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 Widmer, F.
Right arrow Articles by Di Giovanni, G. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Widmer, F.
Right arrow Articles by Di Giovanni, G. D.
Agricola
Right arrow Articles by Widmer, F.
Right arrow Articles by Di Giovanni, G. D.

 Previous Article  |  Next Article 

Appl Environ Microbiol, July 1998, p. 2545-2553, Vol. 64, No. 7
0099-2240/98/$00.00+0

A Highly Selective PCR Protocol for Detecting 16S rRNA Genes of the Genus Pseudomonas (Sensu Stricto) in Environmental Samples

Franco Widmer,1,2,dagger Ramon J. Seidler,2,* Patrick M. Gillevet,3 Lidia S. Watrud,2 and George D. Di Giovanni1,2,Dagger

National Research Council1 and National Health and Ecological Effects Research Laboratory,2 U.S. Environmental Protection Agency, Western Ecology Division, Corvallis, Oregon 97333, and Institute for Bioscience, Bioinformatics, and Biotechnology, George Mason University, Manassas, Virginia 201103

Received 5 June 1997/Accepted 9 April 1998

Pseudomonas species are plant, animal, and human pathogens; exhibit plant pathogen-suppressing properties useful in biological control; or express metabolic versatilities valued in biotechnology and bioremediation. Specific detection of Pseudomonas species in the environment may help us gain a more complete understanding of the ecological significance of these microorganisms. The objective of this study was to develop a PCR protocol for selective detection of Pseudomonas (sensu stricto) in environmental samples. Extensive database searches identified a highly selective PCR primer pair for amplification of Pseudomonas 16S rRNA genes. A protocol that included PCR amplification and restriction analysis, a general cloning and sequencing strategy, and phylogenetic analyses was developed. The PCR protocol was validated by testing 50 target and 14 nontarget pure cultures, which confirmed the selectivity to 100%. Further validation used amplification of target sequences from purified bulk soil DNA followed by cloning of PCR products. Restriction analysis with HaeIII revealed eight different fragmentation patterns among 36 clones. Sequencing and phylogenetic analysis of 8 representative clones indicated that 91.7% of the products were derived from target organisms of the PCR protocol. Three patterns, representing only 8.3% of the 36 clones, were derived from non-Pseudomonas or chimeric PCR artifacts. Three patterns, representing 61.1% of the clones, clustered with sequences of confirmed Pseudomonas species, whereas two patterns, representing 30.6% of the clones, formed a novel phylogenetic cluster closely associated with Pseudomonas species. The results indicated that the Pseudomonas-selective PCR primers were highly specific and may represent a powerful tool for Pseudomonas population structure analyses and taxonomic confirmations.

* Corresponding author. Mailing address: U.S. Environmental Protection Agency, WED, NHEERL, 200 SW 35th St., Corvallis, OR 97333-4902. Phone: (541) 754-4708. Fax: (541) 754-4799. E-mail: seidler{at}mail.cor.epa.gov.

dagger Present address: Institute for Terrestrial Ecology, ETH Zürich, CH-8952 Schlieren, Switzerland.

Dagger Present address: American Waterworks Services Company, Inc., Quality Control and Research Laboratory, Belleville, IL 62220.

Appl Environ Microbiol, July 1998, p. 2545-2553, Vol. 64, No. 7
0099-2240/98/$00.00+0


This article has been cited by other articles:

