To facilitate the discovery of new therapeutics for Burkholderia pseudomallei infections, we have developed cellular reporter screens for inhibitors of B. pseudomallei targets in the surrogate host Pseudomonas aeruginosa. P. aeruginosa strains carrying deletions of essential genes were engineered to be dependent on the IPTG-regulated expression of their B. pseudomallei orthologs on a broad-host-range plasmid. P. aeruginosa genes which are upregulated in response to depletion of each target gene product were fused to the Photorhabdus luminescens luxCDABE operon via pGSV3-lux-SpR to generate reporter strains with increased bioluminescence upon target inhibition. A total of 11 of 19 B. pseudomallei genes complemented deletions of their orthologs in P. aeruginosa. The dependence of growth on IPTG levels varied from complete dependence (ftsQ, gyrA, glmU, secA), to slower growth in the absence of IPTG (coaD, efp, mesJ), to apparently normal growth in the absence of IPTG (ligA, lpxA, folA, ipk). Reporter screening strains have been constructed for three gene targets (gyrA, glmU, secA), and one (gyrA) has been applied to 68,000 compounds resulting in a primary hit rate of 0.5% and a confirmed hit rate of 0.06% including several fluoroquinolones. These results provide proof of principle for surrogate cellular reporter screens as a useful approach to identify inhibitors of essential gene products.
bioluminescence; reporter; screen
SecA ATPase is a critical member of the Sec family, which is important in the translocation of membrane and secreted polypeptides/proteins in bacteria. Small molecule inhibitors can be very useful research tools as well as leads for future antimicrobial agent development. Based on previous virtual screening work, we optimized the structures of two hit compounds and obtained SecA ATPase inhibitors with IC50 in the single digit micromolar range. These represent the first low micromolar synthetic inhibitors of bacterial SecA and will be very useful for mechanistic studies.
SecA, the translocation ATPase of the preprotein translocase, accounts for 0.25% of the total protein in a degU32(Hy) Bacillus subtilis strain in logarithmic phase. The SecA level remained constant irrespective of the demand for exoprotein production but dropped about 12-fold during the late stationary phase. Modulation of the level of functional SecA during the exponential phase of growth affected differently the secretion of levansucrase and α-amylase overexpressed under the control of the sacB leader region. The level of SecA was reduced in the presence of sodium azide and in the div341 thermosensitive mutant at nonpermissive temperatures. Overproduction of SecA was obtained with a multicopy plasmid bearing secA. The gradual decrease of the SecA level reduced the yield of secreted levansucrase with a concomitant accumulation of unprocessed precursor in the cells, while an increase in the SecA level resulted in an elevation of the production of exocellular levansucrase. In contrast, α-amylase secretion was almost unaffected by high concentrations of sodium azide or by very low levels of SecA. Secretion defects were apparent only under conditions of strong SecA deprivation of the cell. These data demonstrate that the α-amylase and levansucrase precursors markedly differ in their dependency on SecA for secretion. It is suggested that these precursors differ in their binding affinities for SecA.
Protein secretion is an essential process for bacterial growth, yet there are few if any antimicrobial agents which inhibit secretion. An in vivo, high-throughput screen to detect secretion inhibitors was developed based on the translational autoregulation of one of the central protein components, SecA. The assay makes use of a SecA-LacZ fusion reporter construct in Escherichia coli which is induced when secretion is perturbed. Several compounds, including two natural product extracts, which had the ability to induce the reporter fusion were identified and the MICs of these compounds for Staphylococcus aureus strain MN8 were found to be ≤128 μg/ml. Enzyme-linked immunosorbent assay, Western blotting, and immunoprecipitation techniques were used to analyze the affects of these compounds on protein secretion. Six representative compounds presented here appear to be bona fide secretion inhibitors but were found to have deleterious effects on membranes. It was concluded that, while the method described here for identifying inhibitors of secretion is valid, screens such as this, which are directed against the membrane-bound portion of a pathway, may preferentially identify compounds which affect membrane integrity.
