Denaturing Gradient Gel Electrophoresis
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Soil Microbial Forensics
- Authors: Tasha M. Santiago-Rodriguez, Raúl J. Cano
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Citation: Santiago-Rodriguez T, Cano R. 2016. Soil microbial forensics. 4(4): doi:10.1128/microbiolspec.EMF-0007-2015
- DOI 10.1128/microbiolspec.EMF-0007-2015
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Abstract:
Soil microbial forensics can be defined as the study of how microorganisms can be applied to forensic investigations. The field of soil microbial forensics is of increasing interest and applies techniques commonly used in diverse disciplines in order to identify microbes and determine their abundances, complexities, and interactions with soil and surrounding objects. Emerging new techniques are also providing insights into the complexity of microbes in soil. Soil may harbor unique microbes that may reflect specific physical and chemical characteristics indicating site specificity. While applications of some of these techniques in the field of soil microbial forensics are still in early stages, we are still gaining insight into how microorganisms may be more robustly used in forensic investigations.
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The Role of Vibrios in Diseases of Corals
- Author: Colin B. Munn
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Citation: Munn C. 2015. The role of vibrios in diseases of corals. 3(4): doi:10.1128/microbiolspec.VE-0006-2014
- DOI 10.1128/microbiolspec.VE-0006-2014
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Abstract:
The tissue, skeleton, and secreted mucus of corals supports a highly dynamic and diverse community of microbes, which play a major role in the health status of corals such as the provision of essential nutrients or the metabolism of waste products. However, members of the Vibrio genus are prominent as causative agents of disease in corals. The aim of this chapter is to review our understanding of the spectrum of disease effects displayed by coral-associated vibrios, with a particular emphasis on the few species where detailed studies of pathogenicity have been conducted. The role of Vibrio shilonii in seasonal bleaching of Oculina patagonica and the development of the coral probiotic hypothesis is reviewed, pointing to unanswered questions about this phenomenon. Detailed consideration is given to studies of V. coralliilyticus and related pathogens and changes in the dominance of vibrios associated with coral bleaching. Other Vibrio-associated disease syndromes discussed include yellow band/blotch disease and tissue necrosis in temperate gorgonian corals. The review includes analysis of the role of enzymes, resistance to oxidative stress, and quorum sensing in virulence of coral-associated vibrios. The review concludes that we should probably regard most—possibly all—vibrios as “opportunistic” pathogens which, under certain environmental conditions, are capable of overwhelming the defense mechanisms of appropriate hosts, leading to rapid growth and tissue destruction.
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Biodiversity of the Surface Microbial Consortia from Limburger, Reblochon, Livarot, Tilsit, and Gubbeen Cheeses
- Authors: Timothy M. Cogan, Stefanie Goerges, Roberto Gelsomino, Sandra Larpin, Markus Hohenegger, Nagamani Bora, Emmanuel Jamet, Mary C. Rea, Jérôme Mounier, Marc Vancanneyt, Micheline Guéguen, Nathalie Desmasures, Jean Swings, Mike Goodfellow, Alan C. Ward, Hans Sebastiani, Françoise Irlinger, Jean-Francois Chamba, Ruediger Beduhn, Siegfried Scherer
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Citation: Cogan T, Goerges S, Gelsomino R, Larpin S, Hohenegger M, Bora N, Jamet E, Rea M, Mounier J, Vancanneyt M, Guéguen M, Desmasures N, Swings J, Goodfellow M, Ward A, Sebastiani H, Irlinger F, Chamba J, Beduhn R, Scherer S. 2014. Biodiversity of the surface microbial consortia from limburger, reblochon, livarot, tilsit, and gubbeen cheeses. 2(1): doi:10.1128/microbiolspec.CM-0010-2012
- DOI 10.1128/microbiolspec.CM-0010-2012
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Abstract:
Comprehensive collaborative studies from our laboratories reveal the extensive biodiversity of the microflora of the surfaces of smear-ripened cheeses. Two thousand five hundred ninety-seven strains of bacteria and 2,446 strains of yeasts from the surface of the smear-ripened cheeses Limburger, Reblochon, Livarot, Tilsit, and Gubbeen, isolated at three or four times during ripening, were identified; 55 species of bacteria and 30 species of yeast were found. The microfloras of the five cheeses showed many similarities but also many differences and interbatch variation. Very few of the commercial smear microorganisms, deliberately inoculated onto the cheese surface, were reisolated and then mainly from the initial stages of ripening, implying that smear cheese production units must have an adventitious “house” flora. Limburger cheese had the simplest microflora, containing two yeasts, Debaryomyces hansenii and Geotrichum candidum, and two