Optimizing the indirect extraction of prokaryotic DNA from soils

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Abstract

The objective of this work was to develop protocols to selectively extract prokaryotic DNA from soils, representative of the whole community, amenable to high-throughput whole genome shotgun sequencing. Prokaryotic cells were extracted from soils by blending, followed by gradient centrifugation. Detergent (sodium deoxycholate) was required for complete dispersion of soil aggregates and detachment of prokaryotic cells from a broad range of soil types. Repeated extractions of a given soil sample were critical to maximize cell yield. Furthermore, cells obtained through repeated extractions captured unique prokaryotic assemblages that would otherwise have been missed in single-pass extractions. DNA was isolated from extracted cells using one of the following treatments: i) lysozyme–SDS–proteinase K (enzymatic) digestion; ii) potassium ethyl xanthogenate digestion; or iii) enzymatic digestion of cells embedded in agarose plugs. In addition, these methods were compared to a commercial bead-beating extraction kit (MoBio UltraClean). Of the indirect DNA extraction methods, plug digestion generated the largest yields (up to 70% of yields obtained by direct DNA extraction) of high-molecular weight DNA (>400 kb). Thus, plug digestion is amenable to large-insert metagenomic library construction and analysis. Comparisons of banding patterns generated by RAPD-PCR and DGGE indicated that sequence composition and inferred community composition of a given extract varied greatly with DNA isolation method. While overall diversity did not change significantly with the cell lysis method, analysis of 16S rRNA gene clone libraries revealed that each extraction procedure produced unique distributions of prokaryotic phyla within the sample population.

Introduction

Soils likely represent the greatest reservoir of biodiversity on the planet. Prokaryotic diversity in soils has been estimated to be three orders of magnitude greater than in all other ecosystems combined (Curtis et al., 2002, Kemp and Aller, 2004). In terms of function, soils and their microbial inhabitants are critical to global biogeochemical cycles including carbon, nitrogen, and phosphorus, which support all other forms of terrestrial diversity. Because of their importance on multiple levels, soils have been the subject of studies in microbial ecology for decades [e.g., (Borneman and Triplett, 1997, Cavigelli et al., 1995, Nunan et al., 2003, Skinner et al., 1952, Skyring and Quadling, 1969, Steffan et al., 1988, Waksman and Woodruff, 1940)]. However, the spatial and temporal heterogeneity of soils, and the complexity of soil chemical and biological characteristics that give rise to such genetic and functional diversity among soil microbiota, also make soils one of the most challenging natural environments for studies of microbial ecology.

The advent of DNA-based techniques, such as PCR amplification of the 16S ribosomal RNA gene (Lane, 1991), has granted new views of prokaryotic diversity by circumventing the requirement of cultivation. Efficient extraction of target DNA is the crucial first step in any DNA-based analysis of soil microbes. While many specific methods for the isolation of prokaryotic DNA from soils have been published [for a review, see (Robe et al., 2003)], the canon of available protocols may be reduced to two general approaches: direct and indirect DNA extraction. Direct extraction involves the in situ lysis of cells followed by extraction and purification of DNA. By contrast, indirect extraction procedures initially separate prokaryotic cells from the soil matrix; cells are then lysed ex situ and the DNA purified. Studies comparing the yield and quality of DNAs extracted from soils by various direct and indirect methods (Krsek and Wellington, 1999, Maron et al., 2006, Roh et al., 2006, Tien et al., 1999) have demonstrated that each extraction approach has specific advantages and disadvantages in terms of DNA yield, purity, and sampling biases, which must be considered in light of the particular experimental goals (Frostegard et al., 1999).

Direct DNA extraction is appropriate if the objective is to characterize the taxonomic diversity of the soil prokaryotic community (Ashby et al., 2007, Fierer et al., 2007, LaMontagne et al., 2003) or the sequence diversity of specific gene sets (Jensen et al., 2000, Tolli and King, 2005, Verhagen et al., 1995). Direct extractions typically provide enough DNA of sufficient quality (i.e., free of inhibitory contaminants) and nominal fragment size that the target gene(s) can be quickly amplified from the resulting mixture of templates. However, different approaches are warranted if the goal is to assess functional diversity, or to establish connections between taxonomy and function.

