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Journal of Bacteriology, July 2007, p. 5090-5100, Vol. 189, No. 14
0021-9193/07/$08.00+0     doi:10.1128/JB.00163-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Identification of Two Mycobacterium smegmatis Lipoproteins Exported by a SecA2-Dependent Pathway{triangledown} ,{dagger}

Henry S. Gibbons,1 Frank Wolschendorf,2 Michelle Abshire,1,{ddagger} Michael Niederweis,2 and Miriam Braunstein1*

Department of Microbiology and Immunology, School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7290,1 Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 352942

Received 31 January 2007/ Accepted 2 May 2007


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ABSTRACT
 
The SecA2 protein is part of a specialized protein export system of mycobacteria. We set out to identify proteins exported to the bacterial cell envelope by the mycobacterial SecA2 system. By comparing the protein profiles of cell wall and membrane fractions from wild-type and {Delta}secA2 mutant Mycobacterium smegmatis, we identified the Msmeg1712 and Msmeg1704 proteins as SecA2-dependent cell envelope proteins. These are the first endogenous M. smegmatis proteins identified as dependent on SecA2 for export. Both proteins are homologous to periplasmic sugar-binding proteins of other bacteria, and both contain functional amino-terminal signal sequences with lipobox motifs. These two proteins appeared to be genuine lipoproteins as shown by Triton X-114 fractionation and sensitivity to globomycin, an inhibitor of lipoprotein signal peptidase. The role of SecA2 in the export of these proteins was specific; not all mycobacterial lipoproteins required SecA2 for efficient localization or processing. Finally, Msmeg1704 was recognized by the SecA2 pathway of Mycobacterium tuberculosis, as indicated by the appearance of an export intermediate when the protein was expressed in a {Delta}secA2 mutant of M. tuberculosis. Taken together, these results indicate that a select subset of envelope proteins containing amino-terminal signal sequences can be substrates of the mycobacterial SecA2 pathway and that some determinants for SecA2-dependent export are conserved between M. smegmatis and M. tuberculosis.


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INTRODUCTION
 
Like all bacteria, mycobacteria possess systems to localize proteins to different subcellular compartments. After synthesis in the cytoplasm, some proteins are exported to the cytoplasmic membrane or cell wall (collectively termed the cell envelope) or to the extracellular environment. Proteins in these external locations can have important activities in nutrient acquisition, maintenance of the cell envelope, or interaction with a host (26). Mycobacteria have several known protein export pathways that localize proteins to extracytoplasmic locations, including the general secretion (Sec) pathway (8, 26, 39), the twin-arginine translocation pathway (TAT) (33, 42, 46), and the specialized ESAT-6 secretion system (ESX-1) (15, 19, 22, 43, 53).

The general Sec export pathway is a highly conserved and essential system of bacteria. In mycobacteria, the Sec pathway is most likely responsible for the majority of protein export beyond the cytoplasm. Years of study, primarily in Escherichia coli, provided advanced understanding of this pathway (for reviews, see references 12 and 34). Proteins exported by the Sec pathway are first synthesized as precursors with recognizable amino-terminal signal sequences. These precursors are translocated in an unfolded state across the cytoplasmic membrane through the concerted action of several Sec proteins. The SecA ATPase has a central role in this export pathway. It recognizes the precursor, in some cases with the assistance of chaperones, and delivers it to the SecYEG pore complex that spans the cytoplasmic membrane. Through repeated cycles of ATP hydrolysis, SecA drives export of the precursor through the SecYEG pore (14, 30). During or immediately following transport across the membrane, the signal sequence is cleaved by one of two signal peptidases to produce the mature protein.

A subset of Sec-exported proteins contains a covalently attached lipid that tethers the mature protein to the cell envelope after export. The amino-terminal signal sequence of these lipoproteins is distinguishable by the presence of a lipobox motif at the signal sequence cleavage site. A lipobox, usually defined as (LV)–3(AST)–2(GA)–1C+1 (32), includes an invariant cysteine at the +1 position of the cleaved mature protein. This cysteine is also the site of lipid modification. The lipoprotein signal sequence is cleaved by a lipoprotein-specific signal peptidase, LspA (57). In E. coli, lipid attachment to the sulfhydryl of the conserved cysteine is a prerequisite for signal sequence cleavage by LspA (58), although a recent study in Listeria monocytogenes revealed that this is not a universal requirement (5). Mycobacterium tuberculosis also possesses a functional LspA, which is important for virulence (3, 47).

Mycobacteria possess two nonredundant homologs of SecA (8, 9). One of the homologs (SecA1) is essential and presumably performs the "housekeeping" role in Sec export; the other homolog (SecA2) is not essential and promotes export of a small subset of proteins (8). A subset of gram-positive bacteria has also been identified as organisms containing two distinct SecA homologs, and similarly, one homolog (SecA2) is not essential and is required for export of a subset of proteins (6, 11, 29). SecA2 has been demonstrated to have a role in the pathogenesis of both M. tuberculosis and L. monocytogenes (9, 27, 28). Examples of proteins identified as proteins exported by various SecA2 systems include extracellular secreted proteins, as well as cell envelope proteins. Some of these SecA2-dependent exported proteins contain recognizable signal sequences, and others do not (2, 6, 9, 11, 28). In the past, we searched for secreted proteins of M. tuberculosis that depend on SecA2 for extracellular release. Comparison of the proteins secreted into culture media by wild-type and {Delta}secA2 mutant strains of M. tuberculosis identified a small number of proteins affected by SecA2 (9). Two of the M. tuberculosis proteins dependent on SecA2 for secretion are superoxide dismutase (SodA) and catalase/peroxidase (KatG), neither of which has a discernible amino-terminal signal sequence.

Here we expanded our search for mycobacterial SecA2-dependent exported proteins to the cell envelope of the fast-growing model organism Mycobacterium smegmatis. We compared subcellular fractions of wild-type and {Delta}secA2 mutant strains of M. smegmatis and identified two proteins, Msmeg1712 and Msmeg1704, that depend on SecA2 for correct localization to the cell envelope. Both of these proteins are homologs of periplasmic sugar-binding proteins with likely roles in sugar uptake. We further demonstrated that both Msmeg1712 and Msmeg1704 are lipoproteins by using, for the first time in mycobacteria, the lipoprotein signal peptidase inhibitor globomycin (13, 58). The role of SecA2 in export of Msmeg1712 and Msmeg1704 is specific, since we noted no global effect on cell envelope proteins or lipoproteins in the {Delta}secA2 mutant. The SecA2 system of M. tuberculosis also recognized Msmeg1704, as shown by a defect in precursor processing when it was expressed in a {Delta}secA2 mutant of M. tuberculosis. These results expand our understanding of the types of substrates exported by SecA2 in mycobacteria.


