Molecular Microbial Ecology

Mark Osborn, Cindy Smith, Mark Osborn, Cindy Smith

• Publisher: CRC Press
• Year: 2004
• eBook ISBN: 9781135320409
• Edition: 1
• Topic: Biological Sciences
• Subtopic: Biology
• Index: Biological Sciences

Microoganisms are distributed across every ecosystem, and microbial transformations are fundamental to the operation of the biosphere. Microbial ecology is the study of this interaction between microorganisms and their environment, and arguably represents one of the most important areas of biological research. Yet for many years our study of microbial flora was severely limited: the primary method of culturing microorganisms on media allowed us to study only between 0.1 and 10% of the total microbial flora in any given environment.

Molecular Microbial Ecology gives a comprehensive guide to the recent revolution in the study of microorganisms in the environment. Details are given on molecular methods for isolating some of the previously uncultured and numerically dominant microbial groups. PCR-based approaches to studying prokaryotic systematics are described, including ribosomal RNA analysis and stable isotope probing. Later chapters cover DNA hybridisation techniques (including fluorescent in situ hybridisation), as well as genomic and metagenomic approaches to microbial ecology. Gathering together some of the world's leading experts, this book provides an invaluable introduction to the modern theory and molecular methods used in studying microbial ecology.

1
Nucleic acid extraction from environmental samples

Annett Milling, Newton C. M.Gomes, Miruna Oros-Sichler, Monika Götz and Kornelia Smalla

1.1 Introduction

Over 20 years ago, the first protocol titled ‘DNA extraction from soil’ was published by Torsvik (1). However, only in the late 1980s/early 1990s, when molecular tools such as nucleic acid hybridization, the polymerase chain reaction (PCR) and DNA cloning and sequencing became increasingly available, more attention was focused on the analysis of DNA extracted from environmental bacteria without prior cultivation. Obviously, the analysis of nucleic acids extracted directly from environmental samples allows the researcher to investigate microbial communities by obviating the limitations of cultivation techniques. The phenomenon that only a small proportion of bacteria can form colonies when tradi-tional plating techniques are used (2) was first described by Staley and Konopka (3) as the great plate anomaly. A further limitation of the cultivation-based studies of microbial communities is that under environmental stress bacteria can enter a state termed ‘viable but non-culturable’ (vbnc), and again these bacteria would not be accessible to traditional cultivation techniques (4, 5). Consequently researchers were attracted by the opportunities afforded by analyzing nucleic acids recovered directly from environmental samples that should be representative of the microbial genomes present in such samples. The analysis of DNA can provide information on the structural diversity of environmental samples, or on the presence or absence of certain functional genes (e.g. genes conferring xenobiotic biodegradative capabilities, antibiotic resistance or plasmid-borne sequences), or to moni-tor the fate of bacteria (including genetically modified organisms) released into an environment. However, 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) (see also Chapter 5).

A large and diverse suite of protocols has been published on nucleic acid extraction from environmental matrices (for review see 6). Two principal approaches exist, each with their own advantages and limitations. The first approach pioneered by Ogram et al. (7) 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 is by far the most frequently utilized method. The advantage of the direct nucleic acid extraction approach is that it is less time-consuming and that a much higher DNA yield is achieved. However, directly extracted DNA often contains consider-able amounts of co-extracted substances such as humic acids that interfere with subsequent molecular analysis (8). Furthermore, a considerable proportion of directly extracted DNA might originate from non-bacterial sources or from free DNA.

In the second approach, the microbial fraction is recovered from the environmental matrix prior to cell lysis and subsequent DNA extraction and purification. The major concern with the so-called indirect or ex situ DNA extraction approach is a differential recovery efficiency of surface-bound cells. Dissociation of cells from surfaces is generally achieved by repeated blending/homogenization steps and differential centrifugation. Thus the indirect method is more time-consuming and prone to contamination. A clear 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. During the last 10 years considerable progress has been made in developing faster and more efficient nucleic acid extraction procedures. While the first protocols for both approaches all required CsCl/ ethidium bromide density gradient ultracentrifugation to purify the DNA and thus were rather tedious and time-consuming (7, 9, 10), the use of 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 (8, 11, 12). However, none of the protocols was suitable for all soil types, in particular for soils and sediments originat-ing from contaminated sites. Only recently have commercial kits for DNA extraction from soils become available and these represent a major breakthrough in view of the simplifica-tion and miniaturization of this crucial method for many cultivation-independent analysis methods. Commercial soil DNA extraction kits can be used to extract DNA in the presence of the environmental matrix and/or from the microbial pellet obtained after efficient dislodgement and centrifugation. Despite the current ease of using commercial kits for nucleic acid extraction, a number of critical factors remain that influence the quantity and quality of nucleic acid extracts, and will be discussed later in this chapter. Whilst DNA extraction seems to work reliably for different matrices, efficient RNA extraction is often still problematic. Thus this chapter will also focus on protocols that enable assessment of the metabolically active microbial fraction within an environmental sample. Furthermore, we demonstrate the utility of 16S/18S rDNA-based molecular fingerprints for comparing different protocols and for illustrating how different methodologies affect the composition of the microbial community recovered.

