Nitrate Respiration of the Hyperthermophile Pyrobaculum aerophilum

University Of California-Los Angeles, Los Angeles CA

Investigators

Abstract

Schroeder Hyperthermophlic archaea inhabit volcanic environments such as hydrothermal vents or hot springs that may resemble conditions when life on earth originated. The hyperthermophilic, marine Archaeon Pyrobaculum aerophilum belongs to a deep branch of the phylogenetic tree suggesting that this organism is relatively ancient. The objective of this research project is to study the mechanism of denitrification in the Archaeon P. aerophilum. Denitrification is a unique prokaryotic pathway that allows the microbe to convert nitrate into dinitrogen gas. It is the only pathway in nature that generates nitrogen gas from fixed N oxides and thus it is essential for the maintenance of the global nitrogen cycle on earth. Respiration of nitrate is coupled to the generation of energy that fuels cell propagation. The pathway is found in both the domain Bacteria and the domain Archaea suggesting that denitrification evolved most likely before the last common ancestor. P. aerophilum represents the oldest denitrifier isolated thus far and provides the opportunity to compare an ancient process with that found in today's modern microbes. The denitrification pathway is well described in Gram-negative bacteria, where two membrane-bound and two soluble enzyme complexes as well as several soluble electron mediating proteins are required. Based on previous results in the PI's laboratory a different mechanism for denitrification is proposed for P. aerophilum: Four membrane-bound enzyme complexes exist that all interact with a menaquinone pool to reduce nitrate to N2 gas. A combination of biochemical, biophysical and molecular techniques will be employed to test this hypothesis and further our understanding of the physiology of hyperthermophlic Archaea. The objectives of this project are to further characterize the nitrate reductase enzyme, which is interesting because of its novel membrane anchor. To obtain a more complete picture of the denitrification pathway, two additional denitrification pathway enzymes, the nitrite reductase and the NO reductase, will be purified and characterized. To facilitate future structural analysis of the nitrite and NO reductases the nir and nor genes will be cloned and overexpressed. Since the DNA sequence of P. aerophilum will be available in the near future a selected set of genes involved in the denitrification pathway will be analyzed for their differential expression in response to selected and defined environmental growth conditions. The results from this research will further our knowledge about respiratory processes that are essential functions of life. The study of an ancient Archaeon may give insight into how electron transfer reactions have evolved. The investigation of the denitrification process in diverse organisms will further our ability to understand, predict and deal with global environmental changes that include the nitrogen cycle.