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MATERIALS AND METHODS
 
Bacterial strains and growth conditions. The mycobacterial strains used in this study are listed in Table 1. E. coli strain DH5{alpha} was used for DNA cloning and was grown in Luria-Bertani medium (Fisher). M. smegmatis strains were grown at 37°C in Middlebrook 7H9 medium (Difco) containing 0.2% glucose, 0.5% glycerol, and 0.1% Tween 80 or in Mueller-Hinton broth (Difco) containing 0.1% Tween 80 to the mid-log phase (optical density at 600 nm [OD600], 0.6 to 1.0) prior to harvesting by centrifugation. M. tuberculosis strains were grown at 37°C in 7H9 medium containing albumin-dextrose-saline enrichment (final concentrations, 5 g/liter bovine serum albumin [fraction V], 2 g/liter dextrose, and 50 mM NaCl), 0.05% Tween 80, plus 0.5% glycerol. When necessary, kanamycin (20 µg/ml for M. smegmatis and M. tuberculosis or 40 µg/ml for E. coli) or hygromycin (50 µg/ml for M. smegmatis or 150 µg/ml for E. coli) was used. Strains expressing hemagglutinin (HA)-tagged Msmeg1712 (Msmeg1712-HA) and Msmeg1704-HA were grown at 30°C.


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  		TABLE 1. Strains used in this study

Reagents. Triton X-100, Triton X-114, and urea (electrophoresis grade) were obtained from Sigma. Triton X-100 was kept as a 10% stock solution in water and mixed with Amberlite MB-150 mixed-bed ion-exchange resin prior to use in two-dimensional (2D) gels. Carrier ampholytes were obtained from Bio-Rad (BioLytes 3-10, 6-8, and 5-7 range). Globomycin was a generous gift from Masatoshi Inukai (Sankyo Corporation and International Union of Health and Welfare, Japan).

Subcellular fractionation. Crude cell wall, membrane, and soluble fractions were prepared by differential ultracentrifugation as described in previously published work (18, 45). Briefly, mycobacterial cultures (250 ml) were harvested by centrifugation at 3,000 x g. Cell pellets (approximately 0.5 g [wet weight]) were washed with phosphate-buffered saline (PBS) and stored at –80°C. For M. tuberculosis, cell pellets were sterilized by gamma-irradiation in a JL Shephard Mark I 137Cs irradiator (Department of Radiobiology, University of North Carolina at Chapel Hill) with a dose of 2.4 megarads. All subsequent steps were performed at 4°C. Pellets were resuspended in 4 ml of breaking buffer (PBS, 1 mM phenylmethylsulfonyl fluoride, 0.6 µg/ml each of DNase and RNase, and a cocktail of protease inhibitors [2 µg/ml each of aprotinin, E-64, leupeptin, and pepstatin A and 100 µg/ml Pefabloc SC]). Cells were lysed by passage through a French pressure cell (five times at 20,000 lb/in2). Unbroken cells were pelleted at 3,000 x g for 20 min to generate a clarified whole-cell lysate, which was centrifuged at 27,000 x g for 30 min to pellet the cell wall. The supernatant was centrifuged at 100,000 x g for 2 h to separate the membrane fraction from the soluble fraction. The cell wall and membrane fractions were washed once in PBS. Protein concentrations were estimated using the Bio-Rad protein assay.

2D-PAGE analysis. Polyacrylamide tube gels (38) in a Bio-Rad minitube cell were used to perform 2D polyacrylamide gel electrophoresis (2D-PAGE) of membrane and cell wall fractions. Tube gels (4% polyacrylamide, 8 M urea, 2% Triton X-100, 0.4% [wt/vol] BioLytes 3-10, 0.8% [wt/vol] each BioLytes 5-7 and 6-8) were polymerized overnight. Fifty to 100 µg of membrane or cell wall protein was resuspended in a 2D-PAGE buffer (8 M urea, 2% Triton X-100, 1% [wt/vol] dithiothreitol, 1% BioLytes 3-10) and loaded into the sample cup of the tube gel apparatus. Proteins were focused in the first dimension for 6 h at 500 V. Tube gels were equilibrated in 4x sodium dodecyl sulfate (SDS)-PAGE loading buffer containing 20 mM dithiothreitol and placed on top of conventional 15% SDS-PAGE minigels with 4% stacking gels. Gels were stained with Coomassie brilliant blue R-250.

Triton X-114 partitioning. The methods used for detergent partitioning experiments were adapted from the methods of Young and Garbe (61). French-pressed whole-cell lysate was diluted to 2 mg/ml in 1 ml of PBS containing 4% (vol/vol) Triton X-114 and a protease inhibitor cocktail (see above). Lipoproteins were extracted for 2 h at 4°C on a rocking platform. Insoluble material was removed by centrifugation at 12,000 x g for 10 min. The insoluble pellet was reextracted with cold PBS-4% Triton X-114. Pooled supernatants were brought to 37°C for 5 min to allow for phase separation and then centrifuged at 12,000 x g for 5 min to partition the phases. The detergent phase was washed twice with PBS and precipitated with 9 volumes of acetone at –20°C overnight. Acetone-precipitated proteins were pelleted at 12,000 x g and washed twice with cold acetone. Proteins were resuspended in SDS-PAGE sample buffer and separated by one-dimensional (1D) SDS-PAGE (1D-PAGE).

Mass spectrometric identification of proteins. Protein spots were cut from a gel, minced, and subjected to in-gel digestion with trypsin using an Investigator Progest (Genomic Solutions, Ann Arbor, MI) robotic digester. Following elution from the gel, the tryptic peptides were lyophilized and resuspended in 50% methanol-0.1% formic acid and spotted (1:1, vol/vol) with a saturated solution of {alpha}-cyano-4-hydroxycinnamic acid in 50% acetonitrile-0.1% trifluoroacetic acid. Spots were analyzed by matrix-assisted laser desorption ionization mass spectrometry and matrix-assisted laser desorption ionization mass spectrometry/mass spectrometry with an ABI 4700 proteomics analyzer (Framingham, MA) in the positive ion mode. Spectra were searched against a database of the M. smegmatis predicted open reading frames (www.tigr.org) using GPS software and the MASCOT (40) search engine. All analyses were performed at the Michael Hooker Proteomics Core Facility at the University of North Carolina at Chapel Hill.

Plasmid construction. Plasmids used in this study are listed in Table 2. Oligonucleotide primers used to amplify DNA sequences for subcloning are listed in Table 3. All plasmid inserts were sequenced and shown to be error free. Plasmids were electroporated into M. smegmatis or M. tuberculosis as previously described (7).


