FIG. 5.

(A) Expression of SdhA and FumB proteins, both regulated positively by Fur, in strains deriving from the MC58 wild type, with or without the fur gene, the NrrF gene and/or the hfq gene. Western blot analysis was performed on total proteins from overnight plating of the strains indicated and stained with antisera raised against the SdhA and FumB proteins. The expression of the Fur protein (17 kDa) and the Hfq protein (11 kDa) were verified in the appropriate strain's lower panel. NMB1870 protein expression was used as a negative control since it is neither Fur nor iron regulated (5). (B) Quantification by S1 nuclease protection assay using probe C and probe A, measuring the relative levels of the sdhC 5′ end of the transcript and the sdhA transcript downstream of the putative base-pairing region. Total RNA was extracted from equivalent cultures from the strains used in the Western blot analyses, and the results of the S1 nuclease assay are shown. (C) RNA analysis by quantitative primer extension of the levels of the sodB transcript in wild type (MC58), Hfq-null mutant (Δhfq), the Fur mutant (MC-Fko), and the double Fur-Hfq mutant (Fko-Hfq) grown to mid-log phase under iron-replete conditions before (+) and after (−) treatment for 15 min with iron chelator.