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Applied and Environmental Microbiology, October 2002, p. 5017-5025, Vol. 68, No. 10
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.10.5017-5025.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Detection and Diversity of Expressed Denitrification Genes in Estuarine Sediments after Reverse Transcription-PCR Amplification from mRNA

Balbina Nogales,* Kenneth N. Timmis, David B. Nedwell, and A. Mark Osborn

Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, CO4 3SQ, United Kingdom

Received 18 April 2002/ Accepted 7 July 2002

The expression of five denitrification genes coding for two nitrate reductases (narG and napA), two nitrite reductases (nirS and nirK), and nitrous oxide reductase (nosZ) was analyzed by reverse transcription (RT)-PCR of mRNA extracted from two sediment samples obtained in the River Colne estuary (United Kingdom), which receives high nitrogen inputs and for which high denitrification rates have been observed. The presence of all five genes in both sediment samples was confirmed by PCR amplification from extracted DNA prior to analysis of gene expression. Only nirS and nosZ mRNAs were detected; nirS was detected directly as an RT-PCR amplification product, and nosZ was detected following Southern blot hybridization. This indicated that active expression of at least the nirS and nosZ genes was occurring in the sediments at the time of sampling. Amplified nirS RT-PCR products were cloned and analyzed by sequencing, and they were compared with amplified nirS gene sequences from isolates obtained from the same sediments. A high diversity of nirS sequences was observed. Most of the cloned nirS sequences retrieved were specific to one site or the other, which underlines differences in the compositions of the bacterial communities involved in denitrifrification in the two sediments analyzed.

* Corresponding author. Present address: Departament de Biologia, Area de Microbiologia, Universitat de les Illes Balears, Crtra. Valldemossa, km 7.5, 07071 Palma de Mallorca, Spain. Phone: 34 971 17 33 35. Fax: 34 971 17 31 84. E-mail: bnogales{at}uib.es.

Applied and Environmental Microbiology, October 2002, p. 5017-5025, Vol. 68, No. 10
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.10.5017-5025.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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