Environmental Molecular Microbiology: Protocols and Applications Chapter Abstracts
Chapter 1
Environmental Molecular Microbiology: Introduction
Paul A. Rochelle
The widespread use of molecular methods has resulted in a dramatic increase in our knowledge of the composition and physiology of microbial communities in the environment. This book is a collection of narrative texts and protocols describing many of these molecular methods and their application to environmental samples. Many of the methods focus on the detection and analysis of DNA, primarily through the use of the polymerase chain reaction (PCR). However, techniques for the analysis of RNA, proteins, and lipids are included, along with the use of DNA analogues such as peptide nucleic acid. The objective of the book is to provide an overview of the various methods and their applications as well as detailed laboratory protocols. Since the analysis of nucleic acids forms the cornerstone of environmental molecular microbiology, a review of the many procedures that have been developed for extracting DNA and RNA is provided. Many of the chapters present an objective description of some of the problems with the methods, including a detailed discussion of PCR biases, artifacts, and contamination. In addition to standard PCR, applications of quantitative PCR, sequence capture, conformation-based analysis, denaturing and thermal gradient gel techniques, gene expression methods, fatty acid profiling, proteomics, and in-situ techniques are all described. The book concludes with a discussion of innovative technologies that may represent the next wave of development for environmental molecular microbiology.
Chapter 2
Extraction of Nucleic Acids from Environmental Samples
Yu-Li Tsai and Paul A. Rochelle
No single technique has been developed which can be used to extract DNA or RNA from all types of environmental sample. In fact, extraction methods often seem to only work for one type of sample or in particular researchers hands. Consequently, many extraction methods have been published. This chapter reviews and compares many of these methods along with techniques used to remove inhibitory compounds. Example protocols for extraction of DNA and RNA from environmental samples are also provided.
Chapter 3
The Polymerase Chain Reaction as a Tool to Investigate Microbial Diversity in Environmental Samples
Philip Hugenholtz and Brett M. Goebel
Our knowledge of the types and distribution of microorganisms in the environment is rudimentary. Traditional enrichment techniques and the pure culture approach to microbiology have offered only a narrow portal into the microbial world. Over the last fifteen years, however, a wealth of novel biodiversity has been discovered through the application of molecular methodologies to microbial ecology. In particular, the cloning and sequencing of small subunit (16S/18S) ribosomal RNA genes (SSU rDNA) amplified directly from environmental samples using the polymerase chain reaction (PCR) has revealed significant, and often novel, biodiversity. PCR-cloning strategies have had an enormous impact on our understanding of microbial phylogeny, diversity and ecology. This chapter provides a critical assessment of the use of the PCR in obtaining SSU rDNA sequence information directly from environmental settings.
Chapter 4
Primer Design for PCR Amplification of Environmental DNA Targets
Julian R. Marchesi
The design and selection of primers is a critical aspect of any project involving PCR. Complete failure of amplification reactions or amplification of non-specific targets can frequently be attributed to poor primers. This chapter focuses on the basic rules of primer design with specific references to amplifying dehalogenase and 16S rRNA gene sequences. Generally, primers should be 16-24 nucleotides long with closely matched melting temperatures (less than 5°C difference) and have at least a 5 base match at the 3'end.
Chapter 5
Direct PCR Detection of Mycobacteria in the Environment using Sequence Capture-PCR
Timothy P. Stinear, Paul D. R. Johnson, and John K. Davies
Mycobacteria are a diverse group of microorganisms with a ubiquitous distribution in the environment. Some are human pathogens while others degrade environmental pollutants. Following recovery of microbial cells from water, sediment, and vegetation samples, DNA was extracted and mycobacterial DNA purified by sequence-capture using a biotin-labeled oligonucleotide targeting the 16S rRNA gene. The approach of sequence-capture is an efficient means of preparing DNA that is free of inhibitor compounds. The sensitivity of subsequent Mycobacterium-specific PCR was 100 to 1,000 cells.
Chapter 6
Protocols for Nucleic Acid-Based Detection and Quantitation of Microorganisms in Water
Knut Rudi and Kjetill S. Jakobsen
There is a demand for new and simplified analytical methods for studying microorganisms in natural aquatic environments. DNA-based methods have emerged as tools for sensitive and specific detection of microbes and the use of DNA or RNA is widely accepted as a means of identifying and distinguishing between different organisms. However, there are still methodological limitations to the application of nucleic acid-based techniques. These include inhibition of enzymatic reactions and the complexity and lack of reproducibility of some of the techniques. In this chapter, two novel approaches are presented that increase the reproducibility and simplicity of nucleic acid-based detection of microorganisms in water. The first part of the chapter is devoted to the description of the methods and protocols, while the second part includes examples of the application of the methods to detect cyanobacteria in environmental samples.
