Charles Kai-Wu Lee

Groundtruthing Next-Gen Sequencing for Microbial Ecology-Biases and Errors in Community Structure Estimates from PCR Amplicon Pyrosequencing

Analysis of microbial communities by high-throughput pyrosequencing of SSU rRNA gene PCR amplicons has transformed microbial ecology research and led to the observation that many communities contain a diverse assortment of rare taxa–a phenomenon termed the Rare Biosphere. Multiple studies have investigated the effect of pyrosequencing read quality on operational taxonomic unit (OTU) richness for contrived communities, yet there is limited information on the fidelity of community structure estimates obtained through this approach. Given that PCR biases are widely recognized, and further unknown biases may arise from the sequencing process itself, a priori assumptions about the neutrality of the data generation process are at best unvalidated. Furthermore, post-sequencing quality control algorithms have not been explicitly evaluated for the accuracy of recovered representative sequences and its impact on downstream analyses, reducing useful discussion on pyrosequencing reads to their diversity and abundances. Here we report on community structures and sequences recovered for in vitro-simulated communities consisting of twenty 16S rRNA gene clones tiered at known proportions. PCR amplicon libraries of the V3–V4 and V6 hypervariable regions from the in vitro-simulated communities were sequenced using the Roche 454 GS FLX Titanium platform. Commonly used quality control protocols resulted in the formation of OTUs with >1% abundance composed entirely of erroneous sequences, while over-aggressive clustering approaches obfuscated real, expected OTUs. The pyrosequencing process itself did not appear to impose significant biases on overall community structure estimates, although the detection limit for rare taxa may be affected by PCR amplicon size and quality control approach employed. Meanwhile, PCR biases associated with the initial amplicon generation may impose greater distortions in the observed community structure.

Adaptations to Submarine Hydrothermal Environments Exemplified by the Genome of Nautilia profundicola

Submarine hydrothermal vents are model systems for the Archaean Earth environment, and some sites maintain conditions that may have favored the formation and evolution of cellular life. Vents are typified by rapid fluctuations in temperature and redox potential that impose a strong selective pressure on resident microbial communities. Nautilia profundicola strain Am-H is a moderately thermophilic, deeply-branching Epsilonproteobacterium found free-living at hydrothermal vents and is a member of the microbial mass on the dorsal surface of vent polychaete, Alvinella pompejana. Analysis of the 1.7-Mbp genome of N. profundicola uncovered adaptations to the vent environment—some unique and some shared with other Epsilonproteobacterial genomes. The major findings included: (1) a diverse suite of hydrogenases coupled to a relatively simple electron transport chain, (2) numerous stress response systems, (3) a novel predicted nitrate assimilation pathway with hydroxylamine as a key intermediate, and (4) a gene (rgy) encoding the hallmark protein for hyperthermophilic growth, reverse gyrase. Additional experiments indicated that expression of rgy in strain Am-H was induced over 100-fold with a 20°C increase above the optimal growth temperature of this bacterium and that closely related rgy genes are present and expressed in bacterial communities residing in geographically distinct thermophilic environments. N. profundicola, therefore, is a model Epsilonproteobacterium that contains all the genes necessary for life in the extreme conditions widely believed to reflect those in the Archaean biosphere—anaerobic, sulfur, H2- and CO2-rich, with fluctuating redox potentials and temperatures. In addition, reverse gyrase appears to be an important and common adaptation for mesophiles and moderate thermophiles that inhabit ecological niches characterized by rapid and frequent temperature fluctuations and, as such, can no longer be considered a unique feature of hyperthermophiles.

The effect of temperature on enzyme activity: new insights and their implications

The two established thermal properties of enzymes are their activation energy and their thermal stability. Arising from careful measurements of the thermal behaviour of enzymes, a new model, the Equilibrium Model, has been developed to explain more fully the effects of temperature on enzymes. The model describes the effect of temperature on enzyme activity in terms of a rapidly reversible active-inactive transition, in addition to an irreversible thermal inactivation. Two new thermal parameters, Teq and ΔHeq, describe the active–inactive transition, and enable a complete description of the effect of temperature on enzyme activity. We review here the Model itself, methods for the determination of Teq and ΔHeq, and the implications of the Model for the environmental adaptation and evolution of enzymes, and for biotechnology.

