We have systematically made a set of precisely defined, single-gene deletions of all nonessential genes in Escherichia coli K-12. Open-reading frame coding regions were replaced with a kanamycin cassette flanked by FLP recognition target sites by using a one-step method for inactivation of chromosomal genes and primers designed to create in-frame deletions upon excision of the resistance cassette. Of 4288 genes targeted, mutants were obtained for 3985. To alleviate problems encountered in high-throughput studies, two independent mutants were saved for every deleted gene. These mutants-the 'Keio collection'-provide a new resource not only for systematic analyses of unknown gene functions and gene regulatory networks but also for genome-wide testing of mutational effects in a common strain background, E. coli K-12 BW25113. We were unable to disrupt 303 genes, including 37 of unknown function, which are candidates for essential genes. Distribution is being handled via GenoBase (http://ecoli.aist-nara.ac.jp/).

/pdf/construction-of-escherichia-coli-k-12-in-frame-single-gene-4c68fx6pb1.pdf

Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection.

KEGG(Kyoto Encyclopedia of Genes and Genomes)〔和文〕 (特集 ゲノム医学の現在と未来--基礎と臨床) -- (データベース)

Random community genomes (metagenomes) are now commonly used to study microbes in different environments. Over the past few years, the major challenge associated with metagenomics shifted from generating to analyzing sequences. High-throughput, low-cost next-generation sequencing has provided access to metagenomics to a wide range of researchers. A high-throughput pipeline has been constructed to provide high-performance computing to all researchers interested in using metagenomics. The pipeline produces automated functional assignments of sequences in the metagenome by comparing both protein and nucleotide databases. Phylogenetic and functional summaries of the metagenomes are generated, and tools for comparative metagenomics are incorporated into the standard views. User access is controlled to ensure data privacy, but the collaborative environment underpinning the service provides a framework for sharing datasets between multiple users. In the metagenomics RAST, all users retain full control of their data, and everything is available for download in a variety of formats. The open-source metagenomics RAST service provides a new paradigm for the annotation and analysis of metagenomes. With built-in support for multiple data sources and a back end that houses abstract data types, the metagenomics RAST is stable, extensible, and freely available to all researchers. This service has removed one of the primary bottlenecks in metagenome sequence analysis – the availability of high-performance computing for annotating the data. http://metagenomics.nmpdr.org

/pdf/the-metagenomics-rast-server-a-public-resource-for-the-1470t0l990.pdf

The Metagenomics RAST Server: A Public Resource for the Automatic Phylogenetic and Functional Analysis of Metagenomes

Publisher Summary This chapter reviews the rates at which Coarse Woody Debris (CWD) is added and removed from ecosystems, the biomass found in streams and forests, and many functions that CWD serves. CWD is an important component of temperate stream and forest ecosystems and is added to the ecosystem by numerous mechanisms, including wind, fire, insect attack, pathogens, competition, and geomorphic processes. Many factors control the rate at which CWD decomposes, including temperature, moisture, the internal gas composition of CWD, substrate quality, the size of the CWD, and the types of organisms involved. The mass of CWD in an ecosystem ideally represents the balance between addition and loss. In reality, slow decomposition rates and erratic variations in input of CWD cause the CWD mass to deviate markedly from steady-state projections. Many differences correspond to forest type, with deciduous-dominated systems having generally lower biomass than conifer-dominated systems. Stream size also influences CWD mass in lotic ecosystems, while successional stage dramatically influences CWD mass in boat aquatic and terrestrial settings. This chapter reviews many of these functions and concludes that CWD is an important functional component of stream and forest ecosystems. Better scientific understanding of these functions and the natural factors influencing CWD dynamics should lead to more enlightened management practices.

