The marine environment represents one of the most promising sources of novel, bioactive natural products. The process used in their discovery has been simplified and accelerated greatly in the 20th century with the advent of new technology, including spectroscopy, mass spectrometry, chromatography, in conjunction with bioassay -guided fractionation. However, more recent developments in genomics and metabolomics suggest that the biosynthetic capacity and interrelationships between organisms has been underappreciated and could likely yield many more important discoveries, both in medical value and in basic biological understanding. Contained in this dissertation are a series of unique experiments that harness the capacity of Matrix Assisted Laser Desorption Ionization (MALDI) to detect multiple metabolites concurrently from a single, small sample. This ability is used to capture the temporal dynamics of biosynthesis and turnover of secondary metabolites from cultured marine filamentous cyanobacteria, both in relation to each other and to primary metabolites. These temporal relationships are useful for improving compound yields from cultured organisms, tracking nitrogen in nutrient cycles, as well as providing an experimental tool to explore secondary metabolism in general. Also contained are the first images of natural products captured by MALDI imaging mass spectrometry, revealing complex chemical microenvironments in marine sponges and the distribution of known bioactive metabolites in marine cyanobacteria. Initial results also suggest that there is differential distribution of metabolites in cultured cyanobacteria. A third series of experiments exploiting the sensitivity of MALDI was conducted on single cells of filamentous marine cyanobacteria that are freed from the sheath and associated heterotrophs. The results reveal that many known natural products are found in single cells, further confirming their origin of biosynthesis; however, it appears not all cells from a filament contain the same metabolites. Nitrogen labeling experiments with these same preparations suggest the biosynthesis of the metabolites that are present occurs at similar rates. The last set of experiments employ Ion Mobility mass spectrometry on various preparations of marine filamentous cyanobacteria and show that this new technology can effectively separate out halogenated metabolites from complex mixtures, a powerful tool for the identification and discovery of bioactive metabolites

The candidate phylum TM7 is globally distributed and often associated with human inflammatory mucosal diseases. Despite its prevalence, the TM7 phylum remains recalcitrant to cultivation, making it one of the most enigmatic phyla known. In this study, we cultivated a TM7 phylotype (TM7x) from the human oral cavity. This extremely small coccus (200-300 nm) has a distinctive lifestyle not previously observed in human-associated microbes. It is an obligate epibiont of an Actinomyces odontolyticus strain (XH001) yet also has a parasitic phase, thereby killing its host. This first completed genome (705 kb) for a human-associated TM7 phylotype revealed a complete lack of amino acid biosynthetic capacity. Comparative genomics analyses with uncultivated environmental TM7 assemblies show remarkable conserved gene synteny and only minimal gene loss/gain that may have occurred as TM7x adapted to conditions within the human host. Transcriptomic and metabolomic profiles provided the first indications, to our knowledge, that there is signaling interaction between TM7x and XH001. Furthermore, the induction of TNF-α production in macrophages by XH001 was repressed in the presence of TM7x, suggesting its potential immune suppression ability. Overall, our data provide intriguing insights into the uncultivability, pathogenicity, and unique lifestyle of this previously uncharacterized oral TM7 phylotype.

Genome sequencing of microorganisms has revealed a greatly increased capacity for natural products biosynthesis than was previously recognized from compound isolation efforts alone. Hence, new methods are needed for the discovery and description of this hidden secondary metabolite potential. Here we show that provision of heavy nitrogen ¹⁵N-nitrate to marine cyanobacterial cultures followed by single-filament MALDI analysis over a period of days was highly effective in identifying a new natural product with an exceptionally high nitrogen content. The compound, named cryptomaldamide, was subsequently isolated using MS to guide the purification process, and its structure determined by 2D NMR and other spectroscopic and chromatographic methods. Bioinformatic analysis of the draft genome sequence identified a 28.7 kB gene cluster that putatively encodes for cryptomaldamide biosynthesis. Notably, an amidinotransferase is proposed to initiate the biosynthetic process by transferring an amidino group from arginine to serine to produce the first residue to be incorporated by the hybrid NRPS-PKS pathway. The maldiisotopic approach presented here is thus demonstrated to provide an orthogonal method by which to discover novel chemical diversity from Nature.

The potential of the diverse chemistries present in natural products (NP) for biotechnology and medicine remains untapped because NP databases are not searchable with raw data and the NP community has no way to share data other than in published papers. Although mass spectrometry (MS) techniques are well-suited to high-throughput characterization of NP, there is a pressing need for an infrastructure to enable sharing and curation of data. We present Global Natural Products Social Molecular Networking (GNPS; http://gnps.ucsd.edu), an open-access knowledge base for community-wide organization and sharing of raw, processed or identified tandem mass (MS/MS) spectrometry data. In GNPS, crowdsourced curation of freely available community-wide reference MS libraries will underpin improved annotations. Data-driven social-networking should facilitate identification of spectra and foster collaborations. We also introduce the concept of 'living data' through continuous reanalysis of deposited data.