Oligonucleotide primers.

<p>Oligonucleotide primers.</p

Binding ability to immobilized human fibrinogen of the wild type (WT) L1 strain, and of isogenic mutant and complemented strains for <i>rgfAC</i>, <i>fbsA</i>, and <i>fbsB</i> genes.

<p>Flat bottomed 96-well polystyrene plates were coated with 21 nM human fibrinogen and 5×10⁶ to 5×10⁸ CFU per ml were added for 90 min at 37°C. Binding ability was calculated from the ratio between the number of bound bacteria and the number of bacteria present in the inoculum. Each experiment was performed at least three times. Boxes are means and bars are standard deviation of the means. The binding values of the mutant strains were significantly lower, at a <i>P</i> value of <0.001, than the values of the L1WT strain and of the corresponding complemented strains carrying <i>rgfAC</i>, <i>fbsA</i>, and <i>fbsB</i> genes on the pP1 plasmid.</p

Prevalence of the <i>fbs</i> genes and of their regulator genes in a collection of 134 isolates belonging to the GBS major clonal complexes.

<p>Prevalence of the <i>fbs</i> genes and of their regulator genes in a collection of 134 isolates belonging to the GBS major clonal complexes.</p

Properties of <i>ΔrgfAC</i> mutant strains.

<p>(A) Fold change in transcription levels of <i>fbsA</i> (filled boxes) and <i>fbsB</i> (open boxes) genes in the isogenic <i>ΔrgfAC</i> mutants as compared to the wild type L1, L2, and L50 strains (WT). The amount of transcripts of each gene was normalized to the amount of <i>gyrA</i> transcripts and expressed relative to the level of transcription in corresponding WT strain. Each experiment was performed at least three times. Boxes are means and bars are standard deviation of the means. (B) Binding ability to immobilized human fibrinogen of the isogenic <i>ΔrgfAC</i> mutants (open boxes) and the WT strains (filled boxes). Flat bottomed 96-well polystyrene plates were coated with 21 nM human fibrinogen and 5×10⁶ to 5×10⁸ CFU per ml were added for 90 min at 37°C. Binding ability was calculated from the ratio between the number of bound bacteria and the number of bacteria present in the inoculum. The level of fibrinogen binding of WT strains is arbitrarily reported as 100 and the fibrinogen-binding levels of the isogenic mutants are relative values. Each experiment was performed at least three times. Boxes are means and bars are standard deviation of the means. * indicates that the binding values of the mutant strains were significantly lower than the values of the corresponding WT strains, at a <i>P</i> value of <0.001.</p

Binding ability to immobilized human fibrinogen of the wild type (WT) <i>S. agalactiae</i> strains and isogenic <i>ΔfbsA</i>, <i>ΔfbsB</i>, <i>ΔfbsAΔfbsB</i> deletion mutants.

<p>Flat bottomed 96-well polystyrene plates were coated with 21 nM human fibrinogen and 5×10⁶ to 5×10⁸ CFU per ml were added for 90 min at 37°C. Binding ability was calculated from the ratio between the number of bound bacteria and the number of bacteria present in the inoculum. The fibrinogen-binding level of WT L1, L2, and L50 strains is arbitrarily reported as 100 and the fibrinogen-binding levels of the various isogenic mutants are relative values. Each experiment was performed at least three times. Boxes are means and bars are standard deviation of the means. * indicates that the binding values of the mutant strains were significantly lower than the values of the corresponding WT strains, at a <i>P</i> value of <0.001. ‡ indicates that the binding values of the <i>ΔfbsB</i> and <i>ΔfbsAΔfbsB</i> mutant strains were significantly lower than the values of <i>ΔfbsA</i> mutant strains, at a <i>P</i> value of <0.001.</p

Schematic representation of the <i>rgf</i> locus.

<p>Open reading frames, direction of transcription and approximate gene sizes are indicated <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014658#pone.0014658-Spellerberg1" target="_blank">[25]</a>. The segment that was deleted in <i>ΔrgfAC</i> mutant is represented as a heavy line below the genes.</p

Transcription levels of <i>fbsA</i> and <i>fbsB</i> genes in three wild type CC17 strains.

<p>The amount of transcripts of <i>fbsA</i> gene (filled boxes) and <i>fbsB</i> gene (open boxes) in L1, L2, and L50 wild type strains was normalized to the amount of <i>gyrA</i> transcripts. Each experiment was performed at least three times. Boxes are means and bars are standard deviation of the means.</p