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Infection and Immunity, October 2001, p. 6348-6363, Vol. 69, No. 10
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.10.6348-6363.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
High Extracellular Levels of Mycobacterium
tuberculosis Glutamine Synthetase and Superoxide Dismutase in
Actively Growing Cultures Are Due to High Expression and
Extracellular Stability Rather than to a Protein-Specific
Export Mechanism
Michael V.
Tullius,
Günter
Harth, and
Marcus A.
Horwitz*
Division of Infectious Diseases, Department
of Medicine, School of Medicine, University of California, Los
Angeles, California 90095-1688
Received 1 March 2001/Returned for modification 9 April
2001/Accepted 26 June 2001
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ABSTRACT |
Glutamine synthetase (GS) and superoxide dismutase (SOD), large
multimeric enzymes that are thought to play important roles in the
pathogenicity of Mycobacterium tuberculosis, are among the
bacterium's major culture filtrate proteins in actively growing cultures. Although these proteins lack a leader peptide, their presence
in the extracellular medium during early stages of growth suggested
that they might be actively secreted. To understand their mechanism of
export, we cloned the homologous genes (glnA1 and
sodA) from the rapid-growing, nonpathogenic
Mycobacterium smegmatis, generated glnA1 and
sodA mutants of M. smegmatis by allelic
exchange, and quantitated expression and export of both mycobacterial
and nonmycobacterial GSs and SODs in these mutants. We also quantitated
expression and export of homologous and heterologous SODs from
M. tuberculosis. When each of the genes was expressed from a multicopy plasmid, M. smegmatis exported
comparable proportions of both the M. tuberculosis and
M. smegmatis GSs (in the glnA1 strain) or
SODs (in the sodA strain), in contrast to previous observations in wild-type strains. Surprisingly, recombinant
M. smegmatis and M. tuberculosis
strains even exported nonmycobacterial SODs. To determine the extent to
which export of these large, leaderless proteins is expression
dependent, we constructed a recombinant M. tuberculosis strain expressing green fluorescent protein (GFP) at
high levels and a recombinant M. smegmatis strain coexpressing the M. smegmatis GS, M. smegmatis SOD, and M. tuberculosis BfrB
(bacterioferritin) at high levels. The recombinant M. tuberculosis strain exported GFP even in early stages of growth
and at proportions very similar to those of the endogenous
M. tuberculosis GS and SOD. Similarly, the recombinant
M. smegmatis strain exported bacterioferritin, a large
(~500-kDa), leaderless, multimeric protein, in proportions comparable
to GS and SOD. In contrast, high-level expression of the large,
leaderless, multimeric protein malate dehydrogenase did not lead to
extracellular accumulation because the protein was highly unstable
extracellularly. These findings indicate that, contrary to
expectations, export of M. tuberculosis GS and SOD in
actively growing cultures is not due to a protein-specific export
mechanism, but rather to bacterial leakage or autolysis, and that the
extracellular abundance of these enzymes is simply due to their high
level of expression and extracellular stability. The same determinants
likely explain the presence of other leaderless proteins in the
extracellular medium of actively growing M. tuberculosis cultures.
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INTRODUCTION |
Mycobacterium
tuberculosis, the primary etiologic agent of tuberculosis, is one
of the world's leading causes of death, killing 2 million persons
annually worldwide (23). New modalities to combat
M. tuberculosis and a greater understanding of the
biology and immunology of this pathogen are high priorities of
tuberculosis research.
The extracellular proteins of M. tuberculosis have been
the focus of many studies investigating their role in host immunity, their potential as vaccine candidates and diagnostic reagents, and more
recently as drug targets (4, 5, 7, 31, 32, 46, 50).
Although sensitive techniques have identified hundreds of proteins in
culture filtrates of M. tuberculosis (36, 56, 62), approximately 12 proteins are released in relatively large amounts (accounting for ~90% of total extracellular protein with a
molecular mass of
10 kDa) and have been designated as the major extracellular proteins (reference 32 and unpublished
data). Although most of the major extracellular proteins have typical leader peptides, the multimeric enzymes glutamine synthetase (GS) and
superoxide dismutase (SOD) do not (29, 30, 71). GS and SOD
are found in the extracellular medium of M. tuberculosis cultures in the early stages of growth, suggesting
that they might be actively secreted by the bacterium. Both of these
enzymes are considered to be strictly intracellular in most other bacteria.
GS is a dodecamer of identical 53-kDa subunits that has a
central role in nitrogen metabolism, catalyzing the synthesis of L-glutamine from L-glutamate, ammonia, and ATP.
Our group has identified GS as a major component of M. tuberculosis culture filtrates (28). We have proposed
that GS may alter the ammonia level (and pH) of the host cell
phagosome, possibly aiding the bacilli in preventing phagosome-lysosome
fusion. We have also proposed that GS may be necessary for synthesis of
poly-L-glutamate-glutamine present in the cell wall
of pathogenic, but not nonpathogenic, mycobacteria.
SOD is ubiquitous in aerobes and is part of the mechanism to protect
the cell from oxidative stress, catalyzing the dismutation of
superoxide to oxygen and hydrogen peroxide (27). In
bacteria, there are two families of SODs: the FeSOD/MnSOD family, whose members do not contain leader peptides, and the Cu,ZnSOD family, whose
members do contain leader peptides (11). The two enzyme families do not share sequence similarities and are likely a result of
convergent evolution. M. tuberculosis contains both a
tetrameric FeSOD (SodA) and a less well characterized Cu,ZnSOD (SodC)
(22, 39, 74, 76). Kusunose et al. first observed that
M. tuberculosis exports large amounts of its FeSOD
(SodA) (39). The Cu,ZnSOD is surface associated;
however, SOD activity detectable in culture filtrates is mainly due to
the presence of SodA. In several gram-negative pathogens, the
periplasmic Cu,ZnSOD (SodC) has been associated with virulence
(18, 25, 58, 72). However, a recent report by Dussurget et
al. has shown that a SodC-deficient M. tuberculosis mutant is not attenuated in a guinea pig model (22). In
humans, SOD may be important to M. tuberculosis
survival in the phagocyte, where the bacterium may encounter
host-generated superoxide and other reactive oxygen species.
Previous studies from this laboratory of recombinant M. tuberculosis GS and SOD in M. smegmatis found that
a large percentage of the recombinant enzymes (>95% GS and 66% SOD)
but a smaller percentage of the Mycobacterium smegmatis
endogenous enzymes (1% GS and 21% SOD) were exported, suggesting that
export of the two M. tuberculosis enzymes relied on
information in the protein sequence and/or structure (29,
30). In this study we now provide evidence that strongly
suggests that GS and SOD are not actively secreted but rather are
released into the extracellular medium as a result of bacterial leakage
or autolysis and that their extracellular abundance is simply dependent
upon their high expression and extracellular stability.
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MATERIALS AND METHODS |
Materials.
Phenol, phenol-CHCl3-isoamyl alcohol
(25:24:1), Superdex-75, and Sephacryl 300HR were purchased from
Amersham Pharmacia Biotech. Reactive Red-120 Sepharose, xanthine
oxidase,
2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT), and gamma-glutamic acid hydroxamate were purchased from Sigma.
RNase (bovine pancreas) and proteinase K were purchased from Boehringer Mannheim.
Bacterial strains and plasmids.
The bacterial strains and
plasmids used in this study are listed in Tables
1 and 2.
M. smegmatis strains were grown on Middlebrook 7H11
agar (Difco) containing 10% (vol/vol) OADC (Becton Dickinson) and
0.5% (vol/vol) glycerol at 37°C or as shaken cultures in Middlebrook 7H9 broth (Difco) supplemented only with 0.2% (vol/vol) glycerol at 28 or 37°C. M. tuberculosis strains were grown on 7H11
agar containing 10% (vol/vol) OADC and 0.5% (vol/vol) glycerol or as unshaken cultures in 7H9 broth supplemented only with 0.2% (vol/vol) glycerol at 37°C in an atmosphere of 5% CO2-95% air.