Ecology of Coarse Woody Debris in Temperate Ecosystems

Use of stable isotope ratios to trace pathways of organic matter among consumers requires knowledge of the isotopic shift between diet and consumer. Variation in trophic shift among consumers can be substantial. For data from the published literature and supplementary original data (excluding fluid-feeding consumers), the mean isotopic shift for C was + 0.5 + 0.13%o rather than 0.0%o, as commonly assumed. The shift for C was higher for consumers analyzed as muscle (+ 1.3 + 0.30%o) than for consumers analyzed whole (+ 0.3 +0.14%o). Among consumers analyzed whole, the trophic shift for C was lower for consumers acidified prior to analysis (-0.2 + 0.21%o) than for unacidified samples ( +0.5 + 0.17%o). For N, trophic shift was lower for consumers raised on invertebrate diets (+ 1.4 + 0.21%o) than for consumers raised on other high-protein diets (+3.3 +0.26%o) and was intermediate for consumers raised on plant and algal diets (+2.2 +0.30%o). The trophic shift for S differed between high-protein (+ 2.0 + 0.65%o) and low-protein diets (-0.5 + 0.56%o). Thus, methods of analysis and dietary differences can affect trophic shift for consumers; the utility of stable isotope methods can be improved if this information is incorporated into studies of trophic relationships. Although few studies of stable isotope ratios have considered variation in the trophic shift, such variation is important because small errors in estimates of trophic shift can result in large errors in estimates of the contribution of sources to consumers or in estimates of trophic position.

Variation in trophic shift for stable isotope ratios of carbon, nitrogen, and sulfur

Stable carbon isotope compositions of organic matter are now widely used to trace carbon flow in ecosystems, and have been instrumental in shaping current perceptions of the importance of terrestrial vegetation to estuarine and coastal marine environments. A general assumption in these and other studies relying on carbon isotope compositions for source identification of organic matter has been that the major biochemical components of plant tissues are isotopically invariant. We report here large differences between the carbon isotope compositions of the polysaccharide and lignin components of a variety of vascular plants, including the salt-marsh grass Spartina alterniflora, and demonstrate that the carbon isotope composition of Spartina detritus gradually changes during biogeochemical processing as polysaccharides are preferentially removed, leaving a material that is relatively enriched in lignin-derived carbon and depleted in 13C.

Depletion of 13 C in lignin and its implications for stable carbon isotope studies