At the core of the bacterial general secretion (Sec) pathway is the SecA ATPase, which powers translocation of unfolded preproteins containing Sec signal sequences through the SecYEG membrane channel. Mycobacteria have two nonredundant SecA homologs: SecA1 and SecA2. While the essential SecA1 handles “housekeeping” export, the nonessential SecA2 exports a subset of proteins and is required for Mycobacterium tuberculosis virulence. Currently, it is not understood how SecA2 contributes to Sec export in mycobacteria. In this study, we focused on identifying the features of two SecA2 substrates that target them to SecA2 for export, the Ms1704 and Ms1712 lipoproteins of the model organism Mycobacterium smegmatis. We found that the mature domains of Ms1704 and Ms1712, not the N-terminal signal sequences, confer SecA2-dependent export. We also demonstrated that the lipid modification and the extreme N terminus of the mature protein do not impart the requirement for SecA2 in export. We further showed that the Ms1704 mature domain can be efficiently exported by the twin-arginine translocation (Tat) pathway. Because the Tat system exports only folded proteins, this result implies that SecA2 substrates can fold in the cytoplasm and suggests a putative role of SecA2 in enabling export of such proteins. Thus, the mycobacterial SecA2 system may represent another way that bacteria solve the problem of exporting proteins that can fold in the cytoplasm.
SecA is a central component of the general secretion system that is essential for bacterial growth and thus an ideal target for the development of antimicrobial agents. A series of fluorescein analogs were first screened against the ATPase activity using the truncated unregulated SecA catalytic domain. Rose Bengal (RB) and Erythrosin B (EB) were found to be potent inhibitors with IC50 values of 0.5 µM and 2 µM, respectively. RB and EB inhibit the catalytic SecA ATPase more than the F1F0-proton ATPase. We used three assays to test the effect of these compounds on full length SecA ATPase: in solution (intrinsic ATPase), in membrane preparation, and translocation ATPase. RB and EB show the following trend in terms of IC50 values: translocation ATPase < membrane ATPase < intrinsic ATPase. Very importantly, the potency of these fluorescein analogs in inhibiting the truncated SecA ATPase correlates with their ability to inhibit the biologically relevant protein translocation activity of SecA. The in vitro translocation of proOmpA precursors into membrane vesicles is strongly inhibited by RB with IC50 of about 0.25 µM, making RB the most potent inhibitor of SecA ATPases and SecA-dependent protein translocation thus far. The ability of these compounds to inhibit SecA directly translates into antibacterial effects as well. Our findings show the value of fluorescein analogs as probes for mechanistic studies of SecA functions, and for the potential development of new antimicrobial agents with SecA as the target.
SecA; ATPase inhibitor; protein translocation; antimicrobial; membrane protein
Mutant strains of Escherichia coli were screened for the ability to grow on L agar plates containing 3.4 or 4.6 mM sodium azide. Most mutants had mutations located in the leucine region, presumably at the azi locus. Two of these mutants were found to have a mutation in the secA gene, but expression of the resistance phenotype also required the presence of upstream gene X. While a plasmid carrying the X-secA mutant gene pair was able to confer azide resistance to a sensitive host, a similar plasmid harboring the wild-type secA allele rendered a resistant strain sensitive to azide, indicating codominance of the two alleles. That azide inhibits SecA is consistent with the fact that SecA has ATPase activity, an activity that is often prone to inhibition by azide.
The SecA protein is present in all bacteria, and it is a central component of the general Sec-dependent protein export pathway. An unusual property of Mycobacterium tuberculosis is the presence of two SecA proteins: SecA1, the essential “housekeeping” SecA, and SecA2, the accessory secretion factor. Here, we report that a ΔsecA2 mutant of M. tuberculosis was defective for growth in the early stages of low-dose aerosol infection of C57BL/6 mice, a time during which the bacillus is primarily replicating in macrophages. Consistent with this in vivo phenotype, we found that the ΔsecA2 mutant was defective for growth in macrophages from C57BL/6 mice. The ΔsecA2 mutant was also attenuated for growth in macrophages from phox−/− mice and from NOS2−/− mice. These mice are defective in the reactive oxygen intermediate (ROI)-generating phagocyte oxidase and the reactive nitrogen intermediate (RNI)-generating inducible nitric oxide synthase, respectively. This indicated a role for SecA2 in the intracellular growth of M. tuberculosis that is independent of protecting against these ROIs or RNIs. Macrophages infected with the ΔsecA2 mutant produced higher levels of tumor necrosis factor alpha, interleukin-6, RNI, and gamma interferon-induced major histocompatibility complex class II. This demonstrated a function for M. tuberculosis SecA2 in suppressing macrophage immune responses, which could explain the role of SecA2 in intracellular growth. Our results provide another example of a relationship between M. tuberculosis virulence and inhibition of the host immune response.
