The dhb operon of Bacillus subtilis encodes the biosynthetic template for the catecholic siderophore 2,3-dihydroxybenzoate-glycine-threonine trimericester bacillibactin

Bacillus subtilis was reported to produce the catecholic siderophore itoic acid (2,3-dihydroxybenzoate (DHB)-glycine) in response to iron deprivation, However, by inspecting the DNA sequences of the genes dhbE, dhbB, and dhbF as annotated by the B, subtilis genome project to encode the synthetase co mplex for the siderophore assembly, various sequence errors within the dhbF gene were predicted and confirmed by re-sequencing. According to the corre cted sequence, dhbF encodes a dimodular instead of a monomodular nonribosom al peptide synthetase, We have heterologously expressed, purified, and assa yed the substrate selectivity of the recombinant proteins DhbB, DhbE, and D hbF, DhbE, a stand-alone adenylation domain of 59.9 kDa, activates, in an A TP-dependent reaction, I)HB, which is subsequently transferred to the free thiol group of the cofactor phosphopantetheine of the bifunctional isochori smate lyase/aryl carrier protein DhbB, The third synthetase, DhbF, is a dim odular nonribosomal peptide synthetase of 264 kDa that specifically adenyla tes threonine and, to a lesser extent, glycine and that covalently loads bo th amino acids onto their corresponding peptidyl carrier domains. To functi onally link the dhb gene cluster to siderophore synthesis, we have disrupte d the dhbF gene. Comparative mass spectrometric analysis of culture extract s from both the wild type and the dhbF mutant led to the identification of a mass peak at mit 881 ([M-H](1-)) that corresponds to a cyclic trimeric es ter of DHB-glycine-threonine.