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September 22, 2019  |  

Precise fecal microbiome of the herbivorous Tibetan antelope inhabiting high-altitude alpine plateau

The metataxonomic approach combining 16S rRNA gene amplicon sequencing using the PacBio technology with the application of the operational phylogenetic unit (OPU) approach, has been used to analyze the fecal microbial composition of the high-altitude and herbivorous Tibetan antelopes. The fecal samples of the antelope were collected in Hoh Xil National Nature Reserve, at an altitude over 4500 m, the largest depopulated zone in Qinghai-Tibetan Plateau, China, where non-native animals or humans may experience life-threatening acute mountain sickness. In total, 104 antelope fecal samples were enrolled in this study, and were clustered into 61,258 operational taxonomic units (OTUs) at an identity of 98.7% and affiliated with 757 OPUs, including 144 known species, 256 potentially new species, 103 potentially higher taxa within known lineages. In addition, 254 comprised sequences not affiliating with any known family, and the closest relatives were unclassified lineages of existing orders or classes. A total of 42 out of 757 OPUs conformed to the core fecal microbiome, of which four major lineages, namely, un-cultured Ruminococcaceae, Lachnospiraceae, Akkermansia and Christensenellaceae were associated with human health or longevity. The current study reveals that the fecal core microbiome of antelope is mainly composited of uncultured bacteria. The most abundant core taxa, namely, uncultured Ruminococcaceae, uncultured Akkermansia, uncultured Bacteroides, uncultured Christensenellaceae, uncultured Mollicutes, and uncultured Lachnospiraceae, may represent new bacterial candidates at high taxa levels, and several may have beneficial roles in health promotion or anti-intestinal dysbiosis. These organisms should be further isolated and evaluated for potential effect on human health and longevity.

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September 22, 2019  |  

Recent insights into the tick microbiome gained through next-generation sequencing.

The tick microbiome comprises communities of microorganisms, including viruses, bacteria and eukaryotes, and is being elucidated through modern molecular techniques. The advent of next-generation sequencing (NGS) technologies has enabled the genes and genomes within these microbial communities to be explored in a rapid and cost-effective manner. The advantages of using NGS to investigate microbiomes surpass the traditional non-molecular methods that are limited in their sensitivity, and conventional molecular approaches that are limited in their scalability. In recent years the number of studies using NGS to investigate the microbial diversity and composition of ticks has expanded. Here, we provide a review of NGS strategies for tick microbiome studies and discuss the recent findings from tick NGS investigations, including the bacterial diversity and composition, influential factors, and implications of the tick microbiome.

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September 22, 2019  |  

Soil microbial communities and elk foraging intensity: implications for soil biogeochemical cycling in the sagebrush steppe.

Foraging intensity of large herbivores may exert an indirect top-down ecological force on soil microbial communities via changes in plant litter inputs. We investigated the responses of the soil microbial community to elk (Cervus elaphus) winter range occupancy across a long-term foraging exclusion experiment in the sagebrush steppe of the North American Rocky Mountains, combining phylogenetic analysis of fungi and bacteria with shotgun metagenomics and extracellular enzyme assays. Winter foraging intensity was associated with reduced bacterial richness and increasingly distinct bacterial communities. Although fungal communities did not respond linearly to foraging intensity, a greater ß-diversity response to winter foraging exclusion was observed. Furthermore, winter foraging exclusion increased soil cellulolytic and hemicellulolytic enzyme potential and higher foraging intensity reduced chitinolytic gene abundance. Thus, future changes in winter range occupancy may shape biogeochemical processes via shifts in microbial communities and subsequent changes to their physiological capacities to cycle soil C and N.© 2017 John Wiley & Sons Ltd/CNRS.

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September 22, 2019  |  

Evaluation of 16S rRNA amplicon sequencing using two next-generation sequencing technologies for phylogenetic analysis of the rumen bacterial community in steers.