In streptococci, the secA2 locus includes genes encoding the following: (i) the accessory Sec components (SecA2, SecY2, and at least three accessory secretion proteins), (ii) two essential glycosyltranferases (GTs) (GtfA and GtfB), (iii) a variable number of dispensable additional GTs, and (iv) a secreted serine-rich LPXTG protein which is glycosylated in the cytoplasm and transported to the cell surface by this accessory Sec system. The secA2 locus of Streptococcus agalactiae strain NEM316 is structurally related to those found in other streptococci and encodes the serine-rich surface protein Srr1. We demonstrated that expression of Srr1 but not that of the SecA2 components and the associated GTs is regulated by the standalone transcriptional regulator Rga. Srr1 is synthesized as a glycosylated precursor, secreted by the SecA2 system, and anchored to the cell wall by the housekeeping sortase A. Srr1 was localized preferentially at the old poles. GtfA and/or GtfB, but not the six additional GTs, is essential for the production of Srr1. These GTs are involved in the attachment of GlcNac and sialic acid to Srr1. Full glycosylation of Srr1 is associated with the cell surface display of a protein that is more resistant to proteolytic attack. Srr1 contributes to bacterial adherence to human epithelial cell lines and virulence in a neonatal rat model. The extent of Srr1 glycosylation by GtfC to -H modulates bacterial adherence and virulence.
Export of protein into the periplasm of Escherichia coli via the general secretory system requires that the transported polypeptides be devoid of stably folded tertiary structure. Capture of the precursor polypeptides before they fold is achieved by the promiscuous binding to the chaperone SecB. SecB delivers its ligand to export sites through its specific binding to SecA, a peripheral component of the membrane translocon. At the translocon the ligand is passed from SecB to SecA and subsequently through the SecYEG channel. We have previously used site-directed spin labeling and electron paramagnetic resonance spectroscopy to establish a docking model between SecB and SecA. Here we report use of the same strategy to map the pathway of a physiologic ligand, the unfolded form of precursor galactose-binding protein, on SecB. Our set of SecB variants each containing a single cysteine, which was used in the previous study, has been expanded to forty-eight residues which cover 49% of the surface of SecB. The residues on SecB involved in contacts were identified as those that, upon addition of the unfolded polypeptide ligand, showed changes in spectral line shape consistent with restricted motion of the nitroxide. We conclude that the bound precursor makes contact with a large portion of the surface of the small chaperone. The sites on SecB that interact with the ligand are compared with the previously identified sites that interact with SecA and a model for transfer of the ligand is discussed.
precursor polypeptide; SecB; export; site-directed spin labeling; chaperone
The SecA molecular nanomachine in bacteria uses energy from ATP hydrolysis to drive posttranslational secretion of pre-proteins through the SecYEG translocon. Cytosolic SecA exists in a dimeric, ‘closed’ state with relatively low ATPase activity. After binding to the translocon, SecA undergoes major conformational rearrangement, leading to a state that is structurally more ‘open’, has elevated ATPase activity, and is active in translocation. The structural details underlying this conformational change in SecA remain incompletely defined. Most SecA crystal structures report on the cytosolic form; only one structure sheds light on a form of SecA that has engaged the translocon. We have used mild destabilization of SecA to trigger conformational changes that mimic those in translocation-active SecA and thus study its structural changes in a simplified, soluble system. Results from circular dichroism, tryptophan fluorescence, and limited proteolysis demonstrate that the SecA conformational reorganization involves disruption of several domain-domain interfaces, partial unfolding of the second nucleotide binding fold (NBF) II, partial dissociation of the helical scaffold domain (HSD) from NBF I and II, and restructuring of the 30 kDa C-terminal region. These changes account for the observed high translocation SecA ATPase activity because they lead to the release of an inhibitory C-terminal segment (called intramolecular regulator of ATPase 1, or IRA1), and of constraints on NBF II (or IRA2) that allow it to stimulate ATPase activity. The observed conformational changes thus position SecA for productive interaction with the SecYEG translocon and for transfer of segments of its passenger protein across the translocon.
