Trace metals in the nitrogen cycle: details

Denitrification (dissimilatory nitrate reduction)

Denitrification is an important chemical transformation in the nitrogen cycle and the principal sink for fixed nitrogen in natural waters. Denitrification occurs as a result of the dissimilatory reduction of nitrogen oxides (nitrate and nitrite), in which nitrogen oxides are used as alternative electron acceptors during respiration by anaerobic bacteria. Denitrification involves a suite of reductase enzymes, all of which require metal cofactors. We shall focus on the nitrate and nitrite reductases.

Dissimilatory nitrate reductases, which contain molybdenum and iron, are usually membrane-bound and oxygen-sensitive. Two major forms of dissimilatory nitrite reductase are found in denitrifying bacteria, one containing iron (heme cd1) and one containing copper (type I and type II); both forms have been crystallographically characterized. Bacterial isolates suggest that the iron-containing enzyme is more common, but methods to assess the abundance in nature of these enzymes and their genes are just being developed.

The major objectives of this project are to establish the relationships between the availability of metals (molybdenum, iron, copper), the diversity and efficiency of enzymes (nitrate and nitrite reductases), and the structure and mechanisms of the enzymes' metal centers. In particular, we wish to understand how the relative availabilities of iron and copper influence the distribution of the different types of nitrite reductase in the environment, and the efficiency of denitrification.

We will begin by investigating the diversity, distribution and abundance in natural system of the genes encoding nitrogen-reducing enzymes. This can be done using techniques similar to those described in the section on siderophore uptake: we'll search for genes in natural samples that resemble known reductase genes. This will require isolating genes for the nitrogen-reducing enzymes in cultured strains ("probes"), obtaining homologous fragments of DNA from environmental samples, sequencing those fragments, making more primers and probes, and developing quantitative hybridization (or PCR) assays for detecting those genes in the environment. (For a brief intro to genetic methods, click here.) Using the resulting DNA sequences, we will express the protein in a suitable vector and perform structural and functional characterizations on the resulting organism.

Once these genetic methods have been developed, we can study how the abundance in field samples of different nitrate reductase genes (particularly the iron and copper forms) varies with changes in nitrogen and trace-metal availability.

Nitrate acquisition (assimilatory nitrate reduction)

The use of nitrate in biosynthesis requires chemical reduction to the level of ammonium. This reduction is catalyzed by assimilatory nitrate and nitrite reductase enzymes. Recent field work has shown that nitrate reductase activity is limited by iron availability. Our objective is to elucidate the relation between trace-element physiology and the activity of nitrate and nitrite reductases in marine autotrophs.

This project will follow the methodology of project d-1 (with the advantages of hindsight). In addition, we will study the cellular cycling of molybdenum and its correlation with the synthesis and degradation of nitrate reductase. Specifically, we wish to know what happens to molybdenum during nitrate-reductase degradation in the dark, and how it is retained within the cell. We hope to gain insight into this process in preliminary experiments using a recently constructed nitrate-reductase-replete strain of Chlamydomonas (the cultured strains of which are usually nitrate-reductase-deficient). This strain has been engineered to resist the antibacterial spectromycin. This antibiotic resistance will make it easy to obtain pure cultures.

Nitrogen fixation

In marine systems, nitrogen is fixed primarily by a single genus of cyanobacteria, Trichodesmium. These organisms are unusual in that they fix N₂ and produce O₂ from photosynthesis within the same cell at the same time. The nitrogenase in Trichodesmium is presumed to contain molybdenum and a lot of iron, as it does in other cyanophytes. Because of the massive iron requirement of the nitrogenase enzyme, some scientists have hypothesized that N₂-fixation in oceans may be limited globally by iron. Two Trichodesmium strains are presently in culture in Cebic laboratories, and the genes for several of the subunits of the multimeric nitrogenase complex have been sequenced.

Our objectives are to examine in Trichodesmium: (1) the relation between the availability of metals (Fe, Mo, V) and N₂-fixation; and (2) the mechanism of protection of nitrogenase from inactivation by O₂. We hypothesize that the nitrogenase is protected by the scavenging of O₂ by an unusually high level of photosynthetic activity.

For the first task, we shall measure in laboratory cultures the effect on N₂-fixation rates of modifying the trace-metal chemistry in the sea-water medium. For the second, we shall use knock-out mutations of photosystem I genes. The relative activities of PS(I) and nitrogenase will be compared in the wild type and the mutant.

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