Several recent publications have revealed the utility of metagenomic libraries in identifying potential connections between taxonomy and function within specific clones (Beja et al., 2000, Beja et al., 2002, Liles et al., 2003, Quaiser et al., 2002). Construction of metagenomic libraries from soil prokaryotic assemblages requires robust methods for the extraction and purification of high-molecular weight prokaryotic DNA. These criteria exclude direct DNA extraction techniques because: 1) DNA released through in situ lysis may bind to clays or organic matter (Frostegard et al., 1999), severely limiting their recovery; 2) DNA from direct extractions necessarily includes eukaryotic (Courtois et al., 2001, Frostegard et al., 1999) and extracellular DNAs (Frostegard et al., 1999, Pietramellara et al., 2009), inflating the perceived DNA yield without necessarily providing additional information on the prokaryotic fraction (which has, ostensibly, been the subject of interest for the majority of molecular microbial ecology studies). Furthermore, eukaryotic genomes are far larger than those of prokaryotes, thus, without some type of screening (such as 16S rRNA PCR) the DNA from eukaryotic cells in total soil DNA extractions can significantly reduce the number of prokaryotic sequences within a metagenome; 3) DNA fragments obtained through direct extractions are rarely larger than 20 kb in size (Krsek and Wellington, 1999, Robe et al., 2003), placing a fundamental limit on the establishment of linkages between taxonomy and function.

By contrast, indirect lysis approaches are required for the construction of metagenomic libraries of soil prokaryotic DNA, because indirect extraction provides for the recovery of large, contiguous DNA fragments (Berry et al., 2003, Bertrand et al., 2005). Indirect lysis approaches therefore facilitate the functional analysis of soil prokaryotic assemblages, and also enable connections to be made between taxonomy and function within contiguous clone inserts. Furthermore, DNAs in such extracts are almost exclusively from prokaryotic sources (Courtois et al., 2001). Major disadvantages associated with indirect DNA extraction are the reduction in DNA yield and the potential decrease in sampling efficiency (i.e., the extent to which the phylogenetic diversity of the sample population represents the diversity of the whole community) relative to direct extraction approaches. The bacterial fraction obtained by single-pass cell extraction has been reported at 25–50% of the total community (Robe et al., 2003). Thus, estimates based on single-pass extractions do not account for potential increases in cell recovery resulting from repeated sequential cell extractions. Furthermore, no data are available regarding improvements in the representation of the phylogenetic diversity of cells through repeated extractions.

In this study, we developed methods for obtaining high-molecular weight (≥400 kb), high-purity, prokaryotic DNA from soils, amenable to the construction of metagenomic libraries. The impacts of repeated sequential extractions on cell recovery and sampling efficiency were also evaluated. After optimization of cell extraction, five cell lysis procedures were used to obtain prokaryotic DNA. The main objective of this work was to evaluate potential taxonomic biases imparted by specific lysis procedures. Evaluations were performed using 16S rRNA PCR-DGGE, as well as through comparisons of sequence data obtained from 16S rRNA gene clone libraries.

Section snippets

Soils

Composite ∼500 g soil samples were collected from the A horizons (0–10 cm) of five different soil types (Table 1). An unclassified agricultural clay loam (AGC) was obtained from a no-till farm field planted to wheat in Sykesville, MD; Glenelg silt loam (GSL) was obtained from a forested site in Rock Creek Regional Park, Rockville, MD; CAL loamy sand was obtained from an eucalyptus forest in La Jolla, CA; BWR loamy sand was collected from a field planted to tomatoes at the Be Wise Ranch,

Recovery of prokaryotic cells from soil

In comparisons based on single-pass extractions, 0.1% sodium deoxycholate (Na d-Ch) and 0.1 M sodium phosphate with 0.005% SDS (Na Phos) provided statistically significant (P < 0.05) increases in cell extraction by microscopic direct counts relative to other extraction buffers for four of the five soils tested (Fig. S1B–E). The exception was AGC soil, for which no significant differences were detected in microscopic direct counts across any of the extraction buffers. For AGC, GSL, CAL, and BWR

Recovery of prokaryotic cells from soil

The efficient extraction of prokaryotic assemblages from soils requires the optimization of three basic parameters: chemical dispersion, physical dispersion, and cell recovery. Recent literature appears to be in agreement that physical dispersion is best achieved through mechanical blending (Courtois et al., 2001, Lindahl, 1996, Maron et al., 2006), since shaking and inversion result in reduced cell yields (Hopkins et al., 1991, Lindahl and Bakken, 1995), and more aggressive treatments such as

Conclusions

Mild detergents are essential for complete dispersion of soil aggregates and detachment of prokaryotic cells from soil surfaces. Sodium deoxycholate proved effective for a broad range of soil types in this study, including loams, loamy sands, and highly organic soils. Repeated extractions of a given soil sample are critical in order to maximize cell yield. Single-pass extractions account for about 50–70% of recoverable cells, while second and third extractions may capture the remaining 30–50%.

