Trachymyrmex septentrionalis ants promote fungus garden hygiene using Trichoderma-derived metabolite cues.

Fungus-growing ants depend on a fungal mutualist that can fall prey to fungal pathogens. This mutualist is cultivated by these ants in structures called fungus gardens. Ants exhibit weeding behaviors that keep their fungus gardens healthy by physically removing compromised pieces. However, how ants detect diseases of their fungus gardens is unknown. Here, we applied the logic of Koch's postulates using environmental fungal community gene sequencing, fungal isolation, and laboratory infection experiments to establish that Trichoderma spp. can act as previously unrecognized pathogens of Trachymyrmex septentrionalis fungus gardens. Our environmental data showed that Trichoderma are the most abundant noncultivar fungi in wild T. septentrionalis fungus gardens. We further determined that metabolites produced by Trichoderma induce an ant weeding response that mirrors their response to live Trichoderma. Combining ant behavioral experiments with bioactivity-guided fractionation and statistical prioritization of metabolites in Trichoderma extracts demonstrated that T. septentrionalis ants weed in response to peptaibols, a specific class of secondary metabolites known to be produced by Trichoderma fungi. Similar assays conducted using purified peptaibols, including the two previously undescribed peptaibols trichokindins VIII and IX, suggested that weeding is likely induced by peptaibols as a class rather than by a single peptaibol metabolite. In addition to their presence in laboratory experiments, we detected peptaibols in wild fungus gardens. Our combination of environmental data and laboratory infection experiments strongly support that peptaibols act as chemical cues of Trichoderma pathogenesis in T. septentrionalis fungus gardens.

Metabolomics Peak Analysis Computational Tool (MPACT): An Advanced Informatics Tool for Metabolomics and Data Visualization of Molecules from Complex Biological Samples.

Untargeted metabolomics is a powerful tool for investigating chemistry of complex biological systems, but its utility is compromised by the presence of uninformative features and the limited efficiency of currently available prioritization tools. More effective filtering and prioritization tools are required to address the challenges of large untargeted metabolomics datasets. Here, we introduce Metabolomics Peak Analysis Computational Tool (MPACT), a new mass spectrometry data analysis platform employing filtering based on multiple modalities, statistical techniques incorporating multilevel replication, and interactive data visualization. We demonstrate application of MPACT to uncover hidden effects of the rare earth element cerium on tunicate-associated bacterium Streptomyces sp. PTY087I2, culminating in characterization of two thiolated compounds including a new cysteine derivative, granaticin C, and granaticin D, recently described as mycothiogranaticin A. While we demonstrate application of MPACT to microbial natural products discovery using an elicitation approach, the platform should be readily adaptable to investigation of multipartite interactions, biomarker detection, small molecules in the environment, and a wide range of other complex sample types.

Genomic and phenotypic characterization of a red-pigmented strain of Massilia frigida isolated from an Antarctic microbial mat.

The McMurdo Dry Valleys of Antarctica experience a range of selective pressures, including extreme seasonal variation in temperature, water and nutrient availability, and UV radiation. Microbial mats in this ecosystem harbor dense concentrations of biomass in an otherwise desolate environment. Microbial inhabitants must mitigate these selective pressures via specialized enzymes, changes to the cellular envelope, and the production of secondary metabolites, such as pigments and osmoprotectants. Here, we describe the isolation and characterization of a Gram-negative, rod-shaped, motile, red-pigmented bacterium, strain DJPM01, from a microbial mat within the Don Juan Pond Basin of Wright Valley. Analysis of strain DJMP01's genome indicates it can be classified as a member of the Massilia frigida species. The genome contains several genes associated with cold and salt tolerance, including multiple RNA helicases, protein chaperones, and cation/proton antiporters. In addition, we identified 17 putative secondary metabolite gene clusters, including a number of nonribosomal peptides and ribosomally synthesized and post-translationally modified peptides (RiPPs), among others, and the biosynthesis pathway for the antimicrobial pigment prodigiosin. When cultivated on complex agar, multiple prodiginines, including the antibiotic prodigiosin, 2-methyl-3-propyl-prodiginine, 2-methyl-3-butyl-prodiginine, 2-methyl-3-heptyl-prodiginine, and cycloprodigiosin, were detected by LC-MS. Genome analyses of sequenced members of the Massilia genus indicates prodigiosin production is unique to Antarctic strains. UV-A radiation, an ecological stressor in the Antarctic, was found to significantly decrease the abundance of prodiginines produced by strain DJPM01. Genomic and phenotypic evidence indicates strain DJPM01 can respond to the ecological conditions of the DJP microbial mat, with prodiginines produced under a range of conditions, including extreme UV radiation.

Bridging the Gap: Plant-Endophyte Interactions as a Roadmap to Understanding Small-Molecule Communication in Marine Microbiomes.

