Tandem mass spectrometry methods to accelerate the identification of phytotoxic metabolites produced by Streptomyces sp. 39 PL.

Natural products isolated by microorganisms are interesting in the search for new compounds with several biological activities. However, low concentration and structural diversity make the isolation a time-consuming step. Tandem mass spectrometry is a well-established technology for the identification and characterization of target microbial natural products due to high sensitivity and selectivity of these experiments. We developed a method employing neutral loss experiments (LC-ESI-MS/MS) to identify luminacins in microbial crude extracts. The luminacins class exhibited conserved fragmentation pattern with loss at 172 Da relative to glycosides fragment and this loss was used in searching for compounds belonging to this class. Therefore, the crude extract produced by Streptomyces sp. 39 PL was analysed and five luminacins were isolated - one is a novel luminacin I at 466 Da.

Microbial Diversity and Chemical Multiplicity of Culturable, Taxonomically Similar Bacterial Symbionts of the Leaf-Cutting Ant Acromyrmex coronatus.

Insects are a highly diverse group, exploit a wide range of habitats, and harbor bacterial symbionts of largely unknown diversity. Insect-associated bacterial symbionts are underexplored but promising sources of bioactive compounds. The community of culturable bacteria associated with the leaf-cutting ant Acromyrmex coronatus (Fabricius) and the diversity of their metabolites produced were investigated. Forty-six phylotypes belonging to Actinobacteria, Firmicutes, and Proteobacteria were identified. The chemical profiles of 65 isolates were further analyzed by LC-MS/MS, and principal components analysis (PCA) was used to group the isolates according to their chemical profiles. Historically, selection of bacterial strains for drug discovery has been based on phenotypic and/or genotypic traits. Use of such traits may well impede the discovery of new compounds; in this study, several indistinguishable phylotypes cultured in identical nutritional and environmental conditions produced completely different chemical profiles. Our data also demonstrated the wide chemical diversity to be explored in insect-associated symbionts.

Single-bacterial genomics validates rich and varied specialized metabolism of uncultivated Entotheonella sponge symbionts.

Marine sponges are prolific sources of unique bioactive natural products. The sponge Theonella swinhoei is represented by several distinct variants with largely nonoverlapping chemistry. For the Japanese chemotype Y harboring diverse complex polyketides and peptides, we previously provided genomic and functional evidence that a single symbiont, the filamentous, multicellular organism "Candidatus Entotheonella factor," produces almost all of these compounds. To obtain further insights into the chemistry of "Entotheonella," we investigated another phylotype, "Candidatus Entotheonella serta," present in the T. swinhoei WA sponge chemotype, a source of theonellamide- and misakinolide-type compounds. Unexpectedly, considering the lower chemical diversity, sequencing of individual bacterial filaments revealed an even larger number of biosynthetic gene regions than for Ca E. factor, with virtually no overlap. These included genes for misakinolide and theonellamide biosynthesis, the latter assigned by comparative genomic and metabolic analysis of a T. swinhoei chemotype from Israel, and by biochemical studies. The data suggest that both compound families, which were among the earliest model substances to study bacterial producers in sponges, originate from the same bacterium in T. swinhoei WA. They also add evidence that metabolic richness and variability could be a more general feature of Entotheonella symbionts.

Tapping the biotechnological potential of insect microbial symbionts: new insecticidal porphyrins.

BACKGROUND: The demand for sustainable agricultural practices and the limited progress toward newer and safer chemicals for use in pest control maintain the impetus for research and identification of new natural molecules. Natural molecules are preferable to synthetic organic molecules because they are biodegradable, have low toxicity, are often selective and can be applied at low concentrations. Microbes are one source of natural insecticides, and microbial insect symbionts have attracted attention as a source of new bioactive molecules because these microbes are exposed to various selection pressures in their association with insects. Analytical techniques must be used to isolate and characterize new compounds, and sensitive analytical tools such as mass spectrometry and high-resolution chromatography are required to identify the least-abundant molecules. RESULTS: We used classical fermentation techniques combined with tandem mass spectrometry to prospect for insecticidal substances produced by the ant symbiont Streptomyces caniferus. Crude extracts from this bacterium showed low biological activity (less than 10% mortality) against the larval stage of the fall armyworm Spodoptera frugiperda. Because of the complexity of the crude extract, we used fractionation-guided bioassays to investigate if the low toxicity was related to the relative abundance of the active molecule, leading to the isolation of porphyrins as active molecules. Porphyrins are a class of photoactive molecules with a broad range of bioactivity, including insecticidal. The active fraction, containing a mixture of porphyrins, induced up to 100% larval mortality (LD50 = 37.7 µg.cm-2). Tandem mass-spectrometry analyses provided structural information for two new porphyrin structures. Data on the availability of porphyrins in 67 other crude extracts of ant ectosymbionts were also obtained with ion-monitoring experiments. CONCLUSIONS: Insect-associated bacterial symbionts are a rich source of bioactive compounds. Exploring microbial diversity through mass-spectrometry analyses is a useful approach for isolating and identifying new compounds. Our results showed high insecticidal activity of porphyrin compounds. Applications of different experiments in mass spectrometry allowed the characterization of two new porphyrins.

Liquid chromatography-tandem mass spectrometry characterization of five new leucinostatins produced by Paecilomyces lilacinus CG-189.

The fungus Paecilomyces lilacinus produces leucinostatins­peptaibiotics that exert a range of biological activities including antimalarial, antiviral, antitumor and phytotoxicity. In this paper, we developed an analytical method employing LC-MS/MS in the precursor ion and product ion scan modes to elucidate five new leucinostatins. Direct Infusion (DI-MS) helped to identify the most abundant leucinostatins: F, D B2, S, A and K. MS/MS analysis using a triple quadrupole operating at different scan modes is a versatile tool to study natural products, especially peptaibiotics. Although DI-MS full-scan analysis is rapid and sensitive, it cannot distinguish between peptide isomers. On the other hand, LC-MS/MS operated in the precursor ion and product ion modes is time consuming, but allows identifying the structure of isomers or isobar in crude extracts.