Microbial Dark Matter: Culturing the Uncultured in Search of Novel Chemotaxonomy

Although \u3c1% of environmental isolates can be cultured, insights into the culturable fraction refine future techniques to unlock the cryptic biodiversity. This cryptic biodiversity is omnipresent within culture-independent surveys of environmental bacterial populations. Subsequently, these elusive organisms are termed “microbial dark matter” due to the intrigue surrounding their potential taxonomic and biosynthetic impacts. To this end of investigating bacterial-specific dark matter, we employed high-throughput culturing techniques to examine Gulf of Mexico sediments and sponges for chemotaxonomically important bacteria. Through stepwise implementation of permissive and selective recovery techniques, we succeeded in recovering over 150 unique bacterial isolates. We observed several isolates that pose as chemotaxonomically interesting, including several strains that belong to critical bioremediation taxa. In addition to these biotechnological-amenable isolates, we have also succeeded in culturing taxa historically attributed with antibacterial discoveries. One such isolate was a previously sponge-associated organism belonging to the rarely cultured Verrucosispora genus. Through extensive comparative genomics, we identified it as a novel species and subsequently named it Verrucosispora sioxanthis sp. nov. with the published genome serving as a resource for continued microbial dark matter investigations. Following continued passaging of the organism, we observed improved growth, biomass, and liquid growth dispersal within each increasingly sub-passaged generation of the isolate. As such, the laboratory attenuation, or domestication, of the organism warranted in-depth whole genomic analyses to generate insight into the increased laboratory sustainability. Through phenotypically-guided bioinformatics, we determined that the domestication was mediated through genome-wide selective pseudo- or non-pseudogenization of genes. As the previously sponge-associated organism evolved with the selection pressures of the static laboratory, we demonstrate that isolate effectively underwent genomic atrophy to resolve into a largely stable, sustainable laboratory-domesticated strain. Collectively, our chemotaxonomic discovery pipeline expands on the culturable bacterial biodiversity and ultimately provides a previously uncultured model organism for initial forays into resolving the microbial dark matter knowledge gap