Andrew S. Whiteley

Rapid method for coextraction of DNA and RNA from natural environments for analysis of ribosomal DNA- and rRNA-based microbial community composition

ABSTRACT A rapid protocol for the extraction of total nucleic acids from environmental samples is described. The method facilitates concomitant assessment of microbial 16S rRNA diversity by PCR and reverse transcription-PCR amplification from a single extraction. Denaturing gradient gel electrophoresis microbial community analysis differentiated the active component (rRNA derived) from the total bacterial diversity (ribosomal DNA derived) down the horizons of an established grassland soil.

RNA stable isotope probing, a novel means of linking microbial community function to Phylogeny

ABSTRACT Identifying microorganisms responsible for recognized environmental processes remains a great challenge in contemporary microbial ecology. Only in the last few years have methodological innovations provided access to the relationship between the function of a microbial community and the phylogeny of the organisms accountable for it. In this study stable-isotope-labeled [13C]phenol was fed into a phenol-degrading community from an aerobic industrial bioreactor, and the 13C-labeled RNA produced was used to identify the bacteria responsible for the process. Stable-isotope-labeled RNA was analyzed by equilibrium density centrifugation in concert with reverse transcription-PCR and denaturing gradient gel electrophoresis. In contradiction with findings from conventional methodologies, this unique approach revealed that phenol degradation in the microbial community under investigation is dominated by a member of the Thauera genus. Our results suggest that this organism is important for the function of this bioreactor.

DNA stable isotope probing

Stable-isotope probing is a method used in microbial ecology that provides a means by which specific functional groups of organisms that incorporate particular substrates are identified without the prerequisite of cultivation. Stable-isotope-labeled carbon (13C) or nitrogen (15N) sources are assimilated into microbial biomass of environmental samples. Separation and molecular analysis of labeled nucleic acids (DNA or RNA) reveals phylogenetic and functional information about the microorganisms responsible for the metabolism of a particular substrate. Here, we highlight general guidelines for incubating environmental samples with labeled substrate and provide a detailed protocol for separating labeled DNA from unlabeled community DNA. The protocol includes a modification of existing published methods, which maximizes the recovery of labeled DNA from CsCl gradients. The separation of DNA and retrieval of unlabeled and labeled fractions can be performed in 4–5 days, with much of the time being committed to the ultracentrifugation step.

The bacterial biogeography of British soils

Despite recognition of the importance of soil bacteria to terrestrial ecosystem functioning there is little consensus on the factors regulating belowground biodiversity. Here we present a multi-scale spatial assessment of soil bacterial community profiles across Great Britain (> 1000 soil cores), and show the first landscape scale map of bacterial distributions across a nation. Bacterial diversity and community dissimilarities, assessed using terminal restriction fragment length polymorphism, were most strongly related to soil pH providing a large-scale confirmation of the role of pH in structuring bacterial taxa. However, while α diversity was positively related to pH, the converse was true for β diversity (between sample variance in α diversity). β diversity was found to be greatest in acidic soils, corresponding with greater environmental heterogeneity. Analyses of clone libraries revealed the pH effects were predominantly manifest at the level of broad bacterial taxonomic groups, with acidic soils being dominated by few taxa (notably the group 1 Acidobacteria and Alphaproteobacteria). We also noted significant correlations between bacterial communities and most other measured environmental variables (soil chemistry, aboveground features and climatic variables), together with significant spatial correlations at close distances. In particular, bacterial and plant communities were closely related signifying no strong evidence that soil bacteria are driven by different ecological processes to those governing higher organisms. We conclude that broad scale surveys are useful in identifying distinct soil biomes comprising reproducible communities of dominant taxa. Together these results provide a baseline ecological framework with which to pursue future research on both soil microbial function, and more explicit biome based assessments of the local ecological drivers of bacterial biodiversity.

