Abstract
The type VI secretion system (T6SS) is widely distributed in pathogenic Proteobacteria. Sequence and structural analysis of T6SS reveals a resemblance to the T4 bacteriophage tail, in which an outer sheath structure contracts an internal tube for injecting nucleic acid into bacterial cells. However, the molecular details of how this phage tail-like T6SS structure is assembled in vivo and executed for exoprotein or effector secretion remain largely unknown. Here, we used a systematic approach to identify T6SS machinery and secreted components and investigate the interaction among the putative sheath and tube components of Agrobacterium tumefaciens. We showed that 14 T6SS components play essential roles in the secretion of the T6SS hallmark exoprotein Hcp. In addition, we discovered a novel T6SS exoprotein, Atu4347, that is dispensable for Hcp secretion. Interestingly, Atu4347 and the putative tube components, Hcp and VgrG, are mainly localized in the cytoplasm but also detected on the bacterial surface. Atu4342 (TssB) and Atu4341 (TssC41) interact with and stabilize each other, which suggests that they are functional orthologs of the sheath components TssB (VipA) and TssC (VipB), respectively. Importantly, TssB interacts directly with the three exoproteins (Hcp, VgrG, and Atu4347), in which Hcp also interacts directly with VgrG-1 on co-purification from Escherichia coli. Further co-immunoprecipitation and pulldown assays revealed these subcomplex(es) in A. tumefaciens and thereby support T6SS functioning as a contractile phage tail-like structure.
Highlights
Protein secretion systems play central roles in export or import of macromolecules across the cell envelope in bacteria
The transcripts of atu4329 and atu4353, genes adjacent to both operons, and housekeeping 16S rRNA gene were expressed at comparable levels in both C58 and Dpro strains (Figure 1B), which suggests that the deletion of this,220-bp intergenic region does not globally affect the expression of genes elsewhere
By co-purification in E. coli, we discovered that Hcp, VgrG-1, and Atu4347 each could interact with the terms of impact on stabilizing Atu4342 (TssB)–TssC41 complex when TssB and TssC41–His were co-expressed in pET22b(+) (Figure 6)
Summary
Protein secretion systems play central roles in export or import of macromolecules across the cell envelope in bacteria. Overexpression of the TssB–TssC proteins, Vibrio cholerae VipA–VipB and Pseudomonas aeruginosa HsiB1–HsiC1, in E. coli produced a cogwheel-like tubular structure [19,20,21]. Both a thinner/extended tube with dense interior and thicker/contracted hollow structure were seen in the cytoplasm of V. cholerae cell by electron cryotomography [22] and immunogold labeling by transmission electron microscopy [23]. The TssB–TssC tubule may wrap around the Hcp tube to form the extended tubule and contract to push the Hcp tube across bacterial membranes from interior cells. How the TssB–TssC tubule accommodates the Hcp tube and coordinates with other T6SS components for T6SS exoprotein or substrate secretion across membranes remain unknown
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