Opportunistic Sampling of Roadkill as an Entry Point to Accessing Natural Products Assembled by Bacteria Associated with Non-anthropoidal Mammalian Microbiomes

Abstract

Few secondary metabolites have been reported from mammalian microbiome bacteria despite the large numbers of diverse taxa that inhabit warm-blooded higher vertebrates. As a means to investigate natural products from these microorganisms, an opportunistic sampling protocol was developed, which focused on exploring bacteria isolated from roadkill mammals. This initiative was made possible through the establishment of a newly created discovery pipeline, which couples laser ablation electrospray ionization mass spectrometry (LAESIMS) with bioassay testing, to target biologically active metabolites from microbiome-associated bacteria. To illustrate this process, this report focuses on samples obtained from the ear of a roadkill opossum (Dideiphis virginiana) as the source of two bacterial isolates (Pseudomonas sp. and Serratia sp.) that produced several new and known cyclic lipodepsipeptides (viscosin and serrawettins, respectively). These natural products inhibited biofilm formation by the human pathogenic yeast Candida albicans at concentrations well below those required to inhibit yeast viability. Phylogenetic analysis of 16S rRNA gene sequence libraries revealed the presence of diverse microbial communities associated with different sites throughout the opossum carcass. A putative biosynthetic pathway responsible for the production of the new serrawettin analogues was identified by sequencing the genome of the Serratia sp. isolate. This study provides a functional roadmap to carrying out the systematic investigation of the genomic, microbiological, and chemical parameters related to the production of natural products made by bacteria associated with non-anthropoidal mammalian microbiomes. Discoveries emerging from these studies are anticipated to provide a working framework for efforts aimed at augmenting microbiomes to deliver beneficial natural products to a host.

Figure 1

Overview of the combined microbiological, chemical, and genomic steps employed to investigate microbiome bacteria derived from roadkill mammals for new natural products and their biosynthetic gene clusters. The methodologic approach illustrated here was developed to support the discovery of new bioactive natural products from non-anthropoidal sources, which in turn could be used for the purpose of engineering new biosynthetic pathway functions into the microbiomes of both humans and agriculturally important animals.

Figure 2

Summary of the bacterial isolates collected from roadkill mammals. (A) Breakdown of the 3659 roadkill microbiome isolates based on the source organisms from which they were derived. (B) Distribution of the isolates based on the locations/orifices on the carcasses from which they were derived. (C) Categorization of the isolate data illustrating the percent contribution of each body site to the overall bacterial collection prepared from the different animal species (note that the colors and categories used to construct the slices within each of the pie charts in panel C are the same as those used for the pie chart in panel B).

Figure 3

Phylogenetic diversity of mammalian microbiome bacteria from different orifices/body sites of a roadkill opossum carcass.

Figure 4

Targeting microbial natural products using LAESIMS. The inset shows bacterial colonies growing on the surface of an agar plate. The plate was placed inside of the LAESIMS chamber for mass spectrometry profiling. A subset of representative colonies was selected (indicated by red arrows), and a virtual grid was laid over these colonies using the instrument’s software to target where laser ablation would occur. The light blue polygons show where mass data were collected from the colonies within the range of m/z 200–2000. The presented mass data were derived from the circled colony (average of several locations taken from the colony and subtracted from mass data obtained from a blank [uncolonized] portion of the plate), which reveals prominent single and doubly charged sodium adduct ions for viscosin (1).

Figure 5

ORTEP rendering of viscosin (1) illustrating the metabolite’s absolute configuration (determined by refinement of the Flack parameter; the water molecule identified near atom N2 has been removed from the figure for clarity; however, the position of the water can be found in the Supporting Information). The numbering system used for this structure reflects the atom assignments used in the Supporting Information and in the Cambridge Structural Database (CCDC 1511786).