Trace metal limitation of denitrification :
We have investigated the trace metal limitations and requirements (Mo, Cd, Fe, Cu) for denitrifying bacteria by quantifying denitrification under trace-metal clean anoxic conditions, using trilaminate incubation bags. Several denitrifying strains were found to be unusually resistant to Cd toxicity. Both Cu and Fe could limit growth and denitrification by denitrifiers, and Cu in particular led to a terminal accumulation of N₂O. Nitrous oxide reductase is a Cu enzyme and Cu limitation appears to act directly at this point in the pathway. We hypothesize that Cu limitation may be involved in controlling the distribution of N₂O in the ocean; regions where N2O accumulates(oxygen minimum zones) are also regions of very low Cu availability. These observations are unusual and significant in that copper is more likely to be toxic than limiting for most microoganisms, and because it may be an example of control of biogeochemical cycles by metals other than iron. Fieldwork is planned for spring of 2003 in the Eastern Tropical South Pacific with experiments designed to detect in situ Cu limitation.

Nitrite reductase
Our research on the biochemistry and molecular biology of denitrification focuses on nitrite reductase, the enzyme that catalyzes the reduction of nitrite to nitric oxide (NO). This is a central enzyme in the pathway, often referred to as the denitrifying enzyme, because it converts fixed N to gaseous N, which is no longer available to most organisms. The two forms of dissimilatory (respiratory) nitrite reductase, the cd-NiR (nirS gene) and Cu-NiR (nirK gene), are distributed across the Bacterial and Archaeal domains in a great diversity of microorganisms.

a) Diversity:
We have retrieved nirK and nirS genes from a large number and variety of microorganisms obtained from marine and sediment environments, from soils and halobenzoate degrading enrichments, and from uncultivated organisms using direct cloning. Phylogenetic analysis of the nir genes showed that with the well known exception of Pseudomonas, both forms of nir are not found within a single genus. The phylogeny of the nir genes is not congruent with the 16S rRNA phylogeny of the organisms, indicating widespread horizontal gene transfer of the components of denitrification. As part of the planned Arabian Sea program, we will determine the relative abundance and activity of nirK and nirS genes in the denitrifying assemblage of the oxygen minimum zone and the role of trace metals in their distribution and activity. The majority of nirS sequences retrieved from a coastal water column oxygen minimum zone of the Arabian Sea (the first denitrifier sequences from a water column environment) were unique; i.e., not closely related to previously known sequences from sediment environments. We also detected a possible relationship between diversity and dentrification rates in this environment.

b) Nitrifying bacteria
Both NiRs are well known in denitrifying bacteria, and the nitric oxide reductase (NoR, which produces N₂O) in denitrifiers has also been characterized. It is also well known that nitrifying bacteria, obligate aerobic autotrophs, also produce both NO and N₂O under some conditions, but the enzymology and genetics of these transformations in nitrifiers was not understood. We discovered a nitrite reductase in nitrifying bacteria that is homologous with the nirK of denitrifiers. Similarly, we also recently discovered a norB gene in nitrifiers which is highly homologous with the norB gene from denitrifiers.

We further investigated the similarities in the genes between denitrifiers and nitrifiers by determining the isotopic effects of the enzymes in whole cell and lysate assays. For the residual nitrite, the isotopic effect is determined by the kind of NiR present, but is not influenced by whether the process is performed by a nitrifier or a denitrifier. The isotopic effect must result from the mode of action of the iron vs. the copper enzyme, and perhaps the degree of exchange that occurs between the cell and the environment. The implication is that the significance of isotopic signatures of inorganic and gaseous nitrogen species in the ocean may be more complicated than previous suspected, if the effects of nitrification and denitrification cannot be distinguished. A model is being developed to help sort out the expected isotopic fractionation in different environmental scenarios.

Assimilatory Nitrate Reductase:
Our goals in this project were to clone and sequence the nitrate reductase (nar) genes from several eukaryotic algae and to investigate trace metal regulation of gene expression. Expression data are to be linked to enzyme assays on cells grown under varying light and metal conditions, to investigate Mo cycling in relation to NR regulation. This work has been slowed by difficulties encountered in cloning the nar gene. We have succeeded in obtaining the complete gene sequence for nar from Dunaliella, the first example from a marine alga, and partial gene sequences of nar from several diatoms. Expression studies to characterize induction of nar have been carried out in Dunaliella and are planned for the diatoms.