FIG. 6.

DNase I footprinting assay of interaction between CcpC_Lm-His₆ and lmo0847. A. A ³²P-labeled DNA fragment corresponding to the putative lmo0847 promoter region was incubated with different amounts of CcpC_Lm-His₆ prior to DNase I digestion. Protein concentrations were as follows: 0 (lane 1), 3.9 (lane 2), 15.6 (lane 3), 62.5 (lane 4), 250 (lane 5), and 1,000 nM (lane 6). Sequence ladders obtained by Sanger sequencing of the same DNA fragment are shown to the left. Arrows show the elements of the putative CcpC binding site identified by genome search, and the vertical line indicates a region protected by CcpC_Lm-His₆ from DNase I digestion. The gray arrowheads show sites of hypersensitivity to DNase I digestion in the presence of CcpC_Lm. B. Effect of citrate on the interaction of CcpC_Lm-His₆ with lmo0847. The same DNA probe as in part A was incubated with CcpC_Lm-His₆ (0 nM, lanes 1 and 6; 62.5 nM, lanes 2 to 5 and 7 to 10) in the presence of citrate (0.5%, lane 3; 0.25%, lane 4; 0.13%, lane 5) or isocitrate (0.5%, lane 8; 0.25%, lane 9; 0.13%, lane 10). The bracket indicates a region where protection by CcpC_Lm-His₆ was inhibited specifically by citrate. The gray arrowheads indicate a site of hypersensitivity to DNase I that was abrogated in the presence of citrate. C. Sequence of the lmo0847 promoter region. The region protected by CcpC_Lm-His₆ from DNase I digestion (positions −135 to −83) is underlined, and the region where CcpC_Lm-His₆ was released by citrate (positions −105 to −83) is indicated by a bracket. The −35 and −10 regions of the putative promoter are shaded in gray, and the translational start codon is in italics.