Systematic Dissection of the Agrobacterium Type VI Secretion System Reveals Machinery and Secreted Components for Subcomplex Formation

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.


Information S1
Plasmid construction and generation of in-frame deletion mutants The plasmid pJQ200KS-ΔtssF was created by ligating the SacI/BamHI-digested tssF PCR product 1 (~500 bp DNA fragment upstream of the tssF open reading frame [ORF]) and the BamHI/XmaI-digested tssF PCR product 2 (~500 bp DNA fragment downstream of the tssF ORF) into SacI/XmaI sites of pJQ200KS and used to generate the deletion mutant ΔtssF (EML1090). The rest of pJQ200KS derivatives (Supplementary Table S1) were created by ligating the XbaI/BamHI-digested PCR product 1 (~500 bp DNA fragments upstream of each target gene) and the BamHI/XmaI-digested PCR product 2 (~500 bp DNA fragments downstream of each target gene) into XbaI/XmaI sites of pJQ200KS and used to generate each of the deletion mutants (Supplementary Tables S1 and S2). For each in-frame deletion mutant confirmed by PCR, at least 2 independent colonies were selected to determine its ability in mediating Hcp secretion.
For complementation, the gene of interest containing its ribosomal-binding sequence (RBS) and ORF was cloned to be driven by a lac promoter on the broad host range vector pRL662 [1]. The PCR products of tssK and clpV genes were digested by HindIII/XbaI and cloned into the same sites of pRL662 to create the plasmids pTssK and pClpV. The PCR-amplified fha and tssE genes were digested by BamHI/XbaI and cloned into the same sites of pRL662 to create the plasmids pFha and pTssE. The remaining genes were amplified with primers described in Supplementary Table S2, and the PCR products were digested by XhoI/XbaI and cloned into the same sites of pRL662 to create the plasmids pTssG, pTssF, pTssC 40 , pTssC 41 , pTssB, pTssA, pHcp, pAtu4346, pAtu4347, pVgrG-1, and pVgrG-2.
To convert each of Δfha and ΔtssC 41 back to the wild type (revertant), pJQ200KS derivatives harboring the fha and tssC 41 genes, including their respective upstream and downstream regions, were used for double crossover. The PCR-amplified products were digested by XbaI/XmaI and cloned into the same sites of pJQ200KS to create the plasmids pJQ200KS-fha, and pJQ200KS-tssC 41 . The resulting revertants were designated as EML2137 & 2138 (fha R-1 and R-2), and EML2141 & 2142 (tssC 41 R-1 and R-2), respectively. The expression vector pET22b(+) was used to overexpress proteins driven by the T7 promoter via IPTG induction in E. coli BL21 (DE3). Each ORF (without stop codon) of ppkA, tssK, fha, tssE, tssC 41 , tssB, tssA, atu4346, atu4347, atu4349, vgrG-1, rpoA, and aopB was PCR-amplified with primers described in Supplementary Table  S2 and cloned into the same sites of pET22b(+) with appropriate enzyme sites. The tssB-tssC 41 fragment (without stop codon) was PCR-amplified with primers described in Supplementary Table S2 and cloned into the same sites of pET22b(+) with appropriate enzyme sites to create the plasmid pET-TssB-TssC 41 -His. The pppA and clpV ORFs (without stop codon) were PCR-amplified, digested by HindIII, and cloned into pET22b(+), which was first digested by NdeI, followed by Klenow repair, and finally digested by HindIII. To construct the pET-N-TssL-His for expressing the N terminus (residues 1 to 255) of TssL, the plasmid pAD-N-TssL [2] was digested by NdeI/XhoI and cloned into the same sites of pET22b(+).
The plasmid pTssB-Strep used in Strep-Tag pull down assay was created by PCR amplifying tssB ORF with primers described in Supplementary Table S2 and cloned into the XhoI/XbaI sites of pRL662.
For the constructs used for yeast two-hybrid, the tssC 41 and tssB ORFs (without stop codon) were PCR-amplified with primers described in Supplementary Table S2, digested by NdeI/BamHI, and cloned into the same sites of pGBKT7 or pGADT7 to create the plasmids pGBKT7-TssC 41 , pGBKT7-TssB, pGADT7-TssC 41 , and pGADT7-TssB, respectively.

Biochemical fractionation
Isolation of A. tumefaciens cellular fractions was as described [2].