Advanced Environmental Biotechnology II

Nucleic acids from the environment

The analysis of nucleic acids extracted directly from environmental samples allows the researcher to investigate microbial communities by avoiding the limitations of cultivation techniques. Only a small proportion of bacteria can form colonies when traditional plating techniques are used.

it would be good to study nucleic acids directly from environmental samples. This would be representative of the microbial genomes in the samples. The analysis of DNA can give information on the structural diversity of environmental samples, or on the presence or absence of certain functional genes (e.g. genes giving xenobiotic biodegradative capabilities, antibiotic resistance or plasmid-borne sequences), or to monitor the fate of bacteria (including genetically modified organisms) released into an environment.

In general the analysis of DNA does not allow conclusions to be drawn on the metabolic activity of members of the bacterial or fungal community or on gene expression. This information might be obtained from analysis of RNA (rRNA or mRNA).

There are many different ways to get nucleic acids from environmental samples. There are two main approaches, each with their own advantages and limitations.

The first is based on direct or in situ lysis of microbial cells in the presence of the environmental matrix (e.g. soil or sediments), followed by separation of the nucleic acids from matrix components and cell debris. This method is by far the most used.

The advantage of the direct nucleic acid extraction approach is that it takes less time and that a much higher DNA yield is achieved. However, directly extracted DNA often contains considerable amounts of co-extracted substances such as humic acids that interfere with later molecular analysis. Also, a large proportion of directly extracted DNA might come from non-bacterial sources or from free DNA.

In the second approach, the microbial fraction is separated from the environmental matrix before cell lysis and then DNA extraction and purification.

The major concern with the so-called indirect or ex situ DNA extraction approach is differences in how easily surface-bound cells can be separated. Dissociation of cells from surfaces is usually done by repeated blending/homogenization steps and differential centrifugation. So the indirect method is more time-consuming and prone to contamination.

An advantage of the indirect approach is that the nucleic acids recovered are less contaminated with co-extracted humic acids and DNA of non-bacterial origin.

Researchers are developing faster and more efficient nucleic acid extraction procedures. The first protocols for both approaches all required CsCl/ethidium bromide density gradient ultracentrifugation to purify the DNA and were rather tedious and time-consuming. Using DNA purification kits based on different kinds of resins considerably improved the purification efficiency and reduced the time needed to obtain DNA suitable for molecular analysis.

However, none of the protocols was suitable for all soil types, in particular for soils and sediments coming from contaminated sites. Only recently have commercial kits for DNA extraction from soils become available. These simplify and miniaturize the method.

Commercial soil DNA extraction kits can be used to extract DNA directly from environmental samples and/or from the microbial pellet obtained after efficiently removing the microbes from the sample and then centrifuging.

Commercial kits for nucleic acid extraction are easy to use, but a number of things remain that influence the quantity and quality of nucleic acid extracts.

DNA extraction seems to work reliably for different samples, but efficient RNA extraction often has problems.

Recovery of cells from environmental samples.

The indirect DNA extraction approach might preferably be used when problematic environmental matrices are to be analyzed, or when cloning large DNA fragments [e.g. to generate bacterial artificial chromosome (BAC) libraries] from soil or sediment DNA where a high proportion of DNA of bacterial origin is crucial.

Different protocols aiming at the representative dislodgement and extraction of surface-attached cells have been published. All these methods have in common that they use repeated homogenization and differential centrifugation.

The protocols differ considerably with respect to the solutions used to break up soil colloids and dislodge surface-attached cells that adhere to surfaces by various bonding mechanisms such as polymers, electrostatic forces and water bridging, and that act to differing degrees.

Homogenization is usually achieved by shaking suspensions with gravel or blending in Stomacher or Waring blenders. In particular, for soils with a high clay content a further purification can be achieved by sucrose/Percoll density gradient centrifugation or flotation of the bacterial fraction on a Nycodenz cushion

Although a complete dislodgement of cells seems to be impossible, it is important that cells that are bound to the surface with different degrees of strength are released with similar efficiency. This can easily be evaluated by using DNA fingerprinting, e.g. denaturing gradient gel electrophoresis to analyze 16S or 18S rDNA fragment profiles amplified from the DNA extracted from the microbial pellet in comparison to profiles generated from directly extracted DNA.

Cell lysis and DNA extraction protocols The efficient disruption of the bacterial and fungal cell walls is crucial for the recovery of representative DNA which reflects the genomes of microbes present in an environmental sample and their relative abundance. Cell lysis can be achieved by mechanical cell disruption and/or by enzymatic or chemical disintegration of cell walls.