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  		TABLE 2. Plasmids used in this study


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  		TABLE 3. Oligonucleotides

(i) Construction of an integrating M. smegmatis secA2 complementation vector. The secA2 gene of M. smegmatis was cloned from pMB208 (8) as a BamHI fragment into similarly digested pMV306.kan to generate pYA810.

(ii) Construction of an Msmeg1712-HA expression vector. A DNA fragment containing the msmeg1712 gene, the upstream 399 bp containing the promoter, and a C-terminal HA tag was amplified from M. smegmatis genomic DNA using an Expand high-fidelity polymerase kit (Roche) with primers Msmeg1712-3 and Msmeg1712-Prom-2. Primer Msmeg1712-3 includes the sequence encoding the HA tag. The fragment was cloned into pCR2.1-TOPO using Topo-TA cloning (Invitrogen) to make pHSG83 and then subcloned as a NotI-HindIII fragment into similarly digested pMV306.kan to make pHSG84. M. smegmatis strains containing pHSG84 were grown in Mueller-Hinton broth to induce expression.

(iii) Construction of an Msmeg1704-HA expression vector. The msmeg1704 gene was cloned by PCR from M. smegmatis genomic DNA using primers Msmeg1704-1 and Msmeg1704-2. The PCR product was cloned into pCR2.1-TOPO to make pHSG56. The gene was then subcloned as a SmaI-EcoRI fragment between the EcoRI and MscI sites of pJSC77 (17) to make pHSG58.

(iv) Construction of a ({Delta}ss)-'Msmeg1712 expression vector. A fragment of the msmeg1712 gene lacking the codons for the predicted signal sequence was PCR amplified with a C-terminal HA epitope tag from genomic DNA using primers Msmeg1712-3 and Msmeg1712-5 and cloned into pCR2.1-TOPO to generate pHSG47. The fragment containing the ({Delta}ss)-'msmeg1712-HA gene was subcloned as a StuI-EcoRI fragment into pMV261.kan cut with MscI and EcoRI to make pHSG54.

(v) Construction of a 19-kDa lipoprotein (LpqH) expression vector. An overlap extension PCR strategy was used to construct a 19-kDa lipoprotein (LpqH) expression vector. A PCR product containing 521 bp of sequence derived from pMV261.kan, including the mycobacterial hsp60 promoter, was amplified using primers Hsp60-Fwd and LpqH-261-1; the latter primer contains 20 bp of sequence derived from the 5' end of the lpqH gene. A second PCR product containing the full-length lpqH gene was amplified from M. tuberculosis genomic DNA using primers LpqH-Fwd and LpqH-Rev. The PCR products were purified with QiaQuick PCR purification (QIAGEN) and then annealed by means of the 20 bp of overlapping sequence and amplified using primers Hsp60-Fwd and LpqH-Rev. The resulting product was cloned into pCR2.1-TOPO to make pHSG65 and then subcloned as a NotI-EcoRI fragment into similarly digested pMV261.kan to make pHSG67.

(vi) Construction of an msmeg1712 promoter fusion to 'lacZ. The 399-bp intergenic region between the msmeg1712 and msmeg1713 genes, along with the first 10 codons of msmeg1712, was amplified by PCR using primers Msmeg1712-Prom-1 and Msmeg1712-Prom-2, cloned into pCR2.1-TOPO to make pHSG61, and subcloned as a BamHI fragment into pYUB76 (4). The resulting vector (pHSG62) has the predicted promoter region and first 10 codons of the msmeg1712 gene fused in frame to the promoterless lacZ reporter gene.

ß-Galactosidase assays. Cultures were grown to saturation and then diluted back and grown to the mid-log phase, and ß-galactosidase activity was measured using o-nitrophenyl-ß-D-galactoside as described by Alland et al. (1).

Immunoblots. Subcellular fractions were boiled in SDS-PAGE sample buffer, separated by 1D-PAGE, and transferred to nitrocellulose (Schleicher & Schuell). The following antibodies were used in immunoblots: anti-HA (Covance) used at a 1:10,000 dilution, anti-PhoA (Research Diagnostics International) used at a 1:20,000 dilution, anti-19-kDa (provided by Douglas Young) used at a 1:20,000 dilution, IT-23/TB71 antibody (obtained from John Belisle through the TB Vaccine Testing and Research Materials Contract) used at a 1:100 dilution to detect PhoS1 (38-kDa protein), and HAT5/IT-64 antibody (obtained from the World Health Organization collection) used at a 1:100 dilution to detect GroEL2. For MspA immunoblots, fractions were boiled in 80% dimethyl sulfoxide prior to SDS-PAGE, and MspA monomers were detected using anti-MspA antiserum at a 1:2,000 dilution (36).

Globomycin treatment of M. smegmatis. Cultures (5 ml) of M. smegmatis were grown to saturation and then diluted to an OD600 of 0.15 and grown for 8 h in the presence of 50 µg/ml globomycin. Whole-cell lysates were prepared by bead beating and separated by SDS-PAGE. Proteins were visualized by immunoblotting. For fractionation experiments with globomycin-treated cells, strains expressing Msmeg1712-HA were grown in 50-ml cultures that were grown overnight to an OD600 of 0.6 to 0.8 in the presence of 20 µg/ml globomycin. Cells were washed and resuspended in 2 ml breaking buffer prior to lysis in a French pressure cell.


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RESULTS
 
Identification of SecA2-dependent proteins in the cell envelope of M. smegmatis. To generate cell wall, cytoplasmic membrane, and soluble fractions of M. smegmatis, we followed the protocol of Roberts et al. that was previously used for fractionation of M. tuberculosis lysed in a French pressure cell (18, 45). In 1D-PAGE analyses (Fig. 1), the protein profile of each fraction was unique, with the soluble fractions resembling the whole-cell lysate. In this study we analyzed the subcellular fractions from three M. smegmatis strains: wild type strain SG025, the {Delta}secA2 mutant SG026, and complemented mutant strain MYA810.


Figure 1
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  		FIG. 1. 1D-PAGE reveals SecA2-dependent differences in protein profiles. M. smegmatis wild-type strain SG025 (WT), {Delta}secA2 mutant SG026 ({Delta}), and complemented {Delta}secA2 mutant MYA810 ({Delta}c) were grown in Mueller-Hinton medium, disrupted in a French press, and fractionated by differential ultracentrifugation. (A) Proteins from whole-cell lysate (WCL), cell wall (CW), membrane (M), and soluble (Sol) fractions were solubilized in SDS-PAGE sample buffer and analyzed on a 10 to 20% gradient 1D-PAGE gel. The arrows indicate the positions of SecA2-dependent protein bands; 120 µg protein was loaded in each lane. (B) Expanded view of the gel region containing SecA2-dependent proteins (arrows). A representative gel from three independent experiments is shown.

