Molecular and stable isotopic characterization of enzymes involved in nitrification and nitrifier-denitrification

Ammonia-oxidizing bacteria are believed to be an important source of the climatically important trace gas nitrous oxide (N₂O). The primary goals of this dissertation were to characterize the genes for enzymes involved in N₂O production in nitrifying bacteria and to establish the groundwork for using stable isotopes to constrain their contribution to N₂O production in the environment.; The genes for nitrite reductase (nirK) and nitric oxide reductase (norB) were identified in marine nitrifying bacteria and were found to be homologous to those in denitrifying bacteria. This observation suggests that they shared a common evolutionary origin and may function similarly in these metabolically distinct organisms. The use of norB gene sequences as genetic probes may also allow for distinction between the roles of nitrifiers and denitrifiers in N₂O production in the environment.; Isotope effects for ammonia oxidation and nitrite reduction in marine nitrifiers were found to be much lower than the corresponding isotope effects for the terrestrial nitrifier Nitrosomonas europaea. Furthermore, concentration dependence of the isotope effect for nitrite reduction suggests that differential substrate availability is a possible explanation for previously recognized isotopic discrepancies between culture and field studies of N ₂O production. More broadly, these results may be used to assess the effect of nitrification processes on nitrogen isotopes of dissolved inorganic nitrogen species (NH₄+, NO₃− and N₂O) in the ocean.; An intriguing correspondence was discovered between nitrogen isotope effects for ammonia monooxygenase and nitrite reductase and the amino acid sequences of these enzymes. Such a relationship has not been observed in other systems, and it raises the possibility of combining genetic information with stable isotope measurements to study the biogeochemical impact of specific groups of ammonia-oxidizing bacteria.; The first method for the oxygen isotopic analysis of nitrate in seawater was developed, standardized, and tested as part of this dissertation. This method, based on the isotopic analysis of N₂O, has many benefits over published freshwater methods, and it opens up opportunities for new insight into the interaction of nitrogen cycle processes in the modern ocean.