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The ideal starting point for investigating, developing, and implementing stable isotope technologies. • Guides researchers through basic, tested, and proven protocols including DNA, RNA, protein, and phospholipid fatty acid (PLFA) SIP, from concept and history through detailed methodology, troubleshooting, and interpretation to optimal and future uses. • Explores important and emerging applications of SIP in environmental microbiology, ranging from bioremediation and gene mining to carbon tracking and gut microflora function. • Examines explorations of further elegant isotope labeling technologies such as Raman-FISH, NanoSIMS, and isotope arrays. • Serves as a valuable resource for environmental microbiology students and researchers and genomics, biotechnology, and medical microbiology professionals.
This book provides definitive methods to perform stable isotope probing (SIP) experiments, covering a wide spectrum of stable isotope techniques used in microbial ecology, such as methods to target and analyze labeled DNA, rRNA, mRNA, protein, and PLFA. Protocols to study stable isotope fractionation by microbial pathways, the analysis of labeled communities with Raman microscopy, Chip-SIM, as well as quantitative SIP (qSIP) and high-resolution SIP (HR-SIP) are also featured. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Stable Isotope Probing: Methods and Protocols provides readers with up-to-date protocols ranging from basic to the most sophisticated applications of SIP and will benefit anyone pursuing this exciting area of study.
Vents and seeps are the epitome of life in extreme environments, but there is much more to these systems than just black smokers or hydrocarbon seeps. Many other ecosystems are characterized by moving fluids and this book provides an overview of the different habitats, their specific conditions as well as the technical challenges that have to be met when studying them. The book provides the current state of the art and will be a valuable resource for everybody that has an interest in such environments.
Marine methane seep habitats represent an important control on the global flux of methane between the subsurface and water column reservoirs. Meta-omics studies have begun to outline community-wide metabolic potential, but expression patterns of proteins that enact sulfate-mediated anaerobic methane oxidation in seeps are poorly characterized. Proteomic stable isotope probing (proteomic SIP) offers an additional layer of information for characterizing phylogenetically specific, functionally relevant activity in mixed microbial communities. Here we applied proteomic SIP to 15NH4+ and CH4 amended seep sediment microcosms in an attempt to track the protein synthesis of slow-growing, low-energy microbial systems. Across all samples, 3495 proteins were identified, 21% of which were 15N-labeled. We observed active synthesis (15N enrichment) of all proteins believed to be involved in sulfate reduction and reverse methanogenesis including methylenetetrahydromethanopterin reductase (Mer). The abundance and phylogenetic range of methyl-coenzyme M reductase (Mcr) orthologs produced during incubation experiments suggests that seeps provide sufficient niches for multiple organisms performing analogous metabolisms. Twenty-eight previously unreported post-translational modifications of McrA were measured, indicating dynamic enzymatic machinery and offering a dimension of functional diversity beyond gene-dictated sequence. RNA polymerase associated with putative sulfur-oxidizing Epsilonproteobacteria and aerobic Gammaproteobacteria were more abundant among pre-incubation proteins, suggesting diminished metabolic activity in long-term anoxic, sulfidic experimental incubations. Twenty-six proteins of unknown function were detected in all proteomic experiments and actively expressed in labeled experiments, suggesting that they play important roles in methane seep ecosystems. The addition of stable isotope probing to environmental proteomics experiments provides a mechanism to begin to assess the degree to which diagnostic meatbolic proteins are long-lived or acively synthesized in complex, slow-growing microbial communities. Our work here demonstrates that sediment-hosted microbial assemblages in marine methane seeps are dynamic, heterogeneous systems with broad functional diversity.
The true extent of prokaryote diversity, encompassing the spectrum of variability among bacteria, remains unknown. Current research efforts focus on understanding why prokaryote diversification occurs, its underlying mechanisms, and its likely impact. The dynamic nature of the prokaryotic world, and continuing advances in the technological tools available make this an important area and hence this book will appeal to a wide variety of microbiologists. Its coverage ranges from studies of prokaryotes in specialized environmental niches to broad examinations of prokaryote evolution and diversity, and the mechanisms underlying them. Topics include: bacteria of the gastrointestinal tract, unculturable organisms in the mouth and in the soil, organisms from extreme environments, the diversity of archaea and their phages, comparative genomics and the emergence of pathogens, the spread of genomic islands between clinical and environmental organisms, minimal genomes needed for life, horizontal gene transfer, phenotypic innovation, and patterns and extent of biodiversity.
"...a number of chapters provide excellent summaries of the modern methods available for studying fungal ecology, along with those more traditional methods that are still extremely valuable...overall it is a hugely valuable compendium of fungal ecology research. It is a must for the library shelf." -Lynne Boddy, Cardiff University, UK, Mycological Research, 2006 "These 44 chapters are an excellent starting point for anyone interested in fungal communities, in the broadest sense of the term. It is a book for dipping into...may be the last comprehensive treatment of fungal communities before the molecular revolution." -Meriel Jones, University of Liverpool, UK, Microbiology Today "... the scope of the work is tremendous. ... Excellent chapters providing overviews of methods ... provide a snap shot of the current approaches used to understand fungal communities at several levels of organization. This book should probably be on the shelf of every student of mycology, and many ecologists too. For all students, this book should be a valuable resource and source of inspiration." -Daniel Henk, Imperial College Faculty of Medicine, London, in Inoculum, Vol. 59, No. 3, May 2008 "Thorough taxonomic and subject indices further aid the reader in navigating through multiple authors’ treatments of subjects of interest." - Anthony Amend, Department of Botany, University of Hawaii at Manoa in Economic Botany, V. 61 In all subjects in science, new findings and the use of new technologies allow us to develop an ever-greater understanding of our world. Expanded and updated coverage in the fourth edition includes: Adds new sections on Integrating Genomics and Metagenomics into Community Analysis, Recent Advances in Fungal Endophyte Research, Fungi in the Built Environment, and Fungal Signaling and Communication Includes a broader treatment of fungal communities in natural ecosystems with in-depth coverage of fungal adaptations to stress and conservation Expands coverage of the influence of climate change on fungi and the role of fungi in organically polluted ecosystems Includes contributions from scientists from 20 nations to illustrate a true global approach for bridging gaps between ecological concepts and mycology
Stabe isotope probe (SIP) incubation studies were performed using 13C-labeled carbon substrates on hyperthermophilic filamentous streamer communities inhabiting Octopus Spring in Yellowstone National Park. Biomass was removed from the outflow stream and incubated at near-in situ conditions with labeled bicarbonate, formate, or acetate. Lipids from the biomass were extracted and analyzed using gas chromatography-mass spectroscopy (GC-MS) and gas chromatography-isotope ratio monitoring-mass spectroscopy(GC-IRMS). We observed incorporation of 13C-labeled acetate into the total biomass, archaeal lipids, and a small number of bacterial lipids, but no incorporation of labeled formate or bicarbonate. During 67 hours of incubation, 0.060 [mu]g of labeled acetate was incorporated by the archaeal and bacterial community. The lack of acetate incorporation by most bacteria, or formate and bicarbonate incorporation by any community member may reflect rates of carbon turnover, the carbon acquisition pathway used, or inhibition under experimental conditions,