Evaluation by 1D-PAGE of fractions prepared from strains grown in rich Mueller-Hinton medium revealed two proteins in both the cell wall and membrane fractions of wild-type M. smegmatis that were diminished or absent in the corresponding fractions of the {Delta}secA2 mutant (Fig. 1). Complementation of the {Delta}secA2 mutation with an integrated plasmid expressing M. smegmatis secA2 restored these proteins to the cell envelope fractions, which demonstrated that the {Delta}secA2 mutation was responsible for their absence in these fractions.

To improve resolution of the SecA2-dependent bands, fractions were separated by 2D-PAGE. Comparison of the 2D profiles of the M. smegmatis cell wall and membrane fractions from the wild-type, {Delta}secA2 mutant, and complemented mutant strains (Fig. 2) revealed two spots in both fractions that were SecA2 dependent. The observed molecular masses (~40 and ~45 kDa for spots 1 and 2, respectively) corresponded to the SecA2-dependent proteins observed in the 1D analysis. A peptide mass fingerprint was generated using matrix-assisted laser desorption ionization-time of flight mass spectrometry. The MASCOT algorithm (40) was used to identify the corresponding coding sequence in the M. smegmatis genome, which was obtained from the Comprehensive Microbial Resource at www.tigr.org (41). Spots 1 and 2 were identified as Msmeg1712 (GenBank accession no. ABK75924) and Msmeg1704 (GenBank accession no. ABK76012) with sequence coverage of 73 and 67%, respectively. Using the LIPOP algorithm (23), both Msmeg1712 and Msmeg1704 were predicted to have amino-terminal signal sequences with a lipobox motif (see Fig. S1A in the supplemental material), which suggested that they are exported lipoproteins subject to signal sequence cleavage during export.


Figure 2
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  		FIG. 2. 2D-PAGE analysis of cell wall and membrane fractions reveals SecA2-dependent proteins. Proteins (50 µg) from the cell wall and membrane fractions derived from the M. smegmatis wild type (WT), {Delta}secA2 mutant ({Delta}), and complemented {Delta}secA2 mutant ({Delta}c) were resolved by 2D-PAGE. The relevant region of the protein profile for each strain is expanded in the boxes on the right. Protein spots that were reproducibly diminished in the {Delta}secA2 mutant and restored in the complemented strain are indicated by arrows. The small boxes mark the expected positions of the SecA2-dependent spots in the {Delta}secA2 mutant. Spots 1 and 2 were identified as Msmeg1712 and Msmeg1704, respectively. Representative gels from one of three independent experiments are shown.

Similarity between Msmeg1712 and Msmeg1704 and predicted function in sugar uptake. Msmeg1712 and Msmeg1704 exhibit 23% identity and 40% similarity to each other on the protein level. Furthermore, both proteins have strong homology to periplasmic monosaccharide-binding proteins of bacteria. Msmeg1712 is 63% similar to YtfQ and 49% similar to RbsB of E. coli, while Msmeg1704 is 80% similar to the ChvE protein of Agrobacterium tumefaciens. YtfQ, RbsB, and ChvE have all been shown to either bind or sense monosaccharides (Richard Horler and Gavin Thomas, personal communication) (10, 31). An additional similarity is the genomic location of msmeg1712 and msmeg1704. They are located in adjacent, convergently transcribed predicted operons along with genes encoding putative transmembrane permeases and ATP-binding proteins. The observed homology to sugar-binding proteins and the genomic organization with ABC transporters strongly suggest that this genomic region functions in monosaccharide binding and uptake (see Fig. S1B in the supplemental material).

Regulation of Msmeg1712 and Msmeg1704 by glucose and glycerol. Unlike our findings with cells grown in Mueller-Hinton broth (Fig. 1 and 2), no obvious differences were found in the subcellular fractions of the wild-type and {Delta}secA2 mutant strains grown in minimal Middlebrook 7H9 medium. Further, although Msmeg1712 and Msmeg1704 were prominent spots in 2D profiles from Mueller-Hinton medium-grown cultures, they were absent in 2D profiles from 7H9 medium-grown cultures (data not shown). Because protein homology predicted that Msmeg1712 and Msmeg1704 have roles in sugar utilization, we hypothesized that expression of msmeg1712 is regulated by sugar composition that is different in 7H9 medium and Mueller-Hinton medium. To test for differential expression in the two media, we constructed plasmid pHSG62, which has the 399-bp upstream region along with the first 10 codons of msmeg1712 transcriptionally and translationally linked to a lacZ reporter. We found that the ß-galactosidase activity produced by wild-type M. smegmatis carrying pHSG62 was approximately fourfold higher in Mueller-Hinton medium than in 7H9 medium. Furthermore, supplementation of Mueller-Hinton medium with glucose and glycerol to mimic the carbohydrate composition of 7H9 medium repressed msmeg1712-'lacZ expression (Fig. 3). Analysis of a wild-type strain containing the promoterless lacZ vector yielded no detectable activity under all conditions. These results demonstrate that the expression of Msmeg1712 is regulated by the carbon source, which is consistent with the proposal that these open reading frames function in sugar catabolism. Notably, the {Delta}secA2 mutation did not have an effect on expression of msmeg1712-'lacZ.


Figure 3
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  		FIG. 3. Carbon source-dependent expression of Msmeg1712. Expression of the promoterless lacZ gene in pYUB76 (far left) or a fusion of the lacZ reporter to the 399-bp region upstream of msmeg1712, containing the putative promoter, was measured in various media by measuring hydrolysis of o-nitrophenyl-ß-D-galactoside. Where indicated, Middlebrook 7H9 or Mueller-Hinton medium was supplemented with 0.2% glucose and 0.5% glycerol. The results are from a representative experiment performed in triplicate. The error bars represent standard deviations. WT, wild type; {Delta}, {Delta}secA2 mutant.

Immunoblot analysis confirms a role for SecA2 in the export of Msmeg1712 and Msmeg1704. To confirm and extend the results obtained from the proteomic analysis, we studied the subcellular distribution of HA epitope-tagged Msmeg1712 and Msmeg1704 proteins expressed in the wild type and the {Delta}secA2 mutant using immunoblotting. Expression of Msmeg1704-HA was achieved by cloning msmeg1704 under the control of the mycobacterial hsp60 promoter (54) and growing the strains in 7H9 medium. Because of difficulty expressing Msmeg1712 from the hsp60 promoter, Msmeg1712-HA was expressed from the native msmeg1712 promoter, and all experiments with this construct were performed with strains grown in Mueller-Hinton medium to obtain expression.