Hygromycin (50 µg ml
1) and/or kanamycin (20 µg
ml
1) were included as appropriate. For the cultures shown
in Fig. 12, glucose and/or additional
(NH4)2SO4 was added to our standard 7H9 medium, which contains 0.2% (vol/vol) glycerol and 3.8 mM (NH4)2SO4. Other supplements are
described in the text.
Escherichia coli DH5
, Salmonella enterica
serovar Typhimurium (ATCC 13311), and Bacillus
subtilis SB168 (ATCC 27689) were grown on Luria-Bertani agar,
Luria-Bertani broth, or Terrific broth at 37°C. Ampicillin (100 µg
ml
1), gentamicin (20 µg ml
1), hygromycin
(250 µg ml
1), and kanamycin (50 µg ml
1)
were included as appropriate.
Recombinant DNA methods.
Plasmid DNA was isolated using
Quantum Prep (Bio-Rad) or Wizard Plus SV (Promega) miniprep kits.
Genomic DNA was isolated from M. smegmatis, M. tuberculosis, E. coli, S. enterica serovar Typhimurium, and B. subtilis by extraction with hot phenol
(65°C, Tris-equilibrated, pH
8) and ethanol precipitation.
The DNA was further purified by sequential digestion with RNase and
proteinase K, additional extractions with phenol and
phenol-CHCl3-isoamyl alcohol (25:24:1), and a final ethanol precipitation.
Electroporation of mycobacteria.
Cells were washed two or
three times with 10% (vol/vol) glycerol and resuspended in 10%
(vol/vol) glycerol to a density of 109 to 1010
cells ml
1. M. smegmatis cells were kept
at 0 to 4°C throughout the washing and electroporation procedures
while M. tuberculosis cells were maintained at room
temperature (68). DNA (1 to 5 µg) was mixed with 400 µl of cells, transferred to a 0.2-cm-gap electroporation cuvette (Bio-Rad), and a single electrical pulse was delivered (2.5 kV,
25 µF, 1,000
). For M. smegmatis, the cells were
immediately transferred to 2 ml of SOC medium (57) (with 5 mM L-glutamine for the glnA1 mutant) and
incubated at 37°C with vigorous shaking for 4 to 6 h before
being plated on selective media. For cells electroporated with
temperature-sensitive pPR27-derived plasmids, the incubation was done
at
32°C. For M. tuberculosis, 600 µl of SOC
medium was added directly to the cells in the cuvette and the cells
were incubated without shaking at 37°C for 18 to 24 h before being
plated on selective media.
Southern and colony hybridizations.
Restriction fragments of
genomic DNA or plasmids were electrophoresed in agarose gels,
transferred to positively charged nylon membranes (Hybond-N+; Amersham
Pharmacia Biotech) in 0.4 M NaOH, and hybridized to specific probes.
Hybridizations were done at 60°C in 6× SSPE-2% skim milk for 15 to
20 h (1× SSPE is 150 mM NaCl, 10 mM
NaH2PO4, 1 mM EDTA [pH 7.4]). Membranes were
washed very stringently at 65°C for 15 to 20 min once with 1×
SSPE-1% skim milk and twice with 0.1× SSPE-1% skim milk before
being exposed to X-ray film for various times. Hybridization and
washing conditions were identical for colony hybridizations. Probes
were radiolabeled to a specific activity of 1 × 108
to 2 × 108 dpm µg
1 with
[
-32P]dCTP by random priming using the Multiprime DNA
Labeling Kit (Amersham Pharmacia Biotech).
Cloning and sequencing of the M. smegmatis glnA1
and sodA genes.
Primers were designed based on the
M. tuberculosis glnA1 sequence (30) and
used to amplify a 1-kb target region encoding ~70% of the
M. smegmatis glnA1 gene from M. smegmatis 1-2c genomic DNA. The amplification product was cloned
into pCR2.1 (Invitrogen), sequenced, and found to be highly similar to
the M. tuberculosis glnA1 gene. The M. smegmatis glnA1 gene fragment was released from pCR2.1 by
EcoRI digestion, gel purified, and used as a probe for the
identification and isolation of its genomic locus in Southern and
colony hybridizations. Likewise, a 0.65-kb EcoRI fragment containing the entire coding region of the M. smegmatis
1-2c sodA gene from a PCR amplification product
(29) was used as a probe for the genomic M. smegmatis 1-2c sodA locus.
M. smegmatis 1-2c genomic DNA was completely digested
with both BamHI and EcoRI and subjected to
Southern analysis. The glnA1 probe hybridized to a 2.2-kb
fragment, and the sodA probe hybridized to a 1.1-kb
fragment. Restriction fragments from 1.7 to 2.4 kb and 0.8 to 1.2 kb
were isolated and ligated into pUC18 digested with BamHI and
EcoRI. The ligation was transformed into E. coli DH5
, and clones were verified for both genes by colony
hybridizations, restriction analysis, and Southern hybridizations. The
plasmids were designated pUC18-Ms-glnA1 and
pUC18-Ms-sodA. For both plasmids, the entire insert was
sequenced in both directions by cycle sequencing with ABI PRISM dye
terminators (Davis Sequencing). One region of M. smegmatis 1-2c sodA exhibited particularly strong
secondary structure and the sequence was determined with the Fidelity
DNA Sequencing System (Oncor) with reaction products resolved on a 40%
formamide-6 M urea gel. The sequences were assembled using the
programs Pregap4 (version 1.0) and Gap4 (version 4.4) from the Staden
Package (13). The sequences were scanned against the
GenBank database using the BLAST programs (3) at the
National Center for Biotechnology Information
(http://www.ncbi.nlm.nih.gov/).
Construction of M. smegmatis glnA1 and
sodA mutants.
A promoterless, nonpolar kanamycin
resistance (Kmr) cassette was constructed using an approach
similar to the one described previously (43), using a
different kanamycin resistance gene, replacing the gram-negative
ribosomal binding site with the M. tuberculosis dnaK
ribosomal binding site and including convenient restriction sites for
exchanging resistance genes (Fig. 1). The cassette was constructed by amplification of the Tn5
kanamycin resistance gene (aphA-2) using pCR2.1 as the
template DNA and contains stop codons in all three reading frames
immediately upstream of the ATG start codon of the aphA-2
gene. Immediately downstream of the aphA-2 gene stop codon
is the sequence for the ribosomal binding site and ATG start codon of
the M. tuberculosis dnaK gene to allow for translation
of the 3' portion of the disrupted gene. The blunt-end PCR product was
cloned into the SmaI site of pUC19. Clones resistant to
ampicillin and kanamycin were selected and were confirmed by
restriction analysis. The cassette was released from pUC19 as a 0.8-kb
fragment by AvaI digestion, and the 5' overhangs were filled
with T4 DNA polymerase.

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FIG. 1.
Nonpolar kanamycin resistance cassette cloned into the
SmaI site of pUC19. The cassette contains the kanamycin
resistance gene (aphA-2) coding region but lacks both a
promoter and a transcriptional terminator. The stop codons in all three
frames immediately upstream of aphA-2 are underlined, as is
the stop codon of the gene. The start codon of aphA-2 and
the start codon provided at the 3' end of the cassette are shown in
boldface type. The 15 nucleotides immediately downstream of the
BclI site are identical to the sequence directly upstream of
the M. tuberculosis dnaK gene (the ribosomal binding
site [rbs] is indicated). NdeI and BclI sites
were included to facilitate cloning of other resistance genes into the
cassette. Abbreviations for restriction sites: H, HindIII;
Sh, SphI; P, PstI; Sl, SalI; X,
XbaI; B, BamHI; K, KpnI; Sc,
SacI; E, EcoRI.
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Unique sites in the coding regions of the M. smegmatis
glnA1 and sodA genomic loci were identified
(XhoI in pUC18-Ms-glnA1 and BstEII in
pUC18-Ms-sodA). The plasmids were digested, and the 5'
overhangs were filled with T4 DNA polymerase. The Kmr
cassette was blunt end ligated into the vectors such that the 3'
portion of glnA1 or sodA was in frame with the
ATG start codon provided at the 3' end of the Kmr cassette
(Fig. 2B and D). Clones were identified
by restriction analysis that had the Kmr cassette in the
same orientation as glnA1 or sodA, allowing for transcription of the kanamycin resistance gene from the
glnA1 or sodA promoter. The disrupted genes were
released from pUC18 by digestion with BamHI and
EcoRI. The EcoRI sites were modified by ligation
to an adapter with multiple restriction sites (designated ECHXB) (Fig.
