Genetic approaches for characterizing soil microbial communities




Rosalind Becker

Introduction

Microorganisms play a fundamental role in the transformation of energy and matter. For this reason, knowledge of microbial diversity is essential to understanding how ecosystems function (Torsvik et al. 1996, van der Heijden et al. 2008). However, genetic approaches for studying soil microbial communities come with a unique set of challenges. In this study two different chemical kits were used to extract genomic DNA from forest soil samples. A methodology for amplifying short DNA sequences from three phyla of fungi and prokaryotes was developed. In order to overcome the challenges associated with sequencing, a DNA library was made to isolate and identify the diversity of fungi amplified by one primer set. Finally, the relative extraction efficiencies of the two different soil DNA extraction kits were examined.

Materials and Methods

Soil DNA was extracted by using either the SoilMaster™ DNA extraction kit by EPICENTRE® Biotechnologies or the PowerSoil® DNA Isolation Kit by Mo Bio Laboratories. The SoilMaster™ DNA extract was also purified using the PowerClean® Soil DNA Clean-Up Kit by Mo Bio Laboratories. Short segments of the ribosomal RNA gene region were PCR amplified using four primer sets to target three phyla of fungi and prokaryotes. The PCR product that produced the strongest band was purified, and the individual amplicons were separated by cloning. One hundred randomly-selected clones were sequenced and compared to the Nucleotide Collection database of GenBank for identification. Radiolabeled PCR products of the two extraction kits, both applied to the same soil sample and amplified with the same set of primers, were run side-by-side on a sequencing gel that separates DNA segments that differ in size by one base. The results were visualized on photographic film to determine the relative extraction efficiencies of the two kits.

Results

PCR could not successfully amplify DNA in the SoilMaster™ DNA extract, nor could it amplify positive control DNA that was diluted in the extract (Fig.1). Even after applying the PowerClean® Soil DNA Clean-Up procedure to the SoilMaster™ DNA extract, only two primer sets could successfully amplify the soil DNA. The PowerSoil® DNA Isolation Kit produced an extract that could be amplified by all four primer sets (Fig.2). Due to the diversity of organisms present in the soil extract, Sanger sequencing could not produce a readable sequence for a single PCR product. Only when the individual amplicons of the PCR product were cloned to form a DNA library and the clones were sequenced independently could Sanger sequencing give a picture of the richness and abundance of soil microorganisms (Fig.3). A total of eleven organisms comprised 83% of the Basidiomycota community present in that soil sample. When the two soil DNA extracts were compared side-by-side on a sequencing gel, it was clear from the banding pattern that the two kits differ in their ability to extract DNA from certain microorganisms (Fig.4).

Discussion

This study demonstrated that soil DNA extraction kits, particularly the SoilMaster™ kit, may leave behind contaminants that inhibit PCR. These inhibitors were so potent that they effectively inhibited PCR at concentrations as low as 1%. Even after applying a clean-up methodology to the SoilMaster™ extract, PCR was still inhibited at higher concentrations of template. When soil DNA is successfully amplified, it is difficult to sequence because it represents many different organisms. When soil PCR products are sequenced by the Sanger method, multiple bases are detected at every step, which prevents the identification of one readable sequence. To overcome this problem, the individual pieces need to be separated from one another by ligation and transformation into cells, creating a DNA library. However, this picture of richness and abundance could be influenced by the kit that was used to extract the DNA. The side-by-side comparison of the two extraction kits on the sequencing gel revealed profound differences in the extraction efficiencies of the two kits. If this variability affects all chemical extraction kits, chemical extraction alone could introduce bias into research by selecting for a particular set of organisms.

Literature Cited

Torsvik, V., R. Sorheim, and J. Goksoyr. 1996. Total bacterial diversity in soil and sediment communitites - a review. Journal of Industrial Microbiology 17:170-178.
van der Heijden, M. G. A., Richard D. Bardgett, and Nico M. van Straalen. 2008. The unseen majority: soil microbes as drivers of plant diversity and productivity in terrestrial ecosystems. Ecology Letters 11:296-310.

Figures


Figure 1-PCR results from SoilMaster™ DNA extract using one set of primers. In this reaction, the positive control was used as template in all wells. The positive control was run in varying dilutions as indicated by the amounts above each well, in either the soil DNA extract or in TE buffer.


Figure 2-PCR results from DNA extracted from soil using the PowerSoil® kit. DNA was successfully amplified with all four primer sets. Large bands and wells with multiple bands indicate the diversity of organisms picked up by each primer set.


Figure 3-The dominant basidiomycetes observed identified from genomic DNA extracted from a soil sample from the New York Botanical Garden. Eleven distinct organisms comprise 83% of the soil microbial community.


Figure 4-Sequencing gel containing PCR products from the SoilMaster™ kit and the PowerSoil® kit (in duplicate). Each band is a different sized piece of the rRNA gene region that was amplified by the primer set. Each band represents a unique organism. The blue arrow indicates a band that is the same for both kits. The red arrows indicate differences in the banding pattern between the two kits.



Abstract

Soil microbial diversity is critically important to ecosystem functioning, but it can be very challenging to study. Microbial genomic DNA was extracted from forest soil samples using two chemical kits. From these extracts, the rRNA gene regions of prokaryotes and three types of fungi were amplified using four sets of primers. PCR was completely inhibited by the extract of one kit. Even after a clean-up procedure was applied, the remaining inhibitors still hindered PCR in several reactions. A second extraction kit produced a relatively clean extract, from which DNA was amplified without clean-up. These kits also varied dramatically in their ability to extract DNA from different organisms in the soil. The many unique sequences from a single PCR product were cloned in a DNA library. This library revealed that eleven unique organisms comprise the majority of the microbial community in the one soil sample. This technique circumvented the problems associated with using Sanger sequencing to read a diversity sequences in one PCR product.

Full Paper

Acknowledgments

This work was supported by the Department of Biological Sciences at Fordham University. Special thanks must be extended to Dr. Rubin for his guidance and uncanny knack for problem solving. Thank you to Alex Bulanov and Xie Xie for their remarkable patience and tireless efforts to help with this project. Thank you to Jessica Arcate Schuler at the New York Botanical Garden for the last-minute soil sample. An extra special thank you to the students of Techniques in Cell and Molecular Biology, past and present, for their advice and support.


This document was last modified 05/19/2011.
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