Comparison of whole-cell lysates revealed equal amounts of HA-tagged protein in the wild-type and {Delta}secA2 mutant strains, ruling out the possibility that the SecA2-dependent differences in our envelope fractions were due to altered expression of the msmeg1712 and msmeg1704 genes (Fig. 4A, top two panels, lanes 1 and 2). Careful comparison of the whole-cell lysates of the wild-type and {Delta}secA2 mutant strains revealed that the HA-tagged protein observed in the {Delta}secA2 mutant migrated slower on 1D-PAGE gels. This was most evident with Msmeg1712-HA (Fig. 4B, lanes 1 and 2). The simplest explanation for the slower-migrating species in the {Delta}secA2 mutant is that uncleaved precursor protein (form P) accumulates in the absence of SecA2, whereas in the wild-type strain Msmeg1712-HA and Msmeg1704-HA are fully processed to the cleaved, lipidated mature form (form M). Evaluation of cell wall, membrane, and soluble fractions revealed additional SecA2-dependent differences for Msmeg1712-HA and Msmeg1704-HA. In the absence of SecA2, the amount of Msmeg1712-HA and Msmeg1704-HA in the cell wall was significantly reduced (Fig. 4A, lanes 3 and 4). The absence of SecA2 also resulted in a reduced abundance of Msmeg1704-HA in the membrane (Fig. 4A, lanes 5 and 6). While the total amount of membrane-associated Msmeg1712-HA did not appear to be affected, the amount of form M was reduced. Slower-migrating species of both Msmeg1712-HA and Msmeg1704-HA were observed in the {Delta}secA2 mutant membranes. These species were quite prominent in the case of Msmeg1712-HA, and two species were detected (Fig. 4B). We termed these aberrant species forms P and X, with form X being the slowest-migrating species. As discussed below, we propose that forms P and X represent uncleaved export intermediates that differ in lipidation. The fact that form X was evident only in the enriched membrane fraction indicated that it is a relatively rare species in the total protein that is specifically associated with membranes.


Figure 4
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  		FIG. 4. SecA2-dependent export of Msmeg1712-HA and Msmeg1704-HA. (A) M. smegmatis wild type (WT) and the {Delta}secA2 mutant ({Delta}) expressing Msmeg1712-HA, Msmeg1704-HA, truncated {Delta}ss-'Msmeg1712-HA, M. tuberculosis 19-kDa lipoprotein, or M. smegmatis alkaline phosphatase (PhoAMsmeg) were subjected to subcellular fractionation. The fractions were separated by 1D-PAGE and analyzed by immunoblotting. The subcellular pattern of GroEL2 and the major M. smegmatis porin (MspA) expressed from the native chromosomal promoters was also evaluated. The material loaded in each lane corresponded to protein derived from the same number of cells, except for Msmeg1712-HA, for which the cell wall (CW) and membrane (M) fractions were loaded with 0.7 cell equivalent relative to the whole-cell lysate (WCL) and soluble (Sol) fractions. In all cases, the same amount of protein was loaded for a given fraction of the wild type and the {Delta}secA2 mutant. Each blot is representative of at least two independent experiments. (B) Comparison of the migration of Msmeg1712-HA forms in selected whole-cell lysate, membrane, or soluble fractions from wild-type and {Delta}secA2 mutant strains: forms M (mature), P (unlipidated, uncleaved precursor), and X (lipidated, uncleaved precursor).

In contrast to the diminished levels of Msmeg1712-HA and Msmeg1704-HA in the envelope fractions derived from the {Delta}secA2 mutant, the soluble fraction of the {Delta}secA2 mutant had an increased amount of Msmeg1712-HA and Msmeg1704-HA (Fig. 4A, lanes 7 and 8) that migrated like form P (Fig. 4B). This is consistent with a role for SecA2 in export of these proteins, in that a lack of SecA2 leads to the accumulation of a putative precursor in the soluble cytosolic fraction. Together, these results demonstrate that SecA2 is required for efficient localization of Msmeg1712 and Msmeg1704 to cell envelope fractions. In addition, the absence of SecA2 results in the accumulation of Msmeg1712-HA and Msmeg1704-HA in the soluble fraction and the appearance of export intermediates.

We also expressed a truncated ({Delta}ss)-'Msmeg1712-HA protein lacking the predicted amino-terminal signal sequence in the wild-type and {Delta}secA2 strains. Analysis of subcellular fractions from these strains showed that the truncated protein was present at equal levels and present exclusively in the soluble fraction of both strains, demonstrating that the signal sequence is required for envelope localization of Msmeg1712. As controls for our fractionation, we also localized GroEL2, a cytoplasmic protein (51), and MspA, a cell wall protein (52), in the fractions by immunoblot analysis. As anticipated, GroEL2 was absent from the envelope fractions and MspA was restricted to the cell wall fraction. This indicates that there was minimal contamination of our envelope fractions with soluble protein and that there was minimal contamination of membrane or soluble fractions with cell wall protein.

Msmeg1712 and Msmeg1704 behave as lipoproteins in Triton X-114 fractionation. To validate the bioinformatic prediction that Msmeg1712 and Msmeg1704 were lipoproteins, we performed Triton X-114 fractionation. Triton X-114 extraction is a commonly used method for the enrichment of lipoproteins, since this subclass of proteins partitions into the detergent phase, whereas soluble proteins remain in the aqueous phase (61). Triton X-114 extraction of whole-cell lysates of the wild type, the {Delta}secA2 mutant, and the complemented mutant revealed multiple proteins in the detergent phase. The majority of these proteins were equally abundant in the Triton X-114 fraction from the wild type and the {Delta}secA2 mutant. However, we routinely detected one ~40-kDa SecA2-dependent protein in the Triton X-114 fraction. Peptide mass fingerprinting identified this protein as Msmeg1712 (Fig. 5A). In some experiments a second SecA2-dependent band with the apparent molecular weight of Msmeg1704 was visible (data not shown).


Figure 5
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  		FIG. 5. Msmeg1712 and Msmeg1704 behave as lipoproteins in Triton X-114 fractionation. Triton X-114 partition experiments were performed with native or epitope-tagged proteins. (A) M. smegmatis wild-type strain (WT), {Delta}secA2 mutant ({Delta}), and complemented {Delta}secA2 mutant ({Delta}c) were grown in Mueller-Hinton broth and subjected to Triton X-114 fractionation. The whole-cell lysate (WCL), Triton X-114 detergent phase (Tx114), and aqueous phase (Aq) were run on a 1D-PAGE gel and stained with Coomassie blue. The arrow indicates the SecA2-dependent band identified as Msmeg1712. (B) Immunoblot analysis of Triton X-114 fractions prepared from the M. smegmatis wild type and the {Delta}secA2 mutant expressing Msmeg1712-HA, Msmeg1704-HA, or 19-kDa lipoprotein. Each blot is representative of three independent experiments.

