Michael S. Robeson

A comprehensive survey of soil acidobacterial diversity using pyrosequencing and clone library analyses

Acidobacteria are ubiquitous and abundant members of soil bacterial communities. However, an ecological understanding of this important phylum has remained elusive because its members have been difficult to culture and few molecular investigations have focused exclusively on this group. We generated an unprecedented number of acidobacterial DNA sequence data using pyrosequencing and clone libraries (39 707 and 1787 sequences, respectively) to characterize the relative abundance, diversity and composition of acidobacterial communities across a range of soil types. To gain insight into the ecological characteristics of acidobacterial taxa, we investigated the large-scale biogeographic patterns exhibited by acidobacterial communities, and related soil and site characteristics to acidobacterial community assemblage patterns. The 87 soils analyzed by pyrosequencing contained more than 8600 unique acidobacterial phylotypes (at the 97% sequence similarity level). One phylotype belonging to Acidobacteria subgroup 1, but not closely related to any cultured representatives, was particularly abundant, accounting for 7.4% of bacterial sequences and 17.6% of acidobacterial sequences, on average, across the soils. The abundance of Acidobacteria relative to other bacterial taxa was highly variable across the soils examined, but correlated strongly with soil pH (R=−0.80, P<0.001). Soil pH was also the best predictor of acidobacterial community composition, regardless of how the communities were characterized, and the relative abundances of the dominant Acidobacteria subgroups were readily predictable. Acidobacterial communities were more phylogenetically clustered as soil pH departed from neutrality, suggesting that pH is an effective habitat filter, restricting community membership to progressively more narrowly defined lineages as pH deviates from neutrality.

Metagenomic and Small-Subunit rRNA Analyses Reveal the Genetic Diversity of Bacteria, Archaea, Fungi, and Viruses in Soil

ABSTRACT Recent studies have highlighted the surprising richness of soil bacterial communities; however, bacteria are not the only microorganisms found in soil. To our knowledge, no study has compared the diversities of the four major microbial taxa, i.e., bacteria, archaea, fungi, and viruses, from an individual soil sample. We used metagenomic and small-subunit RNA-based sequence analysis techniques to compare the estimated richness and evenness of these groups in prairie, desert, and rainforest soils. By grouping sequences at the 97% sequence similarity level (an operational taxonomic unit [OTU]), we found that the archaeal and fungal communities were consistently less even than the bacterial communities. Although total richness levels are difficult to estimate with a high degree of certainty, the estimated number of unique archaeal or fungal OTUs appears to rival or exceed the number of unique bacterial OTUs in each of the collected soils. In this first study to comprehensively survey viral communities using a metagenomic approach, we found that soil viruses are taxonomically diverse and distinct from the communities of viruses found in other environments that have been surveyed using a similar approach. Within each of the four microbial groups, we observed minimal taxonomic overlap between sites, suggesting that soil archaea, bacteria, fungi, and viruses are globally as well as locally diverse.

PyCogent: a toolkit for making sense from sequence

We have implemented in Python the COmparative GENomic Toolkit, a fully integrated and thoroughly tested framework for novel probabilistic analyses of biological sequences, devising workflows, and generating publication quality graphics. PyCogent includes connectors to remote databases, built-in generalized probabilistic techniques for working with biological sequences, and controllers for third-party applications. The toolkit takes advantage of parallel architectures and runs on a range of hardware and operating systems, and is available under the general public license from http://sourceforge.net/projects/pycogent.

Plant host and soil origin influence fungal and bacterial assemblages in the roots of woody plants

Microbial communities in plant roots provide critical links between above‐ and belowground processes in terrestrial ecosystems. Variation in root communities has been attributed to plant host effects and microbial host preferences, as well as to factors pertaining to soil conditions, microbial biogeography and the presence of viable microbial propagules. To address hypotheses regarding the influence of plant host and soil biogeography on root fungal and bacterial communities, we designed a trap‐plant bioassay experiment. Replicate Populus, Quercus and Pinus plants were grown in three soils originating from alternate field sites. Fungal and bacterial community profiles in the root of each replicate were assessed through multiplex 454 amplicon sequencing of four loci (i.e., 16S, SSU, ITS, LSU rDNA). Soil origin had a larger effect on fungal community composition than did host species, but the opposite was true for bacterial communities. Populus hosted the highest diversity of rhizospheric fungi and bacteria. Root communities on Quercus and Pinus were more similar to each other than to Populus. Overall, fungal root symbionts appear to be more constrained by dispersal and biogeography than by host availability.

