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.
authors.affiliation: Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Japan.
authors.forename: Kengo
authors.initials: K
authors.lastname: Kubota
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Imachi
authors.forename: Shuji
authors.initials: S
authors.lastname: Kawakami
authors.forename: Kohei
authors.initials: K
authors.lastname: Nakamura
authors.forename: Hideki
authors.initials: H
authors.lastname: Harada
authors.forename: Akiyoshi
authors.initials: A
authors.lastname: Ohashi
mesh_terms: D002384:Catalysis; D002463:Cell Membrane Permeability; D019286:Endopeptidase K / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D019605:Euryarchaeota / Q000145:classification* / Q000235:genetics / Q000378:metabolism*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008697:Methane / Q000378:metabolism*; D009113:Muramidase / Q000378:metabolism; D010457:Peptidoglycan / Q000378:metabolism; D011135:Polysaccharides, Bacterial / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D010457:Peptidoglycan; D011135:Polysaccharides, Bacterial; D011994:Recombinant Proteins; C051467:peptidoglycan endopeptidase; C073246:methanochondroitin; D009113:Muramidase; D010450:Endopeptidases; D019286:Endopeptidase K; D008697:Methane