Fimbria-associated protein 1 (Fap1) is a high-molecular-mass glycosylated surface adhesin required for fimbria biogenesis and biofilm formation in Streptococcus parasanguinis. The secretion of mature Fap1 is dependent on the presence of SecA2, a protein with some homology to, but with a different role from, SecA. The signals that direct the secretion of Fap1 to the SecA2-dependent secretion pathway rather than the SecA-dependent secretion pathway have not yet been identified. In this study, Fap1 variants containing different domains were expressed in both secA2 wild-type and mutant backgrounds and were tested for their ability to be secreted by the SecA- or SecA2-dependent pathway. The presence or absence of the cell wall anchor domain (residues 2531 to 2570) at the C terminus did not alter the selection of the Fap1 secretion route. The Fap1 signal peptide (residues 1 to 68) was sufficient to support the secretion of a heterologous protein via the SecA-dependent pathway, suggesting that the signal peptide was sufficient for recognition by the SecA-dependent pathway. The minimal sequences of Fap1 required for the SecA2-dependent pathway included the N-terminal signal peptide, nonrepetitive region I (residues 69 to 102), and part of nonrepetitive region II (residues 169 to 342). The two serine-rich repeat regions (residues 103 to 168 and 505 to 2530) were not required for Fap1 secretion. However, they were both involved in the specific inhibition of Fap1 secretion via the SecA-dependent pathway.
The proper extracytoplasmic localization of proteins is an important aspect of mycobacterial physiology and the pathogenesis of Mycobacterium tuberculosis. The protein export systems of mycobacteria have remained unexplored. The Sec-dependent protein export pathway has been well characterized in Escherichia coli and is responsible for transport across the cytoplasmic membrane of proteins containing signal sequences at their amino termini. SecA is a central component of this pathway, and it is highly conserved throughout bacteria. Here we report on an unusual property of mycobacterial protein export—the presence of two homologues of SecA (SecA1 and SecA2). Using an allelic-exchange strategy in Mycobacterium smegmatis, we demonstrate that secA1 is an essential gene. In contrast, secA2 can be deleted and is the first example of a nonessential secA homologue. The essential nature of secA1, which is consistent with the conserved Sec pathway, leads us to believe that secA1 represents the equivalent of E. coli secA. The results of a phenotypic analysis of a ΔsecA2 mutant of M. smegmatis are presented here and also indicate a role for SecA2 in protein export. Based on our study, it appears that SecA2 can assist SecA1 in the export of some proteins via the Sec pathway. However, SecA2 is not the functional equivalent of SecA1. This finding, in combination with the fact that SecA2 is highly conserved throughout mycobacteria, suggests a second role for SecA2. The possibility exists that another role for SecA2 is to export a specific subset of proteins.
secA is translationally regulated by the protein secretion proficiency state of the Escherichia coli cell. This regulation was explored by making signal sequence mutations in the gene upstream of secA, gene X, which promotes secA translational coupling. Gene X signal sequence mutants were constitutive for secA expression, while prlA alleles partially restored secA regulation. These results show that interaction of the pre-gene X protein with the translocon is required for proper secA regulation. Furthermore, gene X signal sequence mutations disrupted secA regulation only in the cis configuration. We propose that nascent pre-gene X protein interacts with the translocon during its secretion to constitute the secretion sensor.
The secA gene product is an autoregulated, membrane-associated ATPase which catalyzes protein export across the Escherichia coli plasma membrane. Previous genetic selective strategies have yielded secA mutations at a limited number of sites. In order to define additional regions of the SecA protein that are important in its biological function, we mutagenized a plasmid-encoded copy of the secA gene to create small internal deletions or duplications marked by an oligonucleotide linker. The mutagenized plasmids were screened in an E. coli strain that allowed the ready detection of dominant secA mutations by their ability to derepress a secA-lacZ protein fusion when protein export is compromised. Twelve new secA mutations were found to cluster into four regions corresponding to amino acid residues 196 to 252, 352 to 367, 626 to 653, and 783 to 808. Analysis of these alleles in wild-type and secA mutant strains indicated that three of them still maintained the essential functions of SecA, albeit at a reduced level, while the remainder abolished SecA translocation activity and caused dominant protein export defects accompanied by secA depression. Three secA alleles caused dominant, conditional-lethal, cold-sensitive phenotypes and resulted in some of the strongest defects in protein export characterized to date. The abundance of dominant secA mutations strongly favors certain biochemical models defining the function of SecA in protein translocation. These new dominant secA mutants should be useful in biochemical studies designed to elucidate SecA protein's functional sites and its precise role in catalyzing protein export across the plasma membrane.
