The dhb Operon of Bacillus subtilisEncodes the Biosynthetic Template for the Catecholic Siderophore 2,3-Dihydroxybenzoate-Glycine-Threonine Trimeric Ester Bacillibactin

Abstract

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 complex for the siderophore assembly, various sequence errors within the dhbF gene were predicted and confirmed by re-sequencing. According to the corrected sequence, dhbF encodes a dimodular instead of a monomodular nonribosomal peptide synthetase. We have heterologously expressed, purified, and assayed the substrate selectivity of the recombinant proteins DhbB, DhbE, and DhbF. DhbE, a stand-alone adenylation domain of 59.9 kDa, activates, in an ATP-dependent reaction, DHB, which is subsequently transferred to the free thiol group of the cofactor phosphopantetheine of the bifunctional isochorismate lyase/aryl carrier protein DhbB. The third synthetase, DhbF, is a dimodular nonribosomal peptide synthetase of 264 kDa that specifically adenylates threonine and, to a lesser extent, glycine and that covalently loads both amino acids onto their corresponding peptidyl carrier domains. To functionally link the dhb gene cluster to siderophore synthesis, we have disrupted the dhbF gene. Comparative mass spectrometric analysis of culture extracts from both the wild type and the dhbF mutant led to the identification of a mass peak at m/z 881 ([M-H] 1؊) that corresponds to a cyclic trimeric ester of DHB-glycine-threonine.

Enterobactin: An archetype for microbial iron transport

“…However, corynebactin (Fig. 4), isolated from Gram-positive Corynebacterium glutamicum (74) and Bacillus subtilis (and then improperly renamed) (75), incorporates a threonine trilactone and glycine spacers, which elongate the three chelating arms as compared with enterobactin. The 12-membered trilactone macrocyclic ring has two stable conformations.…”

Identification and analysis of a siderophore biosynthetic gene cluster from Agrobacterium tumefaciens C58

“…It may also be that Agrobacterium is exposed to iron-limiting conditions when residing in the soil rather than when infecting plants. Many other soilborne bacteria are known to produce siderophores (Cornelis & Matthijs, 2002;Guerinot, 1991;May et al, 2001;Ravel & Cornelis, 2003), suggesting that iron scavenging is important for successful survival in soil. Interestingly, Rosen et al (2003) detected induction of one putative iron transport protein in A. tumefaciens C58, namely AfuC (Atu4786), upon exposure of cells to tomato root extract, suggesting that under some conditions, iron acquisition is important in the plant environment.…”

Genomewide transcriptional changes associated with genetic alterations and nutritional supplementation affecting tryptophan metabolism in Bacillus subtilis

“…For example, genes in the dhb operon that were observed to be down-regulated in experiments 1, 2, 3, and 7 form a single node that includes the following: dbhACEF, ydbL, yuiI, yoeB, yplQ, yukLM, and yvbA. Although ydbL, yuiI, yoeB, yplQ, and yvbA have unknown functions, yukL and yukM are actually part of the dhbF ORF that was recently extended because of sequencing errors (28).…”