Fig. 7.

Development of an iron reporter for symbiotic rhizobium bacteria. (a) Diagram of the bacterial PmbfA:lux reporter and regulation of its expression by the iron response regulator (Irr) which binds the upstream Iron Control Element (ICE). In iron‐replete conditions, Irr binds iron in the form of haem and is degraded, leading to de‐repression (activation) of target gene expression. (b) Expression of the endogenous mbfA gene in free‐living Sinorhizobium meliloti 1021 in response to iron. Bacteria were grown in UMS medium without (−Fe) or with 40 µM iron sulphate (+Fe) until mid‐log phase, then harvested for RNA extraction. mbfA transcript levels were assayed by RT‐qPCR. Values were normalized to the housekeeping gene gapA, and are the mean ± SE of three biological replicates (***, P = 0.0001, Bio‐Rad CFX Maestro software). (c) Sinorhizobium meliloti 1021 carrying the lux plasmid without promoter (P‐:lux); the PmbfA:lux reporter; PmbfA:lux with a mutated ICE motif; or a constitutive lux reporter with the nptII promoter were grown as in (b). Luminescence was measured in a plate reader and corrected for cell density (OD₆₀₀). Values are the mean ± SE of three biological replicates (cell cultures grown in parallel). A.U., arbitrary units. (d) PmbfA:lux activity normalized for constitutive lux activity of the PnptII:lux reporter in response to iron. Error bars represent SE.