Participants:

Biocomplexity: Diversity of urea-hydrolyzing organisms in Chesapeake Bay
People: Jackie L. Collier and Kristopher M. Baker

Most organisms use urease to break down urea
Urea is the single most abundant form of dissolved organic nitrogen present in aquatic ecosystems (even without the addition of urea in runoff from agriculture). Many organisms can use urea as a source of nitrogen by importing urea into the cell cytoplasm, where the enzyme urease releases two ammonium molecules from each urea that can then be assimilated directly into cell biomass. One goal of this project is to investigate the relationships between the rate at which urea-nitrogen is incorporated into biomass at a ‘community’ level and the diversity, abundance, and expression of the genes encoding urease.

Urease molecular biology
Urease (urea amidohydrolase, E.C. 3.5.1.5) gene sequences from organisms as different as bacteria, plants, and animals share significant sequence similarities, even though the main structural sequence of urease is divided into one, two, or three subunits in different types of organisms. We have developed degenerate primers that should be able to amplify the DNA encoding a 200 amino acid region of any urease gene. So far these primers have been successful in amplifying the targeted urease gene fragment from any organism in which urease activity is detectable. The amplified urease gene fragment is well conserved except for one region that exhibits extensive insertion/deletion events. In some eukaryotes, the amplified genomic DNA fragment also includes one or more introns.

Amplifying urease sequences from Chesapeake Bay samples
In addition to testing the primers on several different ‘known’ organisms (e.g., axenic cultures of various algae), we have used the primers to amplify urease gene fragments from four samples collected in the Choptank River at the outset of this project (July 2000). Products of the expected size were easily obtained for all samples, and Terminal Restriction Fragment Length Polymorphism (TRFLP) analysis suggests that a high degree of diversity exists within the products from each sample. To date, about 100 clones from libraries constructed for these four samples have been sequenced.

Identifying organisms based on urease sequences
There is enough phylogenetic information within the amplified urease gene fragment to identify sequences from organisms fairly closely (we currently guess at about the level of ‘genus’) related to the sequence from a ‘known’ organism. However, many of the sequences recovered from the Choptank samples are not closely enough related to sequences from any ‘known’ organisms to allow even a broad identification of the type of organism they came from. This difficulty arises largely from the bias toward pathogenic microorganisms of the urease sequences present in GenBank. Intensive efforts will be focussed on improving the taxonomic representativeness of ‘known’ urease sequences throughout this project.

Following the dynamics of urease sequence ‘types’ in Chesapeake Bay
We are currently in the process of designing and adapting to array hybridization technology oligonucleotide probes to detect some of the major groups of urease sequences that have appeared in the Choptank sequence database so far. The probe library will be expanded and/or modified as new sequences from the Chesapeake are acquired.