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Appl. Environ. Microbiol. doi:10.1128/AEM.02894-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Diversity and abundance of nitrate reductase (narG & napA), and nitrite reductase (nirS and nrfA) genes and transcripts in estuarine sediments

Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, U.K.; Department of Animal and Plant Sciences, University of Sheffield, Western Bank, Sheffield, S10 2TN, U.K.

^* To whom correspondence should be addressed. Email: A.M.Osborn{at}sheffield.ac.uk.

	Abstract

Estuarine systems are the major conduits for the transfer of nitrate from agricultural and other terrestrial-anthropogenic 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, UK, 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 characterised nitrate reduction sequences in soils and offshore marine sediments, and those 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 deltaproteobacteria. A suite of Q-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 Q-RT-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.

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