Isolation and Single Cell Chemical Biological Analysis of Natural Product Producing Cave Microorganisms

Abstract

Microbial secondary metabolites (natural products) constitute a large fraction of pharmaceuticals, with applications as antibiotics and anti‐cancer therapeutics. However, the discovery of new natural product drug leads is increasingly difficult as isolation studies using traditional methods, and from traditional sources, have resulted in progressively fewer novel compounds discovered. Historically, searching for new sources of biodiversity from unique ecosystems has resulted in increased discovery rates. The cave environment, a previously uninvestigated ecosystem, is a promising terrain to discover bacteria with novel natural products because of its unique conditions, which include low light, relatively constant temperature year‐round, abundant oxygen content, and low nutrient content (oligotrophy). We hypothesize that the oligotrophic environment present in caves forces microorganisms to compete or cooperate with each other, which require the development of natural products as communication, offensive, or defensive chemicals. Non‐Streptomyces actinomycetes are a group of bacteria found in caves that represent good candidates for the isolation of new natural products. We acquire bacterial samples by swabbing rock formations and collecting sediment inside a cave. These samples are diluted and incubated on a number of different agar plating conditions. Individual colonies are then isolated and identified by their 16S rRNA sequences. Isolated actinomycetes are stimulated using a battery of chemical and biological conditions known to elicit secondary metabolism (coculture strains, heavy metals, and antibiotics), and then culture extracts are analyzed for possible cellular activity using multiplexed biological assays against mammalian cells. Specifically, extracts are tested using single cell analysis via a flow cytometry platform termed Multiplexed Activity Metabolomics (MAM), which identifies compounds for isolation by analyzing extracts and metabolomic arrays against multiple cell status markers.Support or Funding InformationNational Cancer Institutes R01 CA226833