From molecules to dynamic biological communities

Microbial ecology is flourishing, and in the process, is making contributions to how the ecology and biology of large organisms is understood. Ongoing advances in sequencing technology and computational methods have enabled the collection and analysis of vast amounts of molecular data from diverse biological communities. While early studies focused on cataloguing microbial biodiversity in environments ranging from simple marine ecosystems to complex soil ecologies, more recent research is concerned with community functions and their dynamics over time. Models and concepts from traditional ecology have been used to generate new insight into microbial communities, and novel system-level models developed to explain and predict microbial interactions. The process of moving from molecular inventories to functional understanding is complex and challenging, and never more so than when many thousands of dynamic interactions are the phenomena of interest. We outline the process of how epistemic transitions are made from producing catalogues of molecules to achieving functional and predictive insight, and show how those insights not only revolutionize what is known about biological systems but also about how to do biology itself. Examples will be drawn primarily from analyses of different human microbiota, which are the microbial consortia found in and on areas of the human body, and their associated microbiomes (the genes of those communities). Molecular knowledge of these microbiomes is transforming microbiological knowledge, as well as broader aspects of human biology, health and disease.     

The Ecology of Microbial Communities Associated with Macrocystis pyrifera

Kelp forests are characterized by high biodiversity and productivity, and the cycling of kelp-produced carbon is a vital process in this ecosystem. Although bacteria are assumed to play a major role in kelp forest carbon cycling, knowledge of the composition and diversity of these bacterial communities is lacking. Bacterial communities on the surface of Macrocystis pyrifera and adjacent seawater were sampled at the Hopkins Marine Station in Monterey Bay, CA, and further studied using 454-tag pyrosequencing of 16S RNA genes. Our results suggest that M. pyrifera-dominated kelp forests harbor distinct microbial communities that vary temporally. The distribution of sequence tags assigned to Gammaproteobacteria, Alphaproteobacteria and Bacteriodetes differed between the surface of the kelp and the surrounding water. Several abundant Rhodobacteraceae, uncultivated Gammaproteobacteria and Bacteriodetes-associated tags displayed considerable temporal variation, often with similar trends in the seawater and the surface of the kelp. Bacterial community structure and membership correlated with the kelp surface serving as host, and varied over time. Several kelp-specific taxa were highly similar to other bacteria known to either prevent the colonization of eukaryotic larvae or exhibit antibacterial activities. Some of these kelp-specific bacterial associations might play an important role for M. pyrifera. This study provides the first assessment of the diversity and phylogenetic profile of the bacterial communities associated with M. pyrifera.     

Variability within a pea core collection of LEAM and HSP22, two mitochondrial seed proteins involved in stress tolerance

LEAM, a late embryogenesis abundant protein, and HSP22, a small heat shock protein, were shown to accumulate in the mitochondria during pea (Pisum sativum L.) seed development, where they are expected to contribute to desiccation tolerance. Here, their expression was examined in seeds of 89 pea genotypes by Western blot analysis. All genotypes expressed LEAM and HSP22 in similar amounts. In contrast with HSP22, LEAM displayed different isoforms according to apparent molecular mass. Each of the 89 genotypes harboured a single LEAM isoform. Genomic and RT‐PCR analysis revealed four LEAM genes differing by a small variable indel in the coding region. These variations were consistent with the apparent molecular mass of each isoform. Indels, which occurred in repeated domains, did not alter the main properties of LEAM. Structural modelling indicated that the class A α‐helix structure, which allows interactions with the mitochondrial inner membrane in the dry state, was preserved in all isoforms, suggesting functionality is maintained. The overall results point out the essential character of LEAM and HSP22 in pea seeds. LEAM variability is discussed in terms of pea breeding history as well as LEA gene evolution mechanisms.     

Individuals' diet diversity influences gut microbial diversity in two freshwater fish (threespine stickleback and Eurasian perch)

Vertebrates' diets profoundly influence the composition of symbiotic gut microbial communities. Studies documenting diet‐microbiota associations typically focus on univariate or categorical diet variables. However, in nature individuals often consume diverse combinations of foods. If diet components act independently, each providing distinct microbial colonists or nutrients, we expect a positive relationship between diet diversity and microbial diversity. We tested this prediction within each of two fish species (stickleback and perch), in which individuals vary in their propensity to eat littoral or pelagic invertebrates or mixtures of both prey. Unexpectedly, in most cases individuals with more generalised diets had less diverse microbiota than dietary specialists, in both natural and laboratory populations. This negative association between diet diversity and microbial diversity was small but significant, and most apparent after accounting for complex interactions between sex, size and diet. Our results suggest that multiple diet components can interact non‐additively to influence gut microbial diversity.     

