Fig 6.

Cra and KdpE both directly regulate the espFu promoter region. (A) DNA sequence of the espFu promoter region showing the −35 and −10 positions and the transcription start site. The position of the putative Cra binding site is indicated in boldface. Below this is an alignment of the Cra binding site sequence from the espFu promoter with the consensus binding site sequence of Cra. (B) An espFu-lacZ transcriptional fusion was transformed into wt, ΔkdpE, Δcra, and ΔkdpE Δcra strains. The transcription fusion was also transformed into ΔkdpE and Δcra strains complemented with a low-copy chloramphenicol-resistant plasmid expressing KdpE and Cra, respectively. The beta-galactosidase assay was performed at an OD₆₀₀ of 0.5. (C and D) To investigate direct binding, EMSAs were performed using the espFu probe and increasing concentrations of recombinant Cra (C) or KdpE (D). For KdpE, EMSAs were done in the absence (D, left) or in the presence (D, right) of acetyl phosphate (lithium potassium salt).