Skip to main content
. 2008 Sep 12;190(22):7508–7522. doi: 10.1128/JB.00553-08

FIG. 4.

FIG. 4.

Band shift assay for PecS-DNA binding. (A) Labeled DNA probes, corresponding to promoter regions of rhlA, avrL, and ahpC, were incubated with increasing concentrations of PecS, as indicated at the bottom. The celZ regulatory region was used as a positive control (44), and the mfhR nonspecific fragment derived from the coding region of a transcriptional regulator not controlled by PecS was used as a negative control. (B) Specificity of PecS binding on nipE regulatory region. EMSA shows binding of PecS to the 400-bp nipE regulatory region (region −300 to +100 relative to the translation start site) with an increasing PecS concentration (0, 50, 75, 100, 150, 200, 400, and 800 nM) in the presence of 30 fmol of radiolabeled probe (a). In the reactions mixtures containing 30 fmol of radiolabeled probe and 150 nM PecS, competition assays were performed with the molar excesses of the unlabeled 400-bp nipE regulatory region specific competitor indicated above the lanes (×0, ×5, ×20, and ×50) and with the indicated molar excesses of unlabeled nonspecific competitor DNA derived from a 250-bp fragment of the mfhR coding region (b). Simultaneous titrations were performed with PecS and both 32P-labeled nipE probe and 32P-labeled mfhR probe (c). (C) Specificity of PecS binding on virK regulatory region. EMSA shows binding of PecS to the 440-bp virK regulatory region (region −370 to +70 relative to the translation start site) with an increasing PecS concentration (0, 50, 75, 100, 150, 200, 400, and 800 nM) in the presence of 30 fmol of radiolabeled probe (a). In the reaction mixtures containing 30 fmol of radiolabeled probe and 200 nM PecS, competition assays were performed with molar excesses of the unlabeled 440-bp virK regulatory region specific competitor indicated above the lanes (×0, ×5, ×20, and ×50) and with the indicated molar excesses of unlabeled nonspecific competitor DNA derived from a 250-bp fragment of the mfhR coding region (b). Simultaneous titrations were performed with PecS and both 32P-labeled virK probe and 32P-labeled mfhR probe (c).