Next generation sequencing technologies have vastly changed the approach of sequencing of the 16S rRNA gene for studies in microbial ecology. Three distinct technologies are available for large-scale 16S sequencing. All three are subject to biases introduced by sequencing error rates, amplification primer selection, and read length, which can affect the apparent microbial community. In this study, we compared short read 16S rRNA variable regions, V1-V3, with that of near-full length 16S regions, V1-V8, using highly diverse steer rumen microbial communities, in order to examine the impact of technology selection on phylogenetic profiles. Short paired-end reads from the Illumina MiSeq platform were used to generate V1-V3 sequence, while long “circular consensus” reads from the Pacific Biosciences RSII instrument were used to generate V1-V8 data. The two platforms revealed similar microbial operational taxonomic units (OTUs), as well as similar species richness, Good’s coverage, and Shannon diversity metrics. However, the V1-V8 amplified ruminal community resulted in significant increases in several orders of taxa, such as phyla Proteobacteria and Verrucomicrobia (P < 0.05). Taxonomic classification accuracy was also greater in the near full-length read. UniFrac distance matrices using jackknifed UPGMA clustering also noted differences between the communities. These data support the consensus that longer reads result in a finer phylogenetic resolution that may not be achieved by shorter 16S rRNA gene fragments. Our work on the cattle rumen bacterial community demonstrates that utilizing near full-length 16S reads may be useful in conducting a more thorough study, or for developing a niche-specific database to use in analyzing data from shorter read technologies when budgetary constraints preclude use of near-full length 16S sequencing. Copyright © 2016 Elsevier B.V. All rights reserved.

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September 22, 2019  |  

The gut commensal microbiome of Drosophila melanogaster is modified by the endosymbiont Wolbachia.

Endosymbiotic Wolbachia bacteria and the gut microbiome have independently been shown to affect several aspects of insect biology, including reproduction, development, life span, stem cell activity, and resistance to human pathogens, in insect vectors. This work shows that Wolbachia bacteria, which reside mainly in the fly germline, affect the microbial species present in the fly gut in a lab-reared strain. Drosophila melanogaster hosts two main genera of commensal bacteria-Acetobacter and Lactobacillus. Wolbachia-infected flies have significantly reduced titers of Acetobacter. Sampling of the microbiome of axenic flies fed with equal proportions of both bacteria shows that the presence of Wolbachia bacteria is a significant determinant of the composition of the microbiome throughout fly development. However, this effect is host genotype dependent. To investigate the mechanism of microbiome modulation, the effect of Wolbachia bacteria on Imd and reactive oxygen species pathways, the main regulators of immune response in the fly gut, was measured. The presence of Wolbachia bacteria does not induce significant changes in the expression of the genes for the effector molecules in either pathway. Furthermore, microbiome modulation is not due to direct interaction between Wolbachia bacteria and gut microbes. Confocal analysis shows that Wolbachia bacteria are absent from the gut lumen. These results indicate that the mechanistic basis of the modulation of composition of the microbiome by Wolbachia bacteria is more complex than a direct bacterial interaction or the effect of Wolbachia bacteria on fly immunity. The findings reported here highlight the importance of considering the composition of the gut microbiome and host genetic background during Wolbachia-induced phenotypic studies and when formulating microbe-based disease vector control strategies. IMPORTANCE Wolbachia bacteria are intracellular bacteria present in the microbiome of a large fraction of insects and parasitic nematodes. They can block mosquitos’ ability to transmit several infectious disease-causing pathogens, including Zika, dengue, chikungunya, and West Nile viruses and malaria parasites. Certain extracellular bacteria present in the gut lumen of these insects can also block pathogen transmission. However, our understanding of interactions between Wolbachia and gut bacteria and how they influence each other is limited. Here we show that the presence of Wolbachia strain wMel changes the composition of gut commensal bacteria in the fruit fly. Our findings implicate interactions between bacterial species as a key factor in determining the overall composition of the microbiome and thus reveal new paradigms to consider in the development of disease control strategies.

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September 22, 2019  |  

Improved metagenome assemblies and taxonomic binning using long-read circular consensus sequence data.

