Participants:

Functional Gene Arrays to Study the Nitrogen Cycle
B. B. Ward, Princeton University

Members of the Princeton Biocomplexity Team:
Gaspar Taroncher-Oldenburg
Chris Francis
Greg O’Mullan
Erin Griner

Bacteria and their activity in the environment can be detected in a number of ways, even though the individual cells are very small and cannot be distinguished. Modern methods are based on our abilities to detect and characterize molecules in bacterial cells, which contain important information about who they are and what they do.

In this project, we are mainly interested in genes that encode enzymes that carry out functions related to nitrogen transformations. We are using probes for the DNA that encodes the individual genes and for the RNA that carries the genetic instructions to the protein manufacturing site in the cell. If we extract the nucleic acids from a sample of water and we detect the presence of the functional gene, say the gene that encodes ammonia monooxygense, we can conclude that nitrifying bacteria were present in the sample. If we also detect the messenger RNA for that gene, then we would conclude that the gene is being expressed and that we should be able to measure ammonia oxidation in the sample. Actual measurements of reaction rates of nitrification and denitrification are being carried out by other members of the team (see Kana and Cornwell pages).

Based on results from other environments and substantiated by our preliminary work in Chesapeake Bay and the Choptank River, we know that the microbial assemblage that carries out the various reactions in the nitrogen is very diverse. There are many organisms that perform each of the reactions of interest and they possess a wide variety of different but homologous genes, which encode the enzymes for those reactions. Why is there so much redundancy in the functional groups? Are all of the different kinds present everywhere and all the time? Or are there environmental variables that regulate bacterial activity, such that some members of the group are “turned on” under some conditions and others are active under other conditions?

In order to investigate the distribution and perhaps the activity of all those different genes, we will construct microarrays (“chips”) and macroarrays (membrane filter based) that contain the different versions of each gene. Then by hybridizing the arrays to DNA or RNA extracted from the environmental samples, we will be able to detect which genes are present at various times and locations in the river, bay or ocean.

Genes representing important functions in the nitrogen cycle (nitrite reductase, ammonia monooxygenase, nitrogenase, urease) and the carbon cycle (RuBisCO, carbonic anhydrase) will be incorporated into the arrays.

So far, we have tested small versions of our arrays with a few probes. This image represents a glass slide microarray we tested. The probes (the DNA fragments that were attached to the slide itself) were composed of 70-mer oligonucleotides representing several different versions of some of the genes responsible for ammonia oxidation (amoA), nitrite reduction (nirK and nirS) and nitrogen fixation (nifH).

Another member of the Biocomplexity team, Jon Zehr, is characterizing macroarrays and using them to distinguish nifH genes from various cyanobacteria.

The gene arrays and biogeochemical rate measurements will allow us to link the diverse microbial assemblages to their role in the environment. This requires that we are able to sample and describe the environment at scales ranging from the molecular to the ecosytem , using the sampling tools of oceanographers and the molecular tools of modern biology.