Global Distribution of Polaromonas Phylotypes - Evidence for a Highly Successful Dispersal Capacity

Bacteria from the genus Polaromonas are dominant phylotypes in clone libraries and culture collections from polar and high-elevation environments. Although Polaromonas has been found on six continents, we do not know if the same phylotypes exist in all locations or if they exhibit genetic isolation by distance patterns. To examine their biogeographic distribution, we analyzed all available, long-read 16S rRNA gene sequences of Polaromonas phylotypes from glacial and periglacial environments across the globe. Using genetic isolation by geographic distance analyses, including Mantel tests and Mantel correlograms, we found that Polaromonas phylotypes are globally distributed showing weak isolation by distance patterns at global scales. More focused analyses using discrete, equally sampled distances classes, revealed that only two distance classes (out of 12 total) showed significant spatial structuring. Overall, our analyses show that most Polaromonas phylotypes are truly globally distributed, but that some, as yet unknown, environmental variable may be selecting for unique phylotypes at a minority of our global sites. Analyses of aerobiological and genomic data suggest that Polaromonas phylotypes are globally distributed as dormant cells through high-elevation air currents; Polaromonas phylotypes are common in air and snow samples from high altitudes, and a glacial-ice metagenome and the two sequenced Polaromonas genomes contain the gene hipA, suggesting that Polaromonas can form dormant cells.

Functional shifts in unvegetated, perhumid, recently-deglaciated soils do not correlate with shifts in soil bacterial community composition

Past work in recently deglaciated soils demonstrates that microbial communities undergo shifts prior to plant colonization. To date, most studies have focused on relatively ‘long’ chronosequences with the ability to sample plant-free sites over at least 50 years of development. However, some recently deglaciated soils feature rapid plant colonization and questions remain about the relative rate of change in the microbial community in the unvegetated soils of these chronosequences. Thus, we investigated the forelands of the Mendenhall Glacier near Juneau, AK, USA, where plants rapidly establish. We collected unvegetated samples representing soils that had been ice-free for 0, 1, 4, and 8 years. Total nitrogen (N) ranged from 0.00∼0.14 mg/g soil, soil organic carbon pools ranged from 0.6∼2.3 mg/g soil, and both decreased in concentration between the 0 and 4 yr soils. Biologically available phosphorus (P) and pH underwent similar dynamics. However, both pH and available P increased in the 8 yr soils. Nitrogen fixation was nearly undetectable in the most recently exposed soils, and increased in the 8 yr soils to ∼5 ng N fixed/cm2/h, a trend that was matched by the activity of the soil N-cycling enzymes urease and β-l,4-N-acetyl-glucosa-minidase. 16S rRNA gene clone libraries revealed no significant differences between the 0 and 8 yr soils; however, 8 yr soils featured the presence of cyanobacteria, a division wholly absent from the 0 yr soils. Taken together, our results suggest that microbes are consuming allochtonous organic matter sources in the most recently exposed soils. Once this carbon source is depleted, a competitive advantage may be ceded to microbes not reliant on in situ nutrient sources.

Characterizing microbial communities through space and time

Until recently, the study of microbial diversity has mainly been limited to descriptive approaches, rather than predictive model-based analyses. The development of advanced analytical tools and decreasing cost of high-throughput multi-omics technologies has made the later approach more feasible. However, consensus is lacking as to which spatial and temporal scales best facilitate understanding of the role of microbial diversity in determining both public and environmental health. Here, we review the potential for combining these new technologies with both traditional and nascent spatio-temporal analysis methods. The fusion of proper spatio-temporal sampling, combined with modern multi-omics and computational tools, will provide insight into the tracking, development and manipulation of microbial communities.

