Ebie Y, Miura H, Noda N, Matsumura M, Tsuneda S, Hirata A, Inamori Y
Institute of Applied Biochemistry, University of Tsukuba, Ibaraki, Japan.
Water Sci Technol. 2002;46(1-2):281-8.
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 x 10(8) copies x ml(-1). Additionally, it was calculated that the copy number of amoA in nitrifying bacterial aggregates was 1.0 x 10(12) copies x ml(-1) (5.1 x 10(10) copies x 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.
化能自养型氨氧化细菌进行的氨氧化是生物脱氮过程中的重要一步。第一步转化，即氨氧化为羟胺，由氨单加氧酶（AMO）催化。为了研究氨氧化活性，通过竞争性逆转录（RT）-PCR对编码AMO一个亚基的mRNA（命名为amoA）进行定量。结果，通过竞争性RT-PCR能够检测和定量培养的欧洲亚硝化单胞菌以及硝化细菌聚集体等复杂微生物群落中amoA的表达。据估计，培养的欧洲亚硝化单胞菌中amoA浓度为2.3×10⁸拷贝·ml⁻¹。此外，计算得出硝化细菌聚集体中amoA的拷贝数为1.0×10¹²拷贝·ml⁻¹（5.1×10¹⁰拷贝·mg⁻¹干重）。另一方面，在家庭式伽佩伊-净化槽的天然活性污泥中未检测到amoA的表达，而通过RT-PCR检测到了氨氧化细菌的16S rRNA。然后，将该污泥在无机人工废水中培养四天，通过竞争性RT-PCR使amoA表达增加到可定量的水平。总之，竞争性RT-PCR对于研究amoA的表达是有效的，amoA可作为自养型氨氧化细菌氨氧化活性的指标。
