3.A.23 The Type VI Symbiosis/Virulence Secretory System (T6SS) Family

Some Vibrio cholerae strains do not cause cholera and instead cause human infections. One such strain possesses on its small chromosome a 15 cistron operon that catalyze protein secretion A hydrophobic N-terminal sequence is not required (Pukatzki et al., 2006; Filloux et al., 2008). The protein substrates can be secreted into the extracellular medium, into eukaryotic cells, and into other bacteria. The genes have been named vasA-L (virulence-associated secretion) or the type VI secretion system (T6SS). At least some of the vas genes are required for cytotoxicity by a contact-dependent mechanism. Since major reassembly of a T6SS is necessary after each secretion event, accurate timing and localization of T6SS assembly is important (Lin et al. 2022). While Acinetobacter and Burkholderia thailandensis can assemble T6SS at any site, a significant subset of T6SS assemblies localizes precisely to the site of contact between neighboring bacteria (Lin et al. 2022).  Type VI secretion system dynamics have been modeled as a state-dependent random walk (Miller and Murray 2023).  Type VI secretion system dynamics have been modeled as a state-dependent random walk (Miller and Murray 2023).

The Type VI secretion system (T6SS) is an injection apparatus that uses a spring-like mechanism for effector delivery. The contractile tail is composed of a needle tipped by a sharpened spike and wrapped by the sheath that polymerizes in an extended conformation on the assembly platform or baseplate. Contraction of the sheath propels the needle and effectors associated with it into target cells. The passage of the needle through the cell envelope of the attacker is assured by a dedicated trans-envelope channel complex. This membrane complex (MC) consists of the TssJ lipoprotein, and the TssL and TssM inner membrane proteins. MC assembly is a hierarchized mechanism in which the different subunits are recruited in a specific order: TssJ, TssM and then TssL. Once assembled, the MC serves as a docking station for the baseplate. In enteroaggregative E. coli, the MC is accessorized by TagL, a peptidoglycan-binding (PGB) inner membrane-anchored protein. Santin et al. 2019 showed that the PGB domain is the only functional domain of TagL, and that the N-terminal TMS mediates contact with the TssL transmembrane helix. TagL is recruited to the membrane complex downstream of TssL and is not required for baseplate docking.

Many Gram-negative bacteria have homologous genes as well as potential effector genes. The V. cholerae Vas system may secrete proteins called Hep, VrgG1, VrgG2 and VrgG3. VasK (1181 aas) resembles IcmF (973 aas; AAU26555) of the Legionella pneumophila Dot system throughout most of its length. Similarly, VasF (257 aas) resembles IcmH (261 aas; AAU26556) of the Dot system. Throughout most of its length and like the VasF protein, IcmH shows 4 peaks of hydrophobicity. VasF and IcmH also show sequence similarity with portions of the OmpA/MotB proteins, possibly possessing a peptidoglycan binding domain. While all 15 cistrons of the vas operon are presented under TC# 3.A.23.1.1, it is unlikely that they all function directly in transport. For example, VasG resembles the E. coli ClpB ATP-dependent, stress-induced, heat shock chaperone protein that uses ATP hydrolysis to unfold denatured proteins in conjunction with DnaJK and GrpE. Moreover, VasH resembles a sigma-54-dependent transcriptional activator, and a sigma-54 is encoded downstream of the vas operon. Only VasA, VasF and VasK have been shown to promote virulence. ATP hydroloysis is involved in the energization process (Pukatzki et al. 2006; Basler and Mekalanos 2012).

In Vibrio cholerae, the T6SS secretes three related proteins extracellularly, VgrG-1, VgrG-2, and VgrG-3. Protein structure search algorithms predict that VgrG-related proteins assemble into a trimeric complex that is analogous to that formed by the two trimeric proteins gp27 and gp5 that make up the baseplate 'tail spike' of Escherichia coli bacteriophage T4. VgrG-1 interacts with itself, VgrG-2, and VgrG-3, suggesting that such a complex does form (Pukatzki et al. 2007). Because the phage tail spike protein complex acts as a membrane-penetrating structure as well as a conduit for the passage of DNA into phage-infected cells, the VgrG components of the T6SS apparatus may assemble a 'cell-puncturing device' analogous to phage tail spikes to deliver effector protein domains through membranes of target host cells. As noted above, the VgrGs, share structural features with the cell-puncturing device of the T4 bacteriophage. Possibly, they are used in a similar fashion by bacteria to puncture host cell membranes and insert the T6SS apparatus into the host cytosol. A number of VgrGs contain C-terminal extensions with effector-domains. Thus, the VISP may translocate soluble effectors as well as VgrG effector-domains (Pukatzki et al. 2009). The type VI secretion apparatus and phage tail-associated protein complexes share a common evolutionary origin (Leiman et al. 2009).

