Fig. 1 |. FezB, regulated by Fur and iron, plays a critical role in maintaining iron homeostasis during the transition from iron deficiency to excess.

a, The 5′ untranslated region of fezAB contains two putative Fur boxes and a small transcript ( fezX). The two transcription start sites (TSS1 and TSS2) were mapped by 5′ rapid amplification of complementary DNA ends (5′ RACE). b,c, Expression of fezXAB was quantified by qPCR in WT and fur::CT (b) or in WT following vehicle or 100 μM dipyridyl treatment (c). d,e, The growth kinetics of C. difficile WT, ΔfezB and the complemented strain ΔfezB pJS116-fezB grown under vehicle (BHIS) (d) or during the transition from iron deficiency (100 μM dipyridyl) to excess following addition of 1 mM FeSO₄ (e). f, Intracellular iron was monitored using ICP-MS for 4 h after 1 mM FeSO₄ was added to cultures of C. difficile WT, ΔfezB and ΔfezB pJS116-fezB cells grown in BHIS amended with 100 μM dipyridyl. The data are expressed as ppm per gram of cell pellet. Experiments were conducted three (b–e) or two (f) times independently with similar results. Data are presented as mean ± s.d., n = 6 (b,c); mean ± s.d., n = 6 (d,e); mean ± s.e.m., n = 6 (f). Significant differences were determined by two-tailed t-tests and the P values are denoted in the graphs. OD₆₀₀, optical density at 600 nm.