The ecological and evolutionary aspects of planned introductions of transgenic organisms into the environment are considered in this report. The authors support the timely development of environmentally sound products, such as improved agricultural varieties, fertilizers, pest control agents, and microorganisms for waste treatment, through the use of advanced biotechnology within the context of a scientifically based regulatory policy that encourages innovation without compromising sound environmental management. Economic, social, and ethical concerns also must be weighed along with strictly ecological and evolutionary considerations, but these other issues are beyond the scope of this report. Ecological oversight of planned introductions should be directed at promoting effectiveness while guarding against potential problems. The diversity of organisms that will be modified, functions that will be engineered, and environments that will receive altered organisms makes ecological risk evaluation complex. While we cannot now recommend the complete exemption of specific organisms or traits from regulatory oversight, we support and will continue to assist in the development of methods for scaling the level of oversight needed for individual cases according to objective, scientific criteria, with a goal of minimizing unnecessary regulatory burdens. In this report, we provide a preliminary set of specific criteria for the scaling of regulatory oversight. Genetically engineered organisms should be evaluated and regulated according to their biological properties (phenotypes), rather than according to the genetic techniques used to produce them. Nonetheless, because many novel combinations of properties can be achieved only by molecular and cellular techniques, products of these techniques may often be subjected to greater scrutiny than the products of traditional techniques. Although the capability to produce precise genetic alterations increases confidence that unintended changes in the genome have not occurred, precise genetic characterization does not ensure that all ecologically important aspects of the phenotype can be predicted for the environments into which an organism will be introduced. Many important scientific issues must be considered in evaluating the potential ecological consequences of the planned introduction of genetically engineered organisms into the environment. These include survival and reproduction of the introduced organism, interactions with other organisms in the environment, and effects of the introduced organism on ecosystem function. We encourage the use of small—scale field tests , when justified by previous laboratory and/or greenhouse studies, under conditions that minimize dispersal and under appropriate regulatory oversight. As the biotechnology industry develops, continuing regulatory oversight as well as long—term research and monitoring will be necessary for responsible risk management. Many engineered organisms will probably be less fit than the parent organism, although some important exceptions may arise. Even if an engineered trait reduces an organism's fitness only slightly, may generations may pass before the introduced organisms disappears completely due to decreased fitness. Such persistence is most probable when the turnover rate of populations is very slow. Natural selection will act on genetically engineered organisms, as it does on all others. Selection after the release of the transgenic organism will tend to increase fitness, not decrease it, by reducing the costs associated with the novel traits. If increases in fitness do occur, they will probably increase population growth rate and biological competitiveness, or produce other ecological effects that should be considered in assessing risks. Transfer of engineered genes from the modified organism to other organisms may occur through hybridization in higher organisms, or through conjugation, transduction, or transformation in microorganisms. If lateral transfer occurs, an engineered gene may persist in the natural environment even after the genetically engineered organism itself is no longer present. The available scientific evidence indicates that lateral transfer among microorganisms in nature is neither so rare that we can ignore its occurrence, nor so common that we can assume that barriers crossed by modern biotechnology are comparable to those constantly crossed in nature. Native species, as well as species newly introduced from distant habitats, may become pests. An organism engineered to prosper in a new habitat type, geographic area, or season is effectively an introduced organism in that it will probably enter into new biotic and abiotic interactions. Therefore, regulatory and risk assessment structures that rely on the distinction between ~`native" and ~`non—native" must be used with caution. Concern has frequently been expressed regarding the potential for genetically engineered organisms to displace resident species in the receiving community, particularly microbial species performing key functional roles such as nitrogen fixation of lignin decomposition. Because redundancy of function appears to be common in microbial communities, in many cases there would be little concern over microbial species displacement caused by an introduced transgenic organism. Ecological effects and the geographic ranges of organisms transcend political boundaries; we therefore consider it essential to promote and achieve international coordination of risk assessment and regulation of biotechnology. Special consideration must be given to the protection of rare genetic resources, such as the wild ancestors of domesticated species, and threatened gene pools of other wild species. We urge local, state, national, international cooperation in risk assessment and regulation of the ecological effects of the introduction of transgenic organisms. Evaluating the benefits and risks of biotechnology products requires expertise in many scientific disciplines including molecular biology, genetics, cell biology, evolutionary biology, physiology, population and community ecology, and ecosystem science. For society to realize the full benefits of biotechnology, interdisciplinary research and graduate training programs are needed to expand the expertise of the scientific community at large.

The Planned Introduction of Genetically Engineered Organisms: Ecological Considerations and Recommendations

DISSOLVED organic material in marine and freshwater ecosystems constitutes one of the Earth's largest actively cycled reservoirs for organic matter1. The bacterially mediated turnover of chemically identifiable, low-molecular-mass components of this pool has been studied in detail for nearly three decades, but these compounds constitute less than 20% of the total reservoir2. In contrast, little is known about the fate of the larger, biologically more refractory molecules—including humic substances—which make up the bulk of dissolved organic matter. Here we report results from bacterial bioassays and photochemical studies indicating that exposure to sunlight causes dissolved organic matter to release nitrogen-rich compounds that are biologically available, thus enhancing the bacterial degradation of humic substances. We demonstrate that ammonium is among the nitrogenous compounds released and is produced most efficiently by ultraviolet wavelengths. Photochemical release of ammonium from dissolved organic matter has important implications for nitrogen availability in many aquatic ecosystems, including nitrogen-limited high-latitude environments and coastal oceans, where inputs of terrestrial humic substances are high.