Several Gram-negative bacterial pathogens and symbions have T6SSs (Saier 2006). In Edwardsiella tarda, an E. tarda virulent protein (EVP) gene cluster is involved in protein secretion, and the entire EVP cluster codes for a T6SS. All the 16 EVP genes were mutagenized; the secretion of EvpP was dependent on 13 EVP proteins including EvpC (a homologue of Hcp) and EvpI but not EvpD and EvpJ. The 16 EVP proteins were grouped according to their functions and cellular locations. The first group comprises 11 non-secreted and possibly intracellular apparatus proteins. Among them, EvpO, a putative ATPase which contains a Walker A motif, shows possible interactions with three EVP proteins (EvpA, EvpL and EvpN). The second group includes three secreted proteins (EvpC, EvpI and EvpP). The secretion of EvpC and EvpI is mutually dependent, and they are required for the secretion of EvpP. An interaction between EvpC and EvpP was demonstrated. Two proteins (EvpD and EvpJ) are not required for the T6SS-dependent secretion (Zheng and Leung 2007).

Protein secretion by the T6SS of Vibrio cholerae requires the action of a dynamic intracellular tubular structure that is structurally and functionally homologous to contractile phage tail sheaths. Sheaths of the T6SS cycle between assembly, quick contraction, disassembly and re-assembly (Basler et al. 2012). Whole-cell electron cryotomography showed that the sheaths appear as long tubular structures in either extended or contracted conformations that are connected to the inner membrane by a distinct basal structure. Possibly, contraction of the type VI secretion system sheath provides the energy needed to translocate proteins out of effector cells and into adjacent target cells.

Type 6 secretion dynamics within and between bacterial cells have been measured (Basler and Mekalanos 2012). ClpV imaging provided evidence that P. aeruginosa likely recycles T6SS membrane base plate components and can sense T6SS activity in nearby cells (Basler and Mekalanos 2012). Because T6SS dueling events were spatially and temporally linked, they likely mark the exact location of T6SS translocation of protein components (e.g., VgrG and/or effector proteins) between cells. T6SS dueling may reflect social interactions between heterologous T6SS+ species that coexist in the same niche.  One of the substrates of a T6SS in Dickeya dadantii is an Rhs protein that mediates intercellular competition with other bacteria (Koskiniemi et al. 2013).  This protein carries polymorphic C-terminal toxin domains which are deployed to inhibit the growth of neighboring cells.  They also contain immunity protein domains that neutralize the toxins to protect the producing cells.  In Bacillus subtilis,a  distantly related WapA proteini is exported via the general secretory pathway and delivers the tRNase toxins into neighboring target cells (Koskiniemi et al. 2013).

The T6SS is a dynamic organelle that bacteria use to target prey cells for inhibition via translocation of effector proteins. Opposed T6SS organelles in adjacent sister cells of Pseudomonas aeruginosa give rise to 'T6SS dueling' and likely reflect a biological process that is driven by T6SS antibacterial attack. Basler et al. (2013) have shown T6SS dueling behavior influences the ability of P. aeruginosa to prey upon heterologous bacterial species. T6SS-dependent killing of either Vibrio cholerae or Acinetobacter baylyi is greatly stimulated by T6SS activity occurring in those prey species. Thus, in P. aeruginosa, T6SS organelle assembly and lethal counterattack are regulated by a signal that corresponds to the point of attack of the T6SS apparatus elaborated by a second aggressive T6SS+ bacterial cell. 

