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. 2014 Mar 20;10(3):e1003979. doi: 10.1371/journal.ppat.1003979

Figure 6. Effect of RsaA on capsule formation.

Figure 6

(A) Dot-blot analysis of capsular polysaccharide accumulation in wild-type strains and its corresponding ΔrsaA mutant and complemented strains after growth in BHI for 16 h. Standardized bacterial suspensions were treated with lysostaphine, DNase I and proteinase K, and 1/3 serial dilutions were spotted onto nitrocellulose membranes. Capsular polysaccharides were detected with an anti-CP5 (HG001 and Newman) or CP8 (Becker) antisera. The ΔrsaA mutant produced a higher level of capsular polysaccharides and the ΔmgrA mutant produced a lower level of capsular polysaccharides. The inactivation of both rsaA and mgrA revealed that the repression of capsule production by rsaA achieved via the repression of mgrA. (B) Capsule production in Becker strain and its derivatives grown for 7 h in BHI at 37°C. CP8 expression was determined by indirect immunofluorescence of WT strain, the ΔrsaA mutant strain (LUG2010) and the mutant strain complemented with a plasmid expressing RsaA (LUG2032). The bacteria were labeled with Bodipy-vancomycine and capsule was labeled with an anti-CP8 antibody and a rabbit anti-IgG coupled with a fluorochrome AlexaFluor 647.