Patricia A. Glibert
Jon P. Zehr
University of California, Santa Cruz
Jackie C. Collier
State University of NY, Stony Brook
Mary A. Voytek
United States Geological Survey
George A. Jackson
Texas A&M University
Database management, modeling
Diversity of Denitrifying Bacteria and Their FunctionalGenes
Denitrification is a form of respiration, in which bacteria use oxides of nitrogen rather than oxygen as an electron acceptor. The bacteria which perform this process are mainly heterotrophs, that is, they require organic molecules for their nutrition. They are also usually facultative anaerobes -- they can respire oxygen but in the absence of oxygen, are capable of continuing to respire using oxides of nitrogen. In the process, they reduce these nitrogen compounds through a series of reactions, finally producing dinitrogen gas.
Denitrification is often linked in the environment to nitrification. Nitrification is carried out by a different group of bacteria who oxidize ammonium or nitrite as their sole energy source and assimilate carbon dioxide.
The ability to denitrify is much more widespread in the bacterial world than is the ability to nitrify. Denitrifying bacteria are found in all the major branches of the tree of life. Therefore, if we wish to study them using molecular biology, we need to focus on the genes that are encode the enzymes that are responsible the nitrogen respiration reactions.
In the Biocomplexity project, we are concentrating our efforts on nitrite reductase, the enzyme that first turns soluble ionic nitrogen in the form of nitrite, into a gas, nitric oxide, that is no longer biochemically accessible to most organisms.
Samples have been collected from the water column and sediments of the Choptank River and Chesapeake Bay. We have extracted DNA from some of these samples and are beginning to obtain the DNA sequences from clone libraries of nitrite reductase genes. The PCR primers we used to obtain the clones were derived from denitrifying bacteria in the gamma Proteobacteria, and the clones we have sequenced so far are generally within that group. Nevertheless, the library is very diverse, and few identical clones have been retrieved. The phylogenetic tree below is a very preliminary representation, based on DNA rather than amino acids. The sequences in blue in the tree were all derived from a sediment sample in the upper Choptank River.
Sequences representing nitrite reductase from this library will be used to build gene chips, which we will use to interrogate the environment to learn about how the diversity of denitrifying bacteria varies in time and space. Sequences from other genes that encode other important enzymes in the nitrogen cycle are being produced by other members of the Biocomplexity team and they will also be incorporated into the chips.