Bacterial T6SSs are akin to bacteriophage tails, with VgrG proteins acting as a puncturing device. The P. aeruginosa H1-T6SS is involved in bacterial killing and the delivery of three toxins, Tse1-3. Hachani et al. 2014 demonstrated the independent contribution of the three H1-T6SS co-regulated vgrG genes, vgrG1abc, to bacterial killing. A putative toxin is encoded in the vicinity of each vgrG gene, supporting the concept of specific VgrG/toxin couples. VgrG1c is involved in the delivery of an Rhs protein, RhsP1. The RhsP1 C-terminus carries a toxic activity, from which the producing bacterium is protected by immunity. VgrG1a-dependent toxicity is associated with the PA0093 gene encoding a two-domain protein with a putative toxin domain (Toxin_61) at the C terminus.  VgrG1b-dependent killing is detectable upon complementation of a triple vgrG1abc mutant. The VgrG1b-dependent killing is mediated by PA0099, which has the characteristics of superfamily nuclease 2 toxins. Several additional vgrG genes are encoded on the P. aeruginosa genome and are not linked genetically to other T6SS genes; they encode putative toxins. These associations support the notion of a form of secretion system in which VgrG acts as the carrier (Hachani et al. 2014).

Natural competence for transformation allows horizontal gene transfer and occurs through the uptake of external DNA followed by integration into the genome (see TC# 3.A.11). Borgeaud et al. 2015 showed that the T6SS, which serves as a predatory killing device, is part of the competence regulon in Vibrio cholerae (see TC# 3.A.11.2.3). The T6SS-encoding gene cluster is under positive control by the competence regulators, TfoX and QstR, and is induced by growth on chitinous surfaces (Borgeaud et al. 2015). Live-cell imaging revealed that deliberate killing of nonimmune cells via competence-mediated induction of T6SS releases DNA and makes it accessible for horizontal gene transfer in V. cholerae

The generalized nomenclature for T6SSs uses the Tss nomenclature, TssA - M, for the core constituents of a system (Cianfanelli et al. 2016).  TssJLM form a membrane complex, solved at 11.6 Å, showing a structure with 5-fold symmetry which extends from a substantial base in the cytoplasm and inner membrane, through periplasmic arches, into a periplasmic tip complex contacting the outer membrane.  This complex is assembled sequentially from TssJ, TssM, and TssL.  Large conformational changes on assembly of an active T6SS cause the C-terminus of TssM to cross the outer membrane, forming a transient pore through which the puncturing spike passes (Cianfanelli et al. 2016)

Contractile injection systems mediate bacterial cell-cell interactions by a bacteriophage tail-like structure. In contrast to extracellular systems, the type 6 secretion system (T6SS) is defined by intracellular localization and attachment to the cytoplasmic membrane. Böck et al. 2017 used cryo-focused ion beam milling, electron cryotomography, and functional assays to study a T6SS in Amoebophilus asiaticus The in situ architecture revealed three modules, including a contractile sheath-tube, a baseplate, and an anchor. All modules showed conformational changes upon firing. Lateral baseplate interactions coordinated T6SSs in hexagonal arrays. The system mediated interactions with host membranes and may participate in phagosome escape. Evolutionary sequence analyses predicted that T6SSs are widespread. (Böck et al. 2017). T6SSs and their effectors have been reviewed (Russell et al. 2014; Silverman et al. 2012; Galán and Waksman 2018; Clemens et al. 2018; Lennings et al. 2018; Iacob et al. 2018).

prePAAR is a conserved motif found in over 6000 putative TMS-containing effectors encoded predominantly by 15 genera of Proteobacteria. Based on differing numbers of TMSs, effectors group into two distinct classes that both require a member of the Eag family of T6SS chaperones for export. Co-crystal structures of class I and class II effector TMS-chaperone complexes from Salmonella enterica Typhimurium and Pseudomonas aeruginosa, respectively, revealed that Eag chaperones mimic transmembrane helical packing to stabilize effector TMSs (Ahmad et al. 2020). In addition to participating in the chaperone-TMD interface, prePAAR residues mediate effector-VgrG spike interactions. Thus, mechanisms of chaperone-mediated stabilization and secretion of two distinct families of T6SS membrane protein effectors have been identified.


The reaction catalyzed by most T6SSs is:

Protein and/or DNA (cytoplasm of the donor) → Protein and/or DNA (cytoplasm of the recipient).

This family belongs to the ATP-dependent Clp Protease (Clp) Superfamily.



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Basler, M. and J.J. Mekalanos. (2012). Type 6 secretion dynamics within and between bacterial cells. Science 337: 815.