Acknowledgements

This research was supported by the Office of Science (BER), U.S. Department of Energy, Cooperative Agreement No. De-FC02-02ER63453 and by the J. Craig Venter Institute.

We thank Dr. John Glass and Cynthia Andrews-Pfannkoch for their assistance with soil collection, and C. Andrews-Pfannkoch for providing helpful discussion and resources with regard to specific cell lysis methods. Special thanks are due to Be Wise Ranch, Escondido, CA for soils used in this study, and to Dr. Rebekah R. Helton for

References (72)

  • R.M. MacDonald

    Sampling soil microfloras: dispersion of soil by ion exchange and extraction of specific microorganisms from suspension by elutriation

    Soil Biol. Biochem.

    (1986)
  • P.A. Maron et al.

    Evaluation of quantitative and qualitative recovery of bacterial communities from different soil types by density gradient centrifugation

    Eur. J. Soil Biol.

    (2006)
  • G. Muyzer

    DGGE/TGGE a method for identifying genes from natural ecosystems

    Curr. Opin. Microbiol.

    (1999)
  • N. Nunan et al.

    Spatial distribution of bacterial communities and their relationships with the micro-architecture of soil

    FEMS Microbiol. Ecol.

    (2003)
  • M.H. Rahman et al.

    Physical, chemical and microbiological properties of an Andisol as related to land use and tillage practice

    Soil Till. Res.

    (2008)
  • P. Robe et al.

    Extraction of DNA from soil

    Eur. J. Soil Biol.

    (2003)
  • M.R. Rondon et al.

    The earth’s bounty: assessing and accessing soil microbial diversity

    Trends Biotechnol.

    (1999)
  • C. Verhagen et al.

    Bacterial dichloropropene degradation in soil; screening of soils and involvement of plasmids carrying the dhlA gene

    Soil Biol. Biochem.

    (1995)
  • M.N. Ashby et al.

    Serial analysis of rRNA genes and the unexpected dominance of rare members of microbial communities

    Appl. Environ. Microbiol.

    (2007)
  • S.M. Barns et al.

    Wide distribution and diversity of members of the bacterial kingdom Acidobacterium in the environment

    Appl. Environ. Microbiol.

    (1999)
  • O. Beja et al.

    Bacterial rhodopsin: evidence for a new type of phototrophy in the sea

    Science

    (2000)
  • O. Beja et al.

    Comparative genomic analysis of archaeal genotypic variants in a single population and in two different oceanic provinces

    Appl. Environ. Microbiol.

    (2002)
  • J. Borneman et al.

    Molecular microbial diversity of an agricultural soil in Wisconsin

    Appl. Environ. Microbiol.

    (1996)
  • J. Borneman et al.

    Molecular microbial diversity in soils from eastern Amazonia: evidence for unusual microorganisms and microbial population shifts associated with deforestation

    Appl. Environ. Microbiol.

    (1997)
  • M.D. Burr et al.

    Denaturing gradient gel electrophoresis can rapidly display the bacterial diversity contained in 16S rDNA clone libraries

    Microb. Ecol.

    (2006)
  • C. Carrigg et al.

    DNA extraction method affects microbial community profiles from soils and sediment

    Appl. Microbiol. Biotechnol.

    (2007)
  • M.A. Cavigelli et al.

    Fatty-acid methyl-ester (Fame) profiles as measures of soil microbial community structure

    Plant Soil

    (1995)
  • J.R. Cole et al.

    The Ribosomal Database Project (RDP-II): sequences and tools for high-throughput rRNA analysis

    Nucleic Acids Res.

    (Jan 1, 2005)
  • S. Courtois et al.

    Quantification of bacterial subgroups in soil: comparison of DNA extracted directly from soil or from cells previously released by density gradient centrifugation

    Environ. Microbiol.

    (2001)
  • L.D. Crosby et al.

    Understanding bias in microbial community analysis techniques due to rrn operon copy number heterogeneity

    BioTechniques

    (2003)
  • T.P. Curtis et al.

    Estimating prokaryotic diversity and its limits

    Proc. Natl. Acad. Sci. U.S.A.

    (2002)
  • N. Fierer et al.

    Metagenomic and small-subunit rRNA analyses reveal the genetic diversity of bacteria, archaea, fungi, and viruses in soil

    Appl. Environ. Microbiol.

    (2007)
  • A. Frostegard et al.

    Quantification of bias related to the extraction of DNA directly from soils

    Appl. Environ. Microbiol.

    (1999)
  • Ø. Hammer et al.

    PAST: paleontological statistics software package for education and data analysis

    Paleontol. Electron.

    (2001)
  • Cited by (0)

    View full text