Type-IVC Secretion System: A Novel Subclass of Type IV Secretion System (T4SS) Common Existing in Gram-Positive Genus <em>Streptococcus</em>

<div><p>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 <em>Streptococcus.</em> 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.</p> </div

Genetic organization of T4SS.

<p>For <i>A. tumefaciens</i>, gene names B = <i>virB</i> and D4 = <i>virD4</i>. For <i>L. pneumophila,</i> upper-case gene names = <i>dot</i> and lower-case gene names = <i>icm</i>. Genes in orange correspondents to periplasmic lytic transglycosylase, in green ATPase, in yellow T pilus, in red channel subunit across inner membrane, in blue channel subunit across outer membrane, in grey no T4SS genes or genes with the unclear function.</p

Hypothetical model for Type-IVC secretion system.

<p>The colored parts are the components of the Type-IVC secretion system system. The grey parts are objects present in the canonical T4SS of Gram-negative strains but lost in the Gram-positive Type-IVC secretion system, e.g., the outer membrane, VirB2, and VirB5.</p

The phylogenetic tree.

<p>(A) The neighbor-joining (NJ) tree obtained on the basis of the housekeeping gene <i>rpoB</i>. The red solid circles represent GIs with <i>virB/D</i> clusters. (B) NJ tree obtained on the basis of a concatenated sequence of <i>virB4</i>, <i>virB6</i>, and <i>virD4</i> genes in GI.</p

List of <i>Streptococcus</i> strains with whole genomes used in the current study.

<p>List of <i>Streptococcus</i> strains with whole genomes used in the current study.</p

Detection of ChIP DNAs from WT and F4 strains under low-iron and high-iron conditions.

<p><b>a, d, g, j</b>: positive controls (input). <b>b, e, h, k</b>: negative controls (no antibody). <b>c</b>: high-iron WT. <b>f</b>: low-iron WT. <b>i</b>: high-iron F4. <b>l</b>: low-iron F4. Primer of <i>rpsJ</i> gene (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029572#pone-0029572-t003" target="_blank">Table 3</a>) is included as an additional negative control which codes for a conserved 30S ribosomal S10 protein and is not regulated by Fur <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029572#pone.0029572-Lang1" target="_blank">[32]</a>.</p

Iron-responsive Fur regulon in <i>M. gryphiswaldense</i>.

<p>DP, the comparison of mRNA from WT cells treated with 30 µM iron chelator 2, 2′-dipyridyl (DIPy) and cells added with 60 µM ferric citrate.</p><p><i>fur</i>, the comparison of mRNA expression in the <i>fur</i> mutant (F4) and WT grown under high-iron condition.</p><p>Positive and negative numbers indicate fold increase or decrease, respectively.</p

Primers for ChIP PCR detection.

<p>Primers for ChIP PCR detection.</p

TEM micrographs of WT (A), F4 (B), and F4C (C) strains.

<p>Cells were grown in SLM added with 60 µM ferric citrate for 36 h. Bar, 500 nm.</p

Growth curves measured by OD₅₆₅ of WT, F4, and F4C strains in SLM added with 1 mM H₂O₂.

<p>Experiments were performed in triplicate, and representative results are shown.</p