Basler, M., B.T. Ho, and J.J. Mekalanos. (2013). Tit-for-Tat: Type VI Secretion System Counterattack during Bacterial Cell-Cell Interactions. Cell 152: 884-894.

Basler, M., M. Pilhofer, G.P. Henderson, G.J. Jensen, and J.J. Mekalanos. (2012). Type VI secretion requires a dynamic contractile phage tail-like structure. Nature 483: 182-186.

Böck, D., J.M. Medeiros, H.F. Tsao, T. Penz, G.L. Weiss, K. Aistleitner, M. Horn, and M. Pilhofer. (2017). In situ architecture, function, and evolution of a contractile injection system. Science 357: 713-717.

Borgeaud, S., L.C. Metzger, T. Scrignari, and M. Blokesch. (2015). Bacterial evolution. The type VI secretion system of Vibrio cholerae fosters horizontal gene transfer. Science 347: 63-67.

Chang, Y.W., L.A. Rettberg, D.R. Ortega, and G.J. Jensen. (2017). In vivo structures of an intact type VI secretion system revealed by electron cryotomography. EMBO Rep 18: 1090-1099.

Cianfanelli, F.R., L. Monlezun, and S.J. Coulthurst. (2016). Aim, Load, Fire: The Type VI Secretion System, a Bacterial Nanoweapon. Trends Microbiol. 24: 51-62.

Clemens, D.L., B.Y. Lee, and M.A. Horwitz. (2018). The Type VI Secretion System. Front Cell Infect Microbiol 8: 121.

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Filloux, A., A. Hachani, and S. Bleves. (2008). The bacterial type VI secretion machine: yet another player for protein transport across membranes. Microbiology 154: 1570-1583.

Galán, J.E. and G. Waksman. (2018). Protein-Injection Machines in Bacteria. Cell 172: 1306-1318.

Hachani, A., L.P. Allsopp, Y. Oduko, and A. Filloux. (2014). The VgrG proteins are "à la carte" delivery systems for bacterial type VI effectors. J. Biol. Chem. 289: 17872-17884.

Hachani, A., T.E. Wood, and A. Filloux. (2016). Type VI secretion and anti-host effectors. Curr. Opin. Microbiol. 29: 81-93.

Hurst, M.R., T.R. Glare, and T.A. Jackson. (2004). Cloning Serratia entomophila antifeeding genes--a putative defective prophage active against the grass grub Costelytra zealandica. J. Bacteriol. 186: 5116-5128.

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Koskiniemi, S., J.G. Lamoureux, K.C. Nikolakakis, C. t'Kint de Roodenbeke, M.D. Kaplan, D.A. Low, and C.S. Hayes. (2013). Rhs proteins from diverse bacteria mediate intercellular competition. Proc. Natl. Acad. Sci. USA 110: 7032-7037.

Leiman, P.G. and M.M. Shneider. (2012). Contractile tail machines of bacteriophages. Adv Exp Med Biol 726: 93-114.

Leiman, P.G., M. Basler, U.A. Ramagopal, J.B. Bonanno, J.M. Sauder, S. Pukatzki, S.K. Burley, S.C. Almo, and J.J. Mekalanos. (2009). Type VI secretion apparatus and phage tail-associated protein complexes share a common evolutionary origin. Proc. Natl. Acad. Sci. USA 106: 4154-4159.

Lennings, J., T.E. West, and S. Schwarz. (2018). The Type VI Secretion System 5: Composition, Regulation and Role in Virulence. Front Microbiol 9: 3339.

Lin, L., R. Capozzoli, A. Ferrand, M. Plum, A. Vettiger, and M. Basler. (2022). Subcellular localization of Type VI secretion system assembly in response to cell-cell contact. EMBO. J. 41: e108595.

Miller, J. and P.J. Murray. (2023). Space and time on the membrane: modelling Type VI secretion system dynamics as a state-dependent random walk. R Soc Open Sci 10: 230284.

Pukatzki, S., A.T. Ma, A.T. Revel, D. Sturtevant, and J.J. Mekalanos. (2007). Type VI secretion system translocates a phage tail spike-like protein into target cells where it cross-links actin. Proc. Natl. Acad. Sci. USA 104: 15508-15513.

