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. 2013 Aug 6;288(38):27031–27041. doi: 10.1074/jbc.M113.499772

FIGURE 5.

FIGURE 5.

TssK1 is required for sheath assembly and Hcp1 release. A, absence of TssK1 abolishes assembly of the T6SS sheath. Time-lapse fluorescence microscopy recordings showing sheath dynamics in WT, tssK1 and complemented tssK1 (tssK1+) cells producing TssB1-sfGFP. Individual images were taken every 30 s. Assembly and contraction events are indicated above the time-lapse images. The scale bars are 2 μm. B, absence of TssK1 prevents Hcp1 release. Hcp1HA release was assessed by separating whole cells (C) and culture supernatant (SN) fractions from 2 × 109 WT, tssK1 or complemented tssK1 (tssK1+) cells producing HA-tagged Hcp1. Proteins were separated by 12.5% acrylamide SDS-PAGE and Hcp1 was immunodetected using anti-HA monoclonal antibody (lower panel). The periplasmic TolB protein (immunodetected using an anti-TolB polyclonal antibodies, upper panel) was used as a marker to verify that no lysis occurred.