Discovery and Biosynthesis of Ureidopeptide Natural Products Macrocyclized via Indole N-acylation in Marine Microbulbifer spp. Bacteria.

Commensal bacteria associated with marine invertebrates are underappreciated sources of chemically novel natural products. Using mass spectrometry, we had previously detected the presence of peptidic natural products in obligate marine bacteria of the genus Microbulbifer cultured from marine sponges. In this report, the isolation and structural characterization of a panel of ureidohexapeptide natural products, termed the bulbiferamides, from Microbulbifer strains is reported wherein the tryptophan side chain indole participates in a macrocyclizing peptide bond formation. Genome sequencing identifies biosynthetic gene clusters encoding production of the bulbiferamides and implicates the involvement of a thioesterase in the indolic macrocycle formation. The structural diversity and widespread presence of bulbiferamides in commensal microbiomes of marine invertebrates point toward a possible ecological role for these natural products.

Pseudobulbiferamides: Plasmid-Encoded Ureidopeptide Natural Products with Biosynthetic Gene Clusters Shared Among Marine Bacteria of Different Genera.

Ureidopeptidic natural products possess a wide variety of favorable pharmacological properties. In addition, they have been shown to mediate core physiological functions in producer bacteria. Here, we report that similar ureidopeptidic natural products with conserved biosynthetic gene clusters are produced by different bacterial genera that coinhabit marine invertebrate microbiomes. We demonstrate that a Microbulbifer strain isolated from a marine sponge can produce two different classes of ureidopeptide natural products encoded by two different biosynthetic gene clusters that are positioned on the bacterial chromosome and on a plasmid. The plasmid encoded ureidopeptide natural products, which we term the pseudobulbiferamides (5-8), resemble the ureidopeptide natural products produced by Pseudovibrio, a different marine bacterial genus that is likewise present in marine sponge commensal microbiomes. Using imaging mass spectrometry, we find that the two classes of Microbulbifer-derived ureidopeptides occupy different physical spaces relative to the bacterial colony, perhaps implying different roles for these two compound classes in Microbulbifer physiology and environmental interactions.

Limited Metabolomic Overlap between Commensal Bacteria and Marine Sponge Holobionts Revealed by Large Scale Culturing and Mass Spectrometry-Based Metabolomics: An Undergraduate Laboratory Pedagogical Effort at Georgia Tech.

Sponges are the richest source of bioactive organic small molecules, referred to as natural products, in the marine environment. It is well established that laboratory culturing-resistant symbiotic bacteria residing within the eukaryotic sponge host matrix often synthesize the natural products that are detected in the sponge tissue extracts. However, the contributions of the culturing-amenable commensal bacteria that are also associated with the sponge host to the overall metabolome of the sponge holobiont are not well defined. In this study, we cultured a large library of bacteria from three marine sponges commonly found in the Florida Keys. Metabolomes of isolated bacterial strains and that of the sponge holobiont were compared using mass spectrometry to reveal minimal metabolomic overlap between commensal bacteria and the sponge hosts. We also find that the phylogenetic overlap between cultured commensal bacteria and that of the sponge microbiome is minimal. Despite these observations, the commensal bacteria were found to be a rich resource for novel natural product discovery. Mass spectrometry-based metabolomics provided structural insights into these cryptic natural products. Pedagogic innovation in the form of laboratory curricula development is described which provided undergraduate students with hands-on instruction in microbiology and natural product discovery using metabolomic data mining strategies.

Metabolomics Approaches to Dereplicate Natural Products from Coral-Derived Bioactive Bacteria.

Stony corals (Scleractinia) are invertebrates that form symbiotic relationships with eukaryotic algal endosymbionts and the prokaryotic microbiome. The microbiome has the potential to produce bioactive natural products providing defense and resilience to the coral host against pathogenic microorganisms, but this potential has not been extensively explored. Bacterial pathogens can pose a significant threat to corals, with some species implicated in primary and opportunistic infections of various corals. In response, probiotics have been proposed as a potential strategy to protect corals in the face of increased incidence of disease outbreaks. In this study, we screened bacterial isolates from healthy and diseased corals for antibacterial activity. The bioactive extracts were analyzed using untargeted metabolomics. Herein, an UpSet plot and hierarchical clustering analyses were performed to identify isolates with the largest number of unique metabolites. These isolates also displayed different antibacterial activities. Through application of in silico and experimental approaches coupled with genome analysis, we dereplicated natural products from these coral-derived bacteria from Florida's coral reef environments. The metabolomics approach highlighted in this study serves as a useful resource to select probiotic candidates and enables insights into natural product-mediated chemical ecology in holobiont symbiosis.

Online Liquid Chromatography-Sheath-Flow Surface Enhanced Raman Detection of Phosphorylated Carbohydrates.

Online detection and quantification of three phosphorylated carbohydrate molecules: glucose 1-phosphate, glucose 6-phosphate, and fructose 6-phosphate was achieved by coupling sheath-flow surface enhanced Raman spectroscopy (SERS) to liquid chromatography. The presence of an alkanethiol (hexanethiol) self-assembled monolayer adsorbed to a silver SERS-active substrate helps retain and concentrate the analytes of interest at the SERS substrate to improve the detection sensitivity significantly. Mixtures of 2 µM of phosphorylated carbohydrates in pure water as well as in cell culture media were successfully separated by HPLC, with identification using the sheath-flow SERS detector. The quantification of each analyte was achieved using partial least-squares (PLS) regression analysis and acetonitrile in the mobile phases as an internal standard. These results illustrate the utility of sheath-flow SERS for molecular specific detection in complex biological samples appropriate for metabolomics and other applications.