bacteria, Arthrobacter arilaitensis and Brevibacterium aurantiacum. The microflora of Livarot was the most complicated, comprising 10 yeasts and 38 bacteria, including many gram-negative organisms. Reblochon also had a very diverse microflora containing 8 yeasts and 13 bacteria (excluding gram-negative organisms which were not identified), while Gubbeen had 7 yeasts and 18 bacteria and Tilsit had 5 yeasts and 9 bacteria. D. hansenii was by far the dominant yeast, followed in order by G. candidum, Candida catenulata, and Kluyveromyces lactis. B. aurantiacum was the dominant bacterium and was found in every batch of the 5 cheeses. The next most common bacteria, in order, were Staphylococcus saprophyticus, A. arilaitensis, Corynebacterium casei, Corynebacterium variabile, and Microbacterium gubbeenense. S. saprophyticus was mainly found in Gubbeen, and A. arilaitensis was found in all cheeses but not in every batch. C. casei was found in most batches of Reblochon, Livarot, Tilsit, and Gubbeen. C. variabile was found in all batches of Gubbeen and Reblochon but in only one batch of Tilsit and in no batch of Limburger or Livarot. Other bacteria were isolated in low numbers from each of the cheeses, suggesting that each of the 5 cheeses has a unique microflora. In Gubbeen cheese, several different strains of the dominant bacteria were present, as determined by pulsed-field gel electrophoresis, and many of the less common bacteria were present as single clones. The culture-independent method, denaturing gradient gel electrophoresis, resulted in identification of several bacteria which were not found by the culture-dependent (isolation and rep-PCR identification) method. It was thus a useful complementary technique to identify other bacteria in the cheeses. The gross composition, the rate of increase in pH, and the indices of proteolysis were different in most of the cheeses.
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From Pasteur to Probiotics: A Historical Overview of Cheese and Microbes
- Author: Catherine W. Donnelly
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Source: Cheese and Microbes , pp 1-15
Publication Date :
January 2014
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Abstract:
Nowhere in the microbial world are microorganisms on more magnificent display than on the surfaces or in the interiors of the great cheeses of the world. Cheesemaking is inextricably linked to microbiology, which makes the study of cheeses, their history, and the vast science of cheese and microbes particularly fascinating. Over the past two decades, there has been explosive growth in the U.S. artisan cheese industry. The availability of artisan cheeses, made using traditional practices, has ignited renewed consumer interest in cheesemaking and cheese consumption. This affords a tremendous opportunity to educate a new population of students, scientists, cheesemakers, technologists, and cheese connoisseurs about the essential role which microorganisms play in the process of cheesemaking.
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Biodiversity of the Surface Microbial Consortia from Limburger, Reblochon, Livarot, Tilsit, and Gubbeen Cheeses
- Authors: Timothy M. Cogan, Stefanie Goerges, Roberto Gelsomino, Sandra Larpin, Markus Hohenegger, Nagamani Bora, Emmanuel Jamet, Mary C. Rea, Jérôme Mounier, Marc Vancanneyt, Micheline Guéguen, Nathalie Desmasures, Jean Swings, Mike Goodfellow, Alan C. Ward, Hans Sebastiani, Françoise Irlinger, Jean-François Chamba, Ruediger Beduhn, Siegfried Scherer
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Source: Cheese and Microbes , pp 219-250
Publication Date :
January 2014
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Abstract:
About 145,000 metric tons of smear-ripened cheeses are made in Europe, mainly in France, Belgium, Austria, and Germany. Although relatively small in quantity compared with total European cheese production of almost 9 million metric tons, this amount is important because of the large number of varieties of smear cheeses made, each with its own distinctive flavor, e.g., Port Salut, Pont-l’Évêque, Munster, Vacherin Mont-d'Or, and Taleggio. Smear cheeses are characterized by the development of microbial consortia on the cheese surface early in ripening, composed of yeast and bacteria, which give some of the cheeses (the so-called red smear cheeses) their characteristic red or orange glistening color. This is a direct result of ripening the cheeses at relatively high temperatures (∼14°C) and high relative humidities (>95%), conditions which promote the growth of microorganisms present on the cheese surface. The red or orange color which develops on the surface of many smear cheeses during ripening is due to the production of pigments by the yeast and bacteria growing on the surface. The biochemical activity of the yeast and bacteria are mainly responsible for flavor development in these cheeses. Like every cheese made, smear cheeses also contain high numbers of starter and nonstarter lactic acid bacteria (LAB), but these are found throughout the cheese and are not considered further here.