DNA assembly is a core methodological step in metagenomic pipelines used to study the structure and function within microbial communities. Here we investigate the utility of Pacific Biosciences long and high accuracy circular consensus sequencing (CCS) reads for metagenomic projects. We compared the application and performance of both PacBio CCS and Illumina HiSeq data with assembly and taxonomic binning algorithms using metagenomic samples representing a complex microbial community. Eight SMRT cells produced approximately 94 Mb of CCS reads from a biogas reactor microbiome sample that averaged 1319 nt in length and 99.7% accuracy. CCS data assembly generated a comparative number of large contigs greater than 1?kb, to those assembled from a ~190x larger HiSeq dataset (~18 Gb) produced from the same sample (i.e approximately 62% of total contigs). Hybrid assemblies using PacBio CCS and HiSeq contigs produced improvements in assembly statistics, including an increase in the average contig length and number of large contigs. The incorporation of CCS data produced significant enhancements in taxonomic binning and genome reconstruction of two dominant phylotypes, which assembled and binned poorly using HiSeq data alone. Collectively these results illustrate the value of PacBio CCS reads in certain metagenomics applications.

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September 22, 2019  |  

Multiplex amplicon sequencing for microbe identification in community-based culture collections.

Microbiome analysis using metagenomic sequencing has revealed a vast microbial diversity associated with plants. Identifying the molecular functions associated with microbiome-plant interaction is a significant challenge concerning the development of microbiome-derived technologies applied to agriculture. An alternative to accelerate the discovery of the microbiome benefits to plants is to construct microbial culture collections concomitant with accessing microbial community structure and abundance. However, traditional methods of isolation, cultivation, and identification of microbes are time-consuming and expensive. Here we describe a method for identification of microbes in culture collections constructed by picking colonies from primary platings that may contain single or multiple microorganisms, which we named community-based culture collections (CBC). A multiplexing 16S rRNA gene amplicon sequencing based on two-step PCR amplifications with tagged primers for plates, rows, and columns allowed the identification of the microbial composition regardless if the well contains single or multiple microorganisms. The multiplexing system enables pooling amplicons into a single tube. The sequencing performed on the PacBio platform led to recovery near-full-length 16S rRNA gene sequences allowing accurate identification of microorganism composition in each plate well. Cross-referencing with plant microbiome structure and abundance allowed the estimation of diversity and abundance representation of microorganism in the CBC.

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September 22, 2019  |  

Atmospheric N deposition alters connectance, but not functional potential among saprotrophic bacterial communities.

The use of co-occurrence patterns to investigate interactions between micro-organisms has provided novel insight into organismal interactions within microbial communities. However, anthropogenic impacts on microbial co-occurrence patterns and ecosystem function remain an important gap in our ecological knowledge. In a northern hardwood forest ecosystem located in Michigan, USA, 20 years of experimentally increased atmospheric N deposition has reduced forest floor decay and increased soil C storage. This ecosystem-level response occurred concomitantly with compositional changes in saprophytic fungi and bacteria. Here, we investigated the influence of experimental N deposition on biotic interactions among forest floor bacterial assemblages by employing phylogenetic and molecular ecological network analysis. When compared to the ambient treatment, the forest floor bacterial community under experimental N deposition was less rich, more phylogenetically dispersed and exhibited a more clustered co-occurrence network topology. Together, our observations reveal the presence of increased biotic interactions among saprotrophic bacterial assemblages under future rates of N deposition. Moreover, they support the hypothesis that nearly two decades of experimental N deposition can modify the organization of microbial communities and provide further insight into why anthropogenic N deposition has reduced decomposition, increased soil C storage and accelerated phenolic DOC production in our field experiment. © 2015 John Wiley & Sons Ltd.

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September 22, 2019  |  

Gill bacteria enable a novel digestive strategy in a wood-feeding mollusk.