1.2 Recovery of cells from environmental matrices

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; see Chapter 11] 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 (9, 13–16). All these methods have in common that they use repeated homogenization and differential centrifugation as originally suggested by Faegri et al. (17). However, 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 (e.g. exopolysaccharides or fimbriae), 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 (14). 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 (see Chapter 3), 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.

1.3 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. Most of the published protocols include a combination of these steps. 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. Whereas, in most studies, bead beating was reported to yield the highest amounts of DNA, albeit the DNA produced showed some degree of shearing, some authors favored grinding in the presence of liquid nitrogen (18–20). According to our experience, bead beating is important as a first step before treatment with other cell lysis methods. The length of time and intensity of bead beating, the size of the beads as well as the ratio of beads to soil suspension, and also the content of clay minerals in the soil matrix have all been reported to influence the degree of DNA shearing (12, 21–24). The efficiency of the cell lysis is usually estimated by microscopic examination (18–19, 21, 24). A correlation of lysis effi-ciency and clay content was demonstrated by Zhou et al. (18). We have compared common lysis approaches such as bead beating (cell homogenizer, Braun-Melsungen), Retsch mill, freeze-boiling, grinding in the presence of liquid nitrogen, vortexing (M. Oros-Sichler et al., unpublished data) and also three different extraction and purification protocols (see Figure 1.1). While the quantity and degree of nucleic acid fragmentation was assessed by agarose gel electrophoresis, the influence of the different protocols on the bacterial and fungal diversity was evaluated by denaturing gradient gel electrophoresis (DGGE) analysis of 16S/18S rDNA fragments amplified from the different kinds of DNA. In contrast to our traditional DNA extraction protocol (12, 25), the use of commercial kits is clearly less time-consuming and avoids extraction with phenol and chloroform. In addition, the DNA extracted with the commercial kits less frequently contained PCR-inhibiting substances. A disadvantage may be that the use of rather small amounts of soil (0.25–0.5 g) for DNA extraction, from some environmental matrices, may represent a serious limitation for the recovery of a sufficient quantity of DNA to be representative of that environment (26). Like other authors (27) we found that the yield of DNA recovered per gram of soil depends on the lysis method and on the extraction protocol used. In addition, the 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 whe...

Table of contents

• Cover Page
• Title Page
• Copyright Page
• Contributors
• Abbreviations
• Preface
• 1. Nucleic Acid Extraction from Environmental Samples
• Protocol 1.1: DNA Extraction from Bulk Soil
• Protocol 1.2: BrdU Immunocapture
• Protocol 1.3: Simultaneous DNA/RNA Extraction
• 2. Prokaryotic Systematics: PCR and Sequence Analysis of Amplified 16S rRNA Genes
• Protocol 2.1
• 3. DNA Fingerprinting of Microbial Communities
• Protocol 3.1
• 4. Molecular Typing of Environmental Isolates
• Protocol 4.1: AFLP Analysis
• Protocol 4.2: PCR-RFLP Analysis
• 5. RT-PCR and mRNA Expression Analysis of Functional Genes
• Protocol 5.1: RT-PCR
• Protocol 5.2: RAP-PCR
• 6. Quantitative Real-Rime PCR
• Protocol 6.1
• 7. Stable-Isotope Probing
• Protocol 7.1
• 8. Applications of Nucleic Acid Hybridization In Microbial Ecology
• Protocols
• 9. Fluorescence In Situ Hybridization for the Detection of Prokaryotes
• Protocol 9.1
• 10. Lessons from the Genomes: Microbial Ecology and Genomics
• 11. Metagenomic Libraries from Uncultured Microorganisms
• Protocol 11.1
• 12. A Molecular Toolbox for Bacterial Ecologists: PCR Primers for Functional Gene Analysis
• 13. Molecular Detection of Fungal Communities In Soil
• Protocol 13.1: DGGE Analysis of Ectomycorrhizal Communities In Soil
• 14. Environmental Assessment: Bioreporter Systems
• Case Study
• 15. Bioinformatics and Web Resources for the Microbial Ecologist