Participants:

Diversity of Nitrogenase Genes inChesapeake Bayl
Jon P. Zehr, University of California, Santa Cruz
Grieg Stewart

The biological productivity of ecosystems, including aquatic environments, is dependent upon the availability of nutrients, such as nitrogen, phosphorus, iron, and other elements. Nitrogen is one of the most abundant elements in all life. Ammonium and nitrate are the forms of nitrogen most readily used by most organisms. These forms are often consumed, leaving low concentrations in the environment. Thus, the growth of many organisms in terrestrial and aquatic environments can be “limited” by nitrogen availability.

Nitrogen fixation is the conversion of gaseous dinitrogen to ammonium. Dinitrogen composes about 80% of the Earth’s atmosphere, but is only a source of nitrogen for organisms with the capability for nitrogen fixation. Thus far, only prokaryotes (bacteria and archaea) have been demonstrated to fix nitrogen, or possess the genes that encode the enzyme that catalyzes nitrogen fixation (nitrogenase).

Nitrogenase is likely to be an ancient enzyme. Nitrogenase genes are distributed throughout the prokaryotic kingdom, including representatives of the Archaea as well as the Bacteria including Cyanobacteria. Although the phylogeny of nifH reflects the phylogeny of organisms based on ribosomal RNA genes, there are some differences. One deeply branching cluster is anomalous and is likely to represent an independent line of evolution, and includes some sequences from gram positive organisms, such as Clostridium. Since nitrogenase gene sequences do reflect phylogenetic affiliation, the sequence of nitrogenase genes can be used to identify the types of nitrogen-fixing microorganisms in different habitats.

Unfortunately, it is often difficult to culture organisms from the environment, so it is difficult to determine whether nitrogen fixation is limited by the presence of nitrogen fixing organisms, or other factors that may limit the expression of nitrogen fixation activity (e.g. availability of other nutrients). For this reason, it is important to assess the presence and distribution of nitrogen fixation genes, rather than simply assay the rate of nitrogen fixation. The presence of nitrogen fixation genes can be determined by amplifying the nitrogenase gene using the polymerase chain reaction (PCR). The polymerase chain reaction makes it possible to determine if nitrogen fixing organisms are present, but more importantly, makes it possible to determine what kinds of nitrogen fixing organisms are present. The sequence of the amplified fragment of nitrogenase contains taxonomic information, so phylogenetic analysis of cloned amplified nitrogenase genes provides information on taxonomic identity and diversity.

In the Biocomplexity project, we are assessing the presence and abundance of nitrogen fixing organisms in the Chesapeake Bay and Choptank River. Using the molecular approach we hope to overlay patterns of diversity with fluxes of nutrients, and integrate this information with hydrodynamic features to help explain the interrelationships between environmental factors and microbial activities.