Courtney K. Robinson

Colonization patterns of soil microbial communities in the Atacama Desert

BackgroundThe Atacama Desert is one of the driest deserts in the world and its soil, with extremely low moisture, organic carbon content, and oxidizing conditions, is considered to be at the dry limit for life.ResultsAnalyses of high throughput DNA sequence data revealed that bacterial communities from six geographic locations in the hyper-arid core and along a North-South moisture gradient were structurally and phylogenetically distinct (ANOVA test for observed operating taxonomic units at 97% similarity (OTU0.03), P <0.001) and that communities from locations in the hyper-arid zone displayed the lowest levels of diversity. We found bacterial taxa similar to those found in other arid soil communities with an abundance of Rubrobacterales, Actinomycetales, Acidimicrobiales, and a number of families from the Thermoleophilia. The extremely low abundance of Firmicutes indicated that most bacteria in the soil were in the form of vegetative cells. Integrating molecular data with climate and soil geochemistry, we found that air relative humidity (RH) and soil conductivity significantly correlated with microbial communities’ diversity metrics (least squares linear regression for observed OTU0.03 and air RH and soil conductivity, P <0.001; UniFrac PCoA Spearman’s correlation for air RH and soil conductivity, P <0.0001), indicating that water availability and salt content are key factors in shaping the Atacama soil microbiome. Mineralization studies showed communities actively metabolizing in all soil samples, with increased rates in soils from the southern locations.ConclusionsOur results suggest that microorganisms in the driest soils of the Atacama Desert are in a state of stasis for most of the time, but can potentially metabolize if presented with liquid water for a sufficient duration. Over geological time, rare rain events and physicochemical factors potentially played a major role in selecting micro-organisms that are most adapted to extreme desiccating conditions.

Coordination of frontline defense mechanisms under severe oxidative stress

Complexity of cellular response to oxidative stress (OS) stems from its wide‐ranging damage to nucleic acids, proteins, carbohydrates, and lipids. We have constructed a systems model of OS response (OSR) for Halobacterium salinarum NRC‐1 in an attempt to understand the architecture of its regulatory network that coordinates this complex response. This has revealed a multi‐tiered OS‐management program to transcriptionally coordinate three peroxidase/catalase enzymes, two superoxide dismutases, production of rhodopsins, carotenoids and gas vesicles, metal trafficking, and various other aspects of metabolism. Through experimental validation of interactions within the OSR regulatory network, we show that despite their inability to directly sense reactive oxygen species, general transcription factors have an important function in coordinating this response. Remarkably, a significant fraction of this OSR was accurately recapitulated by a model that was earlier constructed from cellular responses to diverse environmental perturbations—this constitutes the general stress response component. Notwithstanding this observation, comparison of the two models has identified the coordination of frontline defense and repair systems by regulatory mechanisms that are triggered uniquely by severe OS and not by other environmental stressors, including sub‐inhibitory levels of redox‐active metals, extreme changes in oxygen tension, and a sub‐lethal dose of γ rays.

Intricacies of assessing the human microbiome in epidemiologic studies.

PURPOSE In the past decade, remarkable relationships have been documented between dysbiosis of the human microbiota and adverse health outcomes. This review seeks to highlight some of the challenges and pitfalls that may be encountered during all stages of microbiota research, from study design and sample collection, to nucleic acid extraction and sequencing, and bioinformatic and statistical analysis. METHODS Literature focused on human microbiota research was reviewed and summarized. RESULTS Although most studies have focused on surveying the composition of the microbiota, fewer have explored the causal roles of these bacteria, archaea, viruses, and fungi in affecting disease states. Microbiome research is in its relatively early years and many aspects remain challenging, including the complexity and personalized aspects of microbial communities, the influence of exogenous and often confounding factors, the need to apply fundamental principles of ecology and epidemiology, the necessity for new software tools, and the rapidly evolving genomic, technological, and analytical landscapes. CONCLUSIONS Incorporating human microbiome research in large epidemiologic studies will soon help us unravel the intricate relationships that we have with our microbial partners and provide interventional opportunities to improve human health.

Effects of intracellular Mn on the radiation resistance of the halophilic archaeon Halobacterium salinarum

Ionizing radiation (IR) is of particular interest in biology because its exposure results in severe oxidative stress to the cell’s macromolecules. Our recent work with extremophiles supports the idea that IR resistance is most likely achieved by a metabolic route, effected by manganese (Mn) antioxidants. Biochemical analysis of “super-IR resistant” mutants of H. salinarum, evolved over multiple cycles of exposure to high doses of IR, confirmed the key role for Mn antioxidants in the IR resistance of this organism. Analysis of the proteome of H. salinarum “super-IR resistant” mutants revealed increased expression for proteins involved in energy metabolism, replenishing the cell with reducing equivalents depleted by the oxidative stress inflicted by IR. Maintenance of redox homeostasis was also activated by the over-expression of coenzyme biosynthesis pathways involved in redox reactions. We propose that in H. salinarum, increased tolerance to IR is a combination of metabolic regulatory adjustments and the accumulation of Mn-antioxidant complexes.

