-
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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