Diversity of nitrite reductase genes (nirS) in the denitrifying water column of the coastal Arabian Sea

Denitrification often occurs in the water column, underlying zones of intense productivity and decomposition in upwelling regions. In the denitrifying zone off the southwest coast of India, high concentrations of nitrite (>15 μM) and nitrous oxide (>500 nM) have been reported near the sediment-water interface (<80 m). We investigated the chemical and molecular indicators of denitrification along the southwest coast of India during the southwest monsoon season of October 2001. Nitrite reduction to nitric oxide is the key step in the denitrification pathway, and is catalyzed by the enzyme nitrite reductase, which is encoded by the genes nirS and nirK. Here we report the diversity and distribution of nirS genes in relation to nitrite and nitrate distribution in the Arabian Sea coastal denitrifying region. nirS gene fragments were PCR-amplified, cloned, and sequenced from DNA extracted from the water column. Clone libraries were also subjected to restriction fragment length polymorphism (RFLP) and rarefaction analyses. These are the first nitrite reductase sequences reported from a water column denitrifying regime. nirS was amplified from DNA extracted from all water samples in which nitrite was present at high concentrations within the low oxygen waters, but was only rarely amplified from waters containing hydrogen sulfide or from well-oxygenated waters. Phylogenetic analysis grouped 132 nirS Arabian Sea sequences into 12 major clusters. Most of the nirS sequences from the coastal water column did not show a high level of identity with other nirS sequences previously reported from marine and estuarine sediments. Identities of the Arabian Sea sequences to those in the public database ranged from 44 to 99% at the amino acid level. The dominant sequence type from 1 surface sample showed 99% identity to the nirS sequence of the cultivated denitrifier Pseudomonas aeruginosa. Rarefaction analysis, based on both sequence and RFLP data, indicated the highest diversity in a sample in which relatively high nitrite concentrations implied the presence of active denitrification, and the lowest diversity in a surface sample where nitrite was undetectable, suggesting a link between functional diversity and ecosystem chemistry.