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 ⌬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.

β-Lactamase can function as a reporter of bacterial protein export during Mycobacterium tuberculosis infection of host cells

“…In E. coli the 9BlaTEM-1 reporter works with both Sec and Tat signal sequences (Broome-Smith et al, 1990;Stanley et al, 2002). The Sec and Tat pathways appear essential in M. tuberculosis (Braunstein et al, 2001;Saint-Joanis et al, 2006;Sassetti et al, 2003). Therefore, to investigate the mode of export of the ssPlcB-9BlaTEM-1 fusion protein, it was tested in M. In addition to working with the Sec and Tat pathways, the 9BlaTEM-1 reporter has been used with type II and type III secretion systems of Gram-negative bacteria (Charpentier & Oswald, 2004;Sauvonnet & Pugsley, 1996).…”

“…Whole-cell lysates of M. tuberculosis strains were prepared as described previously (Braunstein et al, 2001) with the following modifications. M. tuberculosis cultures were grown in 5 ml volumes to mid-exponential phase.…”

“…Mycobacteria possess two conserved pathways for exporting proteins: the general secretion (Sec) pathway and the twin-arginine translocation (Tat) pathway (Braunstein et al, 2001;Kurtz & Braunstein, 2005;McDonough et al, 2005;Owens et al, 2002;Posey et al, 2006;Saint-Joanis et al, 2006). These systems recognize precursor proteins synthesized with amino-terminal signal sequences and transport them across the cytoplasmic membrane (DeLisa et al, 2003;Mori & Ito, 2001).…”

ADP-dependent Conformational Changes Distinguish Mycobacterium tuberculosis SecA2 from SecA1

“…Several bacterial species, including M. tuberculosis, have been shown to possess an additional SecA protein called SecA2 (7)(8)(9). The mycobacterial secA1 and secA2 genes are genetically distinct and cannot complement each other or deletion of the essential E. coli secA gene (8,10).…”

Bacillus anthracis SlaQ Promotes S-Layer Protein Assembly

“…The genomes of some Gram-positive bacteria harbor two secA genes: one that encodes the canonical SecA ATPase of the protein secretory pathway (23) and another that specifies a paralogue, SecA2, supporting the secretion of only one or a few select substrates (43). SecA2 was first identified in Mycobacterium tuberculosis, where it promotes the transport of proteins across the mycobacterial envelope that aid in the pathogenesis of tuberculosis (44,45). Mycobacterial SecA2 recognizes the mature domains of a few secretory precursors to assist in their secretion, suggesting its primary function may be maintenance of export competence for proteins with the propensity for rapid folding (46).…”