Origins and Diversification of T3SS Figure 5. Genomic organization of the Myxo NF-T3SS. The gene contents of the ‘‘short’’ and ‘‘long’’ Myxo NF-T3SSs are displayed on the 16S rRNA phylogeny of the group. The 16S rRNA maximum likelihood tree was built with RAxML [96] (General time-reversible model + 4-categories- discretized Gamma distribution for rate variations among sites) and chosen from the best of 200 starting trees. The numbers in the tree nodes refer to bootstrap values out of 100 replicates. The identified NF-T3SS genes are represented by boxes containing the last letter of the ‘‘sct’’ gene name, and colored according to the color code of Figure 1A. Dashed transparent boxes correspond to genes showing hits for Hmmer sequence profiles with i- evalue above the 1023 threshold. Empty boxes correspond to genes not annotated in our analysis, and boxes with three dots correspond to several (4 to 16) consecutive non-annotated genes. Proteins encoded in the loci are colored on the NF-T3SS structure drawings, whereas parts of the structure whose corresponding gene could not be found remain unfilled. doi:10.1371/journal.pgen.1002983.g005 Figure 6. Evolutionary history, domain architecture, and genetic organization of the NF-T3SS secretins. (A) Phylogeny of the secretin family. The numbers represent bootstrap support values for branches separating the different types of secretins found in NF-T3SSs (highlighted by colored bars). Gray boxes indicate the main secretin groups. The two genes encoding the Rhizo secretin are indicated in orange and yellow boxes (see panel B). NF-T3SS types are named as in Figure 4. (B) Domain architecture of the secretins related to NF-T3SS secretins. PFAM domains were identified using InterProScan [140], except for the Chlamy-specific N-terminal domain of the NF-T3SS secretin CdsC (burgundy box), and the dashed box ‘‘N-domain’’, which were identified by sequence similarity with the other secretins using Blast. Signal peptides were detected with InterProScan and PsortB [141]. Dotted lines correspond to the boundaries of the alignments of Blast hits. See Text S4 for more analyses of Rhizo secretins. (C) Analysis of the genetic organization of the Rhizo loci mapped on their phylogeny. The phylogeny was inferred from the concatenation of the eight NF-T3SS core genes (Figure 4, Figure S4). Conventions are the same as in Figure 5. Numbers in white boxes correspond to the number of genes separating the block of genes with conserved order (light green box) from the RhcC2 secretin (outside the box), or to the number of consecutive genes not annotated in this study (inside the box). doi:10.1371/journal.pgen.1002983.g006 PLOS Genetics | www.plosgenetics.org 8 September 2012 | Volume 8 | Issue 9 | e1002983