Molecular Microbial Ecology Manual

Frans J. de Bruijn received his Ph.D. (Cellular and Developmental Biology; Microbial Genetics) from Harvard University in 1983. His resume reflects an array of experiences as a teacher, researcher, board member and a plethora of other accomplishments. He is currently Director of Research at the Laboratory for Plant-Microbe Interactions in Toulouse, France.

For a long time microbial ecology has been developed as a distinct field with in Ecology. In spite of the important role of microorganisms in the environ ment, this group of 'invisible' organisms remained unaccessable to other ecologists. Detection and identification of microorganisms remain largely dependent on isolation techniques and characterisation of pure cultures. We now realise that only a minor fraction of the microbial community can be cultivated. As a result of the introduction of molecular methods, microbes can now be detected and identified at the DNA/RNA level in their natural environment. This has opened a new field in ecology: Molecular Microbial Ecology. In the present manual we aim to introduce the microbial ecologist to a selected number of current molecular techniques that are relevant in micro bial ecology. The first edition of the manual contains 33 chapters and an equal number of additional chapters will be added this year. Since the field of molecular ecology is in a continuous progress, we aim to update and extend the Manual regularly and will invite anyone to deposit their new protocols in full detail in the next edition of this Manual.

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SECTION 1: Isolation of microbial nucleic acids.- 1. 1. Extraction of microbial DNA from aquatic sources.- l.1.2. Extraction of microbial DNA from aquatic sources: Freshwater.- 1.1.3. Extraction of microbial DNA from sewage and manure slurries.- 1.1.4. Methods for extracting DNA from microbial mats and cultivated micro-organisms: high molecular weight DNA from French press lysis.- 1.2. Extraction of microhial RNA from aquatic sources.- 1.2.3. Methods for extracting RNA or ribosomes from microbial mats and cultivated microorganisms.- 1.3. Extraction of microhial DNA from hulk soil.- l.3.1. Cell extraction method.- 1.3.3. Extraction of microbial community DNA from soils.- l.3.4. Small scale extraction of DNA from soil with spun column cleanup.- 1.3.5. Gel purification of soil DNA extracts.- 1.4. Extraction of DNA from phytosphere, rhizosphere, rhizoplane.- 1.4.2. Extraction and PCR amplification of DNA from the rhizoplane.- 1.4.3. Extraction of microbial DNA from the phylloplane.- 1.5. Extraction of RNA from bulk soil.- 1.5.1. Direct and simultaneous extraction of DNA and RNA from soil.- 1.6. Nucleic acid extraction from cultures.- 1.6.2. Extraction of ribosomal RNA from microbial cultures.- SECTION 2: Detection of microbial nucleic acid sequences.- 2.2. Preparation of radioactive probes.- 2.2.1. Preparation of radioactive probes.- 2.3. Preparation of non-radioactive probes.- 2.3.1. Detection of nucleic acids by chemiluminescence.- 2.6. Detection of microbial DNA sequences by colony hybridization.- 2.6.1. Detection of microbial DNA sequences by colony hybridization.- 2.7. Detection and quantification of microbial nucleic acid seque Polymerase Chain Reaction (PCR).- 2.7.2. Polymerase chain reaction (peR) analysis of soil microbial DNA.- 2.7.5. Detection of mRNA and rRNA via reverse transcription and peR in soil.- SECTION 3: Identification and classification of microbes usmg DNA and RNA sequences.- 3.1. Partial and complete 16S rDNA sequences, their use in generation of 16S rDN A phylogenetic trees and their implications in molecular ecological studies.- 3.1.1. Partial and complete 16S rONA sequences, their use in generation of 16S rONA phylogenetic trees and their implications in molecular ecological studies.- 3.3. Microbial identification and design of phylogenetic trees hased.- 3.3.1. Amplification of ribosomal RNA sequences.- 3.3.2 Bacterial community fingerprinting of amplified 16S and 16-23S ribosomal DNA gene sequences and restriction endonuclease analysis (ARDRA).- 3.3.4. Investigation of fungal phylogeny on the basis of small ribosomal subunit RNA sequences.- 3.3.5. Sequence Databases.- 3.3.6. In situ identification of micro-organisms by whole cell hybridization with rRNA-targeted nucleic acid probes.- SECTION 4: Detection, identification and classification of microbes usingother methods.- 4.1. Detection, identification and classification of microbes using othermethods.- 4.1.3. Immunofluorescence colony-staining (IFC).- 4.1.8. Fluorescent staining of microbes for total direct counts.- 4.1.9. Slide immunoenzymatic assay (SIA).- SECTION 5: Detection of gene transfer in the environment.- 5.1. Gene transfer by transformation.- 5.1.1. Natural transformation in aquatic environments.- 5.2. Gene tran:,fer by conjugation.- 5.2.3. Detection of gene transfer in the environment: Conjugation in soil.- 5.3. Gene transfer by transduction.- 5.3.2. Phage ecology and genetic exchange in soil.- SECTION 6: Tracking of specific microbes in the environment.- 6.1. Marker genes.- 6.1. 7. Heavy metal resistances in microbial ecosystems.- 6.1.8. Biodegradation genes as marker genes in microbial ecosystems.- 6.2. Designing field and microcosm experiments with GEM's.- 6.2.1. Design of microcosms to provide data reflectin field trials of GEM S.- 6.2.3. Designing release experiments with GEM's in foods: Risk assessment of the use of genetically modified Lactococcus lactis strains in fermented milk products; a case study.