Ferri‐bacillibactin uptake and hydrolysis in Bacillus subtilis

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Abstract
Summary: Upon iron limitation, Bacillus subtilis secretes the catecholic trilactone (2,3‐dihydroxybenzoate‐glycine‐threonine)3 siderophore bacillibactin (BB) for ferric iron scavenging. Here, we show that ferri‐BB uptake is mediated by the FeuABC transporter and that YuiI, a novel trilactone hydrolase, catalyses ferri‐BB hydrolysis leading to cytosolic iron release. Among several Fur‐regulated ABC transport mutants, only ΔfeuABC exhibited impaired growth during iron starvation. Quantification of intra‐ and extracellular (ferri)‐BB in iron‐depleted ΔfeuABC cultures revealed a fourfold increase of the extracellular siderophore concentration, confirming a blocked ferri‐BB uptake in the absence of FeuABC. Ferri‐BB was found to bind selectively to the periplasmic binding protein FeuA (Kd = 57 ± 1 nM), proving high‐affinity transport of the iron‐charged siderophore. During iron starvation, a ΔyuiI mutant displayed impaired growth and strong intracellular (30‐fold) and extracellular (6.5‐fold) (ferri)‐BB accumulation. Kinetic studies in vitro revealed that YuiI hydrolyses both BB and ferri‐BB. While BB hydrolysis led to strong accumulation of the tri‐ and dimeric reaction intermediates, ferri‐BB hydrolysis yielded exclusively the monomeric reaction product and occurred with a 25‐fold higher catalytic efficiency than BB single hydrolysis. Thus, ferri‐BB was the preferred substrate of the YuiI esterase whose gene locus was designated besA.