Vibrio campbellii BAA-1116 (formerly Vibrio harveyi) is a model organism for quorum sensing study and produces the siderophores anguibactin and amphi-enterobactin. This study examined the mechanisms and specificity of siderophore uptake in V. campbellii and V. harveyi, and surveyed the diversity of siderophore production in V. campbellii and V. harveyi strains. The amphi-enterobactin gene cluster of BAA-1116 harbors a gene, named fapA, that is a homologue of genes encoding Fe(III)-siderophore-specific outer membrane receptors. Another strain, V. campbellii HY01, a strain pathogenic to shrimp, also carries this cluster including fapA. Our siderophore bioassay results using HY01-derived indicator strains show that the FapA protein localized in the outer membrane fraction of V. campbellii HY01 is essential for the uptake of Fe(III)-amphi-enterobactin as well as exogenous siderophores, including enterobactin from E. coli, but not vanchrobactin from V. anguillarum RV22 while Fe(III)-amphi-enterobactin can be utilized by V. anguillarum. Electrospray ionization mass spectrometry as well as bioassay revealed that various V. campbellii and V. harveyi strains produce a suite of amphi-enterobactins with various fatty acid appendages, including several novel amphi-enterobactins, and these amphi-enterobactins can be taken up by V. campbellii HY01 via FapA, indicating that amphi-enterobactin production is a common phenotype among V. campbellii and V. harveyi, whereas our previous work, confirmed herein, showed that anguibactin is only produced by V. campbellii strains. These results along with the additional finding that a 2,3-dihydroxybenzoic acid biosynthesis gene, aebA, located in the amphi-enterobactin gene cluster, is essential for both anguibactin and amphi-enterobactin biosynthesis, suggest the possibility that amphi-enterobactin is a native siderophore of V. campbellii and V. harveyi, while the anguibactin system has been acquired by V. campbellii during evolution.

Abstract:The β1α1 loop in the tryptophan biosynthetic enzyme indole-3-glycerol phosphate synthase (IGPS) is important for substrate binding, product release and chemical catalysis. IGPS catalyzes the ring closure of the substrate 1-(o-carboxyphenylamine)-1-dexoyribulose 5-phosphate to form indole-3-glycerol phosphate, involving distinct decarboxylation and dehydration steps. The ring closure step is rate-determining in the thermophilic Sulfolobus sulfataricus enzyme (ssIGPS) at high temperatures. The β1α1 loop is especially important in the dehydration step as it houses the general acid Lys53. We propose that loop dynamics are governed by competing interactions on the N-and C-terminal sides of the loop. We had previously shown that disrupting interactions with the N-terminal side of the loop through the N90A substitution decreases catalytic efficiency, slows down the dehydration step and quenches loop dynamics on the picosecond to millisecond timescales. Here, we show that disrupting interactions on the C-terminal side of the loop through the R64A/D65A substitutions likewise decreases catalytic efficiency, slows down the dehydration step and quenches loop dynamics. Interestingly, the triple substitution R64A/D65A/N90A leads to new µs-ms timescale loop dynamics and makes the ring-closure step rate-determining once again. These results are consistent with a model in which the β1α1 loop is maintained in a structurally dynamic state by these competing interactions, which is important for the dehydration step of catalysis. Competing interactions in other enzymes may likewise keep their loops and other structural elements appropriately mobile.

Amphi-enterobactin is an amphiphilic siderophore isolated from a variety of microbial Vibrio species. Like enterobactin, amphi-enterobactin is a triscatecholate siderophore; however, it is framed on an expanded tetralactone core comprised of four l-Ser residues, of which one l-Ser is appended by a fatty acid and the remaining l-Ser residues are appended by 2,3-dihydroxybenzoate (DHB). Fragments of amphi-enterobactin composed of 2-Ser-1-DHB-FA and 3-Ser-2-DHB-FA have been identified in the supernatant of Vibrio campbellii species. The origin of these fragments has not been determined, although two distinct isomers could exist for 2-Ser-1-DHB-FA and three distinct isomers could exist for 3-Ser-2-DHB-FA. The fragments of amphi-enterobactin could originate from hydrolysis of the amphi-enterobactin macrolactone, or from premature release due to an inefficient biosynthetic pathway. Unique masses in the tandem MS analysis establish that certain fragments isolated from the culture supernatant must originate from hydrolysis of the amphi-enterobactin macrolactone, while others cannot be distinguished from premature release during biosynthesis or hydrolysis of amphi-enterobactin. Graphical abstract

The vast majority of bacteria require iron to grow. A significant iron acquisition strategy is the production of siderophores, which are secondary microbial metabolites synthesized to sequester iron(III). Siderophore structures encompass a variety of forms, of which highly modified peptidic siderophores are of interest herein. State‐of‐the‐art genome mining tools, such as antiSMASH (antibiotics & Secondary Metabolite Analysis SHell), hold the potential to predict and discover new peptidic siderophores, including a combinatoric suite of triscatechol siderophores framed on a triserine‐ester backbone of the general class, (DHB‐l/dCAA‐lSer)3 (CAA, cationic amino acid). Siderophores with l/dArg, l/dLys and lOrn, but not dOrn, were predicted in bacterial genomes. Fortuitously the dOrn siderophore was identified, yet its lack of prediction highlights the limitation of current genome mining tools. The full combinatoric suite of these siderophores, which form chiral iron(III) complexes, reveals stereospecific coordination chemistry encoded in microbial genomes. The chirality embedded in this suite of Fe(III)‐siderophores raises the question of whether the relevant siderophore‐mediated iron acquisition pathways are stereospecific and selective for ferric siderophore complexes of a defined configuration.

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