The efficiency of cell lysis protocols might differ considerably depending on the kind of environmental matrix, since compounds within the matrix might have adverse effects, e.g. reduced enzyme activity due to non-optimal pH or ionic conditions, or simply due to a high adsorption capacity. A number of studies compared the efficiency of lysis protocols with respect to DNA yield and fragmentation.

In most studies, bead beating was said to give the highest amounts of DNA, but the DNA produced was broken to some degree. Grinding in the presence of liquid nitrogen might be helpful. Bead beating is an important step before treatment with other cell lysis methods.

The efficiency of the cell lysis is usually estimated by microscopic examination. A correlation of lysis efficiency and clay content was demonstrated. Common lysis approaches such as bead beating and also three different extraction and purification protocols have been compared.

Comparison of four common lysis procedures on the yield and fragmentation of the DNA’Lane M: 1 kb ladder; lane 1: bead beating; lane 2: Retsch mill; lane 3: vortex; lane 4: freeze-boiling cycles (20 min at 63°C followed by 20 min at −20°C).

The use of commercial kits takes less time and avoids extraction with phenol and chloroform. The DNA extracted with the commercial kits less frequently contained PCR-inhibiting substances.

The use of small amounts of soil (0.25–0.5 g) for DNA extraction may be a serious limitation for the recovery of a sufficient quantity of DNA to be representative of that environment. The yield of DNA recovered per gram of soil depends on the lysis method and on the extraction protocol used. Tthe soil type strongly affected the quality (degree of shearing, PCR-inhibiting substances) and the quantity of the DNA.

Although the quantification of DNA based on agarose gels stained with DNA-staining dyes is more complicated than fluorimetric measurements, this approach also provides insights into the degree of DNA shearing.

High molecular weight DNA is an important criterion when evaluating and comparing different protocols because sheared DNA can cause PCR artifacts and is not suitable for direct cloning of large DNA fragments. DNA yields range from 5 to 250 µg g−1 of soil.

The complete removal of co-extracted humic acids is needed. Humic acids were shown to interfere with DNA hybridization, restriction enzyme digestions and PCR amplification. The amount of humic substances strongly depends on the soil type.

DNA extracted using commercial kits is often suitable for use in PCR amplifications without additional purification steps, except when applied to sandy soils. A range of methods has been used to remove co-extracted substances, e.g. silica-based or ion exchange resins.

Immunochemical isolation of DNA from metabolically active cellsUptake of [3H]thymidine has routinely been used for measuring the in situ growth of bacteria in different environments. Bromodeoxyuridine (BrdU) is structurally similar to thymidine, and since it can be incorporated into newly synthesized DNA it is widely used in medical research.

Recently, researchers have shown the potential use of BrdU to detect metabolically active bacteria in microbial communities from lake water, bacterioplankton or in soil. The procedure consists of four steps:

(i) incubation of environmental bacteria (soil or microbial fraction) with BrdU;(ii) extraction of DNA directly from the environmental sample or the microbial fraction;(iii) immunocapture of DNA containing incorporated BrdU by using magnetic beads covered with anti-BrdU antibody;(iv) immunoprecipitation.

The BrdU method was used to study soil bacterial communities responding to environmental stimuli such as the addition of glucose. Soils with or without glucose amendment were mixed with BrdU and incubated at room temperature.

A potential limitation of the BrdU method might be the proportion of bacteria that are capable of incorporating BrdU. The majority of bacteria are thought to take up and incorporate [3H] thymidine into DNA, but this has not been fully investigated for its analogue BrdU.

RNA extraction from environmental matricesMethods for RNA extraction have been less frequently used and are less well known. Due to the short half-life of bacterial messenger RNA as well as the high abundance and persistence of RNases, an unbiased recovery of total RNA is difficult. Considerable effort is required to ensure the absence of RNases.

Different protocols aimed at the simultaneous extraction of RNA and DNA have recently been published. As with DNA extractions, important criteria for the quality and suitability of RNA extraction protocols are the yield, the integrity, and the purity of the RNA.

Co-extracted humic substances and DNA have been reported to affect RNA hybridization results with oligonucleotide probes. Probe hybridization decreased as the concentration of DNA or humic substances increased.

Humic substances and/or high concentrations of co-extracted DNA were shown to result in membrane saturation and consequently reduced amounts of bound RNA. Even the presence of low concentrations of DNA seemed to cause a reduced accessibility of the rRNA target.

Another major concern is that some DNase preparations might contain residual RNase activity and cause partial degradation of rRNA molecules. rRNA is intact when distinct bands of the small and large subunit RNA can be seen after electrophoresis.

Agarose gel electrophoresis of nucleic acids recovered from bulk soil. Comparison of a classical extraction method and commercial kits for extraction and purification of soil DNA.