Shifting carbon flow from roots into associated microbial communities in response to elevated atmospheric CO2

Rising atmospheric CO2 levels are predicted to have major consequences on carbon cycling and the functioning of terrestrial ecosystems. Increased photosynthetic activity is expected, especially for C-3 plants, thereby influencing vegetation dynamics; however, little is known about the path of fixed carbon into soil-borne communities and resulting feedbacks on ecosystem function. Here, we examine how arbuscular mycorrhizal fungi (AMF) act as a major conduit in the transfer of carbon between plants and soil and how elevated atmospheric CO2 modulates the belowground translocation pathway of plant-fixed carbon. Shifts in active AMF species under elevated atmospheric CO2 conditions are coupled to changes within active rhizosphere bacterial and fungal communities. Thus, as opposed to simply increasing the activity of soil-borne microbes through enhanced rhizodeposition, elevated atmospheric CO2 clearly evokes the emergence of distinct opportunistic plant-associated microbial communities. Analyses involving RNA-based stable isotope probing, neutral/phosphate lipid fatty acids stable isotope probing, community fingerprinting, and real-time PCR allowed us to trace plant-fixed carbon to the affected soil-borne microorganisms. Based on our data, we present a conceptual model in which plant-assimilated carbon is rapidly transferred to AMF, followed by a slower release from AMF to the bacterial and fungal populations well-adapted to the prevailing (myco-)rhizosphere conditions. This model provides a general framework for reappraising carbon-flow paths in soils, facilitating predictions of future interactions between rising atmospheric CO2 concentrations and terrestrial ecosystems.

Bacterial Community Structure and Physiological State within an Industrial Phenol Bioremediation System

ABSTRACT The structure of bacterial populations in specific compartments of an operational industrial phenol remediation system was assessed to examine bacterial community diversity, distribution, and physiological state with respect to the remediation of phenolic polluted wastewater. Rapid community fingerprinting by PCR-based denaturing gradient gel electrophoresis (DGGE) of 16S rDNA indicated highly structured bacterial communities residing in all nine compartments of the treatment plant and not exclusively within the Vitox biological reactor. Whole-cell targeting by fluorescent in situ hybridization with specific oligonucleotides (directed to the α, β and γ subclasses of the class Proteobacteria [α-, β-, and γ-Proteobacteria, respectively], theCytophaga-Flavobacterium group, and thePseudomonas group) tended to mirror gross changes in bacterial community composition when compared with DGGE community fingerprinting. At the whole-cell level, the treatment compartments were numerically dominated by cells assigned to theCytophaga-Flavobacterium group and to the γ-Proteobacteria. The α subclassProteobacteria were of low relative abundance throughout the treatment system whilst the β subclass of theProteobacteria exhibited local dominance in several of the processing compartments. Quantitative image analyses of cellular fluorescence was used as an indicator of physiological state within the populations probed with rDNA. For cells hybridized with EUB338, the mean fluorescence per cell decreased with increasing phenolic concentration, indicating the strong influence of the primary pollutant upon cellular rRNA content. The γ subclass of theProteobacteria had a ribosome content which correlated positively with total phenolics and thiocyanate. While members of theCytophaga-Flavobacterium group were numerically dominant in the processing system, their abundance and ribosome content data for individual populations did not correlate with any of the measured chemical parameters. The potential importance of the γ-Proteobacteria and theCytophaga-Flavobacteria during this bioremediation process was highlighted.

Physiological and Community Responses of Established Grassland Bacterial Populations to Water Stress

ABSTRACT The effects of water stress upon the diversity and culturable activity of bacterial communities in the rhizosphere of an established upland grassland soil have been investigated. Intact monoliths were subjected to different watering regimens over a 2-month period to study community adaptation to moisture limitation and subsequent response to stress alleviation following rewetting. Genetic diversity was analyzed with 16S-based denaturing gradient gel electrophoresis (DGGE) of total soil-extracted DNA (rRNA genes) and RNA (rRNA transcripts) in an attempt to discriminate between total and active communities. Physiological response was monitored by plate counts, total counts, and BIOLOG-GN2 substrate utilization analyses. Controlled soil drying decreased the total number of CFU on all the media tested and also decreased the substrate utilization response. Following rewetting of dried soil, culture-based analyses indicated physiological recovery of the microbial population by the end of the experiment. In contrast, DGGE analyses of community 16S rRNA genes, rRNA transcripts and cultured communities did not reveal any changes relating to the moisture regimens, despite the observed physiological effects. We conclude that the imposed moisture regimen modulated the physiological status of the bacterial community and that bacterial communities in this soil are resistant to water stress. Further, we highlight the need for a reexamination of rRNA transcript-based molecular profiling techniques as a means of describing the active component of soil bacterial communities.