SecA is an ATPase and motor protein that drives protein translocation across the bacterial plasma membrane. In Escherichia coli SecA levels are regulated by the secretion needs of the cell utilizing secM, which encodes a secreted protein. Previous studies demonstrated that this regulation requires a translational pause within secM, whose duration regulates the accessibility of the secA Shine-Dalgarno sequence on secM secA mRNA. Here we provide evidence that translocon “pulling” of nascent SecM is what regulates the duration of the secM translational pause, and thus secA expression levels, thereby providing direct support for this model.
An amber mutation in the secA gene of Escherichia coli causes a pleiotropic decrease in the synthesis of secreted proteins, including maltose-binding protein (MBP) and alkaline phosphatase. Reversal of the inhibition of MBP synthesis in secA(Am) strains by signal sequence mutations in the malE gene has been reported. These results suggest a coupling between secretion and translation which involves an interaction between the signal sequence of nascent polypeptides and a cellular secretion machinery. Further analysis reported here indicated that signal sequence mutations of MBP or alkaline phosphatase did not selectively overcome the inhibition of MBP or alkaline phosphatase synthesis in secA(Am) strains. Rather, at a given time in parallel experiments there was substantial variability among closely isogenic secA(Am) strains in the magnitude of the synthesis block; this variability could account for the earlier results. Further experiments suggested that the inhibition of MBP synthesis in secA(Am) strains was caused by depletion of cyclic AMP, leading to decreased transcription of the malE gene. However, the secretion defects in secA(Am) strains were not affected by cyclic AMP levels. Therefore, we conclude that the reduction in MBP synthesis was a secondary consequence of the primary export defect in the secA(Am) strains.
We describe a novel molecular method for the differentiation and identification of 29 mycobacterial species. The target is the secA1 gene that codes for the essential protein SecA1, a key component of the major pathway of protein secretion across the cytoplasmic membrane. A 700-bp region of the secA1 gene was amplified and sequenced from 47 American Type Culture Collection strains of 29 Mycobacterium species as well as from 59 clinical isolates. Sequence variability in the amplified segment of the secA1 gene allowed the differentiation of all species except for the members of the Mycobacterium tuberculosis (MTB) complex, which had identical sequences. A range of 83.3 to 100% interspecies similarity was observed. All species could also be differentiated by their amino acid sequences as deduced from the sequenced region of the secA1 gene, with the exception of the MTB complex. Partial sequences of secA1 from clinical isolates belonging to nine frequently isolated species of mycobacteria revealed a very high intraspecies similarity at the DNA level (typically >99%; range, 96.0 to 100%); all clinical isolates were correctly identified. Comparison of the deduced 233-amino-acid sequences among clinical isolates of the same species showed between 99.6 and 100% similarity. To our knowledge, this is the first time a secretion-related gene has been used for the identification of the species within a bacterial genus.
The export of lipoprotein has been found to be affected in both secA and secY mutants of Escherichia coli which are defective in the secretion of a number of outer membrane and periplasmic proteins. The kinetics of accumulation of prolipoprotein upon a temperature shift to 42 degrees C is indistinguishable from that of pre-OmpA protein accumulation in the secA mutant. In both secA and secY mutants, the accumulated prolipoprotein is unmodified with glyceride and localized in the cytoplasmic membrane. We conclude from these results that the early steps in protein export are common to prolipoprotein and non-lipoprotein precursors. The pathways for the export of these two groups of precursor proteins diverge with regard to the modification and processing reactions which are late events in the export process.
SecA protein synthesis levels were elevated 10- to 20-fold when protein secretion was blocked in secA, secD, and secY mutants or in a malE-lacZ fusion-containing strain but not in a secB null mutant. An active secB gene product was not required to derepress secA, since SecA levels were elevated during protein export blocks in secB secY and secB malE-lacZ double mutants.
Transport of many proteins to extracytoplasmic locations occurs via the general secretion (Sec) pathway. In Escherichia coli, this pathway is comprised of the SecYEG protein conducting channel and the SecA ATPase. SecA plays a central role in binding the signal peptide region of preproteins, directing preproteins to membrane-bound SecYEG and promoting translocation coupled with ATP hydrolysis. Although it is well-established that SecA is crucial for preprotein transport and thus cell viability, its oligomeric state during different stages of transport remains ill defined. We have characterized the energetics of SecA dimerization as a function of salt concentration and temperature and defined the linkage of SecA dimerization and signal peptide binding using analytical ultracentrifugation. The use of a new fluorescence detector permitted analysis of SecA dimerization down to concentrations as low as 50 nM. The dimer dissociation constants are strongly dependent on salt. Linkage analysis indicates that SecA dimerization is coupled to the release of about five ions, demonstrating that electrostatic interactions play an important role in stabilizing the SecA dimer interface. Binding of signal peptide reduces SecA dimerization affinity such that Kd increases about 9-fold from 0.28 μM in the absence of peptide to 2.68 μM in the presence of peptide. The weakening of the SecA dimer that accompanies signal peptide binding may poise the SecA dimer to dissociate upon binding to SecYEG.