3) and the fragments were redigested with
BamHI and cloned into the BamHI site of the
temperature-sensitive allelic exchange vector, pPR27 (51).
Constructions were verified by restriction analysis and designated
pPR27-Ms-glnA1::Kmr and
pPR27-Ms-sodA::Kmr.

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FIG. 2.
Maps of the M. smegmatis 1-2c
glnA1 and sodA genomic loci. Both the
glnA1 (A) and the sodA (C) loci were isolated as
BamHI EcoRI (5'-to-3') genomic
fragments. Both genes have putative transcriptional terminators
(T) directly downstream of their coding regions. The
glnA1 locus contains a truncated open reading frame (orf)
with a high degree of similarity to the M. tuberculosis
Rv1897c gene. The sodA locus contains a truncated open
reading frame upstream of sodA with a predicted protein
sequence that is highly similar (75% identity) to a truncated open
reading frame in the same location in the M. fortuitum
sodA locus (44). The glnA1 and the
sodA loci were cloned into the multiple cloning site of
pNBV1 for expression of GS and SOD in M. smegmatis and
M. tuberculosis. The Kmr cassette (Fig. 1)
was inserted into the unique XhoI site of glnA1
(B) and the BstEII site of sodA (D) to generate
the disrupted loci used for allelic exchange.
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FIG. 3.
ECHXB adapter. EcoRI adapter with several
restriction sites useful for cloning into the multiple cloning site of
pNBV1 and other vectors.
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The two allelic exchange constructs were electroporated into
M. smegmatis 1-2c, and transformants were selected on
7H11 with kanamycin (20 µg ml
1) at 32°C.
Transformants were inoculated into 7H9 with kanamycin (20 µg
ml
1) and 5 mM L-glutamine and incubated at
32°C for 8 h to allow allelic exchange to take place. Cells were
then plated on 7H11 with kanamycin (20 µg ml
1)
supplemented with 10% (wt/vol) sucrose and 5 mM
L-glutamine and incubated at 39°C for 4 days to
counterselect against those cells that retained the plasmid. Out of a
total of 1,500 clones (each), six sodA clones and three
glnA1 clones were selected for further analysis. Four of the
six sodA clones no longer expressed SodA activity. All three
glnA1 clones grew well on plates with added
L-glutamine (5 mM) but did not grow at all on regular 7H11 plates. Genomic DNA was isolated from one sodA and one
glnA1 clone, digested to completion with both
BamHI and EcoRI, and subjected to Southern
analysis (Fig. 4) using the following
radiolabeled DNA probes: the M. smegmatis glnA1 genomic
locus (Fig. 2A), the M. smegmatis sodA genomic locus
(Fig. 2C), vector (pPR27), and the Kmr cassette. For the
glnA1 mutant, the 2.2-kb band that is present in the
wild-type strain was replaced by a 3-kb band indicating insertion of
the 0.8 kb Kmr cassette. Likewise, for the sodA
mutant, the 1.1-kb band that is present in the wild-type strain was
replaced by an ~2-kb band. When probed with just the Kmr
cassette, only the 3- and 2-kb bands (glnA1 and
sodA mutants, respectively) hybridized, and nothing
hybridized in the wild-type DNA (data not shown). Also, the vector
(pPR27) did not hybridize to DNA from the wild-type or mutant strains
(data not shown).

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FIG. 4.
Southern analysis of M. smegmatis glnA1
and sodA mutants. Genomic DNA from M. smegmatis 1-2c (wild type [wt]), M. smegmatis
glnA1, and M. smegmatis sodA strains was digested
with BamHI and EcoRI and probed with the entire
glnA1 locus (Fig. 2A) (A) or the entire sodA
locus (Fig. 2C) (B). M, molecular mass markers in kilobases.
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Construction of recombinant mycobacterial strains.
In
previous studies, the M. tuberculosis glnA1 locus was
cloned into pSMT3 for expression in the M. smegmatis
1-2c wild-type strain (30). This 1.8-kb locus was
transferred as a BamHI
HindIII fragment
(5' to 3') into the multiple cloning site of pNBV1. The M. tuberculosis sodA locus was previously cloned into pNBV1 as a
ClaI
BamHI PCR product (5' to 3'), and
its expression was studied in M. smegmatis 1-2c
(29). This plasmid was used without modification.
The M. smegmatis glnA1 and sodA genomic loci
cloned into pUC18 likely contained sufficient upstream DNA sequence to
encode all of the necessary promoter elements for expression in a
mycobacterial host. Therefore, the loci were released by digestion with
BamHI and EcoRI and the EcoRI site was
modified by ligation to the ECHXB adapter (Fig. 3). The DNA was
digested with XbaI or HindIII and cloned into
the mycobacterial shuttle vector pNBV1 cut with BamHI and
HindIII or BamHI and XbaI so as to
obtain the genes in both orientations on the plasmid. Prior to
electroporation into mycobacteria as described above, all constructs
were confirmed by restriction analysis. The glnA1 plasmids
were electroporated into the glnA1 mutant, and the
sodA plasmids were electroporated into the sodA mutant. Transformants expressed high levels of GS and SOD for both
orientations of the glnA1 and sodA loci,
respectively. One clone of each with the gene cloned (5' to 3') in the
BamHI and HindIII sites was chosen for all
further studies (designated pNBV1-MsGS and
pNBV1-MsSODA).
The gene encoding a UV-optimized green fluorescent protein (GFPuv) was
amplified by PCR from pGFPuv (Clontech), modifying the upstream region
to contain an NdeI site at the ATG start codon and adding a
HindIII site downstream of the stop codon. The PCR product was digested with NdeI and HindIII
and cloned into a pNBV1 construct that contains a modified
M. bovis BCG hsp60 promoter (amplified from
pSMT3) in which the sequence upstream of the ATG start codon was
modified to contain an NdeI site. The gene contains an
internal NdeI site which required first cloning the 3'
portion of the gene into the vector as an
NdeI
HindIII (5' to 3') fragment. A
clone was isolated and digested with NdeI, and the 5'
portion of the gene was cloned as an NdeI fragment.
The remaining genes (M. tuberculosis mdh, M. tuberculosis bfrB, E. coli sodA, E. coli sodB, B. subtilis
sodA, and S. enterica serovar Typhimurium
glnA) were amplified from genomic DNA using the
Advantage-GC2 PCR kit (Clontech) and cloned downstream of the
M. tuberculosis glnA1 promoter. For all of the genes
except for bfrB the same strategy was employed, cloning into
the BsmI site at the 5' end (immediately upstream of the
M. tuberculosis glnA1 GTG start codon) and the
HindIII site at the 3' end. The 5' forward primers
started with the sequence 5'-GCTAGCATTCTGTG with 18 to 20 additional nucleotides corresponding to codons 2 through 8 of the gene being amplified. The BsmI site is
underlined, and the GTG start codon is shown in italics. The 3' reverse
primers incorporated a HindIII site (underlined)
immediately downstream of the gene's stop codon,
5'-CCCAAGCTT, with 21 or 22 additional nucleotides complementary to the 3' end of the gene. Because a BsmI site is present in pNBV1, the 1.8-kb M. tuberculosis glnA1 locus was first transferred into pUC19. The
PCR-amplified genes were cloned downstream of the glnA1
promoter, replacing the glnA1 coding region, and then the
gene plus promoter was transferred to pNBV1 as a
BamHI
HindIII fragment. The B. subtilis sodA gene has an internal HindIII site
that was first removed by PCR mutagenesis.
One of the genes (bfrB) was cloned downstream of an
M. tuberculosis glnA1 promoter modified to contain an
NdeI site at the start codon. There are no NdeI
sites in pNBV1, which allowed for the PCR product to be cloned
downstream of the promoter directly in pNBV1, avoiding the initial
cloning into pUC19 required for the earlier constructs described above.