Bacteria play many important roles in animal digestive systems, including the provision of enzymes critical to digestion. Typically, complex communities of bacteria reside in the gut lumen in direct contact with the ingested materials they help to digest. Here, we demonstrate a previously undescribed digestive strategy in the wood-eating marine bivalve Bankia setacea, wherein digestive bacteria are housed in a location remote from the gut. These bivalves, commonly known as shipworms, lack a resident microbiota in the gut compartment where wood is digested but harbor endosymbiotic bacteria within specialized cells in their gills. We show that this comparatively simple bacterial community produces wood-degrading enzymes that are selectively translocated from gill to gut. These enzymes, which include just a small subset of the predicted wood-degrading enzymes encoded in the endosymbiont genomes, accumulate in the gut to the near exclusion of other endosymbiont-made proteins. This strategy of remote enzyme production provides the shipworm with a mechanism to capture liberated sugars from wood without competition from an endogenous gut microbiota. Because only those proteins required for wood digestion are translocated to the gut, this newly described system reveals which of many possible enzymes and enzyme combinations are minimally required for wood degradation. Thus, although it has historically had negative impacts on human welfare, the shipworm digestive process now has the potential to have a positive impact on industries that convert wood and other plant biomass to renewable fuels, fine chemicals, food, feeds, textiles, and paper products.

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September 22, 2019  |  

Towards long-read metagenomics: complete assembly of three novel genomes from bacteria dependent on a diazotrophic cyanobacterium in a freshwater lake co-culture.

Here we report three complete bacterial genome assemblies from a PacBio shotgun metagenome of a co-culture from Upper Klamath Lake, OR. Genome annotations and culture conditions indicate these bacteria are dependent on carbon and nitrogen fixation from the cyanobacterium Aphanizomenon flos-aquae, whose genome was assembled to draft-quality. Due to their taxonomic novelty relative to previously sequenced bacteria, we have temporarily designated these bacteria as incertae sedis Hyphomonadaceae strain UKL13-1 (3,501,508 bp and 56.12% GC), incertae sedis Betaproteobacterium strain UKL13-2 (3,387,087 bp and 54.98% GC), and incertae sedis Bacteroidetes strain UKL13-3 (3,236,529 bp and 37.33% GC). Each genome consists of a single circular chromosome with no identified plasmids. When compared with binned Illumina assemblies of the same three genomes, there was ~7% discrepancy in total genome length. Gaps where Illumina assemblies broke were often due to repetitive elements. Within these missing sequences were essential genes and genes associated with a variety of functional categories. Annotated gene content reveals that both Proteobacteria are aerobic anoxygenic phototrophs, with Betaproteobacterium UKL13-2 potentially capable of phototrophic oxidation of sulfur compounds. Both proteobacterial genomes contain transporters suggesting they are scavenging fixed nitrogen from A. flos-aquae in the form of ammonium. Bacteroidetes UKL13-3 has few completely annotated biosynthetic pathways, and has a comparatively higher proportion of unannotated genes. The genomes were detected in only a few other freshwater metagenomes, suggesting that these bacteria are not ubiquitous in freshwater systems. Our results indicate that long-read sequencing is a viable method for sequencing dominant members from low-diversity microbial communities, and should be considered for environmental metagenomics when conditions meet these requirements.

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September 22, 2019  |  

Effect of dietary interventions on the intestinal microbiota of Mongolian hosts

The gut microbiota of Mongolian hosts has distinctive characteristics due to their meat- and dairy-oriented daily diets and unique genotype. The aim of the present study was to investigate the effect of switching from the typical high protein and fat Mongolian diets to carbohydrate-rich meals composed principally of wheat, rice and naked oats on the host gut microbiota within 3 weeks. Our study took the advantage of the long sequence reads produced by the PacBio single molecule real-time sequencing technology to enable the profiling of subjects’ gut microbiota communities along the diet intervention to the species precision. We found that the bacterial richness and diversity decreased apparently along the diet intervention. During the diet intervention, the gut microbiota composition displayed no significant difference at phylum level (with major phyla of Firmicutes, Bacteroidetes, Tenericutes and Proteobacteria). The relative abundances of some genera such as Bacteroidetes, Faecalibacterium, Roseburia, Alistipes, Streptococcus, and Oscillospira were significantly altered after the diet switching started. Notably, significant changes were also observed in the proportions of the species Bacteroides dorei, Bacteroides fragilis, Bacteroides thetaiotaomicron, Ruminococcus albus, Ruminococcus faecis, Roseburia faecis and Eubacterium ventriosum. These results have demonstrated that diet and host gut microbiota is closely linked.

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September 22, 2019  |  

Metasecretome phage display.