Pukatzki, S., A.T. Ma, D. Sturtevant, B. Krastins, D. Sarracino, W.C. Nelson, J.F. Heidelberg, and J.J. Mekalanos. (2006). Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system. Proc. Natl. Acad. Sci. USA 103: 1528-1533.

Pukatzki, S., S.B. McAuley, and S.T. Miyata. (2009). The type VI secretion system: translocation of effectors and effector-domains. Curr. Opin. Microbiol. 12: 11-17.

Russell, A.B., S.B. Peterson, and J.D. Mougous. (2014). Type VI secretion system effectors: poisons with a purpose. Nat. Rev. Microbiol. 12: 137-148.

Saier, M.H., Jr. (2006). Protein secretion and membrane insertion systems in gram-negative bacteria. J. Membr. Biol. 214: 75-90.

Santin, Y.G., C.E. Camy, A. Zoued, T. Doan, M.S. Aschtgen, and E. Cascales. (2019). Role and recruitment of the TagL peptidoglycan-binding protein during Type VI secretion system biogenesis. J. Bacteriol. [Epub: Ahead of Print]

Shikuma, N.J., M. Pilhofer, G.L. Weiss, M.G. Hadfield, G.J. Jensen, and D.K. Newman. (2014). Marine tubeworm metamorphosis induced by arrays of bacterial phage tail-like structures. Science 343: 529-533.

Shneider, M.M., S.A. Buth, B.T. Ho, M. Basler, J.J. Mekalanos, and P.G. Leiman. (2013). PAAR-repeat proteins sharpen and diversify the type VI secretion system spike. Nature 500: 350-353.

Silverman, J.M., Y.R. Brunet, E. Cascales, and J.D. Mougous. (2012). Structure and regulation of the type VI secretion system. Annu. Rev. Microbiol. 66: 453-472.

Zheng, J. and K.Y. Leung. (2007). Dissection of a type VI secretion system in Edwardsiella tarda. Mol. Microbiol. 66: 1192-1206.


TC#NameOrganismal TypeExample

Type VI secretion system (T6SS) VasA-L + Vca0107-09; + Vca0123 + Vc1416 (Pukatzki et al., 2006; Pukatzki et al., 2007). This secretion system displays antimicrobial properties (Macintyre et al., 2010).  It functions like contractile tails of phage and penetrates cells with a trimeric VgrG spike protein to which are associated PAAR repeat proteins that sharpen the tip of the spike and are released into the cytoplasm of the target cell (Shneider et al. 2013). Certain Vibrionaceae adapted their antibacterial T6SS to mediate interactions with eukaryotic hosts or predators, promoting their toxicity (Dar et al. 2018).


Vca0107-0109 + VasA-L + Vca0123 + Vc1416 of Vibrio cholerae
Vca0107 (168 aas) (NP_232508)
Vca0108 (492 aas) (NP_232509)
Vca0109 (145 aas) (NP_232510)
VasA (Vca0110; 589 aas; 0-2 C-terminal TMSs) (AAF96024)
VasB (Vca0111; 338 aas; 0 TMSs) (AAF96025)
VasC (Vca0112; 495 aas; 0 TMSs) (AAF96026)
VasD (Vca0113; 158 aas; 1 N-terminal TMS) (AAF96027)
VasE (Vca0114; 444 aas; 0[-19?] TMSs) (AAF96028)
VasF (resembles IcmH) (Vca0115; 257 aas; 1[-4?] TMSs) (AAF96029)
VasG (ClpB) (Vca0116; 869 aas; 0[-3?] TMSs) (AAF96030)
VasH (sigma-54 dep. Tx activator) (Vca0117; 530 aas; 0 TMSs) (AAF96031)
VasI (Vca0118; 227 aas; 1 N-terminal TMS) (AAF96032)
VasJ (Vca0119; 469 aas; 0[-4] TMSs) (AAF96033)
VasK (resembles IcmF) (Vca0120; 1181 aas; 2 N-terminal TMSs) (AAF96034)
VasL (Vca0121; 421 aas; 1 central TMS) (AAF96035) Vc1416/KgrG (1163 aas) (NP_231059)
Vca0123 (/VgrG 1017 aas) (NP_232524)
Possible cell puncture device, VgrG-1, G-2, G-3 of Vibrio cholera Type VII S-5.
VgrG-1 (197 aas) (A1F9V7)
VgrG-2 (694 aas) (A3E8Q2) (like EupI of E. tarda)
VgrG-3 (1017 aas) (B2D7K2)


