This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Skovhus, T. L. Articles by Dahllöf, I. Search for Related Content PubMed PubMed Citation Articles by Skovhus, T. L. Articles by Dahllöf, I. Agricola Articles by Skovhus, T. L. Articles by Dahllöf, I.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, April 2004, p. 2373-2382, Vol. 70, No. 4
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.4.2373-2382.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Real-Time Quantitative PCR for Assessment of Abundance of Pseudoalteromonas Species in Marine Samples

Department of Microbial Ecology, University of Aarhus, Aarhus,¹ Department of Marine Ecology, National Environmental Research Institute, Roskilde, Denmark,³ School of Biotechnology and Biomolecular Sciences and Centre of Marine Biofouling and Bio-Innovation, University of New South Wales, Sydney, New South Wales, Australia²

Received 16 September 2003/ Accepted 15 December 2003

A real-time quantitative PCR (RTQ-PCR) method for measuring the abundance of Pseudoalteromonas species in marine samples is presented. PCR primers targeting a Pseudoalteromonas-specific region of the 16S rRNA gene were tested at three different levels using database searches (in silico), a selection of pure cultures (in vitro), and a combined denaturing gradient gel electrophoresis and cloning approach on environmental DNA (in situ). The RTQ-PCR method allowed for the detection of SYBR Green fluorescence from double-stranded DNA over a linear range spanning six orders of magnitude. The detection limit was determined as 1.4 fg of target DNA (1,000 gene copies) measured in the presence of 20 ng of nontarget DNA from salmon testes. In this study, we discuss the importance of robust post-PCR analyses to overcome pitfalls in RTQ-PCR when samples from different complex marine habitats are analyzed and compared on a nonroutine basis. Representatives of the genus Pseudoalteromonas were detected in samples from all investigated habitats, suggesting a widespread distribution of this genus across many marine habitats (e.g., seawater, rocks, macroalgae, and marine animals). Three sample types were analyzed by RTQ-PCR to determine the relative abundance of Pseudoalteromonas ribosomal DNA (rDNA) compared to the total abundance of eubacterial rDNA. The rDNA fractions of Pseudoalteromonas compared to all Eubacteria were 1.55% on the green alga Ulva lactuca, 0.10% on the tunicate Ciona intestinalis, and 0.06% on the green alga Ulvaria fusca.

This article has been cited by other articles:

• Rao, D., Webb, J. S., Holmstrom, C., Case, R., Low, A., Steinberg, P., Kjelleberg, S. (2007). Low Densities of Epiphytic Bacteria from the Marine Alga Ulva australis Inhibit Settlement of Fouling Organisms. Appl. Environ. Microbiol. 73: 7844-7852 [Abstract] [Full Text]  
• D'Imperio, S., Lehr, C. R., Breary, M., McDermott, T. R. (2007). Autecology of an Arsenite Chemolithotroph: Sulfide Constraints on Function and Distribution in a Geothermal Spring. Appl. Environ. Microbiol. 73: 7067-7074 [Abstract] [Full Text]  
• Mai-Prochnow, A., Webb, J. S., Ferrari, B. C., Kjelleberg, S. (2006). Ecological Advantages of Autolysis during the Development and Dispersal of Pseudoalteromonas tunicata Biofilms. Appl. Environ. Microbiol. 72: 5414-5420 [Abstract] [Full Text]  
• Ando, A., Suzuki, C., Shima, J. (2005). Survival of Genetically Modified and Self-Cloned Strains of Commercial Baker's Yeast in Simulated Natural Environments: Environmental Risk Assessment. Appl. Environ. Microbiol. 71: 7075-7082 [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]  
• Patrauchan, M. A., Sarkisova, S., Sauer, K., Franklin, M. J. (2005). Calcium influences cellular and extracellular product formation during biofilm-associated growth of a marine Pseudoalteromonas sp.. Microbiology 151: 2885-2897 [Abstract] [Full Text]  
• Mai-Prochnow, A., Evans, F., Dalisay-Saludes, D., Stelzer, S., Egan, S., James, S., Webb, J. S., Kjelleberg, S. (2004). Biofilm Development and Cell Death in the Marine Bacterium Pseudoalteromonas tunicata. Appl. Environ. Microbiol. 70: 3232-3238 [Abstract] [Full Text]