglycerol residue, phosphatidyl glycerol, was missing,which in turn results in the lack of the lipid anchor and an Lgtmutant-like release of lipoproteins.

The release of nonmodified lipoproteins may be particularlydeleterious for the pathogen M. tuberculosis, since lipoproteinshave important virulence functions and are involved in cell wallsynthesis (41). The loss of the lipid anchor leads to redistributionfrom the membrane to the medium, and in turn the lack of thelipoproteins may result in alteration of the mycobacterial cell en-velope. Cell envelope alterations were also observed in lgt mutantsof other bacteria. For example, a Bacillus anthracis lgt deletionmutant had a more hydrophilic surface than the wild type, indi-cating a cell wall defect, and this mutant was markedly attenuatedin a mouse spore infection model (23). Growth-deficient pheno-types and lack of immune activation were reported for a Staphy-lococcus aureus lgt mutant (38). Similarly, the M. smegmatis �lgtmutant showed severe growth attenuation in liquid broth andlacks acid fastness, also indicating cell wall alteration. The pheno-types of the �lgt mutant point to a key role of Lgt for correct

lipoprotein function and consequently for proper cell wall integ-rity. Nevertheless, we were able to successfully generate an M.smegmatis lgt deletion strain, perhaps because of the presence of asecond lgt homologue. A single-nucleotide polymorphism wasfound in the gene MSMEG_3278 in the �lgt mutant. The genedoes not have an annotated function and is located in a regionencoding proteins involved in polyamine transport. One gene ofthis transport system (MSMEG_3280) is a lipoprotein and washighly abundant in the secretome of the �lgt mutant. The role ofMSMEG_3278 in the wild type and the �lgt mutant remains to beinvestigated.

Lipoteichoic acid (LTA), a cell wall component of S. aureus, haslong been assumed to stimulate TLR2. However, isolation of LTAfrom S. aureus lgt mutant indicated that LTA preparations fromwild-type S. aureus are frequently contaminated with lipoproteins(13). Numerous mycobacterial lipoglycans are also supposed tostimulate TLR2. The M. smegmatis �lgt mutant will be an invalu-able tool to investigate lipoprotein-independent TLR2 stimula-tion by mycobacterial lipoglycans.

Lgt is essential in M. tuberculosis and thus is even more impor-tant for M. tuberculosis than LspA, the second enzyme of the lipo-protein biosynthesis pathway, which is not essential but is re-quired for full virulence. Lgt inactivation severely affects growthand cell wall properties of M. smegmatis. M. smegmatis possiblycan better compensate for Lgt depletion due to the presence of asecond lgt homologue and due to the eventual accumulation of asuppressor mutation, which obviously occurs only at low fre-quency. There is an urgent need for novel antituberculosis drugsand new potential drug targets, since the antituberculosis drugpipeline is not sufficiently filled and more and more drug-resistantM. tuberculosis strains emerge. Our investigations in M. tubercu-losis indicate that Lgt is a valuable target for generation of antitu-berculosis drugs, while a corresponding M. smegmatis mutant en-ables us to investigate the physiological role of Lgt inmycobacteria.

ACKNOWLEDGMENT

This work was supported by the Swiss National Foundation (31003A-135705).

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