TC#NameOrganismal TypeExample
3.A.23.2.1Type VI secretion system, EvpA-P (Zheng and Leung, 2007)BacteriaEvpA-P of Edwardsiella tarda
EvpA, 171 aas (Q6EE21)
EvpB, 495 aas (Q6EE20)
EvpC, 163 aas (Q6EE19)
EvpD, 407 aas (Q6EE18)
EvpE, 158 aas (Q6EE17)
EvpF, 613 aas (Q6EE16)
EvpG, 341 aas (Q6EE15)
EvpH, 870 aas (Q6EE14)
EvpI, 661 aas (A8YQR5)
EvpJ, 100 aas (A8YQR6)
EvpK, 335 aas (A8YQR7)
EvpL, 235 aas (A8YQR8)
EvpM, 462 aas (A8YQR9)
EvpN, 216 aas (A8YQS0)
EvpO, 1263 aas (A8YQS1)
EvpP, 185 aas (A8YQR4)

TC#NameOrganismal TypeExample

Type VI secretion system TssA - TssG plus TssI - TssM (Cianfanelli et al. 2016).

Type VI secretion system TssA - TssG plus TssI - TssM of Campylobbacter jejuni
TssA, 415 aas
TssB, 161 aas
TssC, 484 aas
TssD, 171 aas
TssE, 130 aas
TssF, 573 aas
TssG, 302 aas
TssI, 838 aas
TssJ, 148 aas
TssK, 465 aas
TssL, 257 aas
TssM, 1175 aa


TC#NameOrganismal TypeExample

Type VI secretion system, TssA - TssH plus TssK - TssM (Cianfanelli et al. 2016).

Type VI secretion system, TssA - TssH plus TssK - TssM of Acinetobacter baumannii
TssA, 364 aas
TssB, 167 aas
TssC, 493 aas
TssD (Hcp), 167 aas
TssE (lysozyme), 158 aas
TssF (ImpG; VasA), 603 aas
TssG, 332 aas
TssH, 894 aas
TssK, 454 aas
TssL, 268 aas
TssM, 1252 aa


TC#NameOrganismal TypeExample

Type VI secretion system, TssB - TssM (Cianfanelli et al. 2016).

TssB - TssM of Geobacullus sulfurreducens
TssB, 161 aas
TssC, 494 aas
TssD, 161 aas
TssE, 135 aas, needle hub
TssF, 577 aas
TssG, 330 aas
TssH, 875 aas
TssI, 697 aas, needle syringe
TssJ, 814 aas
TssK, 463 aas
TssL, 227 aas
TssM, 1154 aa


TC#NameOrganismal TypeExample

To interact with other cells, bacteria use contractile machines that function similarly to membrane-puncturing bacteriophages. The so-called type 6 secretion system (T6SS) functions from inside a bacterial cell. Böck et al. used modern electron microscopy methods and functional assays to resolve the structure and function of a T6SS in the cellular context. They identified three modules and showed large-scale structural changes upon firing. T6SSs are organized in multibarrel gun-like arrays and may contribute to the survival of bacteria inside their host. 

Contractile injection systems (CISs) deliver effectors to mediate bacterial cell-cell interactions. Their structural components are homologous to the contractile tails of phages (1). CISs consist of an inner tube surrounded by a contractile sheath, a spike capping the inner tube, and a baseplate complex at the base of the sheath. Sheath contraction propels the inner tube into the target. Two modes of action divide CISs into “extracellular CISs” (eCISs) and “type 6 secretion” (T6S) systems (T6SSs). eCISs resemble headless phages; they are released into the medium and bind to the target cell surface. Examples are antibacterial R-type bacteriocins (Leiman and Shneider 2012), insecticidal antifeeding prophages (Afps) (Hurst et al. 2004), and metamorphosis-inducing structures (MACs) (Shikuma et al. 2014). By contrast, the T6SS is defined by its cytoplasmic localization and anchoring to the inner membrane (Basler et al. 2012; Hachani et al. 2016); Chang et al. 2017).

