The C-tail anchored TssL subunit, an essential protein of the enteroaggregative Escherichia coli Sci-1 Type VI secretion system, is inserted by YidC

Abstract

Type VI secretion systems (T6SS) are macromolecular complexes present in Gram-negative bacteria. T6SS are structurally similar to the bacteriophage cell-puncturing device and have been shown to mediate bacteria-host or bacteria-bacteria interactions. T6SS assemble from 13 to 20 proteins. In enteroaggregative Escherichia coli (EAEC), one of the subassemblies is composed of four proteins that form a trans-envelope complex: the TssJ outer membrane lipoprotein, the peptidoglycan-anchored inner membrane TagL protein, and two putative inner membrane proteins, TssL and TssM. In this study, we characterized the TssL protein of the EAEC Sci-1 T6SS in terms of localization, topology, and function. TssL is a critical component of the T6SS, anchored to the inner membrane through a single transmembrane segment located at the extreme C-terminus of the protein. We further show that this transmembrane segment is essential for the function of the protein and its proper insertion in the inner membrane is dependent upon YidC and modulated by the Hsp70 homologue DnaK.

Keywords: Hcp; inner membrane; insertion; protein trafficking; topology.

Figure 1

The TssL protein is required for Type VI secretion. (A) Effect of the tssL mutation on Hcp protein release. Hcp_HA release was assessed by separating whole cells (WC) and supernatant (Sn) fractions from WT, tssL, or complemented tssL (tssL^WT) cultures. A total of 2 × 10⁸ cells and the TCA-precipitated material of the supernatant from 5 × 10⁸ cells were subjected to 12.5% acrylamide SDS-PAGE and immunodetected using the anti-HA monoclonal antibody (lower panel) and the anti-TolB polyclonal antibodies (lysis control; upper panel). (B) Effect of the tssL mutation on biofilm formation. Biofilms formed in static cultures of WT, tssL or complemented tssL (tssL^WT) cells were visualized on cover glass by crystal violet staining (upper panel) and quantified using the ethanol-solubilization procedure, relative to the WT EAEC strain (lower graph).

Figure 2

TssL is an integral inner membrane protein. (A) TssL co-fractionates with integral membrane proteins. A fractionation procedure was applied to EAEC cells producing _FlagTssL (T, total fraction), allowing separation between the soluble (S) and membrane (M) fractions. Membranes were then treated with urea to separate peripheral membrane (pM) and integral membrane (iM) proteins. Samples were subjected to 12.5% acrylamide SDS-PAGE and immunodetected with antibodies directed against the EFTu (soluble), TolR (integral inner membrane), and OmpA (integral outer membrane) proteins, and the Flag epitope of TssL. (B) Total membranes from EAEC cells producing _FlagTssL (T) were treated with SLS, and solubilized inner membrane (IM) and insolubilized outer membrane (OM) proteins were separated. Samples from 5 × 10⁸ cells were subjected to 12.5% acrylamide SDS-PAGE and immunodetected with antibodies directed against the TolR (inner membrane), OmpA (outer membrane) proteins, and against the Flag epitope of TssL. (C) Total membranes (T) from EAEC cells producing _FlagTssL were separated on a discontinuous sedimentation sucrose gradient. Collected fractions were analyzed for contents using the anti-AcrA (inner membrane), anti-OmpF (outer membrane), and anti-Flag antibodies, and with a NADH oxidase (inner membrane) activity test (upper graph). Although AcrA is an integral IM protein, its distribution in the sucrose gradient extends to the OM fractions due to its interaction with the OM protein TolC. The positions of the inner and outer membrane-containing fractions are indicated. Molecular weight markers are indicated on the left of each panel.

Figure 3

Topology of the EAEC TssL protein. (A) Accessibility of cysteine residues. Whole EAEC tssL cells producing the _FlagTssL or _FlagTssL-Lt (_FlagTssL carrying a C-terminal CCPGCC motif) were treated (+) or not (−) with the 3-(N-)MPB probe, solubilized, and the TssL proteins were precipitated using agarose beads coupled to M2 anti-FLAG antibody. Precipitated material was subjected to SDS-PAGE and Western blot analysis using anti-FLAG antibody (to detect TssL, upper panel) and streptavidin coupled to alkaline phosphatase (to detect biotinylated TssL, lower panel). Molecular weight markers are indicated on the left. (B) Accessibility of TssL to proteinase K. Whole cells (WC), spheroplasts (Sph.), or purified membranes (Membr.) of tssL EAEC cells producing the _FlagTssL protein were treated (+) or not (−) with proteinase K and subjected to SDS-PAGE and Western-blot analyses using anti-TolR (an in-to-out topology inner membrane protein with the bulk of the protein in the periplasm, lower panel) and anti-FLAG (upper panel) antibodies. (C) Accessibility of TssL to CPY. Purified membranes of tssL EAEC cells producing the _FlagTssL protein (TssL) or a _FlagTssL protein fused to the 211-amino acid periplasmic domain of TagL (TssL-PG) were treated (+) or not (−) with CPY and subjected to SDS-PAGE and Western-blot analyses using anti-TolR (lower panel) and anti-FLAG (upper panel) antibodies. (D) Topology model for the TssL protein at the inner membrane. The positions of the labeled (C-terminus) and unlabeled (C40, C68, C127, C129, and C200) cysteine residues are indicated by filled and open circles, respectively. The membrane boundaries of the transmembrane segment predicted by computer algorithms and identified by the accessibility studies are indicated.

Figure 4

TssL transmembrane anchor is required for function. (A) Hcp protein release. Hcp_HA release (produced from plasmid pHcp_HA) was assessed by separating whole cells (WC) and supernatant (Sn) fractions from cultures of tssL, or tssL cells producing WT TssL (tssL^WT) or TssL deleted of its C-terminal transmembrane anchor (tssL^ΔTM). A total of 2 × 10⁸ cells and the TCA-precipitated material of the supernatant from 5 × 10⁸ cells were subjected to 12.5% acrylamide SDS-PAGE and immunodetected using the anti-HA monoclonal antibody (lower panel) and the anti-TolB polyclonal antibodies (lysis control; upper panel). (B) Biofilm formation. Biofilms formed in static cultures of tssL, tssL^WT, or tssL^ΔTM cells were visualized on cover glass by crystal violet staining (upper panel) and quantified using the ethanol-solubilization procedure, relative to the WT EAEC strain (lower graph).

Figure 5

TssL insertion is dependent upon YidC and DnaK. Soluble proteins (soluble and peripherally associated membrane proteins; S) and integral inner membrane proteins (IM) were collected from various genetic backgrounds (WT-1, MC4100; secA, MM52 grown in restrictive conditions; secB, A443; tat, DADE; ftsY, IY28 grown in restrictive conditions; yidC, FTL10 grown in restrictive conditions; yidC⁺, FTL10 grown in permissive conditions; WT-2, BW25113; dnaKJ, PK101; dnaJ, GP108; dnaJ djlA, GP110; tig, A1091) and subjected to 12.5% acrylamide SDS-PAGE. The localization of the TolR and _FlagTssL proteins was assessed by immunodetection using anti-TolR and anti-FLAG antibodies, respectively. Arrows indicate soluble fractions of strains in which the TssL protein is not properly inserted in the inner membrane. Molecular weight markers are indicated on the left.