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.The molecular analysis of 16S rRNA is now central to studies examining the diversity of microorganisms in the environment. Traditional methods based upon cultivation underestimate diversity considerably, whereas modern molecular methods (PCR, cloning, and sequencing) have provided a greater insight into the extent of prokaryotic diversity (for a review see reference 6). Methodologies for the analyses of a DNA-based phylogeny (using the 16S rRNA gene) are now well established but the direct targeting of 16S rRNA, as a potential indicator of activity (4), has received comparatively less attention, due primarily to the lack of suitable protocols for extraction from natural environments.Methods currently employed for DNA extraction vary widely, from direct methods of in situ lysis to indirect methods of initial cell extraction prior to lysis. In both cases, the methods used often include various combinations of bead beating, detergents, enzymatic lysis, and solvent extractions to obtain a crude preparation of nucleic acid (see, e.g., references 5 and 8). The utility of the published methods varies, particularly in soil systems, since inhibitory compounds such as humic acids and clay minerals are often coextracted. Therefore, additional purification procedures are required for successful PCR amplification. These additional steps can prevent the simultaneous extraction of the labile RNA (3) and reduce DNA yield. Reliable extraction methods have been reported for isolation of RNA from soils (2, 3, 11) and other environments (10), but they typically involve multiple steps for purification, rendering them impractical for processing large numbers of samples. Here we describe the first direct method for the rapid coextraction of RNA and DNA from soil for the comparison of bacterial diversity by 16S rRNA reverse transcription-PCR (RT-PCR) and 16S ribosomal DNA (rDNA)-PCR. To demonstrate the efficacy and reproducibility of the method, we present the denaturing gradient gel electrophoresis (DGGE) analysis of the diversity of bacterial populations in a humified upland soil based on 16S rDNA and 16S rRNA templates.Sampling and extraction protocol. Replicate cores of a brown forest soil (pH 4.5 to 5.0) were collected from the Sourhope Field Experiment Site in the Scottish Borders (United Kingdom) to a depth of 20 to 25 cm. Each replicate core was divided into four horizons characterized by standard nomenclature (Fh, H, Ah upper, and Ah lower). Prior to nucleic acid extraction, all solutions and glassware were rendered RNase free by diethyl pyrocarbonate...

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.

Here we present a standard developed by the Genomic Standards Consortium (GSC) for reporting marker gene sequences—the minimum information about a marker gene sequence (MIMARKS). We also introduce a system for describing the environment from which a biological sample originates. The ‘environmental packages’ apply to any genome sequence of known origin and can be used in combination with MIMARKS and other GSC checklists. Finally, to establish a unified standard for describing sequence data and to provide a single point of entry for the scientific community to access and learn about GSC checklists, we present the minimum information about any (x) sequence (MIxS). Adoption of MIxS will enhance our ability to analyze natural genetic diversity documented by massive DNA sequencing efforts from myriad ecosystems in our ever-changing biosphere.

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 [ 13 C]phenol was fed into a phenol-degrading community from an aerobic industrial bioreactor, and the 13 C-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.Our dependence upon laboratory-based studies of culturable bacterial species is acknowledged to be a primary limitation to progress in microbial ecology (2). Traditionally, it has been difficult to attribute a recognized microbially mediated process to the organism(s) responsible for that process in situ (12). Recently, however, culture-independent approaches to linking microbial community function with the genetic identity of key organisms have begun to emerge (7,18,23). Among the limited number of elegant methodologies capable of identifying microorganisms responsible for particular biogeochemical processes, stable-isotope probing (SIP) holds considerable promise.SIP involves the incorporation of stable-isotope-labeled substrates into cellular biomarkers that can be used to identify organisms assimilating the substrate (6). Stable isotopes were first used in this capacity to identify the microbial community component responsible for acetate oxidation in aquatic sediments (7). A 13 C-labeled acetate pulse was administered to sediments, with the resulting 13 C-enriched polar-lipid-derived fatty acid (PLFA) signature profiles subsequently being preferentially analyzed. Other studies utilizing the same approach have followed suit (9, 24).The utility of PLFA-based SIP may be limited, because resolving PLFA profiles composed of multiple species can be problematic (26). This limitation has been overcome with the demonstration that stable-isotope-labeled DNA can be recovered from total community nucleic acid extractions on the basis of its increased buoyant density and can be used as an alternative, unambiguous biomarker (21, 26, 32).Because DNA-based SIP relies on the isolation of labeled DNA by density centrifugation, the degree of isotopic enrichment is crucial. Of the many factors determining the success of an enrichment, including the duration of the pulse and the presence of unlabeled substrate inherent to the system, the rate of DNA synthesis in situ plays a pivotal role. We speculate that DNA synthesi...