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. 2015 Oct 20;10(10):e0140784. doi: 10.1371/journal.pone.0140784

Fig 4. The S. pyogenes sortase mutant is under selective pressure leading to the loss of M protein expression.

Fig 4

A Wild type D471, sortase mutant AR01 (original stock), and complemented AR01+pAR107, were sonicated to separate the streptococcal chains into single cells, and subsequently plated. Single colonies were picked and grown in TH+Y (containing spectinomycin for AR01+pAR107) at 37°C overnight. Each day, a seed culture was transferred to a new tube for overnight growth, and a sample of the original culture was separated into supernatant and cell pellet, and stored at -80°C until use; the cultures were passaged in this manner six times. The cell pellets were lysed with PlyC. M protein was quantified by capture ELISA as described in the Materials and Methods section. The results represent 13 repeats for D471, 24 repeats for AR01, and 11 repeats for AR01+pAR107. B The relative amount of SfbI in AR01 supernatant and cell lysate was similarly determined by capture ELISA; the results represent 15 repeats. See S6 Fig for sfbI-negative control.