Detection and quantification of expression of amoA by competitive reverse transcription-PCR

Y. Ebie, H. Miura, N. Noda, M. Matsumura, S. Tsuneda, A. Hirata, Y. Inamori

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

Ammonia oxidation by chemolithoautotrophic ammonia-oxidizing bacteria is an important step in the biological nitrogen removal process. The first conversion step, the oxidation of ammonia to hydroxylamine is catalyzed by ammonia monooxygenase (AMO). To investigate the activity of ammonia oxidation, mRNA (designated as amoA) encoding a subunit of AMO was quantified by competitive reverse transcription (RT)-PCR. As a result, it was possible to detect and quantify amoA expression in cultured Nitrosomonas europaea and even complex microbial communities such as nitrifying bacterial aggregates by competitive RT-PCR. It was estimated that amoA concentration in cultured N. europaea was 2.3 × 108 copies.ml-1. Additionally, it was calculated that the copy number of amoA in nitrifying bacterial aggregates was 1.0 × 1012 copies.ml-1 (5.1 × 1010 copies.mg-1-dry weight). On the other hand, amoA expression in the natural activated sludge in a household Gappei-Johkaso was undetectable, whereas 16S rRNA of ammonia-oxidizing bacteria was detected by RT-PCR. Then, four days cultivation of this sludge in inorganic artificial wastewater resulted in increasing amoA expression to a quantifiable amount by competitive RT-PCR. In conclusion, the competitive RT-PCR was effective to investigate the expression of amoA as an indicator of ammonia oxidation activity by autotrophic ammonia-oxidizing bacteria.

Original languageEnglish
Pages (from-to)281-288
Number of pages8
JournalWater Science and Technology
Volume46
Issue number1-2
DOIs
Publication statusPublished - 2002 Jan 1

Keywords

  • Ammonia monooxygenase
  • Ammonia-oxidizing bacteria
  • Nitrogen removal
  • Reverse transcription-PCR
  • amoA

ASJC Scopus subject areas

  • Environmental Engineering
  • Water Science and Technology

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