Two nonredundant SecA homologues function in mycobacteria

Abstract

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 Delta secA2 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.

FIG. 1

Alignment of SecA proteins from B. subtilis, E. coli, and M. smegmatis (SecA1 and SecA2). ABCI (high-affinity ATP-binding site) and ABCII (low-affinity ATP-binding site) are indicated. The lightning bolt indicates the threonine residue that can be mutated to produce azide resistance. Triangles represent the deletion junction points in the ΔsecA1 mutation. The arrows pointing down identify the deletion junction points in the ΔsecA2 mutation.

FIG. 2

Southern analysis of secA1 recombinants in M. smegmatis. (A to D) The relevant BglII and BamHI restriction endonuclease sites are denoted. The MscI probe used in this Southern analysis is represented by vertical lines below the diagram. Panels: A, diagram of the chromosomal wild-type secA1 allele; B, diagram of secA1 single-crossover strain MB526; C, diagram of a chromosomal ΔsecA1 allele; D, diagram of pYUB544, a multicopy plasmid present in merodiploid strains; E, Southern blot of BglII-digested genomic DNAs probed with the secA1 probe. Sizes of fragments are indicated on the left. Lanes: 1, mc²155; 2, MB526; 3, ΔsecA1 recombinant with pYUB544; 4, wild-type recombinant with pYUB544.

FIG. 3

Southern analysis of secA2 recombinants in M. smegmatis. (A to C) BamHI restriction endonuclease sites are denoted. The BamHI-HindIII probe used in this Southern analysis is indicated by vertical bars below the diagram. Panels: A, diagram of the chromosomal wild-type secA2 allele; B, diagram of secA2 single-crossover strain MB573; C, diagram of chromosomal ΔsecA2 allele; D, Southern blot of BamHI-digested genomic DNAs probed with the secA2 probe. Sizes of fragments are indicated on the left. Lanes: 1, mc²155; 2, MB573; 3, ΔsecA2 (mc²2522 produced from MB573); 4, wild-type recombinant produced from MB573.

FIG. 4

Growth defect of the ΔsecA2 mutant on rich medium agar plates. Single colonies of ΔsecA2 mutant mc²2522, wild-type mc²155, and complemented ΔsecA2 attB::secA2 strain mc²2522/pMB162 are shown on Mueller-Hinton plates grown at 37°C.

FIG. 5

The ΔsecA2 mutant is sensitive to overexpression of secA1. (A) ΔsecA2 mutant mc²2522 transformed with various plasmids. Lanes: 1, pMV261; 2, pMB175 (single-copy secA1); 3, pYUB499 (multicopy secA1). (B) mc²155 transformed with various plasmids. Lanes: 1, pMV261; 2, pYUB499. Colonies were grown on Mueller-Hinton agar plates at 37°C.

FIG. 6

Immunoblot analysis of PhoA fusions in wild-type and ΔsecA2 (mc²2522) strains. Whole-cell extracts from individual strains were prepared, and 20-μl volumes were loaded per lane for SDS-PAGE and Western analysis. (A) Anti-PhoA antibodies were used for Western analysis of whole-cell extracts. Lanes: 1, MB509 (mc²155/pMV261); 2, mc²2757 (mc²155/pMB174); 3, MB648 (mc²155/pMB206); 4, MB648 with azide treatment; 5, MB649 (mc²2522/pMB206); 6, MB634 (mc²155/pMB202); 7, MB634 with azide treatment; 8, MB635 (mc²2522/pMB202). (B) Anti-HA Western blot. Lanes: 1, MB648; 2, MB648 with azide treatment; 3, MB649. (C) Anti-PhoA Western blot. Lanes: 1, MB509; 2, mc²2757; 3, MB624 (mc²155/pMB196); 4, MB625 (mc²2522/pMB196). Marker sizes are indicated to the left in kilodaltons. Full-length FbpB-HA-′PhoA and Rv1566c′-′PhoA fusions are indicated by arrows. ′PhoA breakdown products are identified by a bracket near the bottom of the blots. The asterisks indicate cross-reacting products. Strains grown overnight in the presence of sodium azide are indicated below the blot. Plasmid pMB174 expresses ′phoA, pMB206 expresses an fbpB-HA-′phoA fusion, pMB202 expresses an rv1566c-′phoA fusion, and pMB196 expresses a pepA-′phoA fusion.