UC Santa Barbara

Iron is an essential nutrient required for many organisms, however, obtaining ferric iron becomes challenging due to its low solubility. One strategy that bacteria have evolved to obtain iron is the production of siderophores, low molecular weight organic compounds that bind Fe(III) with high affinity. These siderophores coordinate Fe(III) and are taken up by the cell through outer membrane receptor proteins. Iron is then released for utilization by the microbe. This work focuses on the structural characterization of siderophores containing the catecholate Fe(III)-binding functional group found in several bacterial strains.Due to the organization of non-ribosomal peptide synthetases (NRPS) into distinct domains with predictable functions and amino acid substrates, genome mining has enabled the prediction and discovery of many new siderophore structures. The analysis of genome sequences revealed Marinomonas mediterranea MMB-1 possessing two putative siderophore biosynthesis gene clusters, one with high similarity to acinetobactin biosynthesis in Acinetobacter baumannii ATCC 19606, and one with high similarity to turnerbactin biosynthesis in Teredinibacter turnerae T7901. However, analysis of the second biosynthetic gene cluster reveals a two-module NRPS consistent with a triscatechol siderophore (DHB-DAA-LSer). The first module contains an epimerization domain, suggesting production of a D-amino acid in this siderophore, however, a specific amino acid was not predicted by the Stachelhaus code. After bacterial culture isolation and characterization, mediterraneabactin, with the same molecular weight as turnerbactin (m/z 1030.40 [M+H]+ was found. Through derivatization with Marfey’s reagent, the presence of DOrn was established, making mediterraneabactin a diastereomer to turnerbactin with LOrn. The identification of this siderophore with DOrn is novel and completes the combinatoric suite of triscatecholate siderophores. The stereochemical variation has an effect on the chirality around the metal center, which in turn hints at the importance of chirality during the iron uptake process in bacteria. Amphi-enterobactin is an amphiphilic siderophore initially isolated from Vibrio campbellii ATCC BAA-1116 (formerly V. harveyi BAA-1116). . 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). The biosynthesis and structural characterization of amphi-enterobactin has been studied, as well as the outer membrane recognition of the Fe(III)-amphi-enterobactin complex. While it is established that amphi-enterobactins are produced by several Vibrio harveyi and V. campbellii strains, fragments of these amphi-enterobactins composed of 2-Ser-1-DHB-FA and 3-Ser-2-DHB-FA are present in the culture supernatant. Fragments may originate from premature release due to an inefficient biosynthetic pathway, or an enzymatic/non-enzymatic hydrolysis after biosynthesis of the siderophore. Tandem mass spectrometry analysis was used to determine if selected fragments originate from hydrolysis of the amphi-enterobactin macrolactone siderophore. 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.