Type-IVC Secretion System: A Novel Subclass of Type IV Secretion System (T4SS) Common Existing in Gram- Positive Genus Streptococcus Wen Zhang1., Chengbo Rong2., Chen Chen1*, George F. Gao1,2,3* 1 National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention/State Key Laboratory for Infectious Disease Prevention and Control, Beijing, China, 2 CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Science, Beijing, China, 3 Beijing Institutes of Life Science, Chinese Academy of Sciences, Beijing, China Abstract A growing number of pathogens are being found to possess specialized secretion systems which they use in various ways to subvert host defenses. Type IV secretion system (T4SS) is one of versatile secretion systems essential for the virulence and even survival of some bacteria species, and they enable the secretion of protein and DNA substrates across the cell envelope. T4SS was once believed to be present only in Gram-negative bacteria. In this study, we present evidence of a new subclass of T4SS, Type-IVC secretion system and indicate its common existence in the Gram-positive bacterial genus Streptococcus. We further identified that VirB1, VirB4, VirB6 and VirD4 are the minimal key components of this system. Using genome comparisons and evolutionary relationship analysis, we proposed that Type-IVC secretion system is movable via transposon factors and mediates the conjugative transfer of DNA, enhances bacterial pathogenicity, and could cause large- scale outbreaks of infections in humans. Citation: Zhang W, Rong C, Chen C, Gao GF (2012) Type-IVC Secretion System: A Novel Subclass of Type IV Secretion System (T4SS) Common Existing in Gram- Positive Genus Streptococcus. PLoS ONE 7(10): e46390. doi:10.1371/journal.pone.0046390 Editor: Patrick M. Schlievert, University of Iowa Carver College of Medicine, United States of America Received July 12, 2012; Accepted August 29, 2012; Published October 4, 2012 Copyright: 2012 Zhang et al. 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: Our work was supported by the Young Scholar Scientific Research Foundation of China CDC (No. 2010A104), the Priority Project on Infectious Disease Control and Prevention (No. 2008ZX10004–008), National Natural Science Foundation of China (NSFC, Grant No. 81021003), the China Postdoctoral Science Foundation (20100470590) and the National Key Program of Mega Infectious Diseases (Grant No.2013ZX10004-221). There has been no significant financial support for this work that could have influenced its outcome. Competing Interests: The authors have declared that no competing interests exist. * E-mail: gaof@im.ac.cn (GFG); chenchen@icdc.cn (CC) . These authors contributed equally to this work. Introduction A growing number of pathogens are being found to possess specialized secretion systems which they use in various ways to subvert host defenses [1]. Type IV secretion system (T4SS) is one of versatile secretion systems essential for the virulence and even survival of some bacteria species, and they enable the secretion of protein and DNA substrates across the cell envelope [2,3,4,5]. Based on a number of characteristics, including, the organiza- tion of genetic determinants, shared homologies and evolutionary relationships, T4SSs have been divided into several subgroups: Type-IVA, Type-IVB systems and other T4SSs [6,7]. The Agrobacterium tumefaciens Vir system is considered to be the paradigm of Type-IVA, which consists of 12 components, VirB12VirB11 and VirD4 [7]. VirB1, with its lytic transglycosylase subunits, can create holes in the cell wall to enable the movement of T4SS [3,8]. VirB2 and VirB5 are pilus components [3,9,10]. Three cytoplas- mic ATPases, VirB4, VirB11, and VirD4, provide energy for substrate secretion and assist in the assembly of the T4SS [3]. VirD4 is referred to as a coupling protein which recruit the substrates to the T4SS for translocation [11,12]. VirB6 and VirB8 are polytopic inner membrane proteins essential for substrate secretion through the inner membrane in Gram-negative bacteria [3,13], while VirB7-VirB9-VirB10 forms a stable core complex that spans the cell membrane [3,14]. The type-IVB secretion system was initially found in Legionella pneumophila and is composed of 25 genes on two separate regions. Region I contains seven genes (icmV, W and X, and dotA, B, C and D), and Region II contain the other 18 genes (icmT, S, R, Q, P, O, N, M, L, K, E, G, C, D, J, B, F and H) [15]. The majority of these genes were also found in the genome sequences of Coxiella burnetti [15,16]. More recently, a novel lineage of T4SSs classified as ‘‘others’’ have been identified on the genomic island ICEHin1056 of Haemophilus influenza [7,17]. T4SS was once believed to be present only in Gram-negative bacteria but has been found in Gram-positive organisms as well. Our previous work support that in Gram-positive species of Streptococcus suis (S. suis), a GI-type T4SS-like system was identified in a new pathogenicity island (PAI) with a length of 89 kb [18], which was further proven to be a new subgroup of T4SS in this study. S. suis, a Gram-positive species of Streptococcus found in pigs, has recently caused a rash of human infections in China and gained public attention [19,20,21,22]. In these epidemic S. suis isolates related to two recent large-scale outbreaks of human infection in China, we identified this new 89 kb PAI with GI-type T4SS-like system [18]. Our works also showed that this 89K PAI can spontaneously excise to form an extrachromosomal circular product, laterally conjugally transfer to non-89K S. suis recipients through the 89K-encoded GI-type T4SS [23]. Based on genome comparisons and evolution relationship analysis in the current PLOS ONE | www.plosone.org 1 October 2012 | Volume 7 | Issue 10 | e46390