Acid killing assay.

<p>The means and standard deviations for three independent samples are shown. Significant differences between the wild-type and recombinant strains at 45 min were analyzed by one-way ANOVA. **, <i>P</i><0.05; *, <i>P</i><0. 1.</p

Schematic diagram of the <i>fim</i> operon and its flanking regions of <i>S.</i><i>parasanguinis</i> FW213.

<p>The relative location and transcription direction of each ORF are shown. Spaf_0345 and Spaf_0344 are indicated as 0345 and <i>fimR</i>, respectively. The limits of the sequence present in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066163#pone-0066163-g002" target="_blank">Figure 2A</a> are indicated by two vertical arrows. The position of the <i>erm</i> in strain Δ<i>fimR</i> is indicated by an inverted triangle above the gene. The putative terminators for Spaf_0345 and <i>fimR</i> are indicated. The sizes of Spaf_0345 and <i>fimR</i> in nt, predicted molecular weight in kDa and pI of the gene products are shown.</p

Primers used in this study.

a<p>inserted restriction recognition sites are underlined.</p

The regulation of FimR on p<i>_fim</i> expression.

<p>(A) The nt sequence of the 5′ flanking region of <i>fimC</i>. The <i>pepO</i> and <i>fimC</i> are transcribed from the opposite DNA strands, thus the sequence of <i>pepO</i> presented here is the noncoding strand, and the sequence of <i>fimC</i> is the coding strand. The transcription initiation sites (+1) of <i>fimC</i> and <i>pepO</i> are shown by a solid triangle above the sequence, and two open triangles below the sequence, respectively. The putative −10 and −35 sequences of p<i>_fim</i> are shaded. The potential Per box is underlined. The inverted repeat sequences are shown by horizontal arrows above the sequence. The sequence of the probe used in EMSA is boxed. The limits of the deletion derivatives are indicated by the numbers. (B) The CAT activities in wild-type FW213 and Δ<i>fimR</i> harboring various p<i>_fim</i>-<i>cat</i> fusions. All strains were grown in TH. Values shown are means and standard deviations of three independent experiments. All experiments were done in triplicate reactions and negative controls were reactions carried out in the absence of Cm.</p

Effect of Mn²⁺ and Fe²⁺ on p<i>_fim</i> expression.

<p>Wild-type FW213 and Δ<i>fimR</i> harboring p<i>_fim</i>(445 b)-<i>cat</i> were grown in FMC containing 0.01 µM MnCl₂ and 0.1 µM FeSO₄ (I), 0.01 µM MnCl₂ and 50 µM FeSO₄ (II), 50 µM MnCl₂ and 0.1 µM FeSO₄ (III), 50 µM MnCl₂ and 50 µM FeSO₄ (IV). All cultures were supplemented with 1 mM MgSO₄ and 1 mM CaCl₂. Values are means and standard deviations of three independent experiments. Significant differences between samples were determined by two-way ANOVA using SPSS Statistic 17.0. The <i>P</i> values between the wild-type strain and Δ<i>fimR</i> under all four conditions are less than 0.01. <i>P</i> values between condition I and II are indicated in the figure. *, <i>P</i><0.05; **, <i>P</i><0.01.</p

Bacterial strains and plasmids used in this study.

a,r<p>resistance; -, deficiency.</p

The Expression of the <i>fim</i> Operon Is Crucial for the Survival of <i>Streptococcus parasanguinis</i> FW213 within Macrophages but Not Acid Tolerance

<div><p>The acquisition of transition metal ions is essential for the viability and in some cases the expression of virulence genes in bacteria. The <i>fimCBA</i> operon of <i>Streptococcus parasanguinis</i> FW213 encodes a Mn²⁺/Fe²⁺-specific ATP-binding cassette transporter. FimA, a lipoprotein in the system, is essential for the development of endocarditis, presumably by binding to fibrin monolayers on the damaged heart tissue. Recent sequence analysis revealed that Spaf_0344 was homologous to <i>Streptococcus gordonii scaR</i>, encoding a metalloregulatory protein for the Sca Mn²⁺-specific transporter. Based on the homology, Spaf_0344 was designated <i>fimR</i>. By using various <i>fim</i> promoter (p<i>_fim</i>) derivatives fused with a promoterless chloramphenicol acetyltransferase gene, the functions of the <i>cis</i>-elements of p<i>_fim</i> were analyzed in the wild-type and <i>fimR</i>-deficient hosts. The result indicated that FimR represses the expression of p<i>_fim</i> and the palindromic sequences 5′ to <i>fimC</i> are involved in repression of p<i>_fim</i>. A direct interaction between FimR and the palindromic sequences was further confirmed by <i>in vitro</i> electrophoresis gel mobility shift assay and <i>in vivo</i> chromatin immunoprecipitation assay (ChIP)-quantitative real-time PCR (qPCR). The result of the ChIP-qPCR analysis also indicated that FimR is activated by Mn²⁺ and, to a lesser degree, Fe²⁺. Functional analysis indicated that the expression of FimA in <i>S. parasanguinis</i> was critical for wild-type levels of survival against oxidative stress and within phagocytes, but not for acid tolerance. Taken together, in addition to acting as an adhesin (FimA), the expression of the <i>fim</i> operon is critical for the pathogenic capacity of <i>S. parasanguinis</i>.</p></div

EMSA demonstrating the interaction between FimR and p<b><i>_fim</i></b><b>.</b>

<p>Lanes 1 to 4 are reactions containing 0, 20, 40, and 80 µM His-FimR, respectively; lane 5 is reaction containing 80 µM His-FimR and unlabeled <i>tcrB</i>. The positions of the FimR-probe complexes are indicated by triangles.</p

ChIP-qPCR demonstrating the relative quantity of p<i>_fim</i> bound by FimR.

<p>Cells were grown under 0.01 µM MnCl₂ and 0.1 µM FeSO₄ (I), 0.01 µM MnCl₂ and 50 µM FeSO₄ (II), 50 µM MnCl₂ and 0.1 µM FeSO₄ (III), and 50 µM MnCl₂ and 50 µM FeSO₄ (IV). The ΔCq of the sample from III was used as the reference. Significant differences between samples were determined using one-way ANOVA. A significant difference (<i>P</i><0.05) was detected between all pairs of comparison.</p

Growth kinetics of the wild-type <i>S.</i><i>parasanguinis</i>, VT930, Δ<i>fimR</i>, and VT930_Δ<i>fimR</i> grown in TH (A), TH containing 2 mM (B) and 4 mM (C) paraquat.

<p>A representative figure of at least three experiments under each condition is shown.</p