A method for studying protistan diversity using massively parallel sequencing of V9 hypervariable regions of small-subunit ribosomal RNA genes

© 2009 The Authors. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in PLoS ONE 4 (2009): e6372, doi:10.1371/journal.pone.0006372.Massively parallel pyrosequencing of amplicons from the V6 hypervariable regions of small-subunit (SSU) ribosomal RNA (rRNA) genes is commonly used to assess diversity and richness in bacterial and archaeal populations. Recent advances in pyrosequencing technology provide read lengths of up to 240 nucleotides. Amplicon pyrosequencing can now be applied to longer variable regions of the SSU rRNA gene including the V9 region in eukaryotes. We present a protocol for the amplicon pyrosequencing of V9 regions for eukaryotic environmental samples for biodiversity inventories and species richness estimation. The International Census of Marine Microbes (ICoMM) and the Microbial Inventory Research Across Diverse Aquatic Long Term Ecological Research Sites (MIRADA-LTERs) projects are already employing this protocol for tag sequencing of eukaryotic samples in a wide diversity of both marine and freshwater environments. Massively parallel pyrosequencing of eukaryotic V9 hypervariable regions of SSU rRNA genes provides a means of estimating species richness from deeply-sampled populations and for discovering novel species from the environment.This work was supported by grants from the W.M. Keck Foundation and the Woods Hole Center for Oceans and Human Health from the National Institutes of Health and National Science Foundation (NIH/NIEHS 1 P50 ES012742-01 and NSF/OCE 0430724-J) (LAZ and SH)

Contrasting Microbial Community Assembly Hypotheses: A Reconciling Tale from the Río Tinto

The Río Tinto (RT) is distinguished from other acid mine drainage systems by its natural and ancient origins. Microbial life from all three domains flourishes in this ecosystem, but bacteria dominate metabolic processes that perpetuate environmental extremes. While the patchy geochemistry of the RT likely influences the dynamics of bacterial populations, demonstrating which environmental variables shape microbial diversity and unveiling the mechanisms underlying observed patterns, remain major challenges in microbial ecology whose answers rely upon detailed assessments of community structures coupled with fine-scale measurements of physico-chemical parameters.By using high-throughput environmental tag sequencing we achieved saturation of richness estimators for the first time in the RT. We found that environmental factors dictate the distribution of the most abundant taxa in this system, but stochastic niche differentiation processes, such as mutation and dispersal, also contribute to observed diversity patterns.We predict that studies providing clues to the evolutionary and ecological processes underlying microbial distributions will reconcile the ongoing debate between the Baas Becking vs. Hubbell community assembly hypotheses

Quantitative models of in vitro bacteriophage-host dynamics and their application to phage therapy

Phage therapy is the use of bacteriophages as antimicrobial agents for the control of pathogenic and other problem bacteria. It has previously been argued that successful application of phage therapy requires a good understanding of the non-linear kinetics of phage-bacteria interactions. Here we combine experimental and modelling approaches to make a detailed examination of such kinetics for the important food-borne pathogen Campylobacter jejuni and a suitable virulent phage in an in vitro system. Phage-insensitive populations of C. jejuni arise readily, and as far as we are aware this is the first phage therapy study to test, against in vitro data, models for phage-bacteria interactions incorporating phage-insensitive or resistant bacteria. We find that even an apparently simplistic model fits the data surprisingly well, and we confirm that the so-called inundation and proliferation thresholds are likely to be of considerable practical importance to phage therapy. We fit the model to time series data in order to estimate thresholds and rate constants directly. A comparison of the fit for each culture reveals density-dependent features of phage infectivity that are worthy of further investigation. Our results illustrate how insight from empirical studies can be greatly enhanced by the use of kinetic models: such combined studies of in vitro systems are likely to be an essential precursor to building a meaningful picture of the kinetic properties of in vivo phage therapy