Influence of depth and sampling time on bacterial community structure in an upland grassland soil

Abstract Temporal and spatial variation of soil bacterial communities was evaluated with both molecular and metabolic profiling techniques. Soil cores (20 cm deep) were taken from an upland grassland in the Scottish Borders (UK) over 3 days in July 1999, and on single days in October 1999, April 2000, and August 2000. Cores were separated into four 5-cm depths to examine vertical spatial distribution. The 0-5-, 5-10- and 10-15-cm samples represented organic horizons whilst the 15-20-cm depths were from a mineral horizon. The potential metabolic activities were analysed using BIOLOG-GN plates, whereas genotypic diversity was evaluated using molecular profiling of amplified 16S rRNA and 16S rDNA gene fragments (denaturing gradient gel electrophoresis (DGGE)). BIOLOG-GN analysis revealed decreased substrate utilisation in the lowest depths, which was coupled with changes in the DNA and RNA DGGE profiles. Seasonal variation was pronounced in the 5-10-cm and 10-15-cm organic horizons for the July samplings whilst the 15-20-cm depths appeared more stable. Potential factors influencing the observed changes in bacterial communities resulting from soil depth and sampling time are discussed.

Advances in restoration ecology: rising to the challenges of the coming decades

Simultaneous environmental changes challenge biodiversity persistence and human wellbeing. The science and practice of restoration ecology, in collaboration with other disciplines, can contribute to overcoming these challenges. This endeavor requires a solid conceptual foundation based in empirical research which confronts, tests and influences theoretical developments. We review conceptual developments in restoration ecology over the last 30 years. We frame our review in the context of changing restoration goals which reflect increased societal awareness of the scale of environmental degradation and the recognition that inter-disciplinary approaches are needed to tackle environmental problems. Restoration ecology now encompasses facilitative interactions and network dynamics, trophic cascades, and above- and below ground linkages. It operates in a non-equilibrium, alternative states framework, at the landscape scale, and in response to changing environmental, economic and social conditions. Progress has been marked by conceptual advances in the fields of trait-environment relationships, community assembly, and understanding the links between biodiversity and ecosystem functioning. Conceptual and practical advances have been enhanced by applying evolving technologies, including treatments to increase seed germination and overcome recruitment bottlenecks, high throughput DNA sequencing to elucidate soil community structure and function, and advances in satellite technology and GPS tracking to monitor habitat use. The synthesis of these technologies with systematic reviews of context dependencies in restoration success, model based analyses and consideration of complex socio-ecological systems will allow generalizations to inform evidence based interventions. Ongoing challenges include setting realistic, socially acceptable goals for restoration under changing environmental conditions, and prioritizing actions in an increasingly space-competitive world. Ethical questions also surround the use of genetically modified material, translocations, taxon substitutions, and de-extinction, in restoration ecology. Addressing these issues, as the Ecological Society of America looks to its next century, will require current and future generations of researchers and practitioners, including economists, engineers, philosophers, landscape architects, social scientists and restoration ecologists, to work together with communities and governments to rise to the environmental challenges of the coming decades.

Active microbial RNA turnover in a grassland soil estimated using a 13CO2 spike

Rhizosphere microbes are critical to the initial transfer and transformation of root carbon inputs to the soil but our understanding of the activity of these organisms remains constrained by their limited culturability. In this study we combined isotopic 13C tracer and molecular approaches to measure the incorporation of recently assimilated plant C into soil microbial RNA and DNA pools as a means to determine the turnover of the ‘active’ rhizosphere community. This required the development of a method for the extraction, purification and preparation of small-sample soil DNA and RNA (<5 μg C) for isotope analysis. Soil, plant and respired CO2 samples were collected from a 13CO2 pulse-chase experiment at intervals for 20 days post-labelling. The peak of 13C release in soil/root respired CO2 came between 5 and 48 h after 13CO2 pulse-labelling and was followed by a secondary peak of soil heterotroph 13C respiration after 136 h. Results showed that both soil DNA and RNA rapidly incorporated recent photosynthate with greatest 13C found in the ‘active’ microbial RNA fraction reflecting higher rates of microbial RNA turnover. The dilution rate of the pulse derived 13C in RNA-C was used to estimate a microbial RNA turnover of approximately 20% day−1 with a 15–20 day residence time for photosynthate derived 13C in the RNA pool. The findings of this work confirm the rapid transfer of photosynthate C inputs through soil microorganisms to the atmosphere as CO2 and the potential of the biomolecular-isotope tracer approach in soil C research.