Rapid microbial response to the presence of an ancient relic in the Antarctic Dry Valleys

The extreme cold and aridity of the Antarctic McMurdo Dry Valleys have led to the longstanding belief that metabolic rates of soil microbiota are negligible, and that ecosystem changes take place over millennia. Here we report the first direct experimental evidence that soil microbial communities undergo rapid and lasting changes in response to contemporary environmental conditions. Mummified seals, curious natural artifacts found scattered throughout Dry Valleys, alter their underlying soil environment by stabilizing temperatures, elevating relative humidity and reducing ultraviolet exposure. In a unique, multi-year mummified seal transplantation experiment, we found that endemic Dry Valley microbial communities responded to these changes within 3 years, resulting in a sevenfold increase in CO(2) flux and a significant reduction in biodiversity. These findings challenge prevailing ideas about Antarctic Dry Valley ecosystems and indicate that current and future environmental conditions may strongly influence the ecology of the dominant biota in the Dry Valleys.

Local and regional influences over soil microbial metacommunities in the Transantarctic Mountains

The metacommunity concept provides a useful framework to assess the influence of local and regional controls over diversity patterns. Culture-independent studies of soil microbial communities in the McMurdo Dry Valleys of East Antarctica (77° S) have shown that bacterial diversity is related to soil geochemical gradients, while studies targeting edaphic cyanobacteria have linked local diversity patterns to dispersal-based processes. In this study, we increased the spatial extent of observed soil microbial communities to cover the Beardmore Glacier region in the central Transantarctic Mountains (84° S). We used community profiling techniques to characterize diversity patterns for bacteria and the cyanobacterial subcomponent of the microbial community. Diversity partitioning was used to calculate beta diversity and estimate among-site dissimilarity in the metacommunity. We then used variation partitioning to assess the relationship between beta diversity and environmental and spatial gradients. We found that dominant groups in the soil bacterial metacommunity were influenced by gradients in pH and soil moisture at the Transantarctic scale (800 km). Conversely, beta diversity for the cyanobacterial component of the edaphic microbial metacommunity was decoupled from these environmental gradients, and was more related to spatial filters, suggesting that wind-driven dispersal dynamics created cyanobacterial biogeography at a local scale (<3 km).

Diffuse flow environments within basalt- and sediment-based hydrothermal vent ecosystems harbor specialized microbial communities.

Hydrothermal vents differ both in surface input and subsurface geochemistry. The effects of these differences on their microbial communities are not clear. Here, we investigated both alpha and beta diversity of diffuse flow-associated microbial communities emanating from vents at a basalt-based hydrothermal system along the East Pacific Rise (EPR) and a sediment-based hydrothermal system, Guaymas Basin. Both Bacteria and Archaea were targeted using high throughput 16S rRNA gene pyrosequencing analyses. A unique aspect of this study was the use of a universal set of 16S rRNA gene primers to characterize total and diffuse flow-specific microbial communities from varied deep-sea hydrothermal environments. Both surrounding seawater and diffuse flow water samples contained large numbers of Marine Group I (MGI) Thaumarchaea and Gammaproteobacteria taxa previously observed in deep-sea systems. However, these taxa were geographically distinct and segregated according to type of spreading center. Diffuse flow microbial community profiles were highly differentiated. In particular, EPR dominant diffuse flow taxa were most closely associated with chemolithoautotrophs, and off axis water was dominated by heterotrophic-related taxa, whereas the opposite was true for Guaymas Basin. The diversity and richness of diffuse flow-specific microbial communities were strongly correlated to the relative abundance of Epsilonproteobacteria, proximity to macrofauna, and hydrothermal system type. Archaeal diversity was higher than or equivalent to bacterial diversity in about one third of the samples. Most diffuse flow-specific communities were dominated by OTUs associated with Epsilonproteobacteria, but many of the Guaymas Basin diffuse flow samples were dominated by either OTUs within the Planctomycetes or hyperthermophilic Archaea. This study emphasizes the unique microbial communities associated with geochemically and geographically distinct hydrothermal diffuse flow environments.

