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Whole-cell immunocytochemical detection of nitrogenase in cyanobacteria: improved protocol for highly fluorescent cells

2008; Inter-Research Science Center; Volume: 51; Linguagem: Inglês

10.3354/ame01197

1616-1564

Yukiko Taniuchi, Akio Murakami, K. Ohki,

Microbial Fuel Cells and Bioremediation

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 51:237-247 (2008) - DOI: https://doi.org/10.3354/ame01197 Whole-cell immunocytochemical detection of nitrogenase in cyanobacteria: improved protocol for highly fluorescent cells Yukiko Taniuchi1,3, Akio Murakami2, Kaori Ohki1,* 1Department of Marine Bioscience, Faculty of Biotechnology, Fukui Prefectural University, 1-1, Gakuencho, Obama, Fukui 917-0003, Japan 2Kobe University Research Center for Inland Seas, 2746, Iwaya, Awaji, Hyogo 656-2401, Japan 3Present address: Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaoshiung 80424, Taiwan *Corresponding author. Email: kaoriohki@fpu.ac.jp ABSTRACT: An improved immunocytochemical method was developed to detect nitrogenase in single cells of cyanobacteria using a unicellular diazotrophic strain (Gloeothece sp. 68DGA) and a non-diazotrophic strain (Synechocystis sp. PCC6714) as model organisms. Polyclonal antibodies raised against the Fe-protein and the MoFe-protein (α-subunit) of nitrogenase were used as probes. The antigenicity of nitrogenase was maintained for at least 6 mo when the cells were preserved in chilled methanol (–30°C) after paraformaldehyde fixation (3%). The cells were permeabilized for antibody penetration and non-specific binding was prevented by incubation in phosphate-buffered saline containing dimethyl sulfoxide (10%) and normal rabbit serum (15%). Antibody binding was visualized by a horseradish peroxidase-conjugated secondary antibody and the chromogenic substrate 3-3'-diaminobenzidine tetrachloride because of the difficulty in discriminating between fluorescence from additive fluorochrome and natural autofluorescence from cyanobacteria. Almost all cells (>97%) were immunostained when they were grown diazotrophically and expressed nitrogenase (acetylene reduction) activity. Non-specific staining of both the diazotrophic strains grown with combined nitrogen and the non-diazotrophic strain was negligible. Our protocol was able to detect nitrogenase in unicellular and filamentous non-heterocystous strains without modification. KEY WORDS: Diazotrophic unicellular cyanobacteria · Marine cyanobacteria · Immunocytochemical detection · Nitrogenase · Unicellular cyanobacteria Full text in pdf format Supplementary appendix PreviousNextCite this article as: Taniuchi Y, Murakami A, Ohki K (2008) Whole-cell immunocytochemical detection of nitrogenase in cyanobacteria: improved protocol for highly fluorescent cells. Aquat Microb Ecol 51:237-247. https://doi.org/10.3354/ame01197Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 51, No. 3. Online publication date: June 16, 2008 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 2008 Inter-Research.