Photochemical release of biologically available nitrogen from aquatic dissolved organic matter

Specifically radiolabeled [14C-lignin]lignocelluloses and [14C-polysaccharide]lignocelluloses were prepared from a variety of marine and freshwater wetland plants including a grass, a sedge, a rush, and a hardwood. These [14C]lignocellulose preparations and synthetic [14C]lignin were incubated anaerobically with anoxic sediments collected from a salt marsh, a freshwater marsh, and a mangrove swamp. During long-term incubations lasting up to 300 days, the lignin and polysaccharide components of the lignocelluloses were slowly degraded anaerobically to 14CO2 and 14CH4. Lignocelluloses derived from herbaceous plants were degraded more rapidly than lignocellulose derived from the hardwood. After 294 days, 16.9% of the lignin component and 30.0% of the polysaccharide component of lignocellulose derived from the grass used (Spartina alterniflora) were degraded to gaseous end products. In contrast, after 246 days, only 1.5% of the lignin component and 4.1% of the polysaccharide component of lignocellulose derived from the hardwood used (Rhizophora mangle) were degraded to gaseous end products. Synthetic [14C]lignin was degraded anaerobically faster than the lignin component of the hardwood lignocellulose; after 276 days, 3.7% of the synthetic lignin was degraded to gaseous end products. Contrary to previous reports, these results demonstrate that lignin and lignified plant tissues are biodegradable in the absence of oxygen. Although lignocelluloses are recalcitrant to anaerobic biodegradation, rates of degradation measured in aquatic sediments are significant and have important implications for the biospheric cycling of carbon from these abundant biopolymers.

https://aem.asm.org/content/aem/47/5/998.full.pdf

Anaerobic Biodegradation of the Lignin and Polysaccharide Components of Lignocellulose and Synthetic Lignin by Sediment Microflora

Metagenomics, or environmental genomics, has revolutionized our picture of microorganisms in the real world — as opposed to how they behave in laboratory cultivated 'clonal' cultures. A novel example of 'experimental metagenomics' is now reported, involving the creation of a 20-litre microcosm of sea water collected off Sapelo Island in the US state of Georgia. Manipulation of the system shows that this coastal microbial community is dominated by metabolic generalists capable of utilizing a wide variety of organic compounds, rather than by bacterial species that specialize in metabolizing a specific component of the dissolved organic carbon pool. This finding has important implications for identifying taxon–function relationships for carbon cycle-relevant processes and the construction of predictive models of ocean biogeochemistry. Experimental metagenomics is used to show that coastal communities are populated by taxa capable of metabolizing a wide variety of organic carbon compounds. It is concluded that metabolic generalists dominate coastal microbial communities, with important implications for identifying taxon–function relationships for carbon cycle-relevant processes and the construction of predictive models of ocean biogeochemistry. The assimilation and mineralization of dissolved organic carbon (DOC) by marine bacterioplankton is a major process in the ocean carbon cycle1. However, little information exists on the specific metabolic functions of participating bacteria and on whether individual taxa specialize on particular components of the marine DOC pool2. Here we use experimental metagenomics to show that coastal communities are populated by taxa capable of metabolizing a wide variety of organic carbon compounds. Genomic DNA captured from bacterial community subsets metabolizing a single model component of the DOC pool (either dimethylsulphoniopropionate or vanillate) showed substantial overlap in gene composition as well as a diversity of carbon-processing capabilities beyond the selected phenotypes. Our direct measure of niche breadth for bacterial functional assemblages indicates that, in accordance with ecological theory, heterogeneity in the composition and supply of organic carbon to coastal oceans may favour generalist bacteria. In the important interplay between microbial community structure and biogeochemical cycling, coastal heterotrophic communities may be controlled less by transient changes in the carbon reservoir that they process and more by factors such as trophic interactions and physical conditions.

Robert E. Hodson

Papers

Depletion of 13 C in lignin and its implications for stable carbon isotope studies

The Planned Introduction of Genetically Engineered Organisms: Ecological Considerations and Recommendations

Photochemical release of biologically available nitrogen from aquatic dissolved organic matter

Anaerobic Biodegradation of the Lignin and Polysaccharide Components of Lignocellulose and Synthetic Lignin by Sediment Microflora

Bacterial carbon processing by generalist species in the coastal ocean.