INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

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protein with a molecular mass of 10 kDa) and have been designated as the major extracellular proteins (reference 32 and unpublished data).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.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

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 ml¹) and/or kanamycin (20 µg ml¹) 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.

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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 ml¹. 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 µg¹ 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.

Construction of M. smegmatis glnA1 and sodA mutants. A promoterless, nonpolar kanamycin resistance (Km^r) 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.

View larger version (16K): [in this window] [in a new window]   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.

View larger version (27K): [in this window] [in a new window]   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 BamHIEcoRI (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.

View larger version (10K): [in this window] [in a new window]   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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View larger version (41K): [in this window] [in a new window]   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.

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 BamHIHindIII fragment (5' to 3') into the multiple cloning site of pNBV1. The M. tuberculosis sodA locus was previously cloned into pNBV1 as a ClaIBamHI PCR product (5' to 3'), and its expression was studied in M. smegmatis 1-2c (29). This plasmid was used without modification.

View larger version (28K): [in this window] [in a new window]   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.

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.

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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 ml¹) 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 ml¹. 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 min¹ with PBS and MDH was collected as a single, symmetrical peak that eluted very close to the void volume.

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

Top Abstract Introduction Materials and Methods Results Discussion References

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 ml¹) 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 ml¹), 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.

View larger version (47K): [in this window] [in a new window]   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.

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.

View larger version (69K): [in this window] [in a new window]   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.

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.

View larger version (54K): [in this window] [in a new window]   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.

1

View larger version (80K): [in this window] [in a new window]   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.

(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.

View larger version (60K): [in this window] [in a new window]   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.

(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%).

View larger version (40K): [in this window] [in a new window]   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.

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).

View larger version (38K): [in this window] [in a new window]   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.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

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