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INDEX
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Source: Cheese and Microbes
Publication Date :
January 2014
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No descriptions available.
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Bacterial Diversity in Polar Habitats
- Authors: Bronwyn M. Kirby, Samantha Easton, I. Marla Tuffin, Don A. Cowan
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Source: Polar Microbiology: Life in a Deep Freeze , pp 3-31
Publication Date :
January 2012
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Abstract:
This chapter talks about the development of culture-independent, molecular methods that have revolutionized the field and the understanding of molecular ecology. Through the use of these techniques, it is now apparent that the earlier culture-based studies were not a representative reflection of the dominant microorganisms in many psychrophilic habitats. Cyanobacteria present in Dry Valleys mineral soils are considered to be the major primary producers and contribute significantly to microbial diversity. Lithic communities are classified by the specific environmental niche they reside in, and hypoliths, chasmoliths, and cryptoendoliths are further discussed in this chapter. The majority of bacteria isolated from permafrost are aerobic and include a number of coryneforms, endospore formers, sulfate reducers, nitrifying and denitrifying bacteria, and cellulose degraders. The microbial mat bacterial diversity of 10 Dry Valleys lakes was assessed by culturing techniques (heterotrophic growth conditions and fatty acid analysis). Microbial mats from Markham and Ward Hunt Ice Shelves showed species homogeneity in the vertical profile, which has not been seen previously in Antarctic mats, possibly due to differences in mat thickness. The stratified Antarctic mats from the McMurdo Ice Shelf were up to 8 cm thick in places, while the Arctic mats in this study were approximately 2 cm. Using metagenomic methods researchers can assess the diversity of culturable and uncultured organisms, including rare taxa.
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Archaea
- Authors: Thomas D. Niederberger, Ian R. McDonald, S. Craig Cary
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Source: Polar Microbiology: Life in a Deep Freeze , pp 32-61
Publication Date :
January 2012
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Abstract:
Modern molecular PCR-based methods, typically targeting the 16S rRNA gene, have now revolutionized the field of environmental microbiology and have allowed culture-independent surveys of natural in situ microbial communities. These new approaches have unearthed a wide diversity and ubiquitous presence of Archaea in nonextreme environments such as soils, sediments, and oceans. The archaeal domain is split into two major phyla, the Crenarchaeota and Euryarchaeota. In spite of the extreme environmental conditions in the polar regions, through the application of culture-independent 16S rRNA gene-based surveys, Archaea have been found to inhabit a wide range of polar environments. This chapter reviews the current literature describing archaeal presence and diversity in polar and subpolar habitats. Marine and terrestrial ecosystems are discussed individually for Antarctic and Arctic ecosystems, with final sections discussing comparative studies of archaeal communities between polar regions, the potential response and contribution of Archaea to future climate change models, highlights of recent findings, and future research needs. The first wide-ranging PCR-based survey of archaeal 16S rRNA genes in terrestrial Antarctic sites has recently been reported. 16S rRNA gene clone library-based methods were used to analyze archaeal communities from Kirkpatrickia varialosa, Latrunculia apicalis, Mycale acerata, Homaxinella balfourensis, and Sphaerotylus antarcticus. FISH-based studies have shown that Archaea are typically minor components of pelagic microbial communities in Arctic water bodies. The majority of microbes in any given environment are typically recalcitrant to laboratory cultivation, and as such only a handful of Archaea have been isolated from polar environments.
