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Applied and Environmental Microbiology, November 2006, p. 6907-6913, Vol. 72, No. 11
0099-2240/06/$08.00+0     doi:10.1128/AEM.01499-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Application of Pseudomurein Endoisopeptidase to Fluorescence In Situ Hybridization of Methanogens within the Family Methanobacteriaceae

Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba 305-8566, Japan,¹ Research Institute of Genome-Based Biofactory, National Institute of Advanced Industrial Science and Technology Toyohira, Sapporo, Hokkaido 062-8517, Japan,² Center of Molecular Biosciences, University of Ryukus, 1 Senbaru, Nishihara-cho, Okinawa 903-0213, Japan,³ Marine Biotechnology Institute, 3-75-1 Heita, Kamaishi, Iwate 026-0001, Japan,⁴ Fujikasui Engineering Co., Ltd., 1-4-3 Higashigotanda, Shinagawa-ku, Tokyo 141-0022, Japan⁵

Received 29 June 2006/ Accepted 23 August 2006

In situ detection of methanogens within the family Methanobacteriaceae is sometimes known to be unsuccessful due to the difficulty in permeability of oligonucleotide probes. Pseudomurein endoisopeptidase (Pei), a lytic enzyme that specifically acts on their cell walls, was applied prior to 16S rRNA-targeting fluorescence in situ hybridization (FISH). For this purpose, pure cultured methanogens within this family, Methanobacterium bryantii, Methanobrevibacter ruminantium, Methanosphaera stadtmanae, and Methanothermobacter thermautotrophicus together with a Methanothermobacter thermautotrophicus-containing syntrophic acetate-oxidizing coculture, endosymbiotic Methanobrevibacter methanogens within an anaerobic ciliate, and an upflow anaerobic sludge blanket (UASB) granule were examined. Even without the Pei treatment, Methanobacterium bryantii and Methanothermobacter thermautotrophicus cells are relatively well hybridized with oligonucleotide probes. However, almost none of the cells of Methanobrevibacter ruminantium, Methanosphaera stadtmanae, cocultured Methanothermobacter thermautotrophicus, and the endosymbiotic methanogens and the cells within UASB granule were hybridized. Pei treatment was able to increase the probe hybridization ratio in every specimen, particularly in the specimen that had shown little hybridization. Interestingly, the hybridizing signal intensity of Methanothermobacter thermautotrophicus cells in coculture with an acetate-oxidizing H₂-producing syntroph was significantly improved by Pei pretreatment, whereas the probe was well hybridized with the cells of pure culture of the same strain. We found that the difference is attributed to the differences in cell wall thicknesses between the two culture conditions. These results indicate that Pei treatment is effective for FISH analysis of methanogens that show impermeability to the probe.

Published ahead of print on 1 September 2006.