Figure 3.

FruR binding to fruB O1 is indispensable for fruBKA transcription in V. cholerae. (A) Effect of VcFruR binding to each operator on the transcriptional activation of the fru operon was measured by the lacZ reporter assay using a fruR⁺lacZ⁻ strain harboring a plasmid carrying E. coli lacZ transcriptionally fused with the wild-type or mutated fruB promoter (mutated site indicated as m#). Schematic representation of plasmids is shown left, with the lacZ gene and mutation site depicted by a grey arrow and X, respectively. The fruR⁺lacZ⁻ strain harboring the plasmid containing promoter-less lacZ served as a negative control (PV). Indicated strains were grown on glucose (open bars) or fructose (filled bars) and then lysed to measure the β-galactosidase activity as described under ‘Materials and Methods.’ (right panel). Statistical significance was determined using the Student's t-test. (*P< 0.05; **P< 0.01). (B) The transcriptional activation by FruR binding to fruB O1 was further confirmed by measuring the fruB expression in fructose-grown strains carrying the chromosomal duplication of the wild-type or mutated fruR-fruB intergenic sequence. Schematic representation of the strains carrying duplicated regions is shown in the upper panel, with FruR-binding sites and mutated areas depicted by white rectangles and yellow shadings, respectively. The mRNA expression levels of fruB and fruR in the indicated strains grown on fructose were analyzed by qRT-PCR and shown as log₂ values relative to that in the DP strain grown on glucose (lower panel). (C) Growth of DP-B-m# strains on fructose was compared with the DP (black) and DPΔfruR (blue) strains and the strains showing the same growth defect as the DPΔfruR strain were indicated in red. The means and standard deviations of three independent measurements are shown in (A) and (C).