Triton X-114 extraction of a lysate from wild-type M. smegmatis expressing Msmeg1712-HA demonstrated that the tagged protein also partitions into the detergent phase. In comparison, when the protein was expressed in the {Delta}secA2 mutant, less of the protein was present in the Triton X-114 phase and the protein was observed in the aqueous phase, suggesting that there was loss of lipoprotein characteristics (Fig. 5B). The same was true for Msmeg1704-HA, although the effect of SecA2 on localization to the detergent phase was less dramatic, as observed in multiple experiments. As a control for the Triton X-114 extraction, the well-characterized 19-kDa lipoprotein of M. tuberculosis was expressed in M. smegmatis. The 19-kDa lipoprotein partitioned to the detergent phase, as previously described (Fig. 5B) (61). These results suggest that Msmeg1712 and Msmeg1704 are lipoproteins and that SecA2 is required for efficient lipidation and export of these proteins.

Msmeg1712 and Msmeg1704 behave as lipoproteins in the presence of globomycin. To provide additional evidence that Msmeg1712 and Msmeg1704 are lipoproteins, we grew strains expressing the proteins in the presence of globomycin, a specific inhibitor of the lipoprotein signal peptidase (LspA) (13, 56, 57). While globomycin is lethal to gram-negative bacteria, gram-positive bacteria, including Corynebacterium glutamicum, can grow in its presence (25, 49). Since the nonessential LspA protein of M. tuberculosis was previously shown to function in processing of mycobacterial lipoproteins, we reasoned that globomycin could be used in mycobacteria to inhibit lipoprotein processing (3, 47). We confirmed this by treating M. smegmatis expressing the 19-kDa lipoprotein of M. tuberculosis with globomycin. The 19-kDa lipoprotein was previously shown to be subject to LspA cleavage (47). Globomycin did not inhibit growth of M. smegmatis at concentrations up to 50 µg/ml, but it did lead to a shift toward a higher-molecular-weight species for the 19-kDa lipoprotein (Fig. 6B). The globomycin-dependent mobility shift of the 19-kDa lipoprotein demonstrated that globomycin affects lipoprotein processing in mycobacteria. A more pronounced shift was probably masked by the glycosylated nature of the 19-kDa lipoprotein, which complicates resolution of the protein by SDS-PAGE. When wild-type strains of M. smegmatis expressing Msmeg1712-HA or Msmeg1704-HA were treated with globomycin, similar shifts in mobility were observed (Fig. 6B). In contrast, migration of the ({Delta}ss)-'Msmeg1712-HA protein lacking the signal sequence and monomeric MspA, which has a conventional signal sequence lacking a lipobox (36), was not affected by globomycin (Fig. 6B and data not shown). These results provide further support for the hypothesis that Msmeg1712 and Msmeg1704 are lipoproteins and, more specifically, are subject to LspA cleavage.


Figure 6
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  		FIG. 6. Globomycin sensitivity of mycobacterial lipoproteins. (A) Model for lipoprotein processing based on E. coli studies (60). The unlipidated uncleaved precursor (form P), the lipidated uncleaved precursor (form X), and the mature form (form M) are indicated. Not shown is the possibility of further lipid modification of form M at the exposed N terminus. The prolipoprotein diacylglyceryltransferase (Lgt) transfers the lipid moiety to the free sulfhydryl group. Globomycin inhibits the lipoprotein signal peptidase (LspA). (B) M. smegmatis wild type expressing the 19-kDa lipoprotein, Msmeg1712-HA, Msmeg1704-HA, or truncated {Delta}ss-'Msmeg1712-HA was grown in the presence (+) or absence (–) of 50 µg/ml globomycin. Whole-cell lysates were separated by 1D-PAGE and probed with anti-HA or anti-19-kDa antibody. (C) Fractionation of the wild type or {Delta}secA2 mutant expressing Msmeg1712-HA grown in the presence or absence of 20 µg/ml globomycin. Whole-cell lysates (WCL) prepared by French pressure lysis were fractionated into cell wall (CW), membrane (M), and soluble (Sol) fractions. The following amounts of material were loaded so that less abundant species could be visualized in the cell wall and membrane fractions: whole-cell lysate, 15 µg; cell wall, 2.4 µg; membrane, 4.1 µg; and soluble fraction, 11.3 µg. The positions of forms M, X, and P are indicated. The boxed region identifies the minor globomycin-sensitive mature species detected in the {Delta}secA2 mutant. The lower panel is a shorter exposure of lanes 9 to 12 showing forms M, X, and P. The blots are representative of two independent experiments.

We also evaluated subcellular fractions of globomycin-treated and untreated strains expressing Msmeg1712-HA. These experiments again revealed globomycin sensitivity of the Msmeg1712-HA protein expressed in wild-type M. smegmatis (Fig. 6C, lanes 1 and 2). In contrast, globomycin had a minimal effect on the {Delta}secA2 mutant, and it specifically did not influence form P or X, indicating that both of these forms are uncleaved precursors. Interestingly, the globomycin-shifted species of the wild type comigrated with form X of Msmeg1712-HA in the {Delta}secA2 mutant (Fig. 6C, lanes 2, 9, 10, 11, and 12). At least in E. coli, lipid modification precedes LspA cleavage (58). Therefore, we propose that the slowest-migrating species in the {Delta}secA2 mutant, form X, and the globomycin-shifted species of the wild type are lipidated, uncleaved precursors (Fig. 6A and C), while the slightly faster-migrating form (form P) is an unlipidated, uncleaved precursor. This experiment also revealed that in the membrane fraction of the {Delta}secA2 mutant there was a minor protein species that comigrated with form M and was globomycin sensitive (Fig. 6C, lanes 11 and 12). Therefore, even in the absence of SecA2 a small portion of Msmeg1712-HA appears to be correctly exported and processed.

