FIG. 2.
Binding of LldR to the NCgl2816-lldD promoter. (A) DNA fragments used to analyze LldRHis binding to the NCgl2816-lldD promoter region. The numbers indicate the ends of the fragments relative to the NCgl2816 transcriptional start site (+1). Binding of LldR to the fragments is indicated by a plus sign, and a lack of binding is indicated by a minus sign. Oligonucleotides used for amplification of the six fragments are listed in Table 2. The sequence above the fragments shows the region between position −13 and position 41 relative to the transcriptional start site. The transcriptional start is indicated by a large letter, the −10 region is italicized, and the half-sites of the consensus operator sequence for FadR-type regulators are underlined (single and double underlining). (B) Purified His-tagged LldR protein was used in 0-, 10- and 50-fold molar excess over DNA fragment F0 (P) before separation by native PAGE and SYBR green I staining. A 175-bp fragment of the NCgl0430 (43 nM) promoter served as a negative control DNA fragment (Co). (C) Subfragments F1, F2, F3, F4, and F5 of NCgl2816-lldD promoter fragment F0 were incubated with an 11-fold molar excess of purified LldR, separated by PAGE, and stained with SYBR green I. A 190-bp fragment of the NCgl2027 promoter (40 nM) served as a control fragment (Co). (D) Subfragment F4 and derived fragments with different mutations in or near the consensus sequence for FadR-type regulators M1, M2, M12, M3, M4, M5, and M6 (P) were incubated with an 11-fold molar excess of LldRHis. Lanes WT, wild type; lanes 1, the nucleotides in panel A underlined with one line were changed by PCR from TGT to GTG (fragment M1); lanes 2, the nucleotides in panel A underlined with two lines were changed by PCR from ACA to TAG (fragment M2); lanes 12, all the underlined nucleotides in panel A were changed by PCR (fragment 12). Changes outside the consensus sequence were introduced into fragments M3 (7 bp upstream; TCA → GCA) (lanes 3), M4 (4 bp upstream; ATT → CAA) (lanes 4), M5 (3 bp downstream; GTT → TGG) (lanes 5), and M6 (7 bp downstream; GGG → TTT) (lanes 6). A PCR product from position −178 to position −14 relative to the NCgl2816-lldD transcriptional start site (46 nM) served as a negative control (lanes Co). Oligonucleotides used for amplification of the fragments are listed in Table 2.