The sequence upstream of and including the GTG start codon,
TTCTGTG, was modified to TTCaTaTG in otherwise identical promoters
(start codons are underlined and lowercase letters indicate added or
modified nucleotides). The forward bfrB primer incorporated
an NdeI site at the ATG start codon and the reverse
bfrB primer incorporated a HindIII site as
described above.
Recombinant plasmids carrying both sodA and glnA1
as well as a third gene were constructed by first cloning the
M. smegmatis sodA locus into the BamHI and
XbaI (5'-to-3') sites of pNBV1-MsGS to yield a
plasmid (designated pNBV1-MsGS-MsSODA) containing
both M. smegmatis glnA1 and sodA in opposite
orientations. A 3.3-kb HindIII fragment from
pNBV1-MsGS-MsSODA, containing the entire sequence
of both loci, was cloned into the HindIII site of
pNBV1-MDH and pNBV1-BFRB (Fig. 5).

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FIG. 5.
Maps of the mycobacterial expression constructs. All of
the constructs were cloned into the HindIII and
BamHI or XbaI sites of the multiple cloning site
of pNBV1. (A) The M. tuberculosis mdh gene was cloned
downstream of the M. tuberculosis glnA1 promoter (pGS).
The following genes were cloned in the same way as mdh:
E. coli sodA, E. coli sodB, B. subtilis sodA, and
S. enterica serovar Typhimurium glnA. (B) The
M. tuberculosis bfrB gene was cloned downstream of an
M. tuberculosis glnA1 promoter (pGS') modified to have
an NdeI site at the start codon. (C) The UV-optimized GFP
gene (gfpuv) was cloned downstream of an M. bovis BCG hsp60 promoter (pHSP60') modified to contain
an NdeI site at the start codon. (D and E) A 3.3-kb
HindIII fragment containing the M. smegmatis
glnA1 and sodA loci was cloned into the
HindIII site of pNBV1-MDH (A) or pNBV1-BFRB (B). The
M. smegmatis glnA1 and sodA genes were
transcribed from their own promoters.
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Plasmids were electroporated into mycobacteria as described above.
Recombinant M. smegmatis and M. tuberculosis strains are listed in Table 2.
Analysis of M. smegmatis and M. tuberculosis recombinant strains.
Cultures were typically
inoculated with cells from 7H9 cultures in late log phase or early
stationary phase to give a calculated A550 of
0.01 to 0.02 (100-fold dilution). When the cultures were intended for
analysis of early-log-phase extracellular proteins, the cells were
washed in fresh media before inoculation to avoid carryover of
extracellular proteins. Because some proteins were expressed at higher
levels at 28 than 37°C in M. smegmatis, particularly malate dehydrogenase (MDH), all M. smegmatis cultures
were grown at 28°C except where indicated. When single time points of
a culture were analyzed, the cultures were grown to late log phase or
early stationary phase prior to harvest. Aliquots (10, 20, 40, or 100 ml) were removed for analysis and centrifuged. The supernate was filtered (Acrodisc PF 0.8/0.2 µm filter; Pall Corporation) and concentrated to 100 to 200 µl with a Centricon Plus-20 concentrator (5000 or 8000 MWCO; Millipore), and the concentrate was then diluted to
0.5 or 1.0 ml with phosphate-buffered saline (PBS). The cell pellet was
washed by resuspending in 5 ml of PBS, centrifuging, and decanting the
supernatant fluid. The cells were stored frozen at
80°C or used
immediately. Cells were lysed by resuspending in 5 ml of PBS and
sonicating twice for 2 min each (M. smegmatis) or once
for 4 min (M. tuberculosis) on ice with a Heat Systems Ultrasonics W-375 sonicator (50% pulse; maximum setting with a microtip). Cellular debris was removed by centrifugation and the cleared lysate was filtered (pore size, 0.2 µm).
Enzyme assays.
GS was assayed by the transfer reaction
(73). Enzyme (100 µl) was added to 900 µl of assay mix
(prewarmed to 37°C) to give the following concentrations of
components: 30 mM L-glutamine, 60 mM NH2OH, 20 mM arsenate, 20 mM imidazole, 1 mM MnCl2, and 0.4 mM ADP at
pH 7.0. The reaction was incubated at 37°C for 10 to 120 min
(depending on activity) and stopped by the addition of 250 µl of 8%
(wt/vol) trichloroacetic acid, 2 N HCl, and 3.3% (wt/vol)
FeCl3. The samples were centrifuged for 1 min to remove precipitated protein, and the absorbance was read at 540 nm. Blank reactions, which substituted PBS for enzyme, were subtracted. A
standard curve was prepared from gamma-glutamic acid hydroxamate. A
unit is defined as the amount of enzyme that catalyzes the formation of
1 µmol of gamma-glutamic acid hydroxamate per min under the assay conditions.
SOD was assayed using a modified version of the method described by
Ukeda et al. (66). Enzyme (100 µl) was added to 800 µl
of assay mix, and the reaction was initiated by the addition of
xanthine oxidase (100 µl; 19 mU ml
1 in PBS containing 1 mg of bovine serum albumin ml
1) to give the following
concentration of components: 50 mM NaH2PO4 (pH
8.0), 200 mM NaCl, 0.1 mM EDTA, 0.1 mM xanthine, 25 µM XTT, and 1.9 mU of xanthine oxidase per ml. The reaction was monitored continuously at 470 nm at room temperature (25°C) and was linear for
12 min. One unit is defined as the amount of SOD that inhibits by 50%
the reduction of XTT by superoxide.
MDH was assayed by adding 100 µl of enzyme to 900 µl of assay mix
to give the following concentrations of components: 50 mM NaH2PO4 (pH 7.5), 200 mM NaCl, 1 mM KCN, 0.2 mM
NADH, and 2 mM oxaloacetate. The reaction was monitored continuously at
340 nm for 2 to 3 min during which time the reaction was linear. Blank reactions in which oxaloacetate was omitted were subtracted. KCN at 1 mM was useful in inhibiting respiratory chain oxidation of NADH
(17). For M. smegmatis strains
overexpressing the M. tuberculosis MDH, lysates were
diluted 100-fold in PBS immediately before assay. A unit is defined as
the amount of enzyme that catalyzes the consumption of 1 µmol of NADH
per min under the assay conditions (
= 6,300 M
1
cm
1).
SDS-polyacrylamide gel electrophoresis (PAGE) analysis.
Culture filtrates and cell lysates were analyzed on sodium dodecyl
sulfate (SDS)-12.5% polyacrylamide gels. The gels were stained with
colloidal Coomassie brilliant blue G-250 (47).
SOD activity gels.
Culture filtrates and cell lysates
were electrophoresed on 15% nondenaturing polyacrylamide gels at 80 V
for 16 to 20 h at room temperature. Gels were negatively stained
for SOD activity as described in reference 10.
Immunoblotting.
Culture filtrates and cell lysates of
M. tuberculosis pNBV1-GFPuv were electrophoresed on an
SDS-12.5% polyacrylamide gel and transferred to a nitrocellulose
membrane. Strips containing GS, SOD, and GFPuv antigens were probed
separately with rabbit polyclonal antibodies specific for the
M. tuberculosis GlnA1 (diluted 1:2,500)
(28), the M. tuberculosis SodA (diluted
1:2,500) (29), and GFP (diluted 1:1,000) (Molecular
Probes). The membranes were subsequently incubated with horseradish
peroxidase-conjugated goat anti-rabbit antibodies (diluted
1:10,000; Bio-Rad), a chemiluminescent substrate (SuperSignal
West Pico; Pierce) was added, and the proteins were visualized on X-ray film.
Gel and immunoblot analysis.
Gels were photographed wet and
the negatives were scanned to obtain an 8-bit gray-scale image.
Developed X-ray film from chemiluminescent immunoblotting was scanned
directly. Protein bands were quantitated using the public domain NIH
Image (version 1.62) program (developed at the U.S. National Institutes
of Health and available on the Internet at
http://rsb.info.nih.gov/nih-image/).
