Identifying Potential Nutrient Acquisition Mechanisms of for Long-Term Survival: Adaptive Evolution of Halomonas Isolated from Subseafloor Crustal Fluids

Hans Sebastian ¹ Alberto Robador ¹ Dawson Ray ¹ Angus Angermeyer ² Steven D D'Hondt ³ Julie A Huber ⁴ Steven E Finkel ^1*

• ¹ University of Southern California, Los Angeles, California, United States
• ² Marine Biological Laboratory (MBL), Woods Hole, Massachusetts, United States
• ³ Graduate School of Oceanography, University of Rhode Island, Narragansett, Rhode Island, United States
• ⁴ Woods Hole Oceanographic Institution, Woods Hole, Massachusetts, United States

In nature, microbes frequently survive long periods of time under conditions of nutrient and energy limitation while also facing extremes in temperature, pressure, and competition with other microbes. One low-carbon, cold, and high pressure environment is the subseafloor crustal aquifer, where fluids circulate through old ocean crust. While microbial communities are known to be present in these fluids and contribute to biogeochemical cycling, the survival strategies of microbes in these communities is poorly constrained.. In this study, multiple Halomonas strains were isolated from subseafloor crustal fluids of North Pond, on the Mid-Atlantic Ridge. These organisms are able to grow under laboratory conditions in minimal medium without the addition of carbon sources, as well as in rich nutrient. While strains are highly related in genomic content, each strain has unique mutations and/or undergone genomic rearrangements, suggesting a common ancestor. After serial passage under rich nutrient conditions, we identified mutants that can no longer scavenge scarce nutrients in minimal medium with no added carbon. Genomic sequencing identified genes that appear to be essential for survival under extremely low-nutrient condition, including several hypothetical proteins predicted to function as lipases, peptidases, or nutrient transporters. The application of an adaptive evolution platform selecting for survival and growth under one environmental condition that simultaneously selects against survival in different environments may prove very useful tool for identifying genes and metabolic pathways in a wide variety of complex environments.Bacteria occupy every life-sustaining niche on Earth, yet frequently the mechanisms allowing organisms to succeed are difficult to determine. New tools are needed to identify mechanisms of survival and the genes upon which these metabolism rely. We have developed a novel strategy for identifying genes essential to low-nutrient environments by selecting for cells adapted to highnutrient conditions. These evolved cells have "forgotten" how to thrive in the extremely nutrientrestricted environments from which they were isolated. Since many organisms sampled from these extreme environments are recalcitrant to laboratory manipulation, this approach has the potential to identify important molecular biomarkers that can be used to sample microbial communities, furthering our knowledge of which genes and metabolic pathways contribute to evolutionary fitness.