Structural model of the flagellar and injectisome T3SS, and comparison with structurally related parts of the F0F1-ATP synthase. Components present in both T3SS are listed in the centre, flagellum-specific and injectisome components on the left and right side, respectively. An alternative location for FliH/SctL [27,28] is indicated by dashed lines. Subunits of the F0F1-ATP synthase are listed on the far right side, components that are structurally related to T3SS components are coloured correspondingly. The c-ring of the F0F1-ATP synthase spans the IM, but has been drawn as corresponding to the FlhA/SctV ring in this image [28,29]. Flagellar model based on the study of Minamino & Imada ; injectisome model modified from ; based on cryo-electromicroscopic data ; F0F1-ATP synthase and structural homologies based on Ibuki et al. . Scale bar and relative size of flagellum/injectisome based on Kawamoto et al. .
Proposed scenario for the evolution of T3SS. Adapted from . Loss of flagellum-specific genes (blue) and acquisition of the injectisome-specific (red) genes sctD and sctI lead to a needle-less injectisome-like T3SS that is still present in Myxococcus. Acquisition of sctC secretins from different cellular machineries allowed the formation of contact-dependent injectisome-type T3SS. In addition to the depicted gene acquisitions and losses, family-specific changes occurred, leading to the eight injectisome subfamilies indicated—the more ancestral Chlamydia T3SS and the Rhizobia, Hrp1, Hrp2, Ysc, Desulfovibrio, SPI-1 and SPI-2 injectisomes. In the rhizobial family, a Tad-like secretin has been acquired independently. Gene acquisition is depicted by incoming arrows and solid lines, gene loss is depicted by outgoing arrows and dashed lines. See Abby & Rocha  for details.
Substrate switching events. This image depicts the switching events at the injectisome; the first two switching events occur in all T3SS, whereas the switch upon host cell contact is injectisome-specific. Proteins involved in the respective switching events are denoted; proteins that are exported upon switching are listed on the cytosolic side. Scheme adapted from . Upon completion of the basic T3SS, consisting of the membrane rings, export apparatus and cytosolic components, the export of early T3SS substrates occurs, including the hook/needle, rod and ruler. In the injectisome, formation of the needle is thought to open the previously closed secretin ring. The length of the rod/needle is surveyed by the molecular ruler FliK/SctP and once the correct length is reached, the ruler and the switch protein FlhB/SctU in the export apparatus are involved in switching the substrate specificity. This allows export of the next substrate class, the tip protein in the injectisome and flagellin and its associated proteins in the flagellum. The injectisome is fully assembled at this point, but does not secrete effectors, until contact to a host cell has been established. This signal is thought to be sensed by the needle tip and transferred via the needle, releasing a cytosolic gatekeeper/plug complex with unknown position and allowing effector export. Refer to main text for details and references.
Inputs into the flagellum/the injectisome. Influence of several external cues (shaded boxes) on expression and function of the flagellum (blue, left side) and the injectisome (red, right side). Arrows: positive influence, lines with blocked end: negative influence, lollipops: pleiotropic/unknown influence. Note that this scheme contains data from various organisms under different conditions and not all mechanisms may be present in all species and at all times. FNR, fumarate and nitrate reductase. Refer to main text for references. (Online version in colour.)
Structural components of the flagellar and injectisome T3SS. As the nomenclature for injectisome components is currently species-specific, we will use the unified Sct nomenclature  in this review. Where no Sct name exists, we will use the Ysc nomenclature (denoted by *). Refer to electronic supplementary material, table S1 for a list of names of injectisome components in specific organisms. Grey shading denotes homologous proteins, italics denotes that the protein can be exported by the T3SS. Extra, extracellular location; OM, outer membrane; PP, periplasm; IM, inner membrane; cyto, cytosolic.
degree of similaritya
Filament-capping protein required for proper folding of the flagellin
Flagellin forming the filament
Hook-filament junction proteins connecting the filament and the hook
Pore-forming hydrophobic translocators
Tip protein (hydrophilic translocator)
Hook-capping protein, scaffold protein required for hook assembly
Hook/needle subunit, helical arrangement with 5–6 subunits per turn
Secretin ring in the OM, requires assistance of pilotin lipoprotein for integration in OM
L-ring, lipoprotein, part of bushing
P-ring, part of bushing
Inner rod in the injectisome. Exported T3SS substrate in some systems. Involved in needle anchoring and/or length determination, not required for connection of the membrane rings
Rod-capping protein with muramidase function
Flagellar rod/transmission shaft. Requires functional T3SS export for assembly
Bitopic outer MS ring protein, connecting the secretin (SctC) in the OM and the inner MS ring (SctJ) in the IM
MS ring protein, thought to surround the export apparatus. Large bitopic protein in the flagellum, significantly smaller and predominantly or completely periplasmic in the injectisome
Flagellum-specific IM export apparatus protein regulating FliP during assembly of the flagellum
IM export apparatus proteins, mainly transmembrane helices and periplasmic domains, possibly forming the pore in the IM
IM export apparatus protein with C-terminal cytosolic domain, involved in substrate specificity switch upon hook/needle completion
IM export apparatus protein with large C-terminal cytosolic domain. Present as a multimer, probably forming a nonameric ring
Stalk protein with homology to γ subunit of FoF1-ATP synthase. Also shown to bind empty chaperones (‘chaperone escort’ function). T3SS substrate in some systems
Hexameric ATPase, probably required for chaperone release and substrate unfolding
Negative regulator of ATPase, also shown to interact with stalk and large export apparatus component, part of injectisome sorting platform
Part of the flagellar switch complex controlling the rotation direction (together with FliM and FliN)
Forming the cytosolic C-ring, part of the switch complex in the flagellum and the injectisome sorting platform
Part of the cytosolic C-ring. SctQC is expressed from an internal translation initiation site in SctQ [25,26]. FliM + FliN and SctQ + SctQC form stable complexes (with a 1 : 4 or 1 : 2 ratio, respectively)
Accessory protein interacting with the C-ring, part of injectisome sorting platform.
aDegree of similarity between flagellar and injectisome components in Y. enterocolitica (see electronic supplementary material, table S2 for details): high, E < 10−5; medium, 10−5 < E < 0.01; low, E > 0.01.
bLocated within MS ring, most likely in a membrane-like environment.
Different subfamilies of injectisome T3SS. The names of the subfamilies, selected species containing T3SS of this subfamily, the distribution throughout bacteria and particularities of the subfamily are listed. The SPI-1 subfamily is sometimes referred to as Inv-Mxi-Spa, the SPI-2 subfamily is also called Ssa-Esc. Data compiled from [12,17,38].