, 2011). The isobaric tag for relative and absolute quantitation (iTRAQ) Compound Library concentration and liquid chromatography tandem mass spectrometry (LC-MS/MS) assays were performed by Beijing Protein Innovation (BPI) using previously described methods (Chao et al., 2010). Differentially expressed genes were identified based on at least a twofold expression change and a P-value < 0.05 relative to the WT control. The identified proteins were assigned the appropriate gene numbers by reference to the S. suis strain 05ZYH33 genome (Chen et al., 2007). Where appropriate, the data were analysed using Student's t-test, and a value of P < 0.05 was considered significant. Via genome analysis

of S. suis 05ZYH33, we found that peptides encoded by 05SSU2148 and 05SSU2149 ERK inhibitors exhibit 25% and 30% amino acid sequence identity with the VirR and VirS proteins of C. perfringens strain 13 (Shimizu et al., 2002), respectively, forming a TCS belonging to the widespread LytR/AlgR family. 05SSU2148 and 05SSU2149

were accordingly renamed virR and virS. Sequence analysis of the virRS locus suggested that these overlapping genes are co-transcribed. Like most bacterial response regulators, VirR (237 aa) has a C-terminal helix-turn-helix DNA-binding domain for promoter recognition and transcriptional control of target genes. In its N-terminal region, VirR contains a typical receiver domain that receives the signal from its sensor partner. virS encodes a 435-aa protein with seven N-terminal transmembrane domains (predicted by tmhmm server v. 2.0 software) and a C-terminal Inositol oxygenase transmitter domain that includes a classical histidine kinase and an ATPase motif. A survey of the publicly available complete genome sequences of S. suis revealed the presence of a VirR/VirS homologue in other S. suis isolates, including

the European strain P1/7, the Vietnamese strain BM407 and 7 Chinese strains (98HAH12, SC84, GZ1, A7, ST1, JS14 and SS12). This suggests a wide distribution of the VirR/VirS system among S. suis isolates. Despite the well-characterized function of the VirR/VirS system in C. perfringens infection, the role of this TCS in S. suis remained unclear. To address this issue, an isogenic virRS knockout mutant (ΔvirRS) was constructed in strain 05ZYH33 by allelic replacement with a constitutive spc cassette (Supporting information, Fig. S1). The growth curves of WT and ΔvirRS cultured in THY medium at 37 °C were compared, and no significant difference was observed (Fig. 1). When streaked on THY plates supplemented with 5% sheep blood, ΔvirRS and WT colonies displayed a similar haemolytic phenotype. However, observation with a light microscope revealed that the mean chain length of the ΔvirRS mutant was much shorter than that of WT under the same growth conditions (Fig. 2a).