This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Worden, A. Z.
Right arrow Articles by Binder, B. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Worden, A. Z.
Right arrow Articles by Binder, B. J.
Agricola
Right arrow Articles by Worden, A. Z.
Right arrow Articles by Binder, B. J.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, January 2000, p. 284-289, Vol. 66, No. 1
0099-2240/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

In Situ Hybridization of Prochlorococcus and Synechococcus (Marine Cyanobacteria) spp. with rRNA-Targeted Peptide Nucleic Acid Probes

Alexandra Z. Worden,1,2,* Sallie W. Chisholm,3,4 and Brian J. Binder2

Institute of Ecology1 and Department of Marine Sciences,2 University of Georgia, Athens, Georgia 30602, and Department of Civil and Environmental Engineering3 and Department of Biology,4 Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Received 6 July 1999/Accepted 26 October 1999

A simple method for whole-cell hybridization using fluorescently labeled rRNA-targeted peptide nucleic acid (PNA) probes was developed for use in marine cyanobacterial picoplankton. In contrast to established protocols, this method is capable of detecting rRNA in Prochlorococcus, the most abundant unicellular marine cyanobacterium. Because the method avoids the use of alcohol fixation, the chlorophyll content of Prochlorococcus cells is preserved, facilitating the identification of these cells in natural samples. PNA probe-conferred fluorescence was measured flow cytometrically and was always significantly higher than that of the negative control probe, with positive/negative ratio varying between 4 and 10, depending on strain and culture growth conditions. Prochlorococcus cells from open ocean samples were detectable with this method. RNase treatment reduced probe-conferred fluorescence to background levels, demonstrating that this signal was in fact related to the presence of rRNA. In another marine cyanobacterium, Synechococcus, in which both PNA and oligonucleotide probes can be used in whole-cell hybridizations, the magnitude of fluorescence from the former was fivefold higher than that from the latter, although the positive/negative ratio was comparable for both probes. In Synechococcus cells growing at a range of growth rates (and thus having different rRNA concentrations per cell), the PNA- and oligonucleotide-derived signals were highly correlated (r = 0.99). The chemical nature of PNA, the sensitivity of PNA-RNA binding to single-base-pair mismatches, and the preservation of cellular integrity by this method suggest that it may be useful for phylogenetic probing of whole cells in the natural environment.

* Corresponding author. Mailing address: Institute of Ecology, Ecology Building, University of Georgia, Athens, GA 30602. Phone: (706) 542-7099. Fax: (706) 542-5888. E-mail: azworden{at}arches.uga.edu.

Applied and Environmental Microbiology, January 2000, p. 284-289, Vol. 66, No. 1
0099-2240/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Li, X., Morgenroth, E., Raskin, L. (2008). Quantitative rRNA-Targeted Solution-Based Hybridization Assay Using Peptide Nucleic Acid Molecular Beacons. Appl. Environ. Microbiol. 74: 7297-7305 [Abstract] [Full Text]  
  • Wilks, S. A., Keevil, C. W. (2006). Targeting Species-Specific Low-Affinity 16S rRNA Binding Sites by Using Peptide Nucleic Acids for Detection of Legionellae in Biofilms. Appl. Environ. Microbiol. 72: 5453-5462 [Abstract] [Full Text]  
  • Pernthaler, J., Amann, R. (2005). Fate of Heterotrophic Microbes in Pelagic Habitats: Focus on Populations. Microbiol. Mol. Biol. Rev. 69: 440-461 [Abstract] [Full Text]  
  • Becker, S., Fahrbach, M., Boger, P., Ernst, A. (2002). Quantitative Tracing, by Taq Nuclease Assays, of a Synechococcus Ecotype in a Highly Diversified Natural Population. Appl. Environ. Microbiol. 68: 4486-4494 [Abstract] [Full Text]  
  • Laloui, W., Palinska, K. A., Rippka, R., Partensky, F., Tandeau de Marsac, N., Herdman, M., Iteman, I. (2002). Genotyping of axenic and non-axenic isolates of the genus Prochlorococcus and the OMF-Synechococcus' clade by size, sequence analysis or RFLP of the Internal Transcribed Spacer of the ribosomal operon. Microbiology 148: 453-465 [Abstract] [Full Text]  
  • West, N. J., Schonhuber, W. A., Fuller, N. J., Amann, R. I., Rippka, R., Post, A. F., Scanlan, D. J. (2001). Closely related Prochlorococcus genotypes show remarkably different depth distributions in two oceanic regions as revealed by in situ hybridization using 16S rRNA-targeted oligonucleotides. Microbiology 147: 1731-1744 [Abstract] [Full Text]  
  • Stender, H., Broomer, A. J., Oliveira, K., Perry-O'Keefe, H., Hyldig-Nielsen, J. J., Sage, A., Coull, J. (2001). Rapid Detection, Identification, and Enumeration of Escherichia coli Cells in Municipal Water by Chemiluminescent In Situ Hybridization. Appl. Environ. Microbiol. 67: 142-147 [Abstract] [Full Text]  
  • Chandler, D. P., Stults, J. R., Cebula, S., Schuck, B. L., Weaver, D. W., Anderson, K. K., Egholm, M., Brockman, F. J. (2000). Affinity Purification of DNA and RNA from Environmental Samples with Peptide Nucleic Acid Clamps. Appl. Environ. Microbiol. 66: 3438-3445 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»