Diversity and Abundance of Nitrate Assimilation Genes in the Northern South China Sea

Marine heterotrophic microorganisms that assimilate nitrate play an important role in nitrogen and carbon cycling in the water column. The nasA gene, encoding the nitrate assimilation enzyme, was selected as a functional marker to examine the nitrate assimilation community in the South China Sea (SCS). PCR amplification, restriction fragment length polymorphism (RFLP) screening, and phylogenetic analysis of nasA gene sequences were performed to characterize in situ nitrate assimilatory bacteria. Furthermore, the effects of nutrients and other environmental factors on the genetic heterogeneity of nasA fragments from the SCS were evaluated at the surface in three stations, and at two other depths in one of these stations. The diversity indices and rarefaction curves indicated that the nasA gene was more diverse in offshore waters than in the Pearl River estuary. The phylotype rank abundance curve showed an abundant and unique RFLP pattern in all five libraries, indicating that a high diversity but low abundance of nasA existed in the study areas. Phylogenetic analysis of environmental nasA gene sequences further revealed that the nasA gene fragments came from several common aquatic microbial groups, including the Proteobacteria, Cytophaga–Flavobacteria (CF), and Cyanobacteria. In addition to the direct PCR/sequence analysis of environmental samples, we also cultured a number of nitrate assimilatory bacteria isolated from the field. Comparison of nasA genes from these isolates and from the field samples indicated the existence of horizontal nasA gene transfer. Application of real-time quantitative PCR to these nasA genes revealed a great variation in their abundance at different investigation sites and water depths.