Biochemical and functional characterization of KdpE_DBD.

<p><b>A.</b> Sedimentation velocity analysis of the KdpE_DBD to detect self-association. The c(s) distribution of the KdpE_DBD at 21 (dots), 42 (solid line), and 84 µM (dashes) shows a single species of 1.4 S. No concentration-dependent formation of higher-order species was observed. <b>B.</b> Interaction of KdpE_DBD protein with <i>kdpFABC_BS</i> and <i>ompF_Pro</i> DNA sequences analyzed by EMSA. The triangles represent increasing molar ratios of 1∶0, 1∶1, 1∶2, and 1∶3 of DNA to purified KdpE_DBD. The lower and upper bands represent free DNA and DNA-KdpE_DBD complex, respectively. <b>C. </b><i>In vivo</i> analysis of expression of the β-galactosidase gene fused to <i>kdpFABC_Pro</i>. <i>E. coli</i> RH003 cells lacking the histidine kinase (<i>kdpD</i>) and RR (<i>kdpE</i>) were used to express full-length KdpD alone as well as KdpD combined with KdpE or KdpE_DBD. As described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030102#s2" target="_blank">methods</a>, the cells were grown in K0 (▪) and K10 (□) media prior to analysis of gene expression. Growth in K0 medium mimics stresses resulting from external K⁺ depletion. The β-galactosidase activity expressed as Miller units represents the mean of three independent experiments; error bars represent standard error.