Figure 7.

In vitro and in vivo analysis of the regulatory function of HapX cccA promoter binding motifs. (A–D) SPR co-injection analysis of HapX binding to CBC bound A. fumigatus cccA promoter DNA duplexes carrying mutations within their conserved 5′-RWT-3′ and 5′-TKAN-3′ submotifs. Data are presented as described in figure legend 6. (E) Graphical representation of native or mutated A. fumigatus cccA promoter E. coli lacZ fusions integrated in single copy at the A. fumigatus pksP gene locus. Numbers indicate the positions of the three evolutionary conserved CBC:HapX binding sites (F) Effect of site 1 (-369) mutations on PcccA-lacZ expression after 18 h growth under iron depleted (−Fe) conditions followed by a 3 h shift of identical cultures to iron sufficiency (0.03 mM Fe). Iron dependent PcccA-lacZ expression was determined as β-galactosidase specific activity from soluble cell extracts. The host strains, AfS77 (wt) and ΔhapX, were used as negative controls. Data represent the mean ± SD of three independent biological replicates.