Probing the composition of the microbiome and its association with health and disease states is more accessible than ever due to the rise of affordable sequencing technology. Despite advances in our ability to identify members of symbiont communities, untangling the chemical signaling that they use to communicate with host organisms remains challenging. In order to gain a greater mechanistic understanding of how the microbiome impacts health, and how chemical ecology can be leveraged to advance small-molecule drug discovery from microorganisms, the principals governing communication between host and symbiont must be elucidated. Herein, we review common modes of interkingdom small-molecule communication in terrestrial and marine environments, describe the differences between these environments, and detail the advantages and disadvantages for studies focused on the marine environment. Finally, we propose the use of plant-endophyte interactions as a stepping stone to a greater understanding of similar interactions in marine invertebrates, and ultimately in humans.

Cryptic Species Account for the Seemingly Idiosyncratic Secondary Metabolism of Sarcophyton glaucum Specimens Collected in Palau.

Sarcophyton glaucum is one of the most abundant and chemically studied soft corals with over 100 natural products reported in the literature, primarily cembrane diterpenoids. Yet, wide variation in the chemistry observed from S. glaucum over the past 50 years has led to its reputation as a capricious producer of bioactive metabolites. Recent molecular phylogenetic analysis revealed that S. glaucum is not a single species but a complex of at least seven genetically distinct species not distinguishable using traditional taxonomic criteria. We hypothesized that perceived intraspecific chemical variation observed in S. glaucum was actually due to differences between cryptic species (interspecific variation). To test this hypothesis, we collected Sarcophyton samples in Palau, performed molecular phylogenetic analysis, and prepared chemical profiles of sample extracts using gas chromatography-flame ionization detection. Both unsupervised (principal component analysis) and supervised (linear discriminant analysis) statistical analyses of these profiles revealed a strong relationship between cryptic species membership and chemical profiles. Liquid chromatography with tandem mass spectrometry-based analysis using feature-based molecular networking permitted identification of the chemical drivers of this difference between clades, including cembranoid diterpenes (2R,11R,12R)-isosarcophytoxide (5), (2S,11R,12R)-isosarcophytoxide (6), and isosarcophine (7). Our results suggest that early chemical studies of Sarcophyton may have unknowingly conflated different cryptic species of S. glaucum, leading to apparently idiosyncratic chemical variation.

Shielding the Next Generation: Symbiotic Bacteria from a Reproductive Organ Protect Bobtail Squid Eggs from Fungal Fouling.

The importance of defensive symbioses, whereby microbes protect hosts through the production of specific compounds, is becoming increasingly evident. Although defining the partners in these associations has become easier, assigning function to these relationships often presents a significant challenge. Here, we describe a functional role for a bacterial consortium in a female reproductive organ in the Hawaiian bobtail squid, Euprymna scolopes Bacteria from the accessory nidamental gland (ANG) are deposited into the egg jelly coat (JC), where they are hypothesized to play a defensive role during embryogenesis. Eggs treated with an antibiotic cocktail developed a microbial biomass primarily composed of the pathogenic fungus Fusarium keratoplasticum that infiltrated the JC, resulting in severely reduced hatch rates. Experimental manipulation of the eggs demonstrated that the JC was protective against this fungal fouling. A large proportion of the bacterial strains isolated from the ANG or JC inhibited F. keratoplasticum in culture (87.5%), while a similar proportion of extracts from these strains also exhibited antifungal activity against F. keratoplasticum and/or the human-pathogenic yeast Candida albicans (72.7%). Mass spectral network analyses of active extracts from bacterial isolates and egg clutches revealed compounds that may be involved in preventing microbial overgrowth. Several secondary metabolites were identified from ANG/JC bacteria and egg clutches, including the known antimicrobial lincomycin as well as a suite of glycerophosphocholines and mycinamicin-like compounds. These results shed light on a widely distributed but poorly understood symbiosis in cephalopods and offer a new source for exploring bacterial secondary metabolites with antimicrobial activity.IMPORTANCE Organisms must have strategies to ensure successful reproduction. Some animals that deposit eggs protect their embryos from fouling/disease with the help of microorganisms. Although beneficial bacteria are hypothesized to contribute to egg defense in some organisms, the mechanisms of this protection remain largely unknown, with the exception of a few recently described systems. Using both experimental and analytical approaches, we demonstrate that symbiotic bacteria associated with a cephalopod reproductive gland and eggs inhibit fungi. Chemical analyses suggest that these bacteria produce antimicrobial compounds that may prevent overgrowth from fungi and other microorganisms. Given the distribution of these symbiotic glands among many cephalopods, similar defensive relationships may be more common in aquatic environments than previously realized. Such defensive symbioses may also be a rich source for the discovery of new antimicrobial compounds.