Metasecretome is a collection of cell-surface and secreted proteins that mediate interactions between microbial communities and their environment. These include adhesins, enzymes, surface structures such as pili or flagella, vaccine targets or proteins responsible for immune evasion. Traditional approaches to exploring matasecretome of complex microbial communities via cultivation of microorganisms and screening of individual strains fail to sample extraordinary diversity in these communities, since only a limited fraction of microorganisms are represented by cultures. Advances in culture-independent sequence analysis methods, collectively referred to as metagenomics, offer an alternative approach that enables the direct analysis of collective microbial genomes (metagenome) recovered from environmental samples. This protocol describes a method, metasecretome phage display, which selectively displays the metasecretome portion of the metagenome. The metasecretome library can then be used for two purposes: (1) to sequence the entire metasecretome (using PacBio technology); (2) to identify metasecretome proteins that have a specific function of interest by affinity-screening (bio-panning) using a variety of methods described in other chapters of this volume.

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September 22, 2019  |  

Metagenomic binning of a marine sponge microbiome reveals unity in defense but metabolic specialization.

Marine sponges are ancient metazoans that are populated by distinct and highly diverse microbial communities. In order to obtain deeper insights into the functional gene repertoire of the Mediterranean sponge Aplysina aerophoba, we combined Illumina short-read and PacBio long-read sequencing followed by un-targeted metagenomic binning. We identified a total of 37 high-quality bins representing 11 bacterial phyla and two candidate phyla. Statistical comparison of symbiont genomes with selected reference genomes revealed a significant enrichment of genes related to bacterial defense (restriction-modification systems, toxin-antitoxin systems) as well as genes involved in host colonization and extracellular matrix utilization in sponge symbionts. A within-symbionts genome comparison revealed a nutritional specialization of at least two symbiont guilds, where one appears to metabolize carnitine and the other sulfated polysaccharides, both of which are abundant molecules in the sponge extracellular matrix. A third guild of symbionts may be viewed as nutritional generalists that perform largely the same metabolic pathways but lack such extraordinary numbers of the relevant genes. This study characterizes the genomic repertoire of sponge symbionts at an unprecedented resolution and it provides greater insights into the molecular mechanisms underlying microbial-sponge symbiosis.

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September 22, 2019  |  

Genomic microdiversity of Bifidobacterium pseudocatenulatum underlying differential strain-level responses to dietary carbohydrate intervention.

The genomic basis of the response to dietary intervention of human gut beneficial bacteria remains elusive, which hinders precise manipulation of the microbiota for human health. After receiving a dietary intervention enriched with nondigestible carbohydrates for 105 days, a genetically obese child with Prader-Willi syndrome lost 18.4% of his body weight and showed significant improvement in his bioclinical parameters. We obtained five isolates (C1, C15, C55, C62, and C95) of one of the most abundantly promoted beneficial species, Bifidobacterium pseudocatenulatum, from a postintervention fecal sample. Intriguingly, these five B. pseudocatenulatum strains showed differential responses during the dietary intervention. Two strains were largely unaffected, while the other three were promoted to different extents by the changes in dietary carbohydrate resources. The differential responses of these strains were consistent with their functional clustering based on the COGs (Clusters of Orthologous Groups), including those involved with the ABC-type sugar transport systems, suggesting that the strain-specific genomic variations may have contributed to the niche adaption. Particularly, B. pseudocatenulatum C15, which had the most diverse types and highest gene copy numbers of carbohydrate-active enzymes targeting plant polysaccharides, had the highest abundance after the dietary intervention. These studies show the importance of understanding genomic diversity of specific members of the gut microbiota if precise nutrition approaches are to be realized.IMPORTANCE The manipulation of the gut microbiota via dietary approaches is a promising option for improving human health. Our findings showed differential responses of multiple B. pseudocatenulatum strains isolated from the same habitat to the dietary intervention, as well as strain-specific correlations with bioclinical parameters of the host. The comparative genomics revealed a genome-level microdiversity of related functional genes, which may have contributed to these differences. These results highlight the necessity of understanding strain-level differences if precise manipulation of gut microbiota through dietary approaches is to be realized. Copyright © 2017 Wu et al.

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