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Metagenomic Analysis of Polar Ecosystems
- Authors: Etienne Yergeau, Charles W. Greer
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Source: Polar Microbiology: Life in a Deep Freeze , pp 156-165
Publication Date :
January 2012
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Polar environments can be among the simplest environments on Earth, making them excellent models to understand ecosystem processes and the responses of microorganisms to environmental perturbations. This vulnerability and the rate of the ongoing change make polar environments a priority in climate change and bioremediation studies. This chapter covers studies based on large-scale 16S rRNA gene libraries, environmental microarrays, large-insert clone libraries, and shotgun genome sequencing that are applying metagenomic techniques to characterize and understand polar ecosystems. A recent study at sites ranging from the Falkland Islands all the way to the base of the Antarctic Peninsula used 454 GS FLX Titanium sequencing of the 16S rRNA gene to compare the bacterial community structure and diversity in warmed versus control plots. This study revealed that soil warming induced significant shifts in the major soil bacterial groups like Acidobacteria and α-Proteobacteria, which led to changes in soil respiration. A recent shotgun metagenomic study in the Canadian High Arctic sequenced a permafrost sample and its overlying active-layer soil and focused particularly on genes that might be important for greenhouse gas emissions following permafrost thaw. The only metagenomic study of polar freshwater ecosystems published to date looked at the viral communities of an Antarctic lake. This study revealed that a large proportion of the viral sequences retrieved from the lake were from eukaryotic viruses and not from bacteriophages. Emerging technologies could also be interesting to apply to polar environments. For instance, bioremediation studies could benefit from metabolomics, proteomics, and newly developed reactome arrays.
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Microbial Carbon Cycling in Permafrost
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Source: Polar Microbiology: Life in a Deep Freeze , pp 183-200
Publication Date :
January 2012
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Abstract:
Terrestrial and submarine permafrost is identified as one of the most vulnerable carbon pools on Earth. In some areas, permafrost comprises upwards of 80% ice in the form of large features, such as massive ice sheets many kilometers in length; or on smaller scales, such as ice wedges and ice lenses, and as ice that fills soil pore space. Residual pockets of seawater, from the subsidence of the polar ocean, exist as saturated, salt-rich permafrost environments known as salt lenses or cryopegs. All of these permafrost features sustain microbial communities that contribute to carbon cycling in polar regions. The way in which gas is released from permafrost, i.e., the rate and pathway, determines the ratio of methane and carbon dioxide emitted to the atmosphere. This chapter describes the different carbon pools, carbon fluxes, and freeze-thaw stresses related to microbial activities. It then examines methane-cycling communities in Arctic active-layer and permafrost environments. The fast recovery of the microbial activity during spring suggests that carbon mineralization in thawing Arctic sediment may rapidly respond to warming, resulting in substantial changes in microbial carbon cycling and growth of microbial populations. Environmental sequences from the Laptev Sea coast consist of four specific permafrost clusters. It was hypothesized that these clusters comprise methanogenic Archaea with a specific physiological potential to survive under harsh environmental conditions. A first study on submarine permafrost of the Laptev Sea shelf demonstrated that intact DNA was extractable from late Pleistocene permafrost deposits with an age of up to 111,000 years.
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The Human Microbiome
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Source: Manual of Clinical Microbiology, 10th Edition , pp 188-198
Publication Date :
January 2011
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Abstract:
The human microbiome includes bacteria, viruses, and small eukaryotes, such as fungi, and this chapter focuses on the bacterial members of the microbiome. The Human Microbiome Project (HMP) aims at developing tools and resources for characterization of the human microbiota and to relate this microbiota to human health and disease. The goals of the jumpstart phase have been to sequence 900 reference genomes to provide a catalog of genomes for metagenomic studies, to sample at least 300 healthy adults between 18 and 40 years of age at five body sites, and to develop sequencing and analysis protocols for the samples derived from human subjects. The second phase of the HMP includes human microbiome studies that target particular disease states. In a recent study, four phyla comprised 92.3% of bacterial DNA sequences analyzed from multiple human sources, including hair, oral cavity, skin, genitourinary, and gastrointestinal tract. A study by Pei et al. showed that the distal esophageal microbiomes of four adults had compositions similar to that of the oropharynx, with the exception that no spirochetes were found in the esophagus. The chapter concludes by highlighting that pathogen discovery efforts will be enhanced by new metagenomics strategies, and these studies may uncover single etiologic agents of infections as well as relative shifts in groups of bacterial pathogens that may contribute to human disease.