Chapter 7
Genomic Polymorphisms and the Diversity of Oxygenic Phototrophs
Brett A. Neilan
Rapid and sensitive methods for the detection and genetic characterization of cyanobacteria have been developed based on DNA amplification techniques. This chapter describes the molecular methods that have been used to characterize cyanobacteria and their use as tools to identify toxin-producing strains. Different species and strains were compared using restriction fragment length polymorphism (RFLP) of amplified fragments of the phycocyanin gene and the 16S-23S rRNA internal transcribed spacer.
Chapter 8
Detection of Protozoa in Environmental Water Samples
Paul A. Rochelle
The mission of the approximately 60,000 water utilities in the U.S. and the water industry worldwide is to provide safe and reliable drinking water. In general, this is accomplished with great success most of the time. A large water utility may produce 1 - 2 billion liters of drinking water every day and while the water that leaves the treatment plant is not sterile, it is free of any pathogens that could otherwise lead to diseases. However, on rare occasions outbreaks of disease within communities are caused by drinking water contaminated with viruses, bacteria, or protozoa. The focus of this chapter is the protozoa that most concern the water industry. Giardia duodenalis and Cryptosporidium parvum have been the causative agents of many waterborne disease outbreaks with hundreds of thousands of illnesses, and some deaths, attributed to their presence in drinking water. Both organisms have been the subjects of an intense research effort in the last 10 years. However, so-called emerging pathogens such as the microsporidia may play a more prominent role in the future. This chapter provides details on a variety of protozoa that may be found in environmental water sources. PCR primer sequences for the detection of the most common protozoal pathogens in water are provided along with a detailed discussion of the methods used to "fingerprint" isolates.
Chapter 9
Quantitative Polymerase Chain Reaction Assay
Clifford F. Brunk and Jinliang Li
This chapter describes the use of quantitative PCR for measuring bacterial abundance in environmental samples. The two approaches discussed are: 1) The use of an internal PCR standard constructed to be the same size and have the same sequence as the primary amplification target, but differing from the primary target by 2-3 bases, corresponding to a unique restriction site. This allows the amount of target amplicon to be compared with the internal standard and circumvents the problem of differential amplification efficiencies when using dissimilar targets and standard amplicons. 2) The use of Taqman technology (Applied Biosystems, Foster City, California) with a dual labeled oligonucleotide probe which binds internal to the PCR primers. The detection of Bacteroides is used as an example for both approaches.
Chapter 10
Microbial Community Proteomics
Oladele A. Ogunseitan, James LeBlanc, and E. Dalmasso
Proteomics is defined as an integrative analysis of proteins in biological systems, and it represents an innovative approach to the understanding of how genetic information is transformed into the diversity of function and form displayed in microbial communities. Protocols for extracting proteins from environmental samples are provided along with a discussion of methods available for protein analysis. Innovative and emerging technologies such as mass spectrometry coupled to 2-D polyacrylamide gel electrophoresis and high capacity chip technology are also discussed.
Chapter 11
A Combined Phospholipid and 16S-rDNA PCR-DGGE Analysis to Study Bioremediative Microbial Communities In Situ
Sarah J. Macnaughton and John R. Stephen
With contributions from: Yun-Juan Chang, Gregory A. Davis, George A. Kowalchuk, Ying-Dong Gan, Julia Brüggemann, and David C. White
The extraction, identification, and quantification of phospholipid fatty acids (PLFA) can provide valuable information on the total viable biomass in microbial communities. Information on the overall metabolic or physiological condition of a community can also be obtained. However, nucleic acid-based methods are necessary for fine-scale taxonomic study of microbial communities. Denaturing gradient gel electrophoresis (DGGE) following PCR amplification with primers targeting the small subunit rRNA gene provides a "fingerprint" of microbial communities. Subsequent band identification by DNA sequencing allows fine-scale characterization of specific bacterial members in a community. The combination of lipid analysis with PCR-fingerprinting can enumerate, report on the physiological status, and identify the major components of an actively bioremediating microbial community. This chapter provides protocols for these procedures as used in our laboratories, with much of this taking the form of "trouble shooting" tips. A case study is presented as an example of this combined approach to the study of microbial communities. Milk-whey was injected into pristine soil to simulate oxygen demand attendant to a planned release of BTEX/MTBE laden fuel. The microbial community structures of control soil and the "whey-barrier" are compared 40 days after treatment.
Chapter 12
Single-Strand Conformation Polymorphism (SSCP) for Microbial Community Analysis
Christoph C. Tebbe, Achim Schmalenberger, Sabine Peters, and Frank Schwieger
Single-strand conformation polymorphism (SSCP) is a technique to distinguish DNA molecules of the same size but of different nucleotide sequences using electrophoresis in a non-denaturing polyacrylamide gel. The method can be applied for the cultivation-independent analysis of microbial community diversity in environmental samples, based on PCR-amplified small sub-unit (SSU) rRNA gene sequences from directly extracted DNA. For this purpose, PCR is conducted with a phosphorylated and a non-phosphorylated primer both binding to conserved regions in the SSU rRNA genes. The double stranded PCR products are converted to single strands by lambda exonuclease digestion of the phosphorylated strand. By this means, heteroduplex formation during subsequent electrophoresis, which limited early applications of SSCP for microbial community analysis and which cannot be avoided in T/DGGE based methods, is eliminated. With PCR-SSCP, using this single-strand approach, community-patterns can be obtained from a diversity of environmental samples, such rhizosphere, compost or soil. The patterns in the polyacrylamide can be visualized by silver-staining. Single bands of silver-stained profiles can be cut out of the gels, re-amplified by PCR, and sequenced directly or after cloning in Escherichia coli. Due to its methodological simplicity, PCR-SSCP is a powerful tool for the analysis of microbial communities with a large pool of potential applications in microbial ecology and environmental biotechnology.
