Fig. 2

Ms0535 specifically binds to its own promoter. (a) Bacterial one-hybrid assays. Promoters of the Ms0535 and Ms0540 genes were cloned into the pBXcmT vector, and the Ms0535 gene was cloned into the pTRG vector. A pair of pBXcmT/pTRG plasmids was co-transformed into the reporter strain, and then its growth was tested together with the self-activation controls on a selective medium. Co-transformants containing the pBX-Rv2031/pTRG-Rv3133 plasmids [26] served as positive controls (CK+), and co-transformants containing the empty vectors pBXcmT and pTRG served as negative controls (CK−). (b) Electrophoretic mobility shift assays (EMSAs). The Ms0535p (lanes 1–4) and Ms0540p (lanes 5–8) DNA substrates were co-incubated with various amounts of the Ms0535 protein. The free DNA substrate and DNA-protein complexes are indicated. (c) EMSAs for the specific binding of Ms0535 to its own promoter. Then, 0.05 nM of fluorescein isothiocyanate-labeled Ms0535 promoter DNA substrate was co-incubated with the Ms0535 protein in the absence (lanes 1–5) or presence of non-labeled Ms0535p (0.25-1 nM) (lanes 6–8) or non-labeled Ms0540p (0.25-1 nM) (lanes 9–11). Unlabeled Ms0535 and unlabeled Ms0540 promoter DNA substrates were used to compete with the labeled Ms0535 promoter DNA. The Ms0535 promoter, but not the Ms0540 promoter, inhibited the binding of Ms0535 to the labeled Ms0535 promoter DNA substrate