Amicoumacin B

Amicoumacins are natural products with potent anti-ulcerogenic and anti-bacterial activities, and have been isolated from different Bacillus genera. They belong to a family of 3,4-dihydroisocoumarin derivatives bearing hydroxylated amino acid side chains. The 3,4-dihydroisocoumarin moiety of Amicoumacins has been obtained in two steps from 2-methoxybenzoic acid by combining directed and benzylic metalation strategies. The use of s-BuLi in both steps gave satisfactory and reproducible yields. For the development of an immunoassay (ELISA) of Amicoumacin-related compounds in biological media, the deprotected 3,4-dihydroisocoumarin moiety has been coupled to the BSA carrier protein via a homobifunctional linker deriving from d-tartaric acid. This approach enabled to introduce the hydroxylated portion of Amicoumacin directly during the preparation of hapten-protein conjugates. The coupling ratio was evaluated by mass spectrometry. The hapten-protein conjugate showing the best coupling ratio was used to generate polyclonal immunosera in rabbits. After immunoserum titration, ELISA conditions were set up and specificity tests were performed on solutions of pure parent compounds, semi-purified Amicoumacin B as well as on culture supernatants of strains known for their Amicoumacin production. This immunoassay is suitable for a rapid and simple screening test for the production of Amicoumacins and its related compounds by bacterial strains.

In our study, the anti-quorum sensing (QS) activity of fermentation broth from TRM B-02, a bacterium isolated from Taklimakan desert, was investigated using the biosensor bioassay onChromobacterium violaceumATCC12472. TRM B-02 was 100% similar toBacillus subtilissubsp. Inaquosorum KCTC 13429(T)by genotypic and phenotypic analyses. Based on anti-QS activity tracking, six known amicoumacins, named as AI-77-H (1), AI-77-F (2), amicoumacin B (3), amicoumacin C (4), AI-77-C (5) and bacilosarcins D (6), were isolated and identified. Among them, compounds1-3exhibited a better inhibitory effect onC. violaceumATCC12472. Further research suggested that compounds1-3could significantly down-regulate the expressions of violacein operon A (vioA), vioB, vioD and vioE and up-regulate vioC. Docking experiments indicated that compounds1-3may act as an inhibitor of violacein biosynthetic pathway competitively inhibiting the binding of flavin adenine dinucleotide (FAD) with the vioD enzyme.[Figure: see text].

A novel actinomycete, strain 04-5195(T), that produces amicoumacin B, which targets bone morphogenetic protein-2, was isolated from a soil sample collected in Jinan, Shandong Province, China. Strain 04-5195(T) had morphological, biochemical, physiological and chemotaxonomic properties that were consistent with its classification in the genus Nocardia and it formed a phyletic line in the Nocardia 16S rRNA gene tree. It was evident from the phylogenetic data that strain 04-5195(T) was most closely associated with Nocardia speluncae N2-11(T). However, the two organisms were distinguishable from one another using DNA-DNA relatedness and phenotypic data. The isolate was readily differentiated from other related Nocardia strains by a set of phenotypic properties and by its phylogenetic position. Therefore, it is proposed that the isolate represents a novel species in the genus Nocardia, Nocardia jinanensis sp. nov.; the type strain is 04-5195(T) (=CGMCC 4.3508(T) =DSM 45048(T)).