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. 2009 Apr 10;284(15):9927-36.
doi: 10.1074/jbc.M900325200. Epub 2009 Feb 23.

The Accessory SecA2 System of Mycobacteria Requires ATP Binding and the Canonical SecA1

Nathan W Rigel  1 , Henry S Gibbons, Jessica R McCann, Justin A McDonough, Sherry Kurtz, Miriam Braunstein
Affiliations

The Accessory SecA2 System of Mycobacteria Requires ATP Binding and the Canonical SecA1

Nathan W Rigel et al. J Biol Chem. .

Abstract

In bacteria, the majority of exported proteins are transported by the general Sec pathway from their site of synthesis in the cytoplasm across the cytoplasmic membrane. The essential SecA ATPase powers this Sec-mediated export. Mycobacteria possess two nonredundant SecA homologs: SecA1 and SecA2. In pathogenic Mycobacterium tuberculosis and the nonpathogenic model mycobacterium Mycobacterium smegmatis, SecA1 is essential for protein export and is the "housekeeping" SecA, whereas SecA2 is an accessory SecA that exports a specific subset of proteins. In M. tuberculosis the accessory SecA2 pathway plays a role in virulence. In this study, we uncovered basic properties of the mycobacterial SecA2 protein and its pathway for exporting select proteins. By constructing secA2 mutant alleles that encode proteins defective in ATP binding, we showed that ATP binding is required for SecA2 function. SecA2 mutant proteins unable to bind ATP were nonfunctional and dominant negative. By evaluating the subcellular distribution of each SecA, SecA1 was shown to be equally divided between cytosolic and cell envelope fractions, whereas SecA2 was predominantly localized to the cytosol. Finally, we showed that the canonical SecA1 has a role in the process of SecA2-dependent export. The accessory SecA2 export system is important to the physiology and virulence of mycobacteria. These studies help establish the mechanism of this new type of specialized protein export pathway.