Metabolic labeling of M. tuberculosis
pNBV1-GFPuv.
An actively growing culture of M. tuberculosis pNBV1-GFPuv (10 ml) was diluted into 40 ml of fresh
medium (7H9 with hygromycin at 50 µg ml
1) to give an
initial A550 of ~0.06 and grown for 4 days at
37°C to an A550 of 0.18. Then, a mixture of
[35S]L-methionine and
[35S]L-cysteine (Promix; Amersham Pharmacia
Biotech) was sterile filtered and added to the culture at a final
concentration of 50 µCi ml
1. The culture was incubated
at 37°C for 3 more days, reaching a final A550
of 0.28, during which time 10-ml aliquots were removed at 7, 23, 48, and 72 h. Culture filtrates and cell lysates were prepared as
described above and analyzed by SDS-PAGE. After staining with
Coomassie brilliant blue, the gel was dried and exposed to X-ray film
for various times (16 to 160 h).
Purification and stability of MDH.
M.
smegmatis cells overexpressing the M. tuberculosis
MDH (M. smegmatis glnA1
pNBV1-MsGS-MsSODA-MDH) were grown to stationary phase in 7H9 and harvested by centrifugation. The supernate was sterilized by filtration (pore size, 0.2 µm) and saved at 4°C. The
cells were resuspended in PBS, lysed by sonication, and centrifuged, and the cleared lysate was filtered (pore size, 0.2 µm). The lysate (15 ml) was loaded on a Reactive Red-120 Sepharose column (20 ml, 2.6 by 3.5 cm) and eluted by gravity flow. The column was eluted with 90 ml
of PBS, 60 ml of PBS-1 mM AMP, 30 ml of 0.1× malate buffer, 90 ml of
0.1× malate buffer-1 mM AMP, and finally with 90 ml of 1× malate
buffer-1 mM AMP (1× malate buffer is 50 mM
NaH2PO4, 100 mM L-malic acid, and
500 mM NaCl [pH 7.5]). Fractions were assayed for MDH activity, which
was found only in the final wash with 1× malate buffer. The active
fractions were concentrated with Centricon Plus-20 concentrators (MWCO
8000) and loaded on a Superdex-75 column (1.6 by 55 cm) equilibrated in
PBS. The column was eluted at 1 ml min
1 with PBS and MDH
was collected as a single, symmetrical peak that eluted very close to
the void volume.
Purified MDH (~500 µg in 5 ml) was sterile filtered (pore size, 0.2 µm), added to 195 ml of the sterile culture filtrate (see above), and
incubated at 28°C with shaking (same conditions as for the
M. smegmatis cultures). Aliquots of 20 ml were removed immediately after adding MDH and 1, 2, 3, and 7 days later. The samples
were concentrated to 100 to 200 µl with Centricon Plus-20 concentrators (8000 MWCO) and then diluted to 1.0 ml with PBS. The
concentrated samples were stored at 4°C until day 3, at which point
all of the samples were assayed for MDH and GS activity. On day 7, all
of the samples were analyzed by SDS-PAGE.
Nucleotide sequence accession numbers.
The nucleotide
sequences have been deposited in the GenBank database
(glnA1, GenBank accession number AY008693; sodA,
GenBank accession number AF061031).
 |
RESULTS |
The proportion of GS and SOD exported from M. smegmatis and M. tuberculosis is not dependent
upon the species from which the protein is derived.
Previously, we
found a high level of expression and export of recombinant
M. tuberculosis GS and SOD compared with the endogenous M. smegmatis homologues in a wild-type M. smegmatis host (29, 30). To explore export of these
large, leaderless, multimeric proteins further, we cloned the
M. smegmatis glnA1 and sodA genes and
constructed glnA1 and sodA mutants (see Materials
and Methods), allowing for high-level expression of the M. tuberculosis and M. smegmatis proteins in a null
background. The M. smegmatis SodA was found to be
highly similar (>80% identity) to many mycobacterial SodA proteins as
well as the SodA from Nocardia asteroides
(Mycobacterium fortuitum, accession no. X70914;
Mycobacterium avium, accession no. U11550;
Mycobacterium lepraemurium, accession no. D13288; M. leprae, accession no. X16453; N. asteroides, accession no. U02341; M. tuberculosis,
accession no. AF061030). The M. smegmatis GlnA1 was
most similar (83 to 84% identity) to the M. tuberculosis GlnA1 (accession no. U87280) and M. leprae GlnA1 (accession no. CAC31306), with the next most
similar GlnA proteins (70 to 73% identity) being from
Streptomyces coelicolor (accession no. M23172),
Corynebacterium glutamicum (accession no. Y13221), and
Amycolatopsis mediterranei (accession no. AF050112). In
contrast, the mycobacterial SodA proteins share only 39 to 45%
identity with E. coli and B. subtilis SODs
(Table 3), and the GlnA1 proteins share
~50% identity with S. enterica serovar Typhimurium GlnA
(accession no. M14536).
Expression and export of homologous and heterologous GSs from
the M. smegmatis wild-type and glnA1
strains.
The M. smegmatis glnA1 strain has an
absolute requirement for L-glutamine. Complementation
of the glnA1 strain was achieved by supplying either a
copy of the M. smegmatis glnA1 or M. tuberculosis glnA1 in trans on pNBV1 with expression
driven from their own promoters. Because genes are often poorly
expressed from E. coli promoters in M. smegmatis (8), expression of S. enterica
serovar Typhimurium glnA was driven from the strong
M. tuberculosis glnA1 promoter (Fig. 5A). This
construct was also capable of complementing the glnA1
mutant. The M. smegmatis wild type, glnA1
mutant, and complemented strains were analyzed for expression and
export (extracellular accumulation) of GS by measuring enzyme activity
(Table 4) and by SDS-PAGE analysis (Fig.
6). The M. smegmatis
wild-type strain expressed GS activity at 94 mU per ml of original
culture volume, while virtually no GS activity (0.1 mU
ml
1) was detected for the glnA1 mutant (grown
with 10 mM L-glutamine). Growth of the wild-type strain
with 10 mM L-glutamine did decrease the GS activity to
approximately 20% compared to growth in regular 7H9 but still gave
clearly detectable activity. The level of GS activity in the mutant
strain complemented with the M. smegmatis glnA1
(Ms glnA1 pNBV1-MsGS) or M. tuberculosis glnA1 (Ms glnA1 pNBV1-MtbGS)
was 6- to 13-fold higher than in the wild-type strain. Consistent with
this, bands of much greater intensity (compared with the wild-type
strain) were observed on SDS-PAGE analysis. Even though the S. enterica serovar Typhimurium glnA was expressed from a
strong mycobacterial promoter, the mutant strain complemented with this
gene (Ms glnA1 pNBV1-StGS) had very low levels of
GS activity (4 mU ml
1), but the levels were clearly
detectable above background and no protein band for GS could be
detected by SDS-PAGE analysis. However, this strain grew just as well
as the strains complemented with the mycobacterial glnA1
genes.

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FIG. 6.
GS expression and export in M. smegmatis
wild-type and M. smegmatis glnA1 strains. SDS-PAGE
analysis of culture filtrates (CF) and lysates (L). Tenfold more CF
than L was loaded on the gel (the equivalent of 2 ml versus 0.2 ml of
the original culture volume). The results are representative of three
independent cultures. Arrows indicate the positions of the
M. smegmatis (Ms) and M. tuberculosis
(Mtb) GSs. M, molecular mass markers in kilodaltons.
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|
For all of the strains, the proportion of total GS activity detected in
culture filtrates was quite low (2 to 7%), and no significant
difference in the proportion of the M. smegmatis and M. tuberculosis GSs exported was observed.
