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Abstract
Nitrosomonas europaea is a commonly studied ammonia-oxidizing bacterium, which oxidizes ammonia into nitrite. The process of nitrification is widely studied due to its importance in the nitrogen cycle, and reduction of ammonia released into the environment. Many techniques, including fluorescence in situ hybridization, have been developed to study the diversity and abundance of N. europaea and other nitrifying bacteria, however due to the low growth rates of N. europaea, analysis is extremely time consuming. Compartitive 16S rRNA assays using oligonucleotide probes is a more current method used for identifying and determining the abundance of a particular strain. Nevertheless, detection of ribosomal RNA on a membrane can vary depending on the concentration of the organism, probe binding specificities, and various detection techniques. The goal of this study was to create and characterize a 16S rDNA construct, which would act as a positive control in further comparative 16S rRNA assays. pWL061, containing the first 245 base pairs of the N. europaea 16S rRNA gene, was digested with Xmn 1 and loaded onto a Zeta-Probe membrane in increments of 5, 3 and 1 µg. RNA from N. europaea and Escherichia coli were loaded onto the Zeta-Probe membrane as well. Oligonucleotide probes, Nso190 and Nsm156, which detect ammonium oxidizing β -proteobacteria and Nitrosomonas species respectively, were used to detect nucleic acids immobilized on the membrane. While only the 1 µg slot of 16S rDNA from pWL061 was detected by both probes, Nso190 was found to have a much stronger band intensity for 1 µg of rDNA than Nsm156. Neither E. coli nor N. europaea RNA was detected by either probe.