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

• Published in: Journal of microbiological methods Vol. 72; no. 1; pp. 54 - 59
• Main Authors: Kubota, Kengo, Imachi, Hiroyuki, Kawakami, Shuji, Nakamura, Kohei, Harada, Hideki, Ohashi, Akiyoshi
• Format: Journal Article
• Language: English
• Published: Shannon Elsevier B.V 2008; Elsevier Science
• Subjects: Archaea; Biological and medical sciences; Catalysis; Catalyzed reporter deposition; Cell Membrane Permeability; Endopeptidase K - metabolism; Endopeptidases - metabolism; Euryarchaeota - classification; Euryarchaeota - genetics; Euryarchaeota - metabolism; Fluorescence in situ hybridization; Fundamental and applied biological sciences. Psychology; In Situ Hybridization, Fluorescence - methods; Methane - metabolism; Methanochondroitin; Methanogens; Microbiology; Muramidase - metabolism; Peptidoglycan - metabolism; Polysaccharides, Bacterial - metabolism; Pseudomurein; Pseudomurein endopeptidase PeiW; Recombinant Proteins - metabolism; Sheath; Surface layer; Methanochondroitin; Pseudomurein; Fluorescence in situ hybridization; Methanogens; Pseudomurein endopeptidase PeiW; Surface layer; Catalyzed reporter deposition; Sheath; Evaluation; Molecular hybridization; Enzyme; In situ; Peptidases; Treatment; Endopeptidase; Hydrolases
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2007.10.006

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.