SecA2 does not have a global effect on lipoprotein export. Evaluation of the 1D protein profiles of Triton X-114 extracts suggested that SecA2 does not have a global effect on the export of all lipoproteins (Fig. 5). To test this more directly, we studied the effect of SecA2 on the export and processing of two proven lipoproteins of mycobacteria: the endogenous alkaline phosphatase of M. smegmatis (PhoAMsmeg) (24) and the 19-kDa lipoprotein (16, 61). M. smegmatis wild-type and {Delta}secA2 strains were transformed with pML440 (59), a multicopy plasmid expressing PhoAMsmeg, or with pHSG67, a multicopy plasmid expressing the 19-kDa lipoprotein. Unlike Msmeg1712 and Msmeg1704, the localization of these lipoproteins to subcellular fractions or Triton X-114 fractions was not influenced by SecA2 (Fig. 4A and 5B). Furthermore, neither protein accumulated slower-migrating species in the {Delta}secA2 mutant (Fig. 4). Likewise, MspA, a nonlipidated exported protein with a conventional signal sequence and the major porin of M. smegmatis (52), localized to the cell wall in both the wild-type and mutant strains.

SecA2 in M. tuberculosis can recognize Msmeg1704-HA. To test whether the SecA2 pathway of M. tuberculosis can recognize SecA2-dependent proteins of M. smegmatis, we expressed Msmeg1704-HA in wild-type and {Delta}secA2 M. tuberculosis strains and assayed protein localization by immunoblotting. Subcellular fractions were generated by differential ultracentrifugation as they were with M. smegmatis, and they were probed for GroEL2, a marker for the cytoplasm, Msmeg1704-HA, and the known 19-kDa and PhoS1 lipoproteins, which are envelope associated (16, 51). When Msmeg1704-HA was expressed in M. tuberculosis, its localization pattern was the same as that observed in wild-type M. smegmatis (Fig. 7): present in the cell wall and membrane fractions and absent in the soluble fraction. Unlike what was seen in M. smegmatis, the absence of SecA2 in M. tuberculosis did not have a pronounced effect on localization to the cell wall and membrane fractions. However, the amount of Msmeg1704-HA detected in the soluble fraction increased, as was the case in M. smegmatis. Additionally, in the {Delta}secA2 mutant of M. tuberculosis, a slower-migrating species of Msmeg1704-HA was present that comigrated with the slower species seen in the {Delta}secA2 mutant of M. smegmatis (Fig. 7 and data not shown). This suggests that the SecA2 system of M. tuberculosis is able to recognize the heterologous M. smegmatis protein and contribute to its processing and export. This was not the case with the other mycobacterial lipoproteins tested, namely, the 19-kDa lipoprotein and PhoS1.


Figure 7
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  		FIG. 7. SecA2-dependent processing of Msmeg1704-HA in M. tuberculosis. M. tuberculosis wild-type (H37Rv) (WT) and {Delta}secA2 mutant ({Delta}) strains expressing Msmeg1704-HA were grown in 7H9 medium with albumin-dextrose-saline, disrupted in a French press, and fractionated by differential ultracentrifugation. Proteins were resolved by 1D-PAGE and analyzed by immunoblotting for the presence of GroEL2, Msmeg1704-HA, the 19-kDa lipoprotein, and the PhoS1 lipoprotein. The material loaded in each lane corresponds to the same number of cells. The blots are representative of two independent experiments. WCL, whole-cell lysate; CW, cell wall; M, membrane; Sol, soluble fraction.


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DISCUSSION
 
In this study, we expanded our search for substrates of the mycobacterial SecA2 export system and identified two new SecA2-dependent proteins: Msmeg1712 and Msmeg1704. This work broadens our appreciation of the types of substrates that can be exported by SecA2 of mycobacteria. Both Msmeg1712 and Msmeg1704 are lipoproteins that are localized to the cell envelope by a SecA2-dependent process that requires an amino-terminal signal sequence. In addition to being the first cell envelope proteins identified as SecA2 dependent in mycobacteria, these lipoproteins are the first endogenous SecA2-dependent proteins identified in M. smegmatis. Both of these proteins share homology to monosaccharide-binding proteins. The close proximity on the genome of their coding sequences, their association with genes encoding components of ABC transporters, and the sugar-regulated expression of Msmeg1712 suggest a role for this genomic region in sugar uptake.

A proteomic approach was employed to identify Msmeg1712 and Msmeg1704 as being SecA2 dependent. Since expression of the Msmeg1712-HA and Msmeg1704-HA proteins and of an msmeg1712-'lacZ fusion was the same in wild-type and {Delta}secA2 mutant strains, we concluded that the results of our proteomic experiments reflect a true export defect rather than secondary effects of the {Delta}secA2 mutation on gene expression. Msmeg1712 and Msmeg1704 were identified in both membrane and cell wall fractions. However, with our subcellular fractions we were unable to distinguish whether the proteins are present in both locations or if they are truly membrane associated and detection in the cell wall was due to contamination of the cell wall fraction with membrane. Preparation of uncontaminated cell wall fractions of mycobacteria is difficult (35, 37, 44). In assaying fractions for NADH oxidase activity, a predicted marker of the cytoplasmic membrane, we detected contamination of our cell wall fractions. Although the majority of the total recovered NADH oxidase activity was in the membrane (63%), the cell wall fraction also contained activity (32%). In contrast, there was no evidence that the membrane fraction or cell wall fractions were contaminated with soluble protein. Cell wall (MspA) and soluble (GroEL2) proteins were limited to the appropriate fractions.

In 1D and 2D profiles of membrane and cell wall fractions numerous proteins were visualized, but only Msmeg1712 and Msmeg1704 were clearly seen to be SecA2 dependent. The fact that only a small subset of proteins was identified as SecA2 dependent in M. smegmatis is consistent with findings for other bacteria that have a second SecA homolog, including our own results for M. tuberculosis (6, 9, 11, 28, 29). However, we cannot rule out the possibility that additional SecA2-dependent proteins in M. smegmatis exist. Our 2D-PAGE analysis was limited to proteins with a molecular mass of less than ~100 kDa. In addition, membrane and cell wall fractions are intrinsically difficult to extract and resolve by PAGE. Hydrophobic membrane proteins often precipitate at their isoelectric point and fail to enter the second dimension during 2D-PAGE analysis (21, 48). For these reasons, some SecA2-dependent proteins may have been missed. Finally, our data indicate that growth conditions can dramatically affect the ability to detect SecA2-dependent proteins.

By expressing a truncated ({Delta}ss)-'Msmeg1712-HA protein we showed that the predicted amino-terminal lipoprotein signal sequence of the substrate is required for export. Further, the behavior of Msmeg1712 and Msmeg1704 in Triton X-114 partitioning and globomycin treatment experiments indicates that they are genuine lipoproteins subject to cleavage by LspA. This demonstrates that lipoproteins can be substrates of the mycobacterial SecA2 export system. Notably, there did not appear to be a global role for SecA2 in lipoprotein export or processing; rather, the effect of SecA2 was specific for these two lipoproteins. An interesting similarity is that four predicted lipoproteins are among the exported proteins identified as dependent on SecA2 in L. monocytogenes (28). To our knowledge, globomycin has not previously been used in mycobacteria. Our study revealed that this compound is a useful tool for studying the lipoproteins of mycobacteria.

