Fig. 5.

Cells expressing MdtD(IceT) secrete the CAS-reactive iron chelator citrate. (A) EF341 (ΔentB) is unable to produce siderophores and is CAS-negative (well 1). Expression of MdtD(IceT) (EF342) results in secretion of a CAS-positive iron chelator (well 2), whereas expression of MdtABC (EF336) does not (well 3). (B) The CAS-positive fraction was purified from growth medium and analyzed by ¹H NMR. Peaks indicated by an asterisk correspond to hydrogen atoms bound to the 1 and 3 carbons of citrate (indicated by an asterisk in Inset). (C) Identification of citrate was confirmed by determining the mass of the compound by mass spectrometry. MdtD was therefore renamed IceT. (D) Supernatant from EF345 and EF346 lacking citrate synthase (ΔentB ΔgltA, wells 3 and 4) are not CAS-positive even though EF346 expresses IceT (compare wells 2 and 4). When citrate synthase is expressed from a plasmid (EF347 and EF348, wells 5 and 6), IceT-dependent secretion of the chelator is restored (well 6).