The deep sea remains one of the most understudied ecosystems on earth relative to its size and importance in the global climate system. Knowing both how and how fast organisms are able to evolve in the cold, dark, high-pressure deep ocean is critical for understanding how microbes respond to environmental change in the past, present, and future. I propose an integrated metagenomic, cultivation, and functional biology approach to address this gap for two abundant and geochemically-relevant groups of microbes—the ammonia-oxidizing archaea (AOA) and nitrite-oxidizing bacteria (NOB). I will use long-read sequencing to establish a set of high-quality reference genomes from the mesopelagic and deep ocean. I will then use these genomes to conduct ancestral state reconstruction to identify sets of genes gained and lost at the branch points of key ecotypes. A subset of these genes will then be expressed in heterologous hosts to test enzyme fitness under relevant conditions of temperature and pressure. Finally, a renewed effort at cultivating deep-sea lineages of AOA and NOB using pressure chambers will enable me to answer a suite of physiological questions not answerable from sequence data alone. Together, this work will allow me to test two hypotheses about nitrifiers in the deep ocean: (1) deep-sea AOA and NOB ecotypes have protein variants tuned for higher fitness in the deep sea as opposed to completely novel proteins, and (2) AOA diversification in the deep ocean has been accompanied by simultaneous diversification in co-occurring NOB. The ultimate goal of this work is to identify the specific mechanisms that chemoautotrophic organisms have evolved to cope with the deepsea environment and develop a conceptual model of how their metabolic activities influence the ecology of the entire deep ocean.