Molecular diversity and characterization of nitrite reductase gene fragments (nirK and nirS) from nitrate- and uranium-contaminated groundwater

Nitrate-contaminated groundwater samples were analysed for nir K and nir S gene diversity. The samples differed with respect to nitrate, uranium, heavy metals, organic carbon content, pH and dissolved oxygen levels. A total of 958 nir K and 1162 nir S clones were screened by restriction fragment length polymorphism (RFLP) analysis: 48 and 143 distinct nir K and nir S clones, respectively, were obtained. A single dominant nir K restriction pattern was observed for all six samples and was 83% identical to the Hyphomicrobium zavarzinii nir K gene. A dominant nir S pattern was observed for four of the samples, including the background sample, and was 95% identical to the nir S of Alcaligenes faecalis. Diversity indices for nir K and nir S sequences were not related to any single geochemical characteristic, but results suggested that the diversity of nir K genes was inversely proportional to the diversity of nir S. Principal component analysis (PCA) of the sites based on geochemistry grouped the samples by low, moderate and high nitrate but PCA of the unique operational taxonomic units (OTUs) distributions grouped the samples differently. Many of the sequences were not closely related to previously observed genes and some phylogenetically related sequences were obtained from similar samples. The results indicated that the contaminated groundwater contained novel nir K and nir S sequences, functional diversity of both genes changed in relation to the contaminant gradient, but the nir K and nir S functional diversity was affected differently.