Phylogeography of microbial phototrophs in the dry valleys of the high Himalayas and Antarctica

High-elevation valleys in dry areas of the Himalayas are among the most extreme, yet least explored environments on Earth. These barren, rocky valleys are subjected to year-round temperature fluctuations across the freezing point and very low availability of water and nutrients, causing previous workers to hypothesize that no photoautotrophic life (primary producers) exists in these locations. However, there has been no work using modern biogeochemical or culture-independent molecular methods to test the hypothesis that photoautotrophs are absent from high Himalayan soil systems. Here, we show that although microbial biomass levels are as low as those of the Dry Valleys of Antarctica, there are abundant microbial photoautotrophs, displaying unexpected phylogenetic diversity, in barren soils from just below the permanent ice line of the central Himalayas. Furthermore, we discovered that one of the dominant algal clades from the high Himalayas also contains the dominant algae in culture-independent surveys of both soil and ice samples from the Dry Valleys of Antarctica, revealing an unexpected link between these environmentally similar but geographically very distant systems. Phylogenetic and biogeographic analyses demonstrated that although this algal clade is globally distributed to other high-altitude and high-latitude soils, it shows significant genetic isolation by geographical distance patterns, indicating local adaptation and perhaps speciation in each region. Our results are the first to demonstrate the remarkable similarities of microbial life of arid soils of Antarctica and the high Himalayas. Our findings are a starting point for future comparative studies of the dry valleys of the Himalayas and Antarctica that will yield new insights into the cold and dry limits to life on Earth.

Insights and inferences about integron evolution from genomic data

BackgroundIntegrons are mechanisms that facilitate horizontal gene transfer, allowing bacteria to integrate and express foreign DNA. These are important in the exchange of antibiotic resistance determinants, but can also transfer a diverse suite of genes unrelated to pathogenicity. Here, we provide a systematic analysis of the distribution and diversity of integron intI genes and integron-containing bacteria.ResultsWe found integrons in 103 different pathogenic and non-pathogenic bacteria, in six major phyla. Integrons were widely scattered, and their presence was not confined to specific clades within bacterial orders. Nearly 1/3 of the intI genes that we identified were pseudogenes, containing either an internal stop codon or a frameshift mutation that would render the protein product non-functional. Additionally, 20% of bacteria contained more than one integrase gene. dN/dS ratios revealed mutational hotspots in clades of Vibrio and Shewanella intI genes. Finally, we characterized the gene cassettes associated with integrons in Methylobacillus flagellatus KT and Dechloromonas aromatica RCB, and found a heavy metal efflux gene as well as genes involved in protein folding and stability.ConclusionOur analysis suggests that the present distribution of integrons is due to multiple losses and gene transfer events. While, in some cases, the ability to integrate and excise foreign DNA may be selectively advantageous, the gain, loss, or rearrangment of gene cassettes could also be deleterious, selecting against functional integrases. Thus, such a high fraction of pseudogenes may suggest that the selective impact of integrons on genomes is variable, oscillating between beneficial and deleterious, possibly depending on environmental conditions.

Soil rotifer communities are extremely diverse globally but spatially autocorrelated locally

Bdelloid rotifers are important contributors to biogeochemical cycling and trophic dynamics of both aquatic and terrestrial ecosystems, but little is known about their biogeographic distribution and community structure in terrestrial environments. This lack of knowledge stems from a lack of phylogenetic information and assumptions that microbial eukaryotes are globally distributed and have very limited diversity across vast geographic distances. However, these assumptions have been based more on assessments of their morphology than any measure of their true genetic diversity and biogeographic distribution. We developed specific primers for the cytochrome c oxidase subunit 1 (cox1) gene of bdelloid rotifers and amplified and cloned sequences using a nested sampling scheme that represented local (0–10 m) to global (up to 10,000 km) scales. Using phylogenetic community analyses (UniFrac) and geospatial statistics (semivariograms, mantel tests), we were able to reject the hypothesis that communities of rotifers are the same across even fairly small geographic distances. Bdelloid communities showed highly significant spatial structuring with spatial autocorrelation ranges of 54–133 m, but beyond that distance communities were extremely dissimilar. Furthermore, we show that these spatial patterns are driven not only by changes in relative abundance of phylotypes but also by absolute changes in phylotype occurrence (richness). There is almost no overlap in phylotype [or operational taxonomic unit (OTU)] occurrence between communities at distances beyond the autocorrelation range (~133 m). Such small species ranges, combined with their ubiquity in soils, make it increasingly clear that the biodiversity of bdelloid rotifers (and other less easily dispersed microbes) is much higher than previously thought.