This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hoshino, T. Articles by Inamori, Y. Search for Related Content PubMed PubMed Citation Articles by Hoshino, T. Articles by Inamori, Y. Agricola Articles by Hoshino, T. Articles by Inamori, Y.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, November 2001, p. 5261-5266, Vol. 67, No. 11
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.11.5261-5266.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Direct Detection by In Situ PCR of the amoA Gene in Biofilm Resulting from a Nitrogen Removal Process

Tatsuhiko Hoshino,¹ Naohiro Noda,¹ Satoshi Tsuneda,^1,* Akira Hirata,¹ and Yuhei Inamori²

Department of Chemical Engineering, Waseda University, Shinjuku-ku, Tokyo, 169-8555,¹ and National Institute for Environmental Studies, Tsukuba, Ibaraki 305-0053,² Japan

Received 26 March 2001/Accepted 29 August 2001

Ammonia oxidation is a rate-limiting step in the biological removal of nitrogen from wastewater. Analysis of microbial communities possessing the amoA gene, which is a small subunit of the gene encoding ammonia monooxygenase, is important for controlling nitrogen removal. In this study, the amoA gene present in Nitrosomonas europaea cells in a pure culture and biofilms in a nitrifying reactor was amplified by in situ PCR. In this procedure, fixed cells were permeabilized with lysozyme and subjected to seminested PCR with a digoxigenin-labeled primer. Then, the amplicon was detected with an alkaline phosphatase-labeled antidigoxigenin antibody and HNPP (2-hydroxy-3-naphthoic acid-2'-phenylanilide phosphate), which was combined with Fast Red TR, and with an Alexa Fluor 488-labeled antidigoxigenin antibody. The amoA gene in the biofilms was detected with an unavoidable nonspecific signal when the former method was used for detection. On the other hand, the amoA gene in the biofilms was detected without a nonspecific signal, and the cells possessing the amoA gene were clearly observed near the surface of the biofilm when Alexa Fluor 488-labeled antidigoxigenin antibody was used for detection. Although functional gene expression was not detected in this study, detection of cells in a biofilm based on their function was demonstrated.

---

^* Corresponding author. Mailing address: Department of Chemical Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo, 169-8555, Japan. Phone: 81-3-5286-3210. Fax: 81-3-3209-3680. E-mail: stsuneda{at}mn.waseda.ac.jp.

---

Applied and Environmental Microbiology, November 2001, p. 5261-5266, Vol. 67, No. 11
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.11.5261-5266.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

This article has been cited by other articles:

• Hoshino, T., Yilmaz, L. S., Noguera, D. R., Daims, H., Wagner, M. (2008). Quantification of Target Molecules Needed To Detect Microorganisms by Fluorescence In Situ Hybridization (FISH) and Catalyzed Reporter Deposition-FISH. Appl. Environ. Microbiol. 74: 5068-5077 [Abstract] [Full Text]  
• Chandran, K., Love, N. G. (2008). Physiological State, Growth Mode, and Oxidative Stress Play a Role in Cd(II)-Mediated Inhibition of Nitrosomonas europaea 19718. Appl. Environ. Microbiol. 74: 2447-2453 [Abstract] [Full Text]  
• Tang, Y. Z., Gin, K. Y. H., Lim, T. H. (2005). High-Temperature Fluorescent In Situ Hybridization for Detecting Escherichia coli in Seawater Samples, Using rRNA-Targeted Oligonucleotide Probes and Flow Cytometry. Appl. Environ. Microbiol. 71: 8157-8164 [Abstract] [Full Text]