Construction and Validation of S. suis Mutants

The S. suis strains used in the present study are listed in Table 1. S. suis strain S10 is a virulent isolate from an infected pig, and its genome is 99% identical to the genome of S. suis 2 strain P1/7 (de Greeff et al., 2011 (link)), a sequenced reference strain of which the genome had been annotated previously (Holden et al., 2009 (link)). S. suis was grown at 37°C at 5% atmospheric CO₂ in Todd Hewitt Broth (THB, Thermo Scientific, Oxoid) or on THB plates containing 1.2% of agar (BD). When required the medium was supplemented with spectinomycin (Invitrogen) and/or chloramphenicol (Sigma) at a concentration of 100 and 5 μg/ml, respectively. Insertional deletion mutants of the genes cinA, oppA, and comYC were constructed in S. suis strain S10 by Gene Splicing Overlap Extension PCR (SOE-PCR) and allelic replacement as previously described (Zaccaria et al., 2014 (link)). The primers used for SOE-PCR are shown in Table 1. Successful deletion of the genes was verified by colony PCR using primer combinations based on DNA sequences of the inserted DNA and proximal chromosomal DNA (Table 1) and verified by sequencing of the amplicons. Growth phase was determined by measuring optical density at 600 nm (OD_600nm) using a SpectraMax M5 reader (Molecular Devices LLC).