Evaluation of enzymatic cell treatments for application of CARD-FISH to methanogens

Kengo Kubota; Hiroyuki Imachi; Shuji Kawakami; Kohei Nakamura; Hideki Harada; Akiyoshi Ohashi

doi:10.1016/j.mimet.2007.10.006

Evaluation of enzymatic cell treatments for application of CARD-FISH to methanogens

Kengo Kubota, Hiroyuki Imachi, Shuji Kawakami, Kohei Nakamura, Hideki Harada, Akiyoshi Ohashi

Research output: Contribution to journal › Article › peer-review

32 Citations (Scopus)

Abstract

Several enzymatic permeabilization protocols (utilizing lysozyme, proteinase K, achromopeptidase, or recombinant pseudomurein endopeptidase [PeiW]) were evaluated for application of in situ hybridization with horseradish peroxidase-labeled oligonucleotide probes and catalyzed reporter deposition (CARD-FISH) to methanogens. In this study, twelve methanogens were selected that have typical cell surface structures: pseudomurein, surface layer, methanochondroitin and sheath. Among the treatments tested, PeiW treatment was observed to be the most effective one, although methanogens having a sheath were stained heterogeneously and methanogens having methanochondroitin were not permeabilized. On the other hand, lysozyme, proteinase K, and achromopeptidase treatments were ineffective or caused cell-lysis, resulting in weak or no signals. Applicability of PeiW treatment was further evaluated using an anaerobic granular sludge sample. The detection rate of Archaea by CARD-FISH increased remarkably after the treatment. Based on the results obtained in this study, we propose PeiW treatment as a novel permeabilization method for CARD-FISH application to methanogens.

Original language	English
Pages (from-to)	54-59
Number of pages	6
Journal	Journal of Microbiological Methods
Volume	72
Issue number	1
DOIs	https://doi.org/10.1016/j.mimet.2007.10.006
Publication status	Published - 2008 Jan 1

Keywords

Catalyzed reporter deposition
Fluorescence in situ hybridization
Methanochondroitin
Methanogens
Pseudomurein
Pseudomurein endopeptidase PeiW
Sheath
Surface layer

ASJC Scopus subject areas

Microbiology
Molecular Biology
Microbiology (medical)

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title = "Evaluation of enzymatic cell treatments for application of CARD-FISH to methanogens",

abstract = "Several enzymatic permeabilization protocols (utilizing lysozyme, proteinase K, achromopeptidase, or recombinant pseudomurein endopeptidase [PeiW]) were evaluated for application of in situ hybridization with horseradish peroxidase-labeled oligonucleotide probes and catalyzed reporter deposition (CARD-FISH) to methanogens. In this study, twelve methanogens were selected that have typical cell surface structures: pseudomurein, surface layer, methanochondroitin and sheath. Among the treatments tested, PeiW treatment was observed to be the most effective one, although methanogens having a sheath were stained heterogeneously and methanogens having methanochondroitin were not permeabilized. On the other hand, lysozyme, proteinase K, and achromopeptidase treatments were ineffective or caused cell-lysis, resulting in weak or no signals. Applicability of PeiW treatment was further evaluated using an anaerobic granular sludge sample. The detection rate of Archaea by CARD-FISH increased remarkably after the treatment. Based on the results obtained in this study, we propose PeiW treatment as a novel permeabilization method for CARD-FISH application to methanogens.",

keywords = "Catalyzed reporter deposition, Fluorescence in situ hybridization, Methanochondroitin, Methanogens, Pseudomurein, Pseudomurein endopeptidase PeiW, Sheath, Surface layer",

author = "Kengo Kubota and Hiroyuki Imachi and Shuji Kawakami and Kohei Nakamura and Hideki Harada and Akiyoshi Ohashi",

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T1 - Evaluation of enzymatic cell treatments for application of CARD-FISH to methanogens

AU - Kubota, Kengo

AU - Imachi, Hiroyuki

AU - Kawakami, Shuji

AU - Nakamura, Kohei

AU - Harada, Hideki

AU - Ohashi, Akiyoshi

PY - 2008/1/1

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N2 - Several enzymatic permeabilization protocols (utilizing lysozyme, proteinase K, achromopeptidase, or recombinant pseudomurein endopeptidase [PeiW]) were evaluated for application of in situ hybridization with horseradish peroxidase-labeled oligonucleotide probes and catalyzed reporter deposition (CARD-FISH) to methanogens. In this study, twelve methanogens were selected that have typical cell surface structures: pseudomurein, surface layer, methanochondroitin and sheath. Among the treatments tested, PeiW treatment was observed to be the most effective one, although methanogens having a sheath were stained heterogeneously and methanogens having methanochondroitin were not permeabilized. On the other hand, lysozyme, proteinase K, and achromopeptidase treatments were ineffective or caused cell-lysis, resulting in weak or no signals. Applicability of PeiW treatment was further evaluated using an anaerobic granular sludge sample. The detection rate of Archaea by CARD-FISH increased remarkably after the treatment. Based on the results obtained in this study, we propose PeiW treatment as a novel permeabilization method for CARD-FISH application to methanogens.

AB - Several enzymatic permeabilization protocols (utilizing lysozyme, proteinase K, achromopeptidase, or recombinant pseudomurein endopeptidase [PeiW]) were evaluated for application of in situ hybridization with horseradish peroxidase-labeled oligonucleotide probes and catalyzed reporter deposition (CARD-FISH) to methanogens. In this study, twelve methanogens were selected that have typical cell surface structures: pseudomurein, surface layer, methanochondroitin and sheath. Among the treatments tested, PeiW treatment was observed to be the most effective one, although methanogens having a sheath were stained heterogeneously and methanogens having methanochondroitin were not permeabilized. On the other hand, lysozyme, proteinase K, and achromopeptidase treatments were ineffective or caused cell-lysis, resulting in weak or no signals. Applicability of PeiW treatment was further evaluated using an anaerobic granular sludge sample. The detection rate of Archaea by CARD-FISH increased remarkably after the treatment. Based on the results obtained in this study, we propose PeiW treatment as a novel permeabilization method for CARD-FISH application to methanogens.

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KW - Pseudomurein endopeptidase PeiW

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KW - Surface layer

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