Using the nitrous oxide reductase (nosZ) gene as a molecular marker for tracking denitrifying bacteria in marine and coastal environments.

摘要

Denitrifying bacteria are a phylogenetically diverse group of microbes that reductively respire nitrate or nitrite to N2 or N2O. Because of the importance of denitrifying bacteria in many areas of scientific research, this project was undertaken to develop a molecular tool that would facilitate the study of the ecology of denitrifiers in various natural environments. In the past, studies of denitrifying bacteria often relied upon culturing and isolation techniques. The biases introduced by these methods resulted in an inaccurate picture of the true denitrifier dynamics. Therefore, a denitrifier-specific molecular marker was sought.; The nitrous oxide reductase (nosZ) gene was chosen as the functional gene to be used for detection of denitrifier-specific DNA in natural environments. This gene codes for nitrous oxide reductase, which catalyzes the reduction of nitrous oxide to dinitrogen gas. This gene is largely unique to denitrifying bacteria, and only one form of the enzyme is known, eliminating the need for multiple molecular probes.; An initial set of nosZ-gene specific PCR primers was developed from three nosZ gene sequences. These primers were used to identify and sequence three nosZ sequences from continental shelf sediments. The PCR primers were then redesigned, resulting in a more flexible and useful probe for tracking denitrifiers.; The redesigned PCR primers were used to investigate the diversity of nosZ in continental shelf sediments. Despite a large degree of variation in the gene and its derived protein product, core structural motifs are maintained. Conservation of amino acid residues has lead to the proposition of a location for the CuZ (substrate-binding) site of the enzyme. Denitrifiers in culture collections were found to poorly represent those present in the shelf environment.; These PCR primers were then combined with terminal restriction fragment length polymorphism analysis (T-RFLP) in order to quantify the degree of spatial and temporal heterogeneity in denitrifier assemblages at a continental shelf sediment site. Likely environmental factors that might control such variation were identified. Spatial heterogeneity of denitrifier assemblages in a freshwater and a saltwater marsh were also quantified. Several denitrifiers were found to be common to both marshes, and a subset of these were also identified at the continental shelf site, indicating that there may be denitrifiers in natural environments possessing extremely flexible physiologies. A small suite of common denitrifiers was identified, while the majority were not widely dispersed.