Extraction and Analysis of Microbial Community Nucleic Acids from Environmental Matrices

Jan Dirk van Elsas, Kornelia Smalla, Christoph C. Tebbe 3.1. Introduction

Environmental monitoring on the basis of nucleic acids is increasingly being recognized as an extremely powerful approach, since organisms or genes can be directly assessed, even without prior cultivation. Hence, targets present in unculturable, poorly-culturable or as yet uncultured organisms, which would escape detection when using traditional cultivation-based approaches, are assessable. In the last decade, a number of developments, including the rapid development of nucleic acid detection methodologies, have provided a great thrust to the efforts to assess microbes directly in their natural environment. Areas of study that have been significantly stimulated by nucleic acid based approaches are:

1. Monitoring indigenous microorganisms of interest from an ecological or biotech-nological perspective,

2. The study of natural microbial diversity and of factors that disturb that diversity,

3. The need to monitor genetically modified microorganisms (GMMs) based on their unique nucleic acid sequences,

4. The assessment of the expression cf specific microbial genes in the environment.

The environmental matrices to be sampled and extracted may range from a variety of bulk soils, plant roots (rhizospheres and/or rhizoplanes), leaves (phyllosphere) or interior plant tissue, seeds, rockwool and manure to diverse aquatic systems such as sediments and sewages. A large and diverse suite of protocols for the extraction and analysis of nucleic acids from these environments has been collected in recent texts,1,2 and the reader is referred to these for detailed information. The basic principles of these protocols are inherently very similar, as in most cases the nucleic acids are to be obtained from mixed microbial communities that occur adsorbed to, and heterogeneously dispersed in, the matrix under study. In addition, there are often compelling reasons, e.g., when the persistence of a specific target gene has to be monitored, to also analyze the fractions of the total nucleic acid pool that occur extracellularly.3

There are two different approaches to the extraction of nucleic acids from mixed microbial communities in environmental matrices. The first approach is based on the p-ior separation of microbial cells from the matrix, after which the dislodged cells are lysed and the nucleic acids extracted and further purified (Cell extraction / nucleic acid extraction, or

Tracking Genetically-Engineered Microorganisms, edited by Janet K. Jansson, Jan Dirk van Elsas, Mark J. Bailey. ©2000 EUREKAH.COM.

indirect, approach4,5). The second approach is based on the direct lysis cf microbial cells in the environmental matrix (e.g., soil), followed by separation of the nucleic acids from the matrix and from cell debris and other impurities (Direct lysis approach6). Major differences between the two strategies are the higher nucleic acid yields obtained with the direct lysis approach, coupled to the co-extraction of larger amounts of contaminating substances7. Moreover, the cell extraction/nucleic acid extraction approach in principle allows the removal of a large fraction of the extracellular nucleic acids from the bacterial cells prior to lysis, thus allowing a better assessment of target DNA present inside the micobial cells in the environment.

Since these pioneering studies on DNA extraction from soil,4-7 there has been a considerable methodological development, the main purpose of which was the omission o laborious purification steps, mainly hydroxyapatite column chromatography and cesium chloride (CsCl) gradient purifications, replacing these by faster approaches. Thus, the numerous nucleic acid extraction protocols that are currently in use in different laboratories1-2,8-19 all share a relatively small number of individual extraction and purification steps (Table 3.1).

Table 3.1. Some examples of frequently used steps in nucleic acid extraction and purification protocols

Purpose

Step

Principle

Remarks

Cell lysis

Enzymes and detergents

Breakdown and solubilization of cell envelope components

Very diverse requirements of different cells (e.g., grampositives versus gramnegatives.

Freeze/thaw (heat and cold shocks)

Temperature shocks combined with water crystals, to destabilize membranes

Less efficient for lysis of grampositive bacteria

Grinding with liquid N2

Abrasive action of grinding with soil combined with ice crystals

Efficient for fungal spores and mycelium

Bead beating using small glass beads and high frequency shaking

Mechanical lysis (brute force)

Recognized as highly efficient in lysing a wide range of bacterial and fungal/yeast cells

Microwave oven

Heat induced lysis

Combined with other lysis methods

Sonication

High energy induced lysis

Often not efficient if energy-dissipating materials are present

Extraction and Analysis of Nucleic Acids

31

Purpose Step

Principle

Remarks

Extraction Phenol and precipitation

Denaturing and extractive action on proteins and lipoproteins

Standard method in molecular biology

Chloroform

Denaturing action on proteins and hydrophobes

Standard method in molecular biology

Ethanol or isopropanol precipitations

Removal of salts and solutes

Standard method in molecular biology

Polyethylene glycol (PEG) precipitation

Precipitation/concentration of DNA

Purification CsCl precipitation

Removal of impurities by salting-out effect

KAc/NHUCl precipitations

Precipitation of DNA due to high molarity acetate

Glassmilk sorption

Sorption on glassmilk beads, followed by differential desorption

Highly efficient in removing humic compounds from DNA

Elutip D

Chromatography separation

Method repeated on same extract

Wizard DNA cleanup spin columns

Powerful separation of DNA from humics via chromatography over mini spin column

Sephadex G50/G75/G200

Separation of contaminants, e.g., humics, via gel filtration

Efficient fast method, often combined with other methods

Gel electrophoresis

Charge- and size-related separation of nucleic acids from impurities

Very efficient, but laborious method

PVPP* sorption

Selectively removes humics from DNA/RNA solutions

PVPP needs rigorous acid wash

Hydroxy apatite chromatography

Selective binding of nucleic acids to HAP**, column. Differential elution of DNA or RNA by varying phosphate concentrations

Theoretically very good, but practically difficult. Often low recoveries

* PVPP: polyvinyl poly pyrrolidine;** HAP: hydroxyapatite

* PVPP: polyvinyl poly pyrrolidine;** HAP: hydroxyapatite

Fig. 3.1. Outline of nucleic acid based detection protocols

Fig. 3.1. Outline of nucleic acid based detection protocols

There have even been efforts to simplify and miniaturize soil DNA extraction protocols to a level where their on site use becomes feasible.20 Most of the current protocols have been shown to produce DNA and/or RNA suitable for the analysis of microbial diversity or microbial fate. However, they often differ in the way they release and lyse microbial cells from the environmental matrix, and such (qualitative and quantitative) differences are likely to affect the final analyses performed. Hence, it is key to our understanding of microorganisms in their natural settings as described by molecular methods, that the possible biases introduced by cell extraction and lysis methods are understood.

This chapter will review currently available strategies to recover and purify (1) microbial (bacterial) cells and (2) nucleic acids (DNA and RNA) from environmental matrices, and will then briefly address the use of these nucleic acids in monitoring methods to assess microbial diversity and inoculant fate. Figure 3.1 gives a general outline of these methodologies.

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