Amoebophilus asiaticus is an obligate intracellular bacterial symbiont of amoebae. The Amoebophilus genome does not encode known secretion systems, but it contains a gene cluster with similarities to that of Afps. Böck et al. 2017 reasoned that the Afp-like gene cluster might encode a system that would give insight into T6SS structure, function, and evolution.

Thus, the Amoebophilus Afp-like gene cluster encodes a T6SS (Böck et al. 2017). Sequence analyses indicated a close relationship to eCISs, and the term “T6SS subtype 4” (T6SSiv) was therefore introduced. In contrast to the distant relationships of T6SSi-iii to eCISs and phages that obstruct the reconstruction of an evolutionary path (1, 24), it can be hypothesized that T6SSiv evolved from an Afp/MAC-like eCIS (independently of T6SSi-iii) by the loss of tail fibers, loss of holin, and the establishment of interactions with the cytoplasmic membrane. Alternatively, T6SSiv represents a primordial system from which eCISs, phages, and T6SSi-iii evolved. T6SSiv-like gene clusters were detected in six diverse bacterial phyla. The finding that diverse T6SS subtypes do not share a conserved gene set that would distinguish them from eCISs or phages emphasizes the necessity of an integrative approach to discover and characterize new systems. This situation is reminiscent of type IV secretion systems (3.A.7).

T6SS of Amoebophilus asiaticus:  The proteins listed are those in the T6SS gene cluster although several of them may not be involved in Type 6 secretion.
B3ET73 of 599 aas and 0 TMSs. Like Spike protein (T4 gp5) (Homologous to constituents (i.e., A8YQR5 in 3.A.23.2.1) and other T6SS systems).
B3ET74 of 102 aas and 0 TMSs. Like Tip protein (T4 gp5.4) (Homologous to constituents (i.e., A8YQR6 in 3.A.23.2.1) of T6SSs).
B3ET75 of 131 aas and 0 TMSs. Like Baseplate (T4 gp25). Resembles TC# 1.K.1.1.1; tail lysozyme).
B3ET76 of 831 aas and 0 TMSs. Like Baseplate (T4 gp25). (A part resembles a part of C3L421 in this same system (3.A.23.6.1)).
C3L421 of 1,561 aas and 0 TMSs.  Like Tape measure protein (T4 gp29). (A part resembles a part of B3ET76 in this same system (3.A.23.6.1)).
B3ERV5 of 247 aas and 0 TMSs.  Like Baseplate (T4 gp48)
B3ERV6 of 1258 aas and 0 - 2 TMSs. Like Baseplate (T4 gp6)
B3ERW2 of 1121 aas and 19 TMSs in a 6 = 1 = 1 = 1 =4 + 6 arrangement. Like Baseplate T4 gp27). TC Blast reveals that the first 13 TMSs hit many members of the SSS family (TC# 2.A.1).
B3ERW3 of 314 aas and 0 or 1 TMSs. Of unknown function in T4
B3ET61 of 169 aas and 1 N-terminal TMS.  Like Baseplate (T4 gp53)
B3ET62 of 331 aas and 0 TMSs.  Like glyceraldehyde 3P dehydrogenase.
B3ERD7 of 274 aas and 1 C-terminal TMS. Possibly an IS4-type transposase.
B3ET64 of 347 aas and 1 N-terminal TMS. Like ankyrin.  Has many short (12 - 18 aa) repeats and hits many proteins in TCDB including TC families 1.A.4, 8.A.28 (ankyrin), 9.A.3 (containing ankyrin repeats), 1.I .1, etc.
B3ET65 of 193 aas and 0 TMSs.  Like Tail terminator protein (T4gp15)
B3ET66 of 282 aas and 0 TMSs.  Like an Afp-like protein of unknown function in T4
B3ET67 of 498 aas and 0 TMSs.  Like a tail sheath protein (T4 gp18). Hits TC# 1.K.1.1.1.
B3ET68 of 167 aas and 0 TMSs.  Like an Afp-like protein of unknown function
B3ET69 of 148 aas and 0 TMSs.  Like an Afp-like protein of unknown function
B3ET70 of 154 aas and 0 TMSs.  Like a tube protein (T4gp19)
B3ET71 of 59 aas and 0 TMSs.  Like an Afp protein of unknown function.
B3ET72 of 377 aas and 0 TMSs. Like a baseplate protein (T4 gp54)