SDS-PAGE of <i>S</i>. <i>agalactiae</i> cell lysates followed by western-blotting with conjugated human IgA-horseradish peroxidase.

<p>Lanes 1, 2, 4: the strains that express β antigen; lane 3: strain that doesn’t express β antigen.</p

Schematic representation of the BgrR proteins and the corresponding three-dimensional structures in the strains 168/00 (A) and A49V (B).

<p>The numbers indicate aminoacid positions. Phosphorylation, dimerization and DNA-binding sites are shown.</p

Schematic representation of the <i>bac</i> gene in the strain A909 that expresses β antigen (A), and the <i>bac</i> gene in the strain #06/08 that did not produce β antigen (B).

<p>The numbers indicate nucleotide positions. The signal sequence, C-terminal end, XPZ and LPYTG motifs, and IgA binding and factor H binding domains indicated according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128426#pone.0128426.ref020" target="_blank">20</a>].</p

Natural Mutations in <i>Streptococcus agalactiae</i> Resulting in Abrogation of β Antigen Production

<div><p><i>Streptococcus agalactiae</i> genome encodes 21 two-component systems (TCS) and a variety of regulatory proteins in order to control gene expression. One of the TCS, BgrRS, comprising the BgrR DNA-binding regulatory protein and BgrS sensor histidine kinase, was discovered within a putative virulence island. BgrRS influences cell metabolism and positively control the expression of <i>bac</i> gene, coding for β antigen at transcriptional level. Inactivation of <i>bgrR</i> abrogated <i>bac</i> gene expression and increased virulence properties of <i>S</i>. <i>agalactiae</i>. In this study, a total of 140 strains were screened for the presence of <i>bac</i> gene, and the TCS <i>bgrR</i> and <i>bgrS</i> genes. A total of 53 strains carried the <i>bac</i>, <i>bgrR</i> and <i>bgrS</i> genes. Most of them (48 strains) expressed β antigen, while five strains did not express β antigen. Three strains, in which <i>bac</i> gene sequence was intact, while <i>bgrR</i> and/or <i>bgrS</i> genes had mutations, and expression of β antigen was absent, were complemented with a constructed plasmid pBgrRS(P) encoding functionally active <i>bgrR</i> and <i>bgrS</i> gene alleles. This procedure restored expression of β antigen indicating the crucial regulatory role of TCS BgrRS. The complemented strain A49V/BgrRS demonstrated attenuated virulence in intraperitoneal mice model of <i>S</i>. <i>agalactiae</i> infection compared to parental strain A49V. In conclusion we showed that disruption of β antigen expression is associated with: i) insertion of ISS<i>a4</i> upstream the <i>bac</i> gene just after the ribosomal binding site; ii) point mutation G342A resulting a stop codon TGA within the <i>bac</i> gene and a truncated form of β antigen; iii) single deletion (G) in position 439 of the <i>bgrR</i> gene resulting in a frameshift and the loss of DNA-binding domain of the BgrR protein, and iv) single base substitutions in <i>bgrR</i> and <i>bgrS</i> genes causing single amino acid substitutions in BgrR (Arg187Lys) and BgrS (Arg252Gln). The fact that BgrRS negatively controls virulent properties of <i>S</i>. <i>agalactiae</i> gives a novel clue for understanding of <i>S</i>. <i>agalactiae</i> adaptation to the human.</p></div

Amount of the <i>bac</i> gene positive strains of different serotypes.

<p>Amount of the <i>bac</i> gene positive strains of different serotypes.</p

Schematic representation of the IS<i>Sa4</i> insertion in regulatory region of the <i>bac</i> gene in the strain #8.

<p>The sequences -35, -10, and ribosomal binding site are indicated in boxes.</p

Mortality rates of laboratory mice due to <i>S</i>. <i>agalactiae</i> intra-peritoneal infection.

<p>Mortality rates of laboratory mice due to <i>S</i>. <i>agalactiae</i> intra-peritoneal infection.</p

Predicted <i>pmpF</i> promoter sequence for reference and clinical strains.

<p>Sequences are for reference strains E/Bour and L₂/434, and clinical strains E/537C-05, E/S-141, E/CS-500-96, and L₂. The predicted transcription promoter for <i>pmpF</i> is located within a 100% conserved region of the <i>pmpG/pmpF</i> IGR, where putative -10 and -35 elements are in blue characters and boxed. Potential A/T spacer region is underlined, and the predicted transcription start site is shown in a larger font below a red asterisk. The putative RBS for <i>pmpF</i> is in orange characters, and the putative RNase E cleavage sites are highlighted in grey. Numbers represent positions relative to the start codon of <i>pmpF</i> (highlighted in yellow). The start codon of <i>pmpG</i> is highlighted in blue.</p

Expression profile of the nine <i>pmp</i> genes and <i>ompA</i> throughout the development of <i>C. trachomatis.</i>

<p>Reference strain L₂/434 is represented in panel A and E/Bour in panel B. Values represent the mean±SEM based on three independent experiments for time points of 2, 6, 12, 18, 24, 36, and 48 h post infection. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000878#s2" target="_blank">methods</a> for details.</p

Distribution/Location of the putative RNase E cleavage sites within the <i>pmpFE</i> operon coding sequence.

<p>The sequence is for reference strains E/Bour and L₂/434 and for clinical strains E/537C-05, E/S-141, E/CS-500-96 and L₂. Black vertical lines represent all RNase E cleavage sites conserved among all strains under study; green vertical lines show the ones only conserved among the four “E” strains; orange vertical lines represent those specific solely for both L₂ strains. Numbers represent nucleotide positions relative to the start codon of <i>pmpF.</i></p