Expression and export of homologous and heterologous SODs from
M. smegmatis, M. smegmatis sodA, and
M. tuberculosis.
The sodA mutation in
M. smegmatis did not appear to result in any growth
defect (i.e., the mutant grew well in 7H9 medium). Strains were
constructed from the sodA mutant that expressed the M. smegmatis SodA, M. tuberculosis
SodA, E. coli SodA, E. coli SodB, or
B. subtilis SodA. Transcription of the mycobacterial sodA genes was from their own promoters.
Nonmycobacterial SOD genes were all transcribed from the
M. tuberculosis glnA1 promoter (Fig. 5A), as was done
for the S. enterica serovar Typhimurium glnA. The
plasmids containing the nonmycobacterial SOD genes were also
transformed into the wild-type M. smegmatis to
investigate whether the nonmycobacterial SOD subunits could mix with
the M. smegmatis SodA subunits.
The strains were grown in 7H9 medium, and the culture filtrates and
lysates were analyzed by SDS-PAGE and SOD activity gels (Fig. 7A to
C and Table
5). By SDS-PAGE analysis, the 23-kDa band
for the M. smegmatis SodA was absent from the
sodA strain, as was the major band on the activity gel.
However, there was a small amount of SOD activity in the
sodA mutant that ran slightly higher than the M. smegmatis SodA on the activity gel. This band was not evident in
the wild-type strain or the sodA strain expressing the
M. smegmatis SodA from a plasmid (Ms sodA
pNBV1-MsSODA). However, it may have run differently in the
presence of the M. smegmatis SodA, perhaps forming
hybrids, and been hidden by the larger band. A nearly identical
phenotype was previously observed for an M. smegmatis
sodA mutant resulting from a single crossover event (21). All of the recombinant SODs were expressed at
levels comparable to, or greater than, the endogenous M. smegmatis SodA as judged by SDS-PAGE and SOD activity (Table
5).

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FIG. 7.
SOD expression and export in M. smegmatis wild type, M. smegmatis sodA, and
M. tuberculosis wild-type strains. M. smegmatis strains were analyzed for SOD expression by SDS-PAGE (A)
and SOD activity gel (B and C). M. tuberculosis strains
were also analyzed for SOD expression by SDS-PAGE (D) and SOD activity
gel (E). Tenfold more culture filtrate (CF) than lysate (L), based on
the original culture volume, was loaded on the gels for each analysis
(4 ml of CF and 0.4 ml of L [A and D], 6 ml of CF and 0.6 ml of L [B
and C], and 2 ml of CF and 0.2 ml of L [E]). The results are
representative of two or three independent cultures. Arrows indicate
the positions of the M. smegmatis (Ms) SodA,
M. tuberculosis (Mtb) SodA, M. smegmatis-M. tuberculosis hybrid SODs (Ms-Mtb),
E. coli (Ec) SodA, E. coli SodB,
B. subtilis (Bs) SodA, and the M. smegmatis
SOD activity in the sodA mutant (Ms SodX). M,
molecular mass markers in kilodaltons.
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|
All SODs, recombinant and endogenous, were present in the culture
filtrates of M. smegmatis strains at relatively low
percentages (3 to 15%) of the total expressed. M. tuberculosis and M. smegmatis SodA subunits are
known to mix, forming hybrid enzymes (29, 76). Also, the
E. coli SodA and SodB subunits form a hybrid SOD and
the B. subtilis SodA subunits are capable of forming hybrid SODs with both the E. coli SodA and SodB subunits
(11, 35). However, no mixing of the E. coli and B. subtilis SOD subunits with M. smegmatis SodA subunits was detected (Fig. 7C).
The recombinant M. tuberculosis strain expressing
the M. smegmatis SodA (Mtb
pNBV1-MsSODA) produced the recombinant enzyme at levels even
greater than its own SodA (Fig. 7D and E and Table 5). The
M. smegmatis SodA subunits formed hybrids with the
endogenous M. tuberculosis SodA subunits as observed
previously for M. smegmatis expressing the recombinant
M. tuberculosis SodA (29, 76). The
recombinant M. tuberculosis strain expressing the
E. coli SodB (Mtb pNBV1-EcSODB)
produced the recombinant SOD at levels comparable to its endogenous
SodA. Mixing of the endogenous M. tuberculosis SodA
subunits with the E. coli SodB subunits was not
detected (Fig. 7E), as was previously observed for the M. smegmatis SodA and E. coli SodB subunits in an
M. smegmatis host (Fig. 7C).
All three SODs (M. tuberculosis SodA, M. smegmatis SodA, and E. coli SodB) were present in
the culture filtrate of M. tuberculosis at similar
proportions (14 to 23%) of the total expressed.
The results presented thus far showed that while the absolute amounts
of GS and SOD exported were high relative to other culture filtrate
proteins, the proportion exported represented only a minority of the
total expressed. Moreover, there was no significant difference in
the proportion of GS and SOD exported depending upon whether they
were native to pathogenic mycobacteria, nonpathogenic mycobacteria, or nonmycobacteria. This suggested that
export was largely expression dependent rather than protein specific.
To explore this possibility further, we prepared recombinant
strains of M. smegmatis and M. tuberculosis expressing large amounts of three other leaderless
proteins, all typically intracellular, and studied their expression and export.
The presence of large amounts of GS, SOD, and other leaderless
proteins in the culture filtrate of mycobacteria reflects their high
expression and extracellular stability. (i) Expression of MDH in
M. smegmatis.
The cytoplasmic enzyme lactate
dehydrogenase is frequently assayed as a marker for lysis of bacterial
and eukaryotic cells (41). However, we chose MDH, a Krebs
cycle enzyme that is also expected to be strictly intracellular, as a
marker for autolysis in M. smegmatis primarily because
M. smegmatis does not express an MDH activity
(54) while M. tuberculosis expresses a
high level of activity. This suggested that overexpression of the
M. tuberculosis MDH in M. smegmatis
would be successful and analysis would not be complicated by the
presence of endogenous enzyme. MDH forms either dimers or tetramers of
identical 30- to 35-kDa subunits and has a three-dimensional structure
similar to LDH (45). The M. tuberculosis
mdh gene was cloned downstream of the M. tuberculosis
glnA1 promoter in pNBV1 (Fig. 5A), and a plasmid was constructed
containing the M. smegmatis glnA1, M. smegmatis sodA, and mdh as separate transcriptional units (Fig.
5D) in order to follow MDH activity in recombinant bacteria
overexpressing GS and SOD. This plasmid was transformed into the
M. smegmatis glnA1 mutant, and expression and export of
GS, SOD, and MDH were analyzed by SDS-PAGE and enzyme activity as a
function of growth (Fig. 8). The strain
expressed large amounts of all three enzymes. The percentage of GS and
SOD activity in the culture filtrate increased during growth and
reached a maximum of 15 and 25% after the cells were well into
stationary phase. During the earlier stages of growth (39 to 88 h), the percentages (means ± standard deviations) of
extracellular GS protein (5.6% ± 1.4%; n = 5) and GS
activity (6.2% ± 1.9%; n = 5) were similar to
previous results (Fig. 6 and Table 4). The percentages of extracellular
SOD protein (8.5% ± 0.6%; n = 5) and SOD activity
(13.6% ± 0.6%; n = 5) were also similar to previous
results (Fig. 7 and Table 5). In contrast, although there was a readily
detectable quantity of MDH in the culture filtrate at all time points
(10 to 70 mU per ml of original culture volume), this extracellular MDH
activity represented less than 0.5% of the total MDH activity. The
SDS-PAGE results were consistent, showing no accumulation of MDH in the
culture filtrate.

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FIG. 8.
Expression and export of GS, SOD, and MDH by
M. smegmatis glnA1
pNBV1-MsGS-MsSODA-MDH. (A) Growth of
M. smegmatis glnA1
pNBV1-MsGS-MsSODA-MDH. (B, C, and D) Enzyme
activity of GS, SOD, and MDH in the culture filtrate ( ) and lysate
( ) over time. (E) SDS-PAGE analysis of culture filtrates (CF) and
lysates (L). Tenfold more CF than L, based on the original culture
volume, was loaded on the gels (at 39 and 47 h, 4 ml of CF and 0.4 ml of L; at 63 to 189 h, 2 ml of CF and 0.2 ml of L). The
positions of the M. smegmatis GS, M. smegmatis SOD, and the M. tuberculosis MDH are
indicated. M, molecular mass markers in kilodaltons.
