The structure of ToxT. (A) Ribbon diagram of ToxT showing α-helices (gold), β-strands (cyan), and loops (dark red). The bound cis-palmitoleate is shown in stick form with carbons in green and oxygens in red. The N and C termini are indicated. Helices and strands are numbered according to their topological connectivity in the full-length protein. Note that residues 101–110 are disordered in the structure, as indicated by the loop ends on the left side of the molecule. (B) Close up of the cis-palmitoleate binding region showing interactions of the carboxylate head group with side chains.

Pairwise SSM alignments of the DBDs of ToxT (3GBG), AraC (2K9S), and MarA (1BL0). ToxT is represented in silver, AraC in blue, and MarA in dark red. Helix α7 and the DNA-binding helices α6 and α9 are labeled. Arrows indicate the relative direction each DNA-binding helix is pointing. The asterisk indicates the distorted section of helix α6 on ToxT.

Model for the regulation of ToxT by monounsaturated fatty acids. Fatty acid bound ToxT is in a “closed” conformation, which cannot bind to DNA. Release of the fatty acid results in an “open” conformation that can bind to DNA. In the “open” conformation the N-terminal domain is free to move in relation to the C-terminal domain, and is able to dimerize with another ToxT at an adjacent toxbox. The linker is sufficiently flexible to allow dimerization on DNA in either direct or inverted orientations.

Ribbon diagrams of ToxT and the N- and C-terminal domains of AraC. The models are colored by a rainbow effect with blue at the N terminus and red at the C terminus. The arabinose bound in the N-terminal domain of AraC is shown in stick form. PDB accession numbers are indicated for the three structures.

Effects of fatty acids on tcp and ctx expression. (A) and (B) β-galactosidase activity of tcp-lacZ and ctx-lacZ fusion constructs respectively. Cells were grown in LB pH 6.5 at 30 °C for 18 hours +/- the indicated fatty acids at 0.02% dissolved in methanol. The inset shows TcpA expression by Western blot under the same conditions in the corresponding lanes (C—control with methanol; PA—sodium palmitate; POA—palmitoleic acid; OA—oleic acid). (C) EMSA showing a specific interaction between ToxT and a 40-base-pair segment of the tcp promoter. All lanes contain 0.0025 μM labeled probe. Lane 1, free DNA; lane 2, 0.2 μM ToxT; lane 3, 0.2 μM ToxT with a 70-fold molar excess of cold competing DNA; lane 4, 0.2 μM ToxT with a 70-fold molar excess of a 42-base-pair nonspecific DNA cold competitor. (D) EMSA showing the effect of several fatty acids on ToxT/DNA interactions. All lanes contain 0.0025 μM labeled probe. Lane 1, free DNA; lane 2, 0.125 μM ToxT; lane 3, 0.2 μM ToxT; lane 4, 0.25 μM ToxT; lane 5, 0.25 μM ToxT with methanol; lane 6, 0.25 μM ToxT with 0.002% palmitic acid; lane 7, 0.25 μM ToxT with 0.002% palmitoleic acid; lane 8, 0.25 μM ToxT with 0.002% oleic acid.