Chapter 13
Bacterial Community Profiling Using DGGE or TGGE Analysis
Holger Heuer, Gabriele Wieland, Jens Schönfeld, Annett Schönwälder, Newton C. M. Gomes, and Kornelia Smalla
Denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) are similar techniques that allow PCR products of the same length but of different sequence composition to be separated in gradient gels according to the melting behaviour of the DNA. DGGE involves separation of the double-stranded amplification products in a linearly increasing gradient of formamide and urea while TGGE achieves resolution with a linearly increasing temperature gradient. The techniques are ideal for analysing PCR products amplified from 16S rRNA genes in complex bacterial communities. D/TGGE analysis generates a community fingerprint, but since individual bands can be recovered and analysed following D/TGGE analysis, sequence information can also be obtained. This allows a thorough analysis of microbial communities on several levels ranging from the community structure of dominant populations using conserved sequence primers, to phylogenetic sequence analysis of single bands generated by individual community members. This chapter describes procedures and applications of D/TGGE using the analysis of 16S rDNA fingerprints from bacterial aquifer communities augmented with a strain of Pseudomonas stutzeri with specific attention paid to the V6 hyper-variable region of the 16S rRNA gene. Limitations of the techniques are also described.
Chapter 14
Differential Display of Prokaryotic Messenger RNA and Application to Soil Microbial Communities
James T. Fleming, Aaron Nagel, James Rice, and Gary S. Sayler
Differential display (DD) and RNA arbitrarily primed PCR (RAP-PCR) are similar techniques for amplifying mRNA for identification of differentially expressed genes. The methods differ in the types of primers used for reverse transcription and amplification. This chapter discusses the advantages and disadvantages of the techniques and provides detailed protocols for identification of differential gene expression in microbial communities in soil.
Chapter 15
Fluorescent PNA Probes for In Situ Bacterial Identification
Heather Perry-O'Keefe and Jens J. Hyldig-Nielsen
Peptide nucleic acid (PNA) is a class of synthetic molecules comprising a polyamide backbone with attached nucleobases. PNA probes bind to DNA targets via standard base-pairing but the physical properties of PNA give it many advantages compared to DNA probes. PNA probes are typically shorter than their DNA counterparts and hybridize to their targets very rapidly. Hybridization of PNA probes to DNA is not dependent on salt concentration and the neutral charged backbone makes PNA probes particularly suitable for in situ hybridization. This chapter describes the use of fluorescently labeled PNA probes for detection and identification of bacteria by in situ hybridization
Chapter 16
Nucleic Acid-Based In Situ Analyses of Microbial Diversity and Physiology
Bronwyn R. Robertson and Brett A. Neilan
Molecular microbial ecology is a relatively new field in environmental microbiology, but it is one in which the enormous potential to learn about bacteria at the single cell and community level is enticing to many researchers. The idea that, using nucleic acid-based technology, one could determine bacterial phylogenetics, genetic capabilities, and cellular activity has led to the rapid development of methodologies for such purposes. Even more exciting to the microbial ecologist is the fact that many of these techniques can be carried out on microbial communities in situ. Such investigations provide a far better view of which bacteria are present, what they are doing, and how they interact with one another, than has previously been possible. Such investigations are inherent to any valid ecological study, the observations of life as it occurs and interacts in nature. The following chapter reviews the development of nucleic acid-based in situ methods as they have been applied to studies in environmental microbiology over the last 10 years. We have attempted to follow the progression and refinement of methods in a somewhat chronological manner, beginning with rRNA targeted in situ hybridization, which made it possible to identify bacteria without the need to culture, through to in situ reverse transcriptase PCR technology, whereby the expression of single-copy genes in individual cells can be monitored in natural communities.
Chapter 17
Technology Innovations in Biodetection Systems for Environmental Molecular Microbiology
Darrell P. Chandler
To overcome many of the limitations associated with indirect detection methods, new techniques for the sensitive, specific, and direct detection of nucleic acids are required in order to accurately and quantitatively ascribe phenotype/function to uncultivated microorganisms. However, if advanced diagnostic and detection systems are going to be applied in environmental microbiology, future "biodetection" technologies and systems must be developed not from the point of view of the detector, but from the unique aspects of the environmental sample and the entire analytical process. This chapter highlights recent advances in nucleic acid-based technologies, and look towards future advances that may address the broad needs and conditions imposed by environmental molecular microbiology.
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