Characterizing Staphylococcal Swarming Dynamics

A colony spreading assay was performed as previously described by Kaito et al and Tsompanidou et al, who showed a hyper-spreading phenotype of srtA KO strain [26] (link), [27] (link). In brief, Tryptic Soy Broth (TSB) (BD, BBL, Etten-Leur, the Netherlands) was supplemented with 0.24% Agar powder (BD, Difco, Etten-Leur, the Netherlands) to prepare TSA soft agar. Fifteen ml TSA soft agar was poured into plates with 8 cm diameter and dried for 20 min. In parallel, SH1000 WT and isogenic srtA S. aureus KO strains OD_{600 nm} 0.05 were cultured during 24 hrs in TSB medium upon addition of either i) medium, ii) K(FITC)LPETG-amide or iii) K(FITC)EGTLP-amide (final volume of 50 µl), the final substrate concentration was 2 mM, 1 mM and 0.5 mM. After 24 hrs incubation 2 µl of individual cultures were gently spotted on the TSA soft agar plates and dried for 15 min. The plates were incubated during 20 hrs at 37°C. After incubation the spreading zones were examined and pictures were taken.