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Taxonomy and Classification of Bacteria
- Author: Peter A. R. Vandamme
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Source: Manual of Clinical Microbiology, 10th Edition , pp 213-227
Publication Date :
January 2011
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Abstract:
The process of species delineation in bacterial systematics has undergone drastic modifications as the species concept evolved in parallel with technical progress. The early classification concept was replaced by theories of so-called natural concepts, which were the phenetic and phylogenetic classifications. In the former, relationships between bacteria were based on the overall similarity of phenotypic and genotypic characteristics. The species is the most important and, at the same time, the central element of bacterial taxonomy. The tree of life, based on comparative small-subunit rRNA studies, comprises three lines of descent that are nowadays referred to as the domains Bacteria, Archaea, and Eucarya. Researchers have reported on the place for 16S rRNA gene sequence analysis and DNA-DNA reassociation in the present species definition in bacteriology. In spite of its limitations, rRNA sequence analysis is now commonly used for the identification of bacteria. The determination of the cell wall composition has traditionally been important for gram-positive bacteria. The majority of bacteria in routine diagnostic laboratories will continue to be identified by classical methods, as these methods are adequate, inexpensive, readily available and easy to handle. The present view of classification reflects the best science of this time. The same was true in the past, when only data from morphological and biochemical analyses were available. The main perspective in bacterial taxonomy is that technological progress will dominate and drastically influence methodology, as it always has.
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The Gut Microbiota: Ecology and Function
- Authors: Benjamin P. Willing, Janet K. Jansson
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Source: The Fecal Bacteria , pp 39-65
Publication Date :
January 2011
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Abstract:
This chapter focuses on the microbial ecology of the gut, with emphasis on information gleaned from recent molecular studies. Most attention has been devoted to bacterial components of the gut microbiota and, thus, they are the focus of this chapter. The first metagenomic study of the human gut resulted in 78 million base pairs of DNA sequences from two American individuals. This study cataloged the combined gene complement of the microbiome, including functional genes. A section discusses some dramatic differences that have been observed in the gut microbiota of infants that are fed formula compared to infants that are breast-fed. An interesting example of the importance of host physiology in shaping the composition of the microbiota was shown in reciprocal transplantations of gut microbiota between mice and zebrafish. The chapter primarily discusses Crohn's disease (CD) because of the large number of recent reports that have focused on the correlation of the gut microbiota to this particular disease. Therefore, the increased production of butyrate resulted in greater host responses to colonization. Although this model gave some new insights into the complex ecology of the gut microbiota, it is yet unclear whether the interactions observed between Eubacterium rectale and Bacteroides thetaiotaomicron are representative of common interactions between Bacteroidetes and Firmicutes. The study of genetically matched twins and defined model systems are examples of approaches that have promise to help define diagnostic targets and disease biomarkers.
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DNA Stable Isotope Probing
- Authors: Yin Chen, J. Colin Murrell
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Source: Stable Isotope Probing and Related Technologies , pp 3-24
Publication Date :
January 2011
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Abstract:
The major obstacle for 15N-based DNA stable isotope probing (DNA-SIP) was insufficient separation of 15N-labeled and unlabeled DNA due to the lower nitrogen content in DNA compared with its carbon content. Extracted DNA can be loaded onto a cesium chloride (CsCl) gradient for isopycnic centrifugation and separation of labeled “heavy” DNA from unlabeled background DNA (“light” DNA). DNA-SIP experiments need to be implemented carefully in order to maximize achievable information and to avoid misinterpretation of resulting data. Using DNA-SIP, a study demonstrated the possibility that Candidate phylum TM7 is involved in toluene degradation in soils. Methanol, formaldehyde, and ammonium are the final products of the RDX degradation pathway. It is obvious that microorganisms that only use nitro-nitrogen in the ring-labeled 15N3-RDX would not be seen in this study. In a study, the authors used DNA-SIP to analyze the active methanotroph population in a peatland from the United Kingdom. By using DNA-SIP, the authors demonstrated that Methylocellaand Methylocystisare probably the most active methane utilizers in this peatland. DNA-SIP can be used in combination with metagenomics in a focused way to investigate the function of a subpopulation of environmental microorganisms. It is predicted that this approach will be adopted by more researchers in the near future. The 16S rRNA gene sequences retrieved from the SIP experiments were used to design specific probes targeting 16S rRNA gene of Acidovorax spp. and Pseudomonas spp.