Major Histocompatibility Complex class IIb polymorphism influences gut microbiota composition and diversity

Animals harbour diverse communities of symbiotic bacteria, which differ dramatically among host individuals. This heterogeneity poses an immunological challenge: distinguishing between mutualistic and pathogenic members of diverse and host‐specific microbial communities. We propose that Major Histocompatibility class II (MHC) genotypes contribute to recognition and regulation of gut microbes, and thus, MHC polymorphism contributes to microbial variation among hosts. Here, we show that MHC IIb polymorphism is associated with among‐individual variation in gut microbiota within a single wild vertebrate population of a small fish, the threespine stickleback. We sampled stickleback from Cedar Lake, on Vancouver Island, and used next‐generation sequencing to genotype the sticklebacks’ gut microbiota (16S sequencing) and their MHC class IIb exon 2 sequences. The presence of certain MHC motifs was associated with altered relative abundance (increase or decrease) of some microbial Families. The effect sizes are modest and entail a minority of microbial taxa, but these results represent the first indication that MHC genotype may affect gut microbiota composition in natural populations (MHC‐microbe associations have also been found in a few studies of lab mice). Surprisingly, these MHC effects were frequently sex‐dependent. Finally, hosts with more diverse MHC motifs had less diverse gut microbiota. One implication is that MHC might influence the efficacy of therapeutic strategies to treat dysbiosis‐associated disease, including the outcome of microbial transplants between healthy and diseased patients. We also speculate that macroparasite‐driven selection on MHC has the potential to indirectly alter the host gut microbiota, and vice versa.     

Genetic variant in TP63 on locus 3q28 is associated with risk of lung adenocarcinoma among never-smoking females in Asia

A recent genome-wide association study (GWAS) of subjects from Japan and South Korea reported a novel association between the TP63 locus on chromosome 3q28 and risk of lung adenocarcinoma (p = 7.3 × 10(-12)); however, this association did not achieve genome-wide significance (p ≤ 10(-7)) among never-smoking males or females. To determine if this association with lung cancer risk is independent of tobacco use, we genotyped the TP63 SNPs reported by the previous GWAS (rs10937405 and rs4488809) in 3,467 never-smoking female lung cancer cases and 3,787 never-smoking female controls from 10 studies conducted in Taiwan, Mainland China, South Korea, and Singapore. Genetic variation in rs10937405 was associated with risk of lung adenocarcinoma [n = 2,529 cases; p = 7.1 × 10(-8); allelic risk = 0.80, 95% confidence interval (CI) = 0.74-0.87]. There was also evidence of association with squamous cell carcinoma of the lung (n = 302 cases; p = 0.037; allelic risk = 0.82, 95% CI = 0.67-0.99). Our findings provide strong evidence that genetic variation in TP63 is associated with the risk of lung adenocarcinoma among Asian females in the absence of tobacco smoking.     

Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms

DNA sequencing continues to decrease in cost with the Illumina HiSeq2000 generating up to 600 Gb of paired-end 100 base reads in a ten-day run. Here we present a protocol for community amplicon sequencing on the HiSeq2000 and MiSeq Illumina platforms, and apply that protocol to sequence 24 microbial communities from host-associated and free-living environments. A critical question as more sequencing platforms become available is whether biological conclusions derived on one platform are consistent with what would be derived on a different platform. We show that the protocol developed for these instruments successfully recaptures known biological results, and additionally that biological conclusions are consistent across sequencing platforms (the HiSeq2000 versus the MiSeq) and across the sequenced regions of amplicons.     

Impact of training sets on classification of high-throughput bacterial 16s rRNA gene surveys

Taxonomic classification of the thousands–millions of 16S rRNA gene sequences generated in microbiome studies is often achieved using a naïve Bayesian classifier (for example, the Ribosomal Database Project II (RDP) classifier), due to favorable trade-offs among automation, speed and accuracy. The resulting classification depends on the reference sequences and taxonomic hierarchy used to train the model; although the influence of primer sets and classification algorithms have been explored in detail, the influence of training set has not been characterized. We compared classification results obtained using three different publicly available databases as training sets, applied to five different bacterial 16S rRNA gene pyrosequencing data sets generated (from human body, mouse gut, python gut, soil and anaerobic digester samples). We observed numerous advantages to using the largest, most diverse training set available, that we constructed from the Greengenes (GG) bacterial/archaeal 16S rRNA gene sequence database and the latest GG taxonomy. Phylogenetic clusters of previously unclassified experimental sequences were identified with notable improvements (for example, 50% reduction in reads unclassified at the phylum level in mouse gut, soil and anaerobic digester samples), especially for phylotypes belonging to specific phyla (Tenericutes, Chloroflexi, Synergistetes and Candidate phyla TM6, TM7). Trimming the reference sequences to the primer region resulted in systematic improvements in classification depth, and greatest gains at higher confidence thresholds. Phylotypes unclassified at the genus level represented a greater proportion of the total community variation than classified operational taxonomic units in mouse gut and anaerobic digester samples, underscoring the need for greater diversity in existing reference databases.     

AIDS and associated malignancies

INTRODUCTION It has been over twenty years since the onset of the AIDS epidemic, and in spite of the tremendous progress made towards the understanding of the disease, the virus that causes the disease and the development of highly ef- fective anti-retrov…