Insights into microbial metabolism

Abstract

Nitrogen fixation (catalyzed by the enzyme nitrogenase), cellular respiration (completed through the Tricarboxylic Acid (TCA) cycle) and mercury detoxification (through mercury methylation) are three metabolic processes used by a wide variety of microorganisms, but that also have far reaching impacts on nutrient cycling in the environment. Roseiflexus castenholzii has been found to have a unique nitrogenase gene cluster encoding several nitrogenase homologs, including the structural proteins NifH and NifDK and the radical SAM protein, NifB, necessary for cofactor biosynthesis. However, the genome of R. castenholzii lacks the suite of nitrogenase accessory proteins necessary for nitrogen fixation. To investigate the metabolic role of these nitrogenase homologs, expression and purification protocols were developed that aid in the biochemical characterization of these proteins. Synechococcus sp. PCC 7002 encodes three novel TCA proteins, contrary to previous studies that indicated these phototrophs have incomplete TCA cycles. Expression, purification and preliminary crystallization trials were completed on the three novel TCA proteins in order to gain insight into the structure of the proteins which will elucidate the mechanism of each novel enzyme and provide evidence into the novel TCA cycle utilized by these cyanobacteria. The third project presented examines the role of microorganisms in metabolizing mercury, producing methylmercury and providing an entry point for methylmercury into the food chain in Yellowstone National Park (YNP). In this project, environmental samples were enriched for a sulfate reducing organism and a culture containing three sulfate reducing bacteria (SRB) has been established. The SRB that are present and active in the enrichment samples are known to reduce sulfate and may be responsible for the presence of methyl mercury in algal mats that bioaccumulates through the food chain in YNP. The enrichment of SRB in this culture will enable the identification and characterization of the organisms that are capable of methylating mercury in hydrothermal systems. Collectively, the results presented herein increase the knowledge base of three metabolic processes used by microorganisms: nitrogen fixation, cellular respiration through the TCA cycle and mercury detoxification; these results will contribute to a broader understanding of how these processes have evolved and their impacts on the environment.