Phylogenetic and Functional Substrate Specificity for Endolithic Microbial Communities in Hyper-Arid Environments

Under extreme water deficit, endolithic (inside rock) microbial ecosystems are considered environmental refuges for life in cold and hot deserts, yet their diversity and functional adaptations remain vastly unexplored. The metagenomic analyses of the communities from two rock substrates, calcite and ignimbrite, revealed that they were dominated by Cyanobacteria, Actinobacteria, and Chloroflexi. The relative distribution of major phyla was significantly different between the two substrates and biodiversity estimates, from 16S rRNA gene sequences and from the metagenomic data, all pointed to a higher taxonomic diversity in the calcite community. While both endolithic communities showed adaptations to extreme aridity and to the rock habitat, their functional capabilities revealed significant differences. ABC transporters and pathways for osmoregulation were more diverse in the calcite chasmoendolithic community. In contrast, the ignimbrite cryptoendolithic community was enriched in pathways for secondary metabolites, such as non-ribosomal peptides (NRP) and polyketides (PK). Assemblies of the metagenome data produced population genomes for the major phyla found in both communities and revealed a greater diversity of Cyanobacteria population genomes for the calcite substrate. Draft genomes of the dominant Cyanobacteria in each community were constructed with more than 93% estimated completeness. The two annotated proteomes shared 64% amino acid identity and a significantly higher number of genes involved in iron update, and NRPS gene clusters, were found in the draft genomes from the ignimbrite. Both the community-wide and genome-specific differences may be related to higher water availability and the colonization of large fissures and cracks in the calcite in contrast to a harsh competition for colonization space and nutrient resources in the narrow pores of the ignimbrite. Together, these results indicated that the habitable architecture of both lithic substrates- chasmoendolithic versus cryptoendolithic – might be an essential element in determining the colonization and the diversity of the microbial communities in endolithic substrates at the dry limit for life.

Ultra-high throughput multiplexing and sequencing of >500 bp amplicon regions on the Illumina HiSeq 2500 platform

Amplification, sequencing and analysis of the 16S rRNA gene affords characterization of microbial community composition. As this tool has become more popular and projects have grown in size and scope, greater sample multiplexing is becoming necessary while maintaining high quality sequencing. Here, modifications to the Illumina HiSeq 2500 platform are described that afford greater multiplexing and 300 bp paired-end reads of higher quality than produced by the current Illumina MiSeq platform. To improve the feasibility and flexibility of this method, a 2-Step PCR amplification protocol is also described that allows for targeting of different amplicon regions, thus improving amplification success from low bacterial bioburden samples.

O06.4 Higher levels of a cytotoxic protein, vaginolysin, in lactobacillus-deficient community state types in the vaginal mucosa

Introduction Bacterial cytotoxic proteins, such as vaginolysin (VLY) produced by Gardnerella vaginalis, are thought to be virulence factors that in vitro alter cell integrity and local immunity. VLY may play a significant role in bacterial vaginosis (BV), therefore we assessed whether G. vaginalis dominant vaginal microbiota and immune markers were associated with higher concentrations of VLY in reproductive-age women. Methods Forty women self-collected mid-vaginal swabs in a cross-sectional study. Microbial communities were characterised by 16S rRNA gene amplicon sequence analysis. VLY (ng/ml) was detected by ELISA and normalised by a cube root transformation. Absolute bacterial abundance of G. vaginalis (log10 transformed) was estimated by multiplying its relative abundance by the sample total bacterial burden estimated by qPCR. Pro-inflammatory immune markers were quantified by Luminex and categorised above and below the median. Multivariate linear regression models evaluated factors associated with VLY abundance and controlled for confounders, including smoking and history of vaginal douching. Results Vaginal microbiota clustered into 3 community state types (CSTs); 2 dominated by Lactobacillus spp. (Lactibacillus iners (CST-III) or L. crispatus (CST-I)), and one lacking Lactobacillus spp. and characterised by BV-associated bacteria including G. vaginalis and Atopobium vaginae (CST-IV). In the multivariate analysis, CST-IV, G. vaginalis bacterial load, a high Nugent Gram stain score, and a more basic vaginal pH (all p<0.03) were positively associated with increasing concentrations of VLY. TNF-α, TGF-β and IL-8 were inversely associated with VLY but only TNF-α remained significant in multivariate analysis (p=0.01). Conclusion This study confirms that vaginal microbiota lacking lactobacilli, as well as other clinical indicators of BV, were associated with higher concentrations of VLY in vivo. Inflammatory markers were inversely associated with VLY. Because VLY may alter the vaginal microbiota and local inflammation, the role of VLY in BV warrants further evaluation.