Microbial Evolution at High Pressure: Deep Sea and Laboratory Studies

Elevated hydrostatic pressures are present in deep-sea and deep-Earth environments where this physical parameter has influenced the evolution and characteristics of life. Piezophilic (high-pressure-adapted) microbes have been isolated from diverse deep-sea settings, and would appear likely to occur in deep-subsurface habitats as well. In order to discern the factors enabling life at high pressure my research group has explored these adaptations at various levels, most recently including molecular analyses of deep-sea trench communities, and through the selective evolution of the model microbe Escherichia coli in the laboratory to progressively higher pressures. Much of the field work has focused on the microbes present in the deeper portions of the Puerto Rico Trench (PRT)and in the Peru-Chile Trench (PCT), from 6-8.5 km below the sea surface (~60-85 megapascals pressure). Culture-independent phylogenetic data on the Bacteria and Archaea present on particles or free-living, along with data on the microeukarya present was complemented with genomic analyses and the isolation and characterization of microbes in culture. Metagenomic analyses of the PRT revealed increased genome sizes and an overrepresentation at depth of sulfatases for the breakdown of sulfated polysaccharides and specific categories of transporters, including those associated with the transport of diverse cations or carboxylate ions, or associated with heavy metal resistance. Single-cell genomic studies revealed several linneages which recruited to the PRT metagenome far better than existing marine microbial genome sequences. analyses. Novel high pressure culture approaches have yielded new piezophiles including species preferring very low nutrient levels, those living off of hydrocarbons, and those adapted to various electron donor/electron acceptor combinations. In order to more specifically focus on functions enabling life at increased pressure selective evolution experiments were performed with Escherichia coli during laboratory cultivation. More than 60 subcultures were obtained at progressively increasing hydrostatic pressures ranging from 28 - 62 megapascals. A strain isolated from the 63rd subculture displayed dramatically improved growth over the parental strain at 59 megapascals but reduced growth rate relative to the parental strain at atmospheric pressure. The mutant also produced far more unsaturated fatty acids than its parent and also acquired the ability to upregulate these fatty acids species at elevated pressure. Solexa sequencing revealed mutations within an operon (acpP operon) governing unsaturated fatty acid production, and these have been examined as a function of generation at high pressure. These and other results indicate that a large number and variety of microbes are adapted to life at high pressure, that the selective constraints of pressure increases up to ~60 megapascals are not so severe as to preclude the rapid evolution to a piezotolerant phenotype, and that the production of increased levels of unsaturated fatty acids correlates with adaptation to this stressor. This work was supported by grants from the National Science Foundation (EF-0801793 and EF-0827051) and the National Aeronautics and Space Administration (NASA SSC NNX10AR13G).