Furthermore, in order to determine whether other MarR family and TetR family proteins such as SAR2349 from S. aureus and . IcaR DNA1 probe duplex of 1 mM was pre-incubated with 2 mM TcaR (dimer) at room temperature for 15 min before mixing with increasing concentration of GC33 ssDNA, followed by the same procedure as described in the legend to Figure 1B . (F) Competition experiment was carried out to compare the binding strength of TcaR to ssDNA and to dsDNA. In the EMSA analysis, 1 mM IcaR DNA1 probe duplex was pre-incubated with 1 mM GC33 ssDNA fragment for 15 min at room temperature before mixing with TcaR protein of increasing concentration. (G) The effects of pH to the TcaR binding to ssDNA GC33 in EMSA experiment. Molar ratio of GC33:TcaR was 1:2. (H) EMSA analysis of mutant TcaR protein binding to ssDNA (GC33) and dsDNA (IcaR DNA1) fragments with different protein-DNA ratio. (I) EMSA analysis of the binding of TcaR protein to ssDNA(GC33) in the present of different types of antibiotics. GC33 ssDNA probe of 1 mM was preincubated with 2 mM TcaR (dimer) at room temperature for 15 min before mixing with 2 mM or 20 mM antibiotics, followed by the same procedure as described in the legend to Figure 1B IcaR from S. epidermidis have the ssDNA binding ability, we conducted a series of SPR experiments to analyze the binding ability of SAR2349 and IcaR proteins to GC33 ssDNA ( Figure 2C ). The result shows that not all MarR family proteins have this ssDNA binding ability, thus pointing to the specific ssDNA-binding feature of TcaR. In addition, we have previously demonstrated that antibiotics appear to antagonize the ssDNA binding activity of TcaR ( Figure 1I ). Therefore, a measurement for the effect of kanamycin and ampicillin to the GC33 ssDNA binding affinity of TcaR is conducted using surface plasmon resonance to confirm the result. As seen in Figure 2D , the affinity between TcaR and GC33 ssDNA is shown by a decrease in RU values in the presence of antibiotics. This was especially apparent with kanamycin, which yield the lower binding capacity. Taken together, we demonstrate that TcaR shows a higher binding affinity to ssDNA than to dsDNA, and several antibiotics could regulate the ssDNA binding activity of TcaR.