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RNA Stable Isotope Probing
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Source: Stable Isotope Probing and Related Technologies , pp 25-36
Publication Date :
January 2011
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Abstract:
This chapter focuses on RNA-based stable isotope probing (RNA-SIP) that was developed to take advantage of the features of RNA that make it an excellent biomarker for linking environmental processes—rapid turnover rates independent of cellular replication, coupled to in-depth phylogenetic information within the molecule itself. The primary aim of an RNA extraction protocol for RNA-SIP is to generate over 1µg of quantifiable RNA. Nucleic acids appear in almost all gradient fractions as revealed by high-sensitivity methods for detecting them, such as reverse transcription PCR (RT-PCR) or PCR. The chapter also talks about the RNA-SIP- directed investigation that ultimately led to the isolation of a novel Thauera strain responsible for the observed phenol degradation and to confirmation that it could be used to revive sludge that had lost phenol-degrading activity. These findings changed one's understanding of the microbes responsible for phenol degradation in aerated sludge, revealed the pitfalls of both conventional culturing and basic molecular approaches, and highlighted the importance of methods linking function with phylogeny. Manefield used RNA-SIP to compare the communities dominating the assimilation of carbon from phenol in near-identical wastewater treatment bioreactors that were operated in the same manner but differed in wastewater treatment performance. This study revealed that Acidovorax species were responsible for phenol degradation and the poor performance of one reactor was associated with two different Acidovorax populations, while the strong performance of the other was associated with a single dominant Acidovorax lineage.
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Stable Isotope Probing and Metagenomics
- Authors: Lee J. Pinnell, Trevor C. Charles, Josh D. Neufeld
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Source: Stable Isotope Probing and Related Technologies , pp 97-114
Publication Date :
January 2011
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Abstract:
Two promising culture-independent approaches that have been employed to assess the function and metabolic potential of uncultivated microorganisms are stable isotope probing (SIP) and metagenomics. This chapter discusses the methodology of metagenomics within the context of DNA stable isotope probing (DNA-SIP), and provides a description of the possible limitations and how these limitations can be overcome, summarizes the combined DNA-SIP and meta-genomic studies to date, and highlights future directions. The chapter also focuses on metagenomics as it relates to SIP and highlights some of the methodological considerations for cloning and characterization of labeled DNA from active and uncultivated microorganisms. A study using SIP and metagenomics with increasingly low substrate concentrations to characterize marine methylotrophs involved in C1 cycling of surface seawater was a proof-of-concept approach that utilized multiple displacement amplification (MDA) for the first time in association with DNA-SIP and metagenomics. The study also demonstrated that DNA-SIP employing near-in situ substrate concentrations may be used because the resulting low yields of DNA are still amenable to metagenomic analysis through MDA amplification. The combination of SIP, MDA, and metagenomics provides powerful access to the genomes of active-but-uncultivated microorganisms. An alternative approach for combining SIP and metagenomics is to profile the purified 13C-labeled DNA with high-throughput sequencing of cloned DNA fragments. DNA-SIP paired with metagenomics is expected to yield invaluable insight into the uncultured microbial world as the techniques become increasingly commonplace, isotopes become increasingly available and affordable, and experiments become increasingly well designed.
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Stable Isotope Probing Techniques Using H2 18O
- Author: Egbert Schwartz
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Source: Stable Isotope Probing and Related Technologies , pp 115-128
Publication Date :
January 2011
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Abstract:
This chapter reviews the use of 18O-water in stable isotope probing (SIP) studies. Research groups made important contributions to understanding of adenosine triphosphate (ATP) production in mitochondria and bacteria. Richards and Boyer, employing 18O-water, showed that oxygen atoms from water can be transferred to DNA inside E. coli cells. Subsequently, it was shown that 18O-water may also be used to label DNA formed in soil. Water is a small molecule and therefore can rapidly diffuse throughout the soil environment, so that the label is relatively homogenously distributed throughout the sample and all soil organisms are exposed to similar concentrations of label. However, it is unlikely that the label will be completely homogenously distributed in soil. SIP with 18O-water may also be used to study the impact of environmental conditions such as temperature or pH on microbial population dynamics in soil. Finally, SIP with 18O-water is suitable for studies on the impact of complex nutrient sources on microbial population dynamics in soil. For instance, it is well known that plant litter quality, often gauged by measuring the lignin to nitrogen ratio, affects decomposition rates and therefore multiple nutrient cycles in soil.
