Virulent Brucella suis Biovar 2 Strain Characterization

The characteristics of the bacterial strains and plasmids are shown in Supplementary Table 1. The virulent B. suis biovar 2 (bv2) strain CITA 198 (henceforth Bs2WT) was selected for this study because the reference bv2 strain B. suis Thomsen is attenuated in mice (Aragón-Aranda et al., 2020 (link)). The choline-containing media used were tryptic soy broth (TSB, Scharlau, Barcelona, Spain), the same medium with 15% agar (TSA) or Blood Agar Base (BAB; Oxoid, United Kingdom), supplemented when necessary with 50 μg/ml kanamycin (Km), or/and 5% sucrose or/and 0.2% activated charcoal (Sigma) (see below). The choline-free medium was the lactate-glutamate-glycerol-vitamins medium of Gerhardt (1958) (link) supplemented with 1 mM methionine (a Bs2WT requirement; Zúñiga-Ripa, unpublished results) (henceforth mGSM). E. coli strains were cultured on TSA-Km (50 μg/ml), supplemented with 1 mM diaminopimelic acid (DAP [Sigma]) in the case of E. coli β2150. All strains were stored at −80°C either in skimmed milk (Scharlau, Barcelona, Spain) or in TSB containing 0.5% yeast extract and 7% dimethylsulfoxide. Work was performed under Biosafety Level 3 (BSL-3) conditions.