Diversity and Abundance of Nitrate Reductase Genes ( narG and napA ), Nitrite Reductase Genes ( nirS and nrfA ), and Their Transcripts in Estuarine Sediments

Abstract

Estuarine systems are the major conduits for the transfer of nitrate from agricultural and other terrestrialanthropogenic sources into marine ecosystems. Within estuarine sediments some microbially driven processes (denitrification and anammox) result in the net removal of nitrogen from the environment, while others (dissimilatory nitrate reduction to ammonium) do not. In this study, molecular approaches have been used to investigate the diversity, abundance, and activity of the nitrate-reducing communities in sediments from the hypernutrified Colne estuary, United Kingdom, via analysis of nitrate and nitrite reductase genes and transcripts. Sequence analysis of cloned PCR-amplified narG, napA, and nrfA gene sequences showed the indigenous nitrate-reducing communities to be both phylogenetically diverse and also divergent from previously characterized nitrate reduction sequences in soils and offshore marine sediments and from cultured nitrate reducers. In both the narG and nrfA libraries, the majority of clones (48% and 50%, respectively) were related to corresponding sequences from delta-proteobacteria. A suite of quantitative PCR primers and TaqMan probes was then developed to quantify phylotype-specific nitrate (narG and napA) and nitrite reductase (nirS and nrfA) gene and transcript numbers in sediments from three sites along the estuarine nitrate gradient. In general, both nitrate and nitrite reductase gene copy numbers were found to decline significantly (P < 0.05) from the estuary head towards the estuary mouth. The development and application, for the first time, of quantitative reverse transcription-PCR assays to quantify mRNA sequences in sediments revealed that transcript numbers for three of the five phylotypes quantified were greatest at the estuary head.

Archaeal ammonia oxidizers and nirS-type denitrifiers dominate sediment nitrifying and denitrifying populations in a subtropical macrotidal estuary

“…The abundance of nirS genes detected in this study is consistent with that of the only other comparable study of denitrifier marker genes in an estuarine sediment (Smith et al, 2007). Although we did not examine transcript abundance or diversity, and as such did not resolve that fraction of the community actively expressing the genes Figure 7 Contribution of major T-RFs to total AOA diversity by estuary section averaged across all months.…”

“…However, unlike the study of Smith et al (2007), which showed that decrease in nirS gene abundance with distance down an estuary, possibly related to the nitrate and ammonia gradient, there was no significant difference in nirS abundance between sections, suggesting that different factors influence the structure of the denitrifier community in the Fitzroy river. It is likely that the large changes in temperature, flow rate and salinity in the river during the year, combined with the rapid growth rates of the denitrifier community, may explain the seasonal succession seen in the Fitzroy given the apparent link between a number of denitrifier T-RFs and salinity.…”

Response of denitrification genes nirS, nirK, and nosZ to irrigation water quality in a Chinese agricultural soil

“…Therefore, it is difficult to determine the diversity of nitrate-reducing communities using 16S rRNA gene approaches. Fortunately, genes that encode key enzymes (nitrate-, nitrite-, nitric oxide-, and nitrous oxide reductases) in nitrate reduction processes have been established as molecular markers (Smith et al 2007). These functional genes include nitrite reductase genes (nirS and nirK), nitrous oxide reductase gene (nosZ), and nitric oxide reductase gene (norB).…”

Characterization of the microbial community in a lotic environment to assess the effect of pollution on nitrifying and potentially pathogenic bacteria

“…PCR amplifications were carried out as previously described. All the primers used in this study were fully characterised in previous studies, in which they were used as primers or fluorescent in situ hybridisation (FISH) probes (Amann et al, 1990;Mobarry et al, 1996;Hovanec et al, 1998;Watanabe et al, 2001;Mohan et al, 2004;Geets et al, 2007;Smith et al, 2007). The PCR products were separated by electrophoresis on a 1% agarose gel and visualised by staining with 0.5 mg of ethidium bromide per mL of gel.…”