  • Zouache, K., Voronin, D., Tran-Van, V., Mavingui, P. (2009). Composition of Bacterial Communities Associated with Natural and Laboratory Populations of Asobara tabida Infected with Wolbachia. Appl. Environ. Microbiol. 75: 3755-3764 [Abstract] [Full Text]  
  • Narisawa, N., Haruta, S., Arai, H., Ishii, M., Igarashi, Y. (2008). Coexistence of Antibiotic-Producing and Antibiotic-Sensitive Bacteria in Biofilms Is Mediated by Resistant Bacteria. Appl. Environ. Microbiol. 74: 3887-3894 [Abstract] [Full Text]  
  • Kim, J.-S., Crowley, D. E. (2007). Microbial Diversity in Natural Asphalts of the Rancho La Brea Tar Pits. Appl. Environ. Microbiol. 73: 4579-4591 [Abstract] [Full Text]  
  • Lenaerts, J., Lappin-Scott, H. M., Porter, J. (2007). Improved Fluorescent In Situ Hybridization Method for Detection of Bacteria from Activated Sludge and River Water by Using DNA Molecular Beacons and Flow Cytometry. Appl. Environ. Microbiol. 73: 2020-2023 [Abstract] [Full Text]  
  • Danovaro, R., Luna, G. M., Dell'Anno, A., Pietrangeli, B. (2006). Comparison of Two Fingerprinting Techniques, Terminal Restriction Fragment Length Polymorphism and Automated Ribosomal Intergenic Spacer Analysis, for Determination of Bacterial Diversity in Aquatic Environments. Appl. Environ. Microbiol. 72: 5982-5989 [Abstract] [Full Text]  
  • Pesaro, M., Widmer, F. (2006). Identification and Specific Detection of a Novel Pseudomonadaceae Cluster Associated with Soils from Winter Wheat Plots of a Long-Term Agricultural Field Experiment. Appl. Environ. Microbiol. 72: 37-43 [Abstract] [Full Text]  
  • Opelt, K., Berg, G. (2004). Diversity and Antagonistic Potential of Bacteria Associated with Bryophytes from Nutrient-Poor Habitats of the Baltic Sea Coast. Appl. Environ. Microbiol. 70: 6569-6579 [Abstract] [Full Text]  
  • Pesaro, M., Nicollier, G., Zeyer, J., Widmer, F. (2004). Impact of Soil Drying-Rewetting Stress on Microbial Communities and Activities and on Degradation of Two Crop Protection Products. Appl. Environ. Microbiol. 70: 2577-2587 [Abstract] [Full Text]  
  • Spilker, T., Coenye, T., Vandamme, P., LiPuma, J. J. (2004). PCR-Based Assay for Differentiation of Pseudomonas aeruginosa from Other Pseudomonas Species Recovered from Cystic Fibrosis Patients. J. Clin. Microbiol. 42: 2074-2079 [Abstract] [Full Text]  
  • Rediers, H., Vanderleyden, J., De Mot, R. (2004). Azotobacter vinelandii: a Pseudomonas in disguise?. Microbiology 150: 1117-1119 [Full Text]  
  • Cho, J.-C., Tiedje, J. M. (2000). Biogeography and Degree of Endemicity of Fluorescent Pseudomonas Strains in Soil. Appl. Environ. Microbiol. 66: 5448-5456 [Abstract] [Full Text]  
  • Martín, M., Mengs, G., Plaza, E., Garbi, C., Sánchez, M., Gibello, A., Gutierrez, F., Ferrer, E. (2000). Propachlor Removal by Pseudomonas Strain GCH1 in an Immobilized-Cell System. Appl. Environ. Microbiol. 66: 1190-1194 [Abstract] [Full Text]  
  • Widmer, F., Shaffer, B. T., Porteous, L. A., Seidler, R. J. (1999). Analysis of nifH Gene Pool Complexity in Soil and Litter at a Douglas Fir Forest Site in the Oregon Cascade Mountain Range. Appl. Environ. Microbiol. 65: 374-380 [Abstract] [Full Text]  
  • De Vos, D., Bouton, C., Sarniguet, A., De Vos, P., Vauterin, M., Cornelis, P. (1998). Sequence Diversity of the oprI Gene, Coding for Major Outer Membrane Lipoprotein I, among rRNA Group I Pseudomonads. J. Bacteriol. 180: 6551-6556 [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»