The Non-Flagellar Type III Secretion System Evolved from the Bacterial Flagellum and Diversified into Host-Cell Adapted Systems Sophie S. Abby1,2*, Eduardo P. C. Rocha1,2 1 Institut Pasteur, Microbial Evolutionary Genomics, De´partement Ge´nomes et Ge´ne´tique, Paris, France, 2 CNRS, UMR3525, Paris, France Abstract Type 3 secretion systems (T3SSs) are essential components of two complex bacterial machineries: the flagellum, which drives cell motility, and the non-flagellar T3SS (NF-T3SS), which delivers effectors into eukaryotic cells. Yet the origin, specialization, and diversification of these machineries remained unclear. We developed computational tools to identify homologous components of the two systems and to discriminate between them. Our analysis of .1,000 genomes identified 921 T3SSs, including 222 NF-T3SSs. Phylogenomic and comparative analyses of these systems argue that the NF- T3SS arose from an exaptation of the flagellum, i.e. the recruitment of part of the flagellum structure for the evolution of the new protein delivery function. This reconstructed chronology of the exaptation process proceeded in at least two steps. An intermediate ancestral form of NF-T3SS, whose descendants still exist in Myxococcales, lacked elements that are essential for motility and included a subset of NF-T3SS features. We argue that this ancestral version was involved in protein translocation. A second major step in the evolution of NF-T3SSs occurred via recruitment of secretins to the NF-T3SS, an event that occurred at least three times from different systems. In rhizobiales, a partial homologous gene replacement of the secretin resulted in two genes of complementary function. Acquisition of a secretin was followed by the rapid adaptation of the resulting NF-T3SSs to multiple, distinct eukaryotic cell envelopes where they became key in parasitic and mutualistic associations between prokaryotes and eukaryotes. Our work elucidates major steps of the evolutionary scenario leading to extant NF-T3SSs. It demonstrates how molecular evolution can convert one complex molecular machine into a second, equally complex machine by successive deletions, innovations, and recruitment from other molecular systems. Citation: Abby SS, Rocha EPC (2012) The Non-Flagellar Type III Secretion System Evolved from the Bacterial Flagellum and Diversified into Host-Cell Adapted Systems. PLoS Genet 8(9): e1002983. doi:10.1371/journal.pgen.1002983 Editor: Mark Achtman, Environmental Research Institute, University College Cork, Ireland Received April 11, 2012; Accepted August 9, 2012; Published September 27, 2012 Copyright: 2012 Abby, Rocha. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by the CNRS, the Institut Pasteur, and the ERC (grant EVOMOBILOME, number 281605). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: sophie.abby@pasteur.fr Introduction Microbial protein secretion facilitates environmental exploita- tion and manipulation [1,2]. The most frequent bacterial motility machinery, the flagellum, uses a type 3 secretion system (T3SS) to secrete its extracellular components. The non-flagellar type 3 protein secretion system (NF-T3SS), often named injectisome, is homologous to the flagellum and also acts as a T3SS. This system is used to secrete its extracellular components and to deliver effectors into host cells. The NF-T3SS is an important virulence factor for animal pathogens within Salmonella, Escherichia, Chlamydia and Yersinia and plant pathogens within Xanthomonas, Ralstonia or Burkholderia (for reviews see [3–8]). Some genomes encode several NF-T3SSs. In Burkholderia pseudomallei discrete NF-T3SSs are involved in animal and plant pathogenesis [9–11], and in Salmonella enterica two NF-T3SSs are used in different phases of infection [12]. NF-T3SSs are exposed at the cell surface and are therefore targeted by the immune response. As a result, NF-T3SSs are being studied as targets for vaccines, e.g. for protection against Shigella [13], and for new antibacterial drugs [14]. They have also been used to deliver vaccine antigens to the cytosol of eukaryotic cells [15,16]. NF-T3SSs have been most thoroughly studied for their role in antagonistic associations between pathogens and their hosts, but they also play an important role in mutualistic associations between bacteria and insects [17], plants [18], or fungi [19]. Phylogenetic evidence indicates that NF-T3SSs originated before most multicellular eukaryotes, possibly to favor interactions with early unicellular eukaryotes [20]. Each model NF-T3SS has its unique nomenclature in the literature. Hence, we use a unifying nomenclature throughout this publication [3], naming the NF-T3SS core components with the prefix sct for secretion and cellular translocation, followed by the suffixes used in the Yersinia Ysc system (see Table S1 for correspondence with other systems). When no unique name has been proposed we use the name of the Yersinia system by default, unless specifically specified. To avoid ambiguity, we follow Desvaux et al. [21] and use ‘‘translocation’’ for transport through a lipid bilayer and ‘‘secretion’’ for transport from the interior to the exterior of the cell. We use the term ‘‘delivery’’ to refer to the active transport from the interior of one cell to the cytosol of a second cell. The NF-T3SS is a complex protein structure that spans the cytoplasmic and outer membranes of bacteria and the cell envelope of the eukaryotic host to deliver effectors directly into PLOS Genetics | www.plosgenetics.org 1 September 2012 | Volume 8 | Issue 9 | e1002983