Eurythermalism and the temperature dependence of enzyme activity

The “Equilibrium Model” has provided new tools for describing and investigating enzyme thermal adaptation. It has been shown that the effect of temperature on enzyme activity is not only governed by ∆G‡cat and ∆G‡inact but also by two new intrinsic parameters, ∆Heq and Teq, which describe the enthalpy and midpoint, respectively, of a reversible equilibrium between active and inactive (but not denatured) forms of enzyme. Twenty‐one enzymes from organisms with a wide range of growth temperatures were characterized using the Equilibrium Model. Statistical analysis indicates that Teq is a better predictor of growth temperature than enzyme stability (∆G‡inact). As expected from the Equilibrium Model, ∆Heq correlates with catalytic temperature tolerance of enzymes and thus can be declared the first intrinsic and quantitative measure of enzyme eurythermalism. Other findings shed light on the evolution of psychrophilic and thermophilic enzymes. The findings suggest that the description of the Equilibrium Model of the effect of temperature on enzyme activity applies to all enzymes regardless of their temperature origins and that its associated parameters, ∆Heq and Teq, are intrinsic and necessary parameters for characterizing the thermal properties of enzymes and their temperature adaptation and evolution.—Lee C. K., Daniel, R. M., Shepherd, C., Saul, D., Cary, S. C., Danson, M. J., Eisenthal, R., Peterson M. E. Eurythermal‐ism and the temperature dependence of enzyme activity FASEB J. 21, 1934–1941 (2007)

Application of an unmanned aerial vehicle in spatial mapping of terrestrial biology and human disturbance in the McMurdo Dry Valleys, East Antarctica

The McMurdo Dry Valleys of Antarctica are a unique yet threatened polar biome. Cyanobacterial mats form a large part of the standing biomass in the McMurdo Dry Valleys and are therefore an indicator of ecosystem productivity and health. They are, however, patchily distributed, and this has hampered spatial ecology studies due to the logistical challenges of ground-based field sampling. Here, we report the application of remote sensing using a fixed-wing unmanned aerial vehicle (UAV) and GIS spatial mapping to identify cyanobacterial mats, estimate their extent and discriminate between different mat types. Using the Spalding Pond area of Taylor Valley as a test site, we were able to identify mats on soil surfaces within the hyporheic zone, as well as benthic mats below the water surface. The mapping also clearly identified the footprint of campsites and walking trails on soils, and we highlight the potential of this technique in monitoring human impact in this fragile ecosystem.

Diversity and Distributional Patterns of Ciliates in Guaymas Basin Hydrothermal Vent Sediments

Little is known about protists at deep‐sea hydrothermal vents. The vent sites at Guaymas Basin in the Gulf of California are characterized by dense mats of filamentous pigmented or nonpigmented Beggiatoa that serve as markers of subsurface thermochemical gradients. We constructed 18S rRNA libraries to investigate ciliate assemblages in Beggiatoa mats and from bare sediments at the Guaymas vent site. Results indicated a high diversity of ciliates, with 156 operational taxonomic units identified in 548 sequences. Comparison between mat environments demonstrated that ciliate and bacterial assemblages from pigmented mats, nonpigmented mats, and bare sediments were significantly different and highly correlated with bacterial assemblages. Neither bacterial nor ciliate assemblages were correlated with environmental factors. The most abundant ciliates at Guaymas were more likely to be represented in clone libraries from other hydrothermal, deep‐sea, and/or anoxic or microaerophilic environments, supporting the hypothesis that these ciliate species are broadly distributed. The orange mat environment included a higher proportion of ciliate sequences that were more similar to those from other environmental studies than to cultured ciliate species, whereas clone libraries from bare sediments included sequences that were the most highly divergent from all other sequences and may represent species that are endemic to Guaymas.

Influence of soil properties on archaeal diversity and distribution in the McMurdo Dry Valleys, Antarctica

Archaea are the least understood members of the microbial community in Antarctic mineral soils. Although their occurrence in Antarctic coastal soils has been previously documented, little is known about their distribution in soils across the McMurdo Dry Valleys, Victoria Land. In this study, terminal-restriction fragment length polymorphism (t-RFLP) analysis and 454 pyrosequencing were coupled with a detailed analysis of soil physicochemical properties to characterize archaeal diversity and identify environmental factors that might shape and maintain archaeal communities in soils of the three southern most McMurdo Dry Valleys (Garwood, Marshall, and Miers Valley). Archaea were successfully detected in all inland and coastal mineral soils tested, revealing a low overall richness (mean of six operational taxonomic units [OTUs] per sample site). However, OTU richness was higher in some soils and this higher richness was positively correlated with soil water content, indicating water as a main driver of archaeal community richness. In total, 18 archaeal OTUs were detected, predominately Thaumarchaeota affiliated with Marine Group 1.1b (> 80% of all archaeal sequences recovered). Less abundant OTUs (2% of all archaeal sequences) were loosely related to members of the phylum Euryarchaeota. This is the first comprehensive study showing a widespread presence and distribution of Archaea in inland Antarctic soils.