Evaluation of the environmental specificity of fluorescence in situ hybridization (FISH) using fluorescence-activated cell sorting (FACS) of probe (PSE1284)-positive cells extracted from rhizosphere soil

Gougoulias, C. and Shaw, L. J. (2012) Evaluation of the environmental specificity of fluorescence in situ hybridization (FISH) using fluorescence-activated cell sorting (FACS) of probe (PSE1284)-positive cells extracted from rhizosphere soil. Systematic and Applied Microbiology, 35 (8). pp. 533-540. ISSN 0723-2020 doi: 10.1016/j.syapm.2011.11.009 (Special Issue: Fluorescence In Situ Hybridization (FISH))

Abstract/Summary

We explicitly tested for the first time the ‘environmental specificity’ of traditional 16S rRNAtargeted fluorescence in situ hybridization (FISH) through comparison of the bacterial diversity actually targeted in the environment with the diversity that should be exactly targeted (i.e. without mismatches) according to in silico analysis. To do this, we exploited advances in modern Flow Cytometry that enabled improved detection and therefore sorting of sub-micron-sized particles and used probe PSE1284 (designed to target Pseudomonads) applied to Lolium perenne rhizosphere soil as our test system. The 6-carboxyfluorescein (6-FAM)-PSE1284-hybridised population, defined as displaying enhanced green fluorescence in Flow Cytometry, represented 3.51±1.28% of the total detected population when corrected using a nonsense (NON-EUB338) probe control. Analysis of 16S rRNA gene libraries constructed from Fluorescence Activated Cell Sorted (FACS) -recovered fluorescent populations (n=3), revealed that 98.5% (Pseudomonas spp. comprised 68.7% and Burkholderia spp. 29.8%) of the total sorted population was specifically targeted as evidenced by the homology of the 16S rRNA sequences to the probe sequence. In silico evaluation of probe PSE1284 with the use of RDP-10 probeMatch justified the existence of Burkholderia spp. among the sorted cells. The lack of novelty in Pseudomonas spp. sequences uncovered was notable, probably reflecting the well-studied nature of this functionally important genus. To judge the diversity recorded within the FACS-sorted population, rarefaction and DGGE analysis were used to evaluate, respectively, the proportion of Pseudomonas diversity uncovered by the sequencing effort and the representativeness of the Nycodenz® method for the extraction of bacterial cells from soil.

Item Type	Article
URI	https://reading-clone.eprints-hosting.org/id/eprint/24954
Item Type	Article
Refereed	Yes
Divisions	Science > School of Archaeology, Geography and Environmental Science > Department of Geography and Environmental Science Science > School of Archaeology, Geography and Environmental Science > Earth Systems Science Interdisciplinary centres and themes > Soil Research Centre
Publisher	Elsevier
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