Evaluation of enzymatic cell treatments for application of CARD-FISH to methanogens.
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.
Kubota
Kengo
K
Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Japan.
Imachi
Hiroyuki
H
Kawakami
Shuji
S
Nakamura
Kohei
K
Harada
Hideki
H
Ohashi
Akiyoshi
A
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
D010457:Peptidoglycan; D011135:Polysaccharides, Bacterial; D011994:Recombinant Proteins; C051467:peptidoglycan endopeptidase; C073246:methanochondroitin; D009113:Muramidase; D010450:Endopeptidases; D019286:Endopeptidase K; D008697:Methane