The {Delta}secA2 mutation resulted in an export defect for Msmeg1712-HA and Msmeg1704-HA such that the amount of these proteins present in the cell wall, membrane, and Triton X-114 fractions was significantly reduced, while the amount of these proteins increased in the soluble and aqueous fractions. Forms P and X were also observed in the {Delta}secA2 mutant. Additional studies are required to define the molecular identities of the form P, X, and M species observed in this study. However, the simplest explanation is that forms P and X represent uncleaved export intermediates and form M is a mature processed lipoprotein. We propose that the faster-migrating form P is an unlipidated uncleaved precursor and that the slower-migrating form X, which comigrated with the globomycin-elicited product in wild-type M. smegmatis, is a lipidated uncleaved precursor. Form M was sensitive to globomycin, and it comigrated with the truncated ({Delta}ss)-'Msmeg1712-HA, which is consistent with this species being mature and processed by the LspA peptidase (Fig. 6C).

A model to explain our findings is as follows. The efficient delivery to and transit through the membrane of Msmeg1712 and Msmeg1704 are dependent on SecA2. In the absence of SecA2, a reduced amount of precursor reaches the membrane-bound export machinery and instead accumulates in the cytosol (form P). The residual export that occurs in the {Delta}secA2 mutant is inefficient; less protein is correctly localized or processed. A minor amount of globomycin-sensitive form M and intermediates (forms P and X) are found in the membrane of the {Delta}secA2 mutant (Fig. 4B and 6C). The detection of export intermediates in the membrane suggests that they are in the process of engaging the export machinery to cross the membrane. At least in E. coli, lipid modification requires a functional Sec pathway and is thought to occur during or after translocation (20). Thus, one explanation for the appearance of form X is that it is a normally transient export intermediate that accumulates during inefficient export in the {Delta}secA2 mutant. Due to the presence of a recognizable Sec signal sequence on these substrates, the housekeeping SecA1 protein is an attractive candidate for carrying out the residual export. However, it is also possible that the residual export occurs independently of SecA1. While the model described above seems plausible, our data cannot rule out other more complex possibilities. For example, SecA2 may function in the export of the specific lipoproteins, as well as participate directly in their lipidation.

Given the close homology of SecA1 and SecA2, it is tempting to speculate that SecA2 works with the general Sec translocase (SecYEG) to promote export of a subset of signal sequence-containing proteins. The signal sequence-containing proteins may span a continuum from those completely independent of SecA2 and exclusively reliant on SecA1 to those with an absolute requirement for SecA2 with no contribution of SecA1. In the middle of this continuum would be proteins that are exported optimally when both SecA1 and SecA2 are present. Consequently, we believe that Msmeg1712 and Msmeg1704 have as-yet-undefined features that render them heavily dependent on SecA2 for initial recognition and translocation through the SecYEG pore. However, it is also possible that molecules other than the known components of the Sec apparatus are involved in exporting SecA2-dependent substrates.

In line with the idea that SecA2 can promote export of some signal sequence-containing proteins are the results of a prior study with M. smegmatis in which the effect of SecA2 on signal sequence-containing PhoA (E. coli alkaline phosphatase) fusion proteins was evaluated (8). In this study, the absence of SecA2 resulted in a partial export defect for some, but not all, of the fusion proteins tested. However, in M. tuberculosis the SecA2-dependent proteins identified are secreted and lack recognizable signal sequences (9). An intriguing possibility is that the role of SecA2 in the secretion of nonconventional substrates by M. tuberculosis is in the export to the cell envelope of a specific signal sequence-containing protein, which is itself a component of a novel secretion system. We are currently searching for SecA2-dependent cell envelope proteins of M. tuberculosis, which may help define such a pathway. Alternatively, SecA2 in M. tuberculosis could have a more direct role in the secretion of nonconventional proteins through the SecYEG channel or through a novel pathway.

Here we also showed that the SecA2 system of M. tuberculosis can recognize some signal sequence-containing proteins, as shown by the appearance of an export intermediate of Msmeg1704-HA in the {Delta}secA2 mutant of M. tuberculosis. As in M. smegmatis, this export defect was limited to Msmeg1704-HA among the lipoproteins tested, suggesting that at least some of the molecular determinants for SecA2-dependent export are shared between M. tuberculosis and M. smegmatis. Even though M. tuberculosis lacks an obvious homolog of Msmeg1712 or Msmeg1704, its genome contains four genes encoding proteins annotated as periplasmic sugar-binding proteins and genes encoding as many as 99 proteins that contain predicted lipoprotein signal sequences (55). The lipoprotein fraction is an attractive candidate for our ongoing search for SecA2-dependent proteins of M. tuberculosis. The results of the current study should aid our efforts to identify SecA2-dependent proteins of M. tuberculosis by emphasizing the need to examine lipoproteins, cell envelope proteins, and multiple growth conditions.


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ACKNOWLEDGMENTS
 
Our thanks go to Adrienne Cox (Department of Radiobiology, University of North Carolina at Chapel Hill) for the use of the 137Cs irradiator. We also thank John Belisle and Karen Dobos (Colorado State University) for many helpful discussions and technical advice, Douglas Young (Imperial College of London, United Kingdom) for 19-kDa lipoprotein antiserum, Masatoshi Inukai (International Union of Health and Welfare, Japan) for providing globomycin, and Cameron Scarlett of the Michael Hooker Proteomics Core Facility (University of North Carolina at Chapel Hill) for constructing the M. smegmatis MASCOT database. We also thank members of the Braunstein lab for critical reading of the manuscript.

This research was supported by NIH grant AI054540 to M.B. H.S.G. was supported by a postdoctoral fellowship from the Heiser Foundation of the New York Community Trust and by NIH Training Grant in Infectious Disease Pathogenesis AI007151.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, CB#7290, 804 Mary Ellen Jones, University of North Carolina, Chapel Hill, NC 27599-7290. Phone: (919) 966-5051. Fax: (919) 962-8103. E-mail: Miriam_Braunstein{at}med.unc.edu Back

{triangledown} Published ahead of print on 11 May 2007. Back

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

{ddagger} Present address: Department of Microbiology, University of Virginia Charlottesville, VA 22908. Back


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Journal of Bacteriology, July 2007, p. 5090-5100, Vol. 189, No. 14
0021-9193/07/$08.00+0     doi:10.1128/JB.00163-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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