SecA; signal peptide; analytical ultracentrifugation; thermodynamic linkage
The Bacillus subtilis secA homolog, div, was cloned and expressed at a variety of different levels in wild-type and secA mutant strains of Escherichia coli. Analysis of Div function showed that it could not substitute for SecA despite being present at a wide range of concentrations at or above the physiological level. Location of regions of functional similarity between the two proteins using div-secA chimeras revealed that only the amino-terminal ATP-binding domain of Div could functionally substitute for the corresponding region of SecA. The role of this domain was revealed by subcellular localization experiments that demonstrated that in both B. subtilis and E. coli Div had cytoplasmic, peripheral, and integral membrane distributions similar to those of its SecA homolog and that an intact ATP-binding domain was essential for regulating integration of this protein into the plasma membrane. These results suggest strongly that the previously observed cycle of membrane binding, insertion, and deinsertion of SecA protein (A. Economou and W. Wickner, Cell 78:835-843, 1994) is common to these two bacteria, and they demonstrate the importance of the conserved ATP-binding domain in promoting this cycle.
The export of proteins from their site of synthesis in the cytoplasm across the inner membrane is an important aspect of bacterial physiology. Because the location of extracytoplasmic proteins is ideal for host-pathogen interactions, protein export is also important to bacterial virulence. In bacteria there are conserved protein export systems that are responsible for the majority of protein export: the general secretion (Sec) pathway and the twin-arginine translocation (Tat) pathway. In some bacteria, there are also specialized export systems dedicated to exporting specific subsets of proteins. In this review, we discuss a specialized export system that exists in some Gram-positive bacteria and mycobacteria – the accessory Sec system. The common element to the accessory Sec system is an accessory SecA protein called SecA2. Here we present our current understanding of accessory Sec systems in Streptococcus gordonii, Streptococcus parasanguinis, Mycobacterium smegmatis, Mycobacterium tuberculosis, and Listeria monocytogenes, making an effort to highlight apparent similarities and differences between the systems. We also review the data showing that accessory Sec systems can contribute to bacterial virulence.
Bacterial protein secretion is catalyzed by the SecYEG protein-conducting channel complexed with the SecA ATPase motor. To gain insight into the SecA-SecYEG interaction we used peptide arrays, thermodynamic quantitation, mutagenesis and functional assays. Our data reveal that: a. SecA binds with low affinity on several, peripheral, exposed SecYEG sites. This largely electrostatic association is modulated by temperature and nucleotides. b. Binding sites cluster in five major binding “regions”: three that are exclusively cytoplasmic and two that reach the periplasm. c. Both the aminoterminal and carboxyterminal regions of SecA participate in binding interactions and share some sites. d. Several of these sites are essential for translocase catalysis. Our data provide residue-level dissection of the SecYEG-SecA interaction. Two models of assembly of SecA on dimeric SecYEG are discussed.
The Sec-dependent translocation pathway that involves the essential SecA protein and the membrane-bound SecYEG translocon is used to export many proteins across the cytoplasmic membrane. Recently, several pathogenic bacteria, including Mycobacterium tuberculosis, were shown to possess two SecA homologs, SecA1 and SecA2. SecA1 is essential for general protein export. SecA2 is specific for a subset of exported proteins and is important for M. tuberculosis virulence. The enzymatic activities of two SecA proteins from the same microorganism have not been defined for any bacteria. Here, M. tuberculosis SecA1 and SecA2 are shown to bind ATP with high affinity, though the affinity of SecA1 for ATP is weaker than that of SecA2 or Escherichia coli SecA. Amino acid substitution of arginine or alanine for the conserved lysine in the Walker A motif of SecA2 eliminated ATP binding. We used the SecA2(K115R) variant to show that ATP binding was necessary for the SecA2 function of promoting intracellular growth of M. tuberculosis in macrophages. These results are the first to show the importance of ATPase activity in the function of accessory SecA2 proteins.