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Figures

FIGURE 1.
FIGURE 1.
Both M. tuberculosis and M. smegmatis secA2 can complement the macrophage growth defect of a M. tuberculosis ΔsecA2 mutant. Murine bone marrow-derived macrophages were infected at a multiplicity of infection of 1.0 with the following strains: H37Rv (wild type), mc23112 (ΔsecA2 mutant), mc23112 complemented with wild type M. tuberculosis secA2 encoded on pMB162 (ΔsecA2/Mtb secA2), and mc23112 complemented with wild type M. smegmatis secA2 encoded on pYA810 (ΔsecA2/Msm secA2). Colony forming units (CFU) were determined by plating macrophage lysates. The infection was performed with triplicate wells for each strain. The error bars represent ± standard deviation of the mean. The data are representative of two independent experiments. *, p < 0.05.
FIGURE 2.
FIGURE 2.
Complementation of a smooth colony phenotype of the M. tuberculosis ΔsecA2 mutant. A, wild type and ΔsecA2 mutant M. tuberculosis strains were transformed with pMV306.kan (empty vector), wild type M. tuberculosis secA2 encoded on pMB162 (Mtb secA2), or wild type M. smegmatis secA2 encoded on pYA810 (Msm secA2). B, wild type and ΔsecA2 mutant M. tuberculosis strains were transformed with Walker Box mutant secA2 alleles from M. tuberculosis encoded on pNR7 (secA2 K115R), or M. smegmatis secA2 K129R encoded on pNR25 (secA2 K129R). All transformants were grown on 7H10 plates containing 0.05% Tween 80 and photographed after 3 weeks of growth at 37 °C.
FIGURE 3.
FIGURE 3.
M. smegmatis secA2 K129R fails to complement and exacerbates M. smegmatis ΔsecA2 mutant phenotypes. A, WT, ΔsecA2 mutant (Δ), ΔsecA2 mutant complemented with wild type secA2 (Δ/WT), and ΔsecA2 mutant expressing secA2 K129R (Δ/KR) M. smegmatis strains were transformed with vectors expressing HA-tagged Msmeg1704 (pNR36) or Msmeg1712 (pNR35). Whole cell lysates (WCL) were prepared from each strain and used to prepare cell wall (CW), membrane (MEM), and SOL fractions. Protein derived from an equal number of starting cells was analyzed by SDS-PAGE and immunoblot using anti-HA antibodies. B, M. smegmatis colonies grown on Mueller Hinton agar for 4 days at 37 °C. Shown from left to right are wild type M. smegmatis (WT) and ΔsecA2 mutant (Δ) strains carrying empty vectors, the ΔsecA2 mutant expressing wild type secA2 (Δ/WT), the ΔsecA2 mutant expressing secA2 K129R (Δ/KR), and wild type M. smegmatis expressing secA2 K129R (WT/KR). Colonies of the Δ/KR strain appeared upon extended incubation. C, the same strains in B were tested for sensitivity to 0.15 m sodium azide spotted on a filter disc. The size of the zone of clearing reports on azide sensitivity of each strain.
FIGURE 4.
FIGURE 4.
SecA2 K129R is dosage-dependent. Wild type and ΔsecA2 mutant M. smegmatis strains were transformed with pNR54 encoding inducible secA2 K129R under control of the Tet ON promoter. Strains were grown in Mueller Hinton broth with different concentrations of Atc (closed circle, 0 ng/ml; open circle, 100 ng/ml; closed triangle, 200 ng/ml). Growth of wild type (A) and ΔsecA2 mutant (B) was monitored by measuring A600 nm. At 21 h post-inoculation, serial dilutions of the wild type (C) and ΔsecA2 mutant (D) from each culture in the growth curve were plated onto Mueller Hinton plates, all of which contained 500 ng/ml Atc. Shown are colonies on plates incubated at 37 °C for 3–4 days. Even after extended incubation, colonies did not appear from cultures treated with 200 ng/ml Atc during liquid growth.
FIGURE 5.
FIGURE 5.
Subcellular localization of SecA1, SecA2, and SecA2 K129R in M. smegmatis. Cultures of WT (A), ΔsecA2 mutant (Δ, B), ΔsecA2 mutant complemented with secA2 (Δ/WT, C), and ΔsecA2 mutant expressing secA2 K129R (Δ/KR, D) were used to prepare whole cell lysate (WCL), SOL, and cell envelope (ENV) fractions. Protein derived from an equal number of cells was analyzed using anti-SecA1 and anti-SecA2 antibodies. The cell wall porin MspA and the cytoplasmic chaperone GroEL are controls for the fractionation. Percent localization to a given fraction for SecA1 (E) and SecA2 (F) was determined by quantitative immunoblot analysis of the fractions and is reported as the percentage of the total (SOL fraction plus cell envelope). The error bars indicate the standard error of the mean of three independent replicates for each strain.
FIGURE 6.
FIGURE 6.
Expression of SecA1 and SecA2 is required for export of Msmeg1712-HA. (A) Strain MSE10 (WT/conditional secA1), was either left untreated or treated with 600 ng/ml Atc for 21 h to deplete SecA1. Cell wall (CW), membrane (MEM), and SOL subcellular fractions were prepared from whole cell lysate (WCL) of these cultures. For each fraction, protein derived from an equal number of starting cells was analyzed by SDS-PAGE and immunoblot. The percentage of total Msmeg1712-HA localized to a given fraction from ±Atc-treated MSE10 (B) or ±Atc-treated JM693 (C)(ΔsecA2/conditional secA1) cultures was quantified using phosphorimaging and is reported as the percentage of the total (cell wall plus membrane plus SOL). The results are the mean of three independent experiments ± standard error.*, p < 0.05.
FIGURE 7.
FIGURE 7.
Model to explain SecA2 function and the phenotypes of Walker Box secA2 alleles. SecA2 exports select proteins across the cytoplasmic membrane through a mechanism involving SecA1 and a membrane-embedded translocase. Amino acid substitution in the ATP-binding site of SecA2, as in SecA2 K129R, results in a dominant negative SecA2 protein trapped at the translocase and compromising an essential process.
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References

    1. Finlay, B. B., and Falkow, S. (1997) Microbiol. Mol. Biol. Rev. 61 136–169 - PMC - PubMed
    1. Abdallah, A. M., Gey van Pittius, N. C., Champion, P. A., Cox, J., Luirink, J., Vandenbroucke-Grauls, C. M., Appelmelk, B. J., and Bitter, W. (2007) Nat. Rev. Microbiol. 5 883–891 - PubMed
    1. Gerlach, R. G., and Hensel, M. (2007) Int. J. Med. Microbiol. 297 401–415 - PubMed
    1. Braunstein, M., Brown, A. M., Kurtz, S., and Jacobs, W. R., Jr. (2001) J. Bacteriol. 183 6979–6990 - PMC - PubMed
    1. Braunstein, M., Espinosa, B., Chan, J., Belisle, J. T., and Jacobs, W. R. J. (2003) Mol. Microbiol. 48 453–464 - PubMed

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