Ferri-bacillibactin uptake and hydrolysis in Bacillus subtilis.
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 DeltafeuABC exhibited impaired growth during iron starvation. Quantification of intra- and extracellular (ferri)-BB in iron-depleted DeltafeuABC 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 DeltayuiI 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.
Miethke
Marcus
M
Department of Chemistry, Philipps-Universität Marburg, D-35032 Marburg, Germany. marahiel@chemie.uni-marburg.de
Klotz
Oliver
O
Linne
Uwe
U
May
Jürgen J
JJ
Beckering
Carsten L
CL
Marahiel
Mohamed A
MA
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D001412:Bacillus subtilis / Q000201:enzymology / Q000378:metabolism*; D004950:Esterases / Q000235:genetics / Q000378:metabolism; D004952:Esters / Q000378:metabolism*; D017353:Gene Deletion; D006868:Hydrolysis; D007501:Iron / Q000378:metabolism*; D007502:Iron Chelating Agents / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D004952:Esters; D007502:Iron Chelating Agents; D009842:Oligopeptides; C430721:bacillibactin; D007501:Iron; D004950:Esterases