Gram-positive bacteria, it is not a surprise that proteins contributing to the assembly of the secretion channel across outer membranes (VirB7, VirB9 and VirB10) in type-IVA secretion systems have not been found in Type-IVC secretion system. The other components in 89K PAI may also play role in substrates transport but the determination of their functions requires further investigation. We propose that VirB1, VirB4, VirB6 and VirD4 are the minimal key component of Type-IVC secretion system (Figure 2). The function of these four proteins (VirB1, VirB4, VirB6, and VirD4) for the secretion of DNA has been proven by making individual knockouts of T4SS gene homologues (DvirB1-89K, DvirB4-89K, DvirB6-89K and DvirD4-89K) [23]. Our previous results showed that no transconjugants were obtained when DvirB4-89K, DvirB6-89K and DvirD4-89K were used as donors. And the transfer frequency of DvirB1-89K (9.261027) was six-fold less than that of wild type strains [23]. Our other experiments in experimental infection of mice showed that knockout of VirB4 and VirD4 in this Type-IVC secretion system also eliminated the lethality of the highly virulent strain and impaired its ability to trigger host immune response [30]. Mobility of Type-IVC Secretion System The phylogenetic tree of the housekeeping gene rpoB clearly showed that Type-IVC secretion system is movable, for strains with and without Type-IVC secretion system co-existing in several branches (Figure 3A). The popular existence of Type-IVC secretion system could not be only caused by mutations. HT between species underlie the presence of GIs with T4SS in Streptococcus. Figure 3B further indicates that the movement of type- IVC secretion system occurs more easily within the strains of a species than that between species. Our previous experimental work also proved that type-IVC secretion system was movable. In S. suis 05ZYH33, 89-kb GI that could spontaneously excise to form an extrachromosomal circular product and lateral transfer from a donor to a recipient cell at a frequency of 1026 transconjugants/donor [23]. The HT of type-IVC secretion system is under the help of transposons. In the nearby DNA sequences of 15 Type-IVC secretion system in Streptococcus sp., 9 of 15 (60%) were identified with Tn916 transposons (Figure S2). Although without Tn916 nearby Type-IVC secretion systems, two strains (S. pneumoniae CGSP14 and S. pyogenes MGAS2096) harbored Tn916 residues, whereas the other two strains (S. pneumoniae Hungary19A and S. agalactiae 2603V) had Tn916 at another position on the chromo- some (Figure S2). Of the 4 strains without Tn916, 3 were found to have other transposons, like Tn5252 and Tn5421. Considering the surprisingly high correlation between transposons and Type-IVC secretion system, we could reasonably assume that some mobile elements necessary for the HT of T4SS were introduced by transposons. These mobile elements help T4SS move between cells, and the T4SS enables transposons to cross the barriers between donor and recipient cells. We believe that a random confusion between a transposon and minimal T4SS led to the formation of Type-IVC secretion system. Type-IVC secretion system gradually accumulated in Streptococcus species by HT, and an increasing number of strains harbor this system. During transport, the loss or acquisition of variable elements in this Type- IVC secretion system is ongoing. Further experimental work is necessary to support our hypothesis and answer questions regarding when the random event leading to the formation of Type-IVC secretion system occurred and whether HT of this system occurs between different species. Novel Type-IVC Secretion System in Streptococcus Function of Type-IVC Secretion System in Pathogenicity of Streptococcus The Type-IVC secretion system could enhance bacterial pathogenicity and mediate the injection of virulent proteins into host cells. Our previous study showed that the two component system SalK/R within the 89-kb island controls the virulence of the highly pathogenic strain S. suis 2 [23]. Recently, using NimbleGen tiling arrays, Zhu and colleagues found this 89-kb fragment in 9 other virulent S. suis 2 lineages, all of which were sampled from two recent large-scale outbreaks of human infection in China [31]. The 89-kb GI which include Tn916 with tetracycline-resistance genes of the S. suis 2 strain was proved could laterally transfer to other S. suis 2 strains with the help of Type-IVC secretion system [23]. Knockout of the 2 key components (VirD4 and VirB4) of the S. suis 2 T4SS system eliminated the lethality of the highly virulent strain and impaired its ability to trigger host immune response in experimental infection of mice [30]. All of this evidence together suggests that Type-IVC secretion system contributes to the pathogenicity of S. suis 2, particularly in the two outbreaks of S. suis 2 infections in China. Conclusions In this paper, we present evidence that the GI-type T4SS-like system we experimentally defined earlier in S. suis is unexpectedly popular in the genus Streptococcus based on an analysis of deposited genome sequences. It always located in a GI with abnormal GC content. VirB1, VirB4, VirB6 and VirD4 are the minimal key component of this system in Streptococcus. We propose that this system in Gram-positive bacteria is a new subclass of T4SS (Type- IVC secretion system). Further, it is movable with the help of transposon factors, which could mediate the conjugative transfer of plasmid DNA/transposons and enhance bacterial pathogenicity. Supporting Information Figure S1 Strain numbers in which virB1–virB11 and virD4 genes were identified. The blue columns are the number of Streptococcus strains with virB/D genes, whereas the red columns are the number of Streptococcus strains with virB/D clusters. (DOC) Figure S2 Genome islands with T4SS in 10 strains of Streptococcus compared to the 89-kb GI in S. suis 05ZYH33. GC%, locations of the virB/D genes, Tn916, and other important genes in GI are shown. A) S. suis SC84; B) S. suis BM407 (1000794,1091159); C) S. suis BM407 (499472,585444); D) S. pneumoniae P1031; E) S. pneumoniae G54; F) S. pneumoniae ATCC 700669; G) S. pneumoniae CGSP14; H) S. agalactiae 2603V R; I) S. agalactiae NEM316; J) S. pyogenes MGAS2096. Genes with varying functions are presented in different colors. (DOC) Table S1 List of virB/D clusters identified in 14 Streptococcus strains. ID: virB/D cluster ID in a genome; S: start site of virB/D gene in genome; E: end site of virB/D gene in genome; D: direction of virB/D gene. (DOC) Table S2 List of Streptococcus strains with draft genomes used in this study. ‘‘+’’ indicates that there is an identified virB/D gene cluster in this strain. (DOC) Detailed Material and Methods. Protocol S1 (DOC) PLOS ONE | www.plosone.org 6 October 2012 | Volume 7 | Issue 10 | e46390