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However, after 3 weeks in stationary phase, by which time the
enzyme would have been expected to have been released extracellularly by autolysis, only 0.2% of the total MDH activity was present in the
culture filtrate, while intracellular MDH activity was at 96% of its
maximum value (data not shown). This suggested that MDH was not stable
in the extracellular medium. Therefore, we examined the stability of
the recombinant M. tuberculosis MDH in culture
filtrate. We partially purified MDH from a cell lysate of M. smegmatis glnA1 pNBV1-MsGS-MsSODA-MDH, added
the purified MDH to the sterile culture filtrate from the same strain,
and incubated the mixture under the same conditions as a growing
culture. Aliquots were removed at various times, analyzed by SDS-PAGE, and assayed for MDH and GS activity (Fig.
9). MDH was clearly unstable in 7H9
culture filtrate, while the amounts of other proteins such as GS and
SOD remained constant over the course of 1 week at 28°C. Further
experiments showed that MDH had a half-life of 2.3 to 3 h in
culture filtrate or fresh 7H9 medium at 28°C, and the addition of a
combination of protease inhibitors (2 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, and 1 µM pepstatin A) or bovine serum
albumin (10 mg ml
1) did little to improve stability (data
not shown).

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FIG. 9.
Stability of MDH in 7H9 culture filtrate. (A) SDS-PAGE
analysis of purified MDH (lane 1) and its stability after addition to
culture filtrate. An amount equivalent to 2 ml of the original culture
volume was loaded for each culture filtrate time point. The positions
of the M. smegmatis GS, M. smegmatis
SOD, and the M. tuberculosis MDH are indicated. The
purified MDH was judged to be 85% pure by analysis with NIH Image
software (version 1.62). (B) The percentage of initial activity of MDH
and GS is listed beneath each lane, with the activity at day zero
defined to be 100%. Abbreviations: ND, not determined; M, molecular
mass markers in kilodaltons.
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(ii) Expression of bacterioferritin in M. smegmatis.
Because of the significant and unexpected
instability of MDH in culture filtrates, we sought a more stable
protein for use as an intracellular marker for M. smegmatis. Ferritins and bacterioferritins are large
(~450-kDa), highly stable, multimeric, iron storage proteins composed
of 24 subunits of ~19 kDa (6). M. tuberculosis has two bacterioferritin genes (bfrA and
bfrB) that do not encode leader peptides. One of the
bacterioferritin genes, bfrB, was cloned downstream of the
M. tuberculosis glnA1 promoter (Fig. 5B), and a plasmid
was constructed containing the M. smegmatis glnA1, M. smegmatis sodA, and M. tuberculosis bfrB as
separate transcriptional units (Fig. 5E), as was done for
mdh. This plasmid was transformed into the glnA1
mutant, and expression and export of GS, SOD, and BfrB were analyzed by
SDS-PAGE as a function of growth (Fig.
10). In contrast to MDH, BfrB was
detected in the culture filtrate at levels comparable to GS and SOD at
all time points analyzed, even in early log phase. Furthermore, BfrB in the culture filtrate likely exists as a functional 24-subunit multimer
based on analysis of culture filtrate proteins in which BfrB largely
coeluted with GS (molecular mass, ~640 kDa) on a Sephacryl-300HR size exclusion column (data not shown). Thus, in
addition to GS, another very large and typically intracellular protein
was released by M. smegmatis cultures into the growth medium.

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FIG. 10.
Expression and export of GS, SOD, and BfrB by
M. smegmatis glnA1
pNBV1-MsSODA-MsGS-BFRB. (A) Growth of
M. smegmatis glnA1
pNBV1-MsGS-MsSODA-BFRB. (B) SDS-PAGE analysis of
culture filtrates (CF) and lysates (L). Tenfold more CF than L, based
on the original culture volume, was loaded on the gels (at 19 and
39 h, 8 ml of CF and 0.8 ml of L; at 47 h, 4 ml of CF and 0.4 ml of L; at 67 and 87 h, 2 ml of CF and 0.2 ml of L). The
percentages of extracellular GS, SOD, and BfrB (listed below the gel)
were determined with NIH Image software (version 1.62). Arrows indicate
the positions of the M. smegmatis GS, M. smegmatis SOD, and the M. tuberculosis BfrB
(BfrB). M, molecular mass markers in kilodaltons.
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(iii) Expression of GFP in M. tuberculosis.
We chose the nonbacterial GFP as an intracellular marker for
M. tuberculosis because we anticipated that (i) it
would be expected to remain strictly intracellular since it lacks a
leader peptide, (ii) it would not be exported by M. tuberculosis based on similarities to endogenous proteins since it
is not a bacterial protein, and (iii) it would persist in the culture
medium if released since it is highly stable (14).
M. tuberculosis was transformed with a plasmid
containing the gene for GFPuv downstream of the M. bovis BCG hsp60 promoter (Fig. 5C), a strong
mycobacterial promoter that has been successfully used to express GFP
in mycobacteria (20, 37). High level expression of GFPuv
was obtained, and the cells were bright green when examined under
long-wavelength UV light. Expression and export of the recombinant
GFPuv along with the endogenous M. tuberculosis GlnA1
and M. tuberculosis SodA was analyzed by SDS-PAGE and
immunoblotting as a function of growth (Fig.
11). All three proteins were clearly
present in the M. tuberculosis culture filtrate during
all stages of growth and at similar percentages (GS, 8 to 14%; GFPuv,
11 to 18%; SOD, 8 to 15%). Expression of GFPuv had no noticeable
toxicity on the cells, and the export of SodA by the GFPuv-expressing
strain was consistent with previous results obtained with three
different M. tuberculosis strains not expressing GFPuv
(Fig. 7 and Table 5) (SodA protein, 21.0% ± 1.7%; SodA activity,
14.7% ± 0.6%).

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FIG. 11.
Expression and export of GS, SOD, and GFPuv by
M. tuberculosis pNBV1-GFPuv. (A) Growth of
M. tuberculosis pNBV1-GFPuv. (B) SDS-PAGE
analysis of culture filtrates (CF) and lysates (L). Tenfold more CF
than L, based on the original culture volume, was loaded on the gels
(at 10 days; 15 ml of CF and 1.5 ml of L; at 15 days, 6 ml of CF and
0.6 ml of L; at 22 to 38 days, 2 ml of CF and 0.2 ml of L). (C to E)
Immunoblot analysis of GS, GFPuv, and SOD. (F) Autoradiogram of CF and
L from M. tuberculosis pNBV1-GFPuv metabolically
labeled with [35S]L-methionine and
[35S]L-cysteine. The bacteria were grown for
4 days, radiolabel was added, and aliquots were removed for analysis 7, 23, 48, and 72 h later. The culture was actively growing over the
72-h period, increasing in turbidity (A550) from
0.18 to 0.28. Twentyfold more CF than L was loaded on the gel (the
equivalent of 2 ml versus 0.1 ml of original culture volume). Arrows
indicate the positions of the M. tuberculosis GS,
M. tuberculosis SOD, and GFPuv. M, molecular mass
markers in kilodaltons.
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Because all three proteins appeared in the culture filtrate even during
the early stages of growth, the kinetics of export were investigated by
metabolic labeling of M. tuberculosis pNBV1-GFPuv with
[35S]L-methionine and
[35S]L-cysteine. Export of GFPuv and the
endogenous M. tuberculosis GlnA1 was analyzed over a
3-day period (Fig. 11F). By 7 h, the cell lysate proteins were
fairly heavily labeled, whereas the culture filtrate was nearly devoid
of labeled proteins with the exception of the 30/32-kDa complex
(antigen 85 complex) and 23.5-kDa (MPT64) major secretory antigens.
