Requirement for YaeT in the outer membrane assembly of autotransporter proteins

Abstract

Autotransporters constitute the largest group of secreted proteins in gram-negative bacteria. Autotransporter secretion involves the insertion of a carboxy-terminal beta barrel into and the translocation of an amino-terminal domain across the outer membrane. Here, we demonstrate that secretion of autotransporters from several organisms requires the outer membrane assembly factor YaeT.

FIG. 1.

Secretion of S. flexneri autotransporters IcsA and SepA is dependent on YaeT. (A) YaeT is essential for growth in S. flexneri. Growth curves of wild-type S. flexneri 2457T (squares) and the 2457T YaeT depletion strain (circles) under replete conditions (0.2% arabinose in LB; filled symbols) or depletion conditions (0.1% fucose in LB; open symbols) at 37°C. Time zero represents the start of growth in depleted versus replete medium; 180 min represents the point of back-dilution of cultures into the same medium. (B) Levels of YaeT and Skp in cell pellets of the wild-type or YaeT depletion strain after 3 h of growth under repletion or depletion conditions, with FtsZ as a loading control. Western blots were probed with YaeT, Skp, or FtsZ antibody. (C and D) Levels of IcsA (C) or SepA (D) in culture supernatants or cell pellets of these strains after growth in repletion or depletion medium for 5 h (3 plus 2 h) as described in the text. Western blots were probed with IcsA, SepA, or FtsZ antibody. The absence of FtsZ in the culture supernatants indicates the absence of cell lysis. The images are representative of those obtained for three or more independent experiments. IcsA′ or SepA′, proteolytically processed mature amino terminus of IcsA or SepA, respectively; IcsA or SepA, mature full-length IcsA or SepA, respectively; ara, 0.2% arabinose; fuc, 0.1% fucose. Molecular size markers are indicated in kilodaltons.

FIG. 2.

DegP-dependent instability of unassembled IcsA following depletion of YaeT in S. flexneri. (A) Growth drops off more rapidly with depletion of both DegP and YaeT than with depletion of YaeT alone. Growth curves of wild-type S. flexneri 2457T (squares), a YaeT depletion strain (circles), a DegP depletion strain (triangles), or a YaeT and DegP double depletion strain (diamonds) under replete conditions (filled symbols) or depletion conditions (open symbols) at 37°C. Growth was as described in the legend of Fig. 1. (B) Levels of IcsA in cell pellets of these strains after growth in repletion or depletion medium for 3 h (1.5 h plus 1.5 h) as described in the text. Western blots were probed with IcsA or FtsZ antibody. (C) Detection of IcsA on the surface of intact bacteria. Indirect immunofluorescence using antibody to IcsA (left panels) and phase-contrast (right panels) images are shown. Bar, 10 um. (D) Heat-modifiable mobility of IcsA. Western blots were probed with IcsA antibody. The two blots were run in parallel using samples that were prepared at the same time. Strains and growth conditions for panels C and D are as in panel B. uIcsA, unfolded IcsA; fIcsA, folded IcsA; IcsA′, proteolytically processed mature amino terminus of IcsA; ara, 0.2% arabinose; fuc, 0.1% fucose. Molecular size markers are indicated in kilodaltons.

FIG. 3.

YaeT is required for the secretion of autotransporters from gram-negative bacilli other than S. flexneri. Levels of AIDA-I (A) or BrkA (B) in cell pellets of E. coli JCM158 or the isogenic YaeT depletion strain (JCM166) carrying an AIDA-I expression plasmid (p-aidA), a BrkA expression plasmid (p-brkA), or an empty vector after growth in repletion or depletion medium for 3 h. Western blots were probed with AIDA-I, BrkA, or FtsZ antibody. (C) Levels of BrkA associated with cell pellets of a YaeT depletion strain after treatment with proteinase K. Western blots were probed with BrkA or β-lactamase antibody. All lanes are from the same blot. AIDA-I or BrkA, mature full-length AIDA-I or BrkA, respectively; AIDA-I′ or BrkA′, proteolytically processed mature amino terminus of AIDA-I or BrkA, respectively; BrkA*, proteinase K cleavage product of BrkA; PK, proteinase K concentration in μg/ml; ara, 0.2% arabinose; fuc, 0.1% fucose. Molecular size markers are indicated in kilodaltons.