Origins and Diversification of T3SS Figure 7. Proposed scenario for the evolution of NF-T3SS. We propose that genes common to the flagellum and the NF-T3SS were present in the flagellar ancestor of NF-T3SS. They are designated as ‘‘T3SS genes’’. Then we detail the evolution of this system to the extant NF-T3SSs. First, the loss of flagellum-specific genes resulted in loss of the motility function. Presumably this system kept the ability to translocate or secrete proteins. The ancestral NF-T3SS experienced a series of gene losses and gains while diversifying to the ancestor of all extant NF-T3SSs. One early lineage derived into the Myxo systems by loss of some genes, notably SctF. Acquisition of secretins and of a few other genes allowed the formation of ancestral contact-dependent protein secretion systems, and the concomitant ability to subvert eukaryotic cells by direct delivery of effectors in their cytosol. Secretins were recruited to the NF-T3SS at least three times from three different cell machineries. Finally, the NF-T3SS quickly diversified and adapted to different host cells. Some components of NF-T3SS, such as the translocon proteins (YopB/YopD), the needle length determinant (SctP) or the needle tip (LcrV), cannot yet be integrated in this schema because of low or undetectable sequence similarity among T3SSs. Structural and sequence similarities were previously noted between translocon proteins of Chlamydia (CopB) and Salmonella (SipB) [142]. In contrast, we found no more than 28% identity over less than half of the protein when aligning CopB with all complete genomes in GenBank after excluding Chlamy proteins (Blastp, e- value.0.05). We also did not find significant sequence homologs of YopB/YopD in the NF-T3SS of Myxo or plant-associated bacteria. Since the translocon is required for protein delivery but not for secretion [35], it might have been acquired after the secretin. Balloons indicate gene losses and accretions. Only genes mentioned in the main text are shown. Abbreviated names of taxa are as in Figure 4. doi:10.1371/journal.pgen.1002983.g007 [53]. Hence, it is conceivable that a needle-like structure might have assembled at the periplasm in the ancestral NF-T3SS even in the absence of an outer membrane channel. We argue that this system was subsequently transferred across taxa prior to indepen- dent acquisitions of a secretin. The atypical Myxo system The early evolution of the NF-T3SS was accompanied by the accretion of new genes that are present in even the most basal clades of the NF-T3SS (Figure 7). These include the structural genes sctD and sctI and probably also system-specific chaperones and regulators, whose evolutionary patterns are obscured by their fast evolutionary rates. The Myxo NF-T3SS was the first group to split from the others (Figure 7). Myxo systems are only present in Cystobacterineae and should have significant adaptive value because they are present in all available genomes of this taxon, show strict vertical inheritance and are highly conserved in sequence and genetic organization. The absence of the secretin and translocon as well as other proteins essential for protein delivery by other NF-T3SSs suggests that the Myxo NF-T3SS is unable to deliver effectors into the cytosol of eukaryotes. This is consistent with Cystobacterineae ecology, which are free-living bacteria that prey on other bacteria [122]. These systems could thus be involved in protein translocation and/or make some sort of periplasmic or extracellular structure. Functional analysis of these systems would be needed to understand their function. Multiple recruitments of secretins to NF-T3SSs Acquisition of the secretin was the next major step in NF-T3SS evolution for which clear evidence is available. Our analysis shows that the secretin was recruited three times: in Chlamy (from T2SS), in the other systems (possibly from T4P) and then again in Rhizo (from Tad systems). This is consistent with the remarkable versatility of secretins as outer-membrane channels [123]. The interaction between the inner membrane rings and the secretin takes place between the C-terminal region of SctD and the N- terminus domains of the secretin [124–127]. Interestingly, the only part of the early secretin RhcC1 that remains in Rhizo after its partial replacement is the N-terminal domain that interacts with SctD (Figure 6, Text S4). The Tad and the RhcC2 secretins lack this N-terminal domain and have a membrane-interaction, ‘‘BON’’ domain [105], that is present in SctD but absent in other secretins. This domain architecture might interfere with a direct interaction between RhcC2 and SctD, which led to the conservation of RhcC1 as a linker between the two. In that case, interactions of the secretin during the assembly of the NF-T3SS PLOS Genetics | www.plosgenetics.org 9 September 2012 | Volume 8 | Issue 9 | e1002983