study, we identified that this GI-type T4SS-like system is a new subgroup T4SS named as Type-IVC secretion system for its clearly different genetic organization with type-IVA and type-IVB in Gram-negative bacteria. VirB1, VirB4, VirB6 and VirD4 were proven to be the minimal key component of type-IVC secretion system. We further identified that type-IVC secretion system is unexpectedly popular in the genus Streptococcus. This system is movable with the help of transposon factors (such as Tn916), which could also mediate the conjugative transfer of DNAs and enhance bacterial pathogenicity. Materials and Methods 1. Genome Sequences of Streptococcus The Streptococcus strains used in the current study are listed in Supplemental Tables. Whole genome sequences of 50 strains of Streptococcus (Table 1) were downloaded from the NCBI ftp (ftp:// ftp.ncbi.nih.gov/genomes). Draft genome sequences of 67 Strepto- coccus strains were also obtained from the NCBI database (Table S2). We manually collected 839 gene sequences of the 12 components of T4SS (virB12virB11 and virD4) from the NCBI and UniprotKB database [24,25]. 2. Genome-wide Search for T4SS Genes and virB/D Cluster To search for genes in the T4SS, a new program (TFSP) was used (http://t4ss.bioinfo-icdc.org/). This program combines the alignment algorithm approaches, prediction of protein functions, and domain evaluation to detect candidate T4SS genes (virB1211 and virD4) in genomes of bacteria (the detailed method is shown in the Protocol S1) with good precision. Based on the locations of identified T4SS genes (virB/D genes), we found virB/D clusters in genomes of Streptococcus. In this study, determination of virB/D clusters conformed to the following criteria: (1) the distance between two nearby virB/D genes is less than 5 kb, (2) the total length of the virB/D cluster is less than 50 kb, and (3) the number of virB/D genes in a virB/D cluster is $3. 3. Identification of Genomic Islands (GI) with virB/D Cluster For co-lineage comparisons, Blast similarity searches were performed using local BLAST software [26]. Based on the Blast results, the GI and its precise location in the genome were determined by the co-lineage comparison between a genome with a virB/D cluster and one without it [27]. The GC content skew of GI was step-analyzed in a window of 2,000 bp by a self-developed Perl program (draw_GC_content.pl) [27]. The function of genes in the GI was annotated by the method of Clusters of Orthologous (COG) [28]. 4. Construction of Phylogenetic Tree rpoB is a housekeeping gene and highly conserved in many bacteria. We used rpoB gene sequences in 50 species to generate a phylogenetic tree of Streptococcus. Multiple sequence alignments of these gene sequences were performed using MEGA [29]. A phylogenetic tree was constructed using the neighbor-joining algorithm in Mega, and 1,000 subsets were generated for bootstrapping re-sampling of the data. Another tree was built based on the concatenated sequences of virB4, virB6, and virD4 genes using the same method. Novel Type-IVC Secretion System in Streptococcus Results A New Subgroup of T4SS in Gram-positive S. suis: Type- IVC Secretion System A new subgroup of T4SS (GI-type T4SS) was identified in Gram-positive strain S. suis [18,23]. Genetic organization repre- sented that this GI-type T4SS in Gram-positive strain S. suis is clearly different with type-IVA, type-IVB and other T4SS in Gram-negative bacteria, thus it is classified as Type-IVC secretion system in this study (Figure 1). Different with other T4SS systems, only 4 proteins (VirB1, VirB4, VirB6, and VirD4) were identified in Type-IVC secretion systems, which mainly work in three fields: (1) transglycosylases (VirB1), working for degrading peptidoglycan outside the plasma membrane of bacteria, could reduce the resistance for the secretion of substrates; (2) ATPases (VirB4, and VirD4) play essential roles in supplying the energy for substrates translocation and apparatus assembly [4]; (3) a gene contributing to the assembly of the secretion channel across inner cell membrane(VirB6). These genes clustered together with the same direction in the chromosome. Those genes correspondent to channel subunit across outer membrane, such as VirB7/VirB9/ VirB10 in type-IVA and DotD/DotC/DotH/DotG/DotF in type-IVB, were lost in type-IVC secretion system (Figure 1 and Figure 2). Popular Existence of Type-IVC Secretion System in Genus Streptococcus To more fully understand the distribution of Type-IVC secretion system in genus Streptococcus, we used bioinformatic methods to predict T4SS genes (Figure S2) in 50 Streptococcus strains with published genome sequences (Table 1). Detailed methods are described in the Method section. 15 Type-IVC secretion systems were identified in 14 of 50 Streptococcus strains (Table S1). S. suis BM407 (NC_012926) has two copies of T4SS, while the other 13 genomes have only one. All 15 Type-IVC secretion systems own clustered virB1-like/virB4/virB6/virD4 or virB4/virB6/virD4 genes in the same direction and order (Table S1 and Figure S2). The Type-IVC secretion system in S. pyogenes MGAS2096 also has homolog of virB2 gene. Our genome analysis represent that, most strains in Streptococcus were identified with virB/D genes (Figure S1) and approximately 28% of all Streptococcus strains have Type-IVC secretion system (Table S1). Additional investigations into the 67 draft genomes of Strepto- coccus strains (Table S2) further supported the belief that Type-IVC secretion system are popular in Streptococcus; in these studies, 19 Type-IVC secretion system were identified (Table S2). Evolutionary Relationship Among Type-IVC Secretion Systems in Genus Streptococcus Among 50 strains of Streptococcus, 14 strains own additional DNA fragments (GI, 50–89 kb) with Type-IVC secretion system by co- lineage comparisons genomic comparison. Compared with the 89 kb PAI of S. suis, the structures of these GIs are significantly variable (Figure S2): Their locations in the genomes are different and genes in these GIs are also variable. However, they retain some common features. First, all the GIs have virB/D gene clusters with similar genes. Second, genes in the virB/D clusters always have the same order and direction. Third, most of the clusters own a transposon, such as Tn916. The phylogenetic tree obtained from the housekeeping gene rpoB clearly showed that Type-IVC secretion system could be present or absent in different strains of a species (Figure 3A). The occurrence of Type-IVC secretion system in these branches could PLOS ONE | www.plosone.org 2 October 2012 | Volume 7 | Issue 10 | e46390