These antigens all contain N-terminal signal peptides and have high
localization indexes (70). Radiolabeled GFPuv and GlnA1 in
the 7-h culture filtrate were close to, or below, detection limits and
accounted for
0.5% of the total metabolically labeled GFPuv and
GlnA1. By 72 h, only 5% of GFPuv and 3% of GlnA1 were present in
the culture filtrate. The 30/32-kDa and 23.5-kDa antigens were very
heavily labeled at this time, and many other proteins were evident in
the culture filtrate. SodA could not be analyzed by this technique
because it is poorly labeled, possessing only two methionine residues
(one of which is cleaved off [29]) and no cysteine
residues. GlnA1 and GFPuv have 13 and 7 total methionine and cysteine
residues, respectively.
These experiments on expression and export of MDH and bacterioferritin
by M. smegmatis and GFPuv by M. tuberculosis demonstrate that proteins accumulate extracellularly
in large amounts if they are abundantly expressed and resistant to
degradation in the extracellular milieu. Also, the metabolic labeling
showed that the export is very likely not an active process because of
the very slow release of GS and GFPuv compared to actively secreted
mycobacterial proteins.
The role of carbon and nitrogen on release of proteins from
M. smegmatis and evidence that autolysis plays a role
in the accumulation of extracellular proteins.
To investigate the
potential role of carbon and nitrogen in protein export, we cultured
M. smegmatis glnA1
pNBV1-MsSODA-MsGS-BFRB for 4 days in 7H9 medium
in the presence of various amounts of added glucose and
(NH4)2SO4. The addition of
both (NH4)2SO4 and glucose to
7H9 medium resulted in a dramatic decrease in extracellular proteins
(Fig. 12A). In fact,
rather modest absolute amounts of the two substances [12.4 mM
(NH4)2SO4 and 0.25% (wt/vol) Glc] were capable of preventing the accumulation of extracellular proteins (Fig. 12A, culture 2). Growth in 7H9 medium with additional
(NH4)2SO4 but without glucose did
not affect the profile of extracellular proteins (Fig. 12B, culture 6).
However, growth in 7H9 medium with 1% (wt/vol) glucose and no
additional (NH4)2SO4 resulted in a much greater level of extracellular proteins than growth in standard 7H9 (Fig. 12C, culture 10). The increase in extracellular proteins was
most likely due to autolysis, because cultures analyzed after 2 days rather than after 4 days of growth showed no difference in
culture filtrate proteins compared with growth in standard 7H9 medium
(data not shown). Glycerol (1%, vol/vol) could substitute for glucose
(1% [wt/vol]), and NH4Cl could substitute for
(NH4)2SO4. MgCl2,
MgSO4, and NaCl could not substitute for a source of
NH4+ (data not shown).

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FIG. 12.
Expression and export of GS, SOD, and BfrB by
M. smegmatis glnA1
pNBV1-MsSODA-MsGS-BFRB under various growth
conditions. (A to C) M. smegmatis glnA1
pNBV1-MsGS-MsSODA-BFRB was grown for 4 days
(cultures reached stationary phase by 2 to 3 days) in 7H9 medium
containing 0.2% (vol/vol) glycerol with various amounts of glucose
and/or additional (NH4)2SO4, and
culture filtrates (CF) and lysates (L) were analyzed by SDS-PAGE.
Tenfold more CF than L was loaded on the gels (the equivalent of 2 ml versus
0.2 ml of original culture volume). Arrows indicate the positions of
the M. smegmatis GS, M. smegmatis SOD,
and the M. tuberculosis BfrB (BfrB). Two to four
independent cultures were grown and analyzed for each of the four
extreme culture conditions (1, 5, 6, and 10) as well as for
culture condition 2, and the results shown are representative. (D)
Graph indicating the glucose and
(NH4)2SO4 concentrations of the
media that were tested in panels A to C. The standard 7H9 medium
(culture 1) contains 82 mM carbon (as 0.2% [vol/vol] glycerol) and
11 mM nitrogen [as 3.8 mM
(NH4)2SO4 and 3.4 mM
L-glutamate]. The
(NH4)2SO4 (AmS) concentration is
stated as the total amount present in the medium for each culture
condition. M, molecular mass markers in kilodaltons.
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After prolonged incubation of the M. smegmatis strain
in stationary phase (10 days) in 7H9 medium containing 12.4 mM
(NH4)2SO4 and 0.25% (wt/vol)
glucose, extracellular proteins again accumulated, resulting in a
culture filtrate protein profile similar to growth for 4 days in
standard 7H9 medium. This suggested that the added nutrients were
eventually consumed after which a portion of the culture lysed (data
not shown). However, after prolonged incubation of the
M. smegmatis strain in stationary phase (10 days)
in 7H9 containing 38 mM (NH4)2SO4
and 1% (wt/vol) glucose, almost no extracellular proteins were
present, presumably because an excess of carbon and nitrogen was
maintained. It was also observed that high-molecular-weight DNA (
12
kb) was present in culture filtrates and was highly correlated with the
presence of extracellular proteins (i.e., DNA was readily detected in
culture filtrates when proteins were present but not when proteins were
absent [data not shown]), providing further evidence that autolysis
was occurring and that increased levels of glucose and nitrogen could
prevent it.
When M. smegmatis glnA1
pNBV1-MsSODA-MsGS-BFRB was grown under the same
conditions as M. tuberculosis (37°C unshaken),
additional glucose and NH4+ still prevented the
release of GS, SOD, and BfrB into the growth medium (data not shown).
However, when M. tuberculosis was grown under the four
extreme culture conditions (cultures 1, 5, 6, and 10 in Fig. 12D) the
extracellular protein profiles of the cultures were very similar; i.e.,
additional carbon and nitrogen in the medium did not prevent the
release of proteins as it did for M. smegmatis. DNA was
readily detected in culture filtrates under both standard conditions
and with additional glucose and NH4+ in the
medium (data not shown), suggesting that autolysis was also occurring
in M. tuberculosis cultures but that it could not be
prevented by the addition of glucose and NH4+.
 |
DISCUSSION |
The culture filtrate proteins of M. tuberculosis
have been the subject of numerous studies (see, for example, references
4, 5, 7, 31, 32, 46, and 50). One often-discussed topic is
to what degree autolysis contributes to the presence of antigens in the
culture filtrate (for a recent discussion, see reference 69). Culture filtrate proteins have largely been defined
as secreted based on their presence in early culture filtrates and/or the lack of an intracellular marker (enzyme activity or antigen) in the
culture filtrate, but only a few studies have determined a localization
index and/or an extracellular percentage for selected culture filtrate
proteins (2, 5, 28-30, 46, 55, 62, 70, 71). Occasionally,
when Hsp65 (GroEL, antigen 82) was not present in the culture filtrate,
it was assumed that minimal autolysis had occurred (2, 62,
76). However, Wiker et al. noted that this antigen is not very
stable (70). In the present study, under conditions in
which substantial amounts of recombinant GFPuv were released by
M. tuberculosis, Hsp65 was virtually undetectable in
culture filtrates despite a high intracellular level (Fig. 11). This
suggests that Hsp65 should not be used as an intracellular marker to
measure autolysis. There is consensus, however, that most of the major
extracellular proteins of M. tuberculosis are encoded
by genes containing leader peptide sequences, and they have been shown
to be greatly enriched in the culture filtrate (70, 71).
With respect to export of leaderless proteins by M. tuberculosis, we previously showed that M. tuberculosis and other pathogenic mycobacteria export abundant
amounts of GS, whereas M. smegmatis and
Mycobacterium phlei export small amounts (28).
Moreover, recombinant M. tuberculosis GS was abundantly
exported by M. smegmatis while very little of the
endogenous M. smegmatis GS was released (30). Others have also identified M. tuberculosis GS in culture filtrates (55, 56, 62,
69). In this study, we found a somewhat lower percentage of
total GS (8 to 14%) in M. tuberculosis culture
filtrates compared with our previous work (33%) and that of Raynaud et
al. (17%) (55). We also found, in contrast to our earlier
work, that abundantly expressed and active M. tuberculosis GS is not released in large proportions by
M. smegmatis. In fact, the M. smegmatis
wild-type strain as well as glnA1 strains expressing high
levels of the M. smegmatis or M. tuberculosis GS, all exported a similar, relatively low,
percentage of total GS (3 to 7%). The large, quantitative