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Stable Isotope Probing Techniques Using 15N
- Author: Daniel H. Buckley
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Source: Stable Isotope Probing and Related Technologies , pp 129-147
Publication Date :
January 2011
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Abstract:
This chapter examines technical considerations associated with the use of 15N in nucleic acid stable isotope probing (SIP) experiments, discusses concerns that should be considered prior to undertaking a 15N-labeling experiment, and provides an overview of different applications of 15N-SIP. The preferable approach is to use quantitative PCR (qPCR) to determine the number of 16S rRNA genes in each gradient fraction. The gene target for qPCR analysis can vary by application, and it may be desirable to use universal 16S rRNA gene-targeted primers or primers that are specific to individual domains, individual subgroups, or genera. In DNA purified by secondary gradient fractionation, nif H genes similar to Methylosinus represented 53% of those recovered while Methylocystis-like sequences represented 17% of those recovered. However, in an experiment in which 15N2-DNA-SIP was used to examine nitrogen-fixing methanotrophs in soil. There are several reasons why 15N-DNA-SIP represents an appealing method for examining nitrogen (N2)-fixing organisms. First, incubations can be carried out at realistic concentrations of substrate, as air can be evacuated from sealed containers and replaced with simulated air containing 15N2. Second, since nitrogen fixation is inhibited in the presence of mineral forms of nitrogen, problems associated with isotope dilution can largely be ignored. Experiments will need to be performed with pure cultures and environmental samples to determine whether 15N-RNA-SIP can be used effectively in microbial ecology studies.
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Stable Isotope Probing and Plants
- Authors: Yahai Lu, Ralf Conrad
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Source: Stable Isotope Probing and Related Technologies , pp 151-163
Publication Date :
January 2011
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This chapter summarizes the applications of stable isotope probing (SIP) technology in plant-soil systems and presents an overview of the progress achieved in the understanding of the plant-soil microbe interactions and their role in ecosystem functioning. The applications of phospholipid fatty acids-based SIP (PLFA-SIP) and then the applications of DNA- and RNA-based SIP in upland soils and flooded rice field soils, respectively, are described. Several studies have exploited PLFA-SIP technology to determine the plant-microbe interactions driven by rhizosphere carbon flow. In these studies, the living plants, either in the field or laboratory, are exposed to 13C-labeled CO2, and the microbial PLFAs are collected from rhizosphere soil. After pulse-labeling of rice plants with 13CO2 in a microcosm, soil samples were divided into rhizosphere and bulk soil, and the bulk soil samples were further partitioned vertically into upper layer and lower layer and horizontally into five layers with an increasing distance from roots. A study performed on grassland soil and on peatland soil, targeted mainly the root symbiont's arbuscular mycorrhizal (AM) fungi and the bacteria possibly associated with them. RNA-SIP revealed that AM fungi were labeled with 13C immediately after plant assimilation, suggesting that AM fungi preferentially used assimilates provided by plants rather than previously fixed carbon. Combining SIP with techniques such as metatranscriptomics, pyrosequencing, and community systems biology, promises a better and deeper understanding of plant-microbe interactions.
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Stable Isotope Probing Techniques and Bioremediation
- Author: Eugene L. Madsen
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Source: Stable Isotope Probing and Related Technologies , pp 165-201
Publication Date :
January 2011
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Abstract:
This chapter summarizes the state of the art for applications of stable isotope probing (SIP) to bio-degradation and bioremediation research. SIP is one of the many emerging tools of inquiry used by environmental microbiologists. The goals of this chapter are to catalog and analyze trends exhibited by the majority of studies published to date that are pertinent to biodegradation and bioremediation. 14C-based phospholipid fatty acid (PLFA)-SIP showed chromatographic profiles related to but distinct from known type II methanotrophs. Using PLFA-SIP analyses, a variety of comparisons were made between forest, shrubland, and pasture soils; type II methanotrophs were dominant in forest and shrubland, while type I methanotrophs dominated pasture soil. When suitable probes are available, fluorescent in situ hybridization (FISH) is an effective means of microscopic identification of microorganisms and can be combined with techniques using radioactive and stable isotopes to identify metabolically active microorganisms. The combined microscopic approaches of FISH and secondary ion mass spectrometry (SIMS) have tremendous potential to aid investigations examining the roles of bacteria in biogeochemical processes and in the biodegradation of organic pollutants. SIP is a means toward the goal of improved pollution-control technology. In general, no single technique or piece of evidence is sufficient to advance the discipline of environmental microbiology. But SIP is inherently heuristic—results have the potential to create new information and hypotheses that can be tested and confirmed with multidisciplinary approaches.