Menu

Auto Tag: Microbiota

September 22, 2019

The Florida manatee (Trichechus manatus latirostris) immunoglobulin heavy chain suggests the importance of clan III variable segments in repertoire diversity.

Manatees are a vulnerable, charismatic sentinel species from the evolutionarily divergent Afrotheria. Manatee health and resistance to infectious disease is of great concern to conservation groups, but little is known about their immune system. To develop manatee-specific tools for monitoring health, we first must have a general knowledge of how the immunoglobulin heavy (IgH) chain locus is organized and transcriptionally expressed. Using the genomic scaffolds of the Florida manatee (Trichechus manatus latirostris), we characterized the potential IgH segmental diversity and constant region isotypic diversity and performed the first Afrotherian repertoire analysis. The Florida manatee has low V(D)J combinatorial diversity (3744 potential combinations) and few constant region isotypes. They also lack clan III V segments, which may have caused reduced VH segment numbers. However, we found productive somatic hypermutation concentrated in the complementarity determining regions. In conclusion, manatees have limited IGHV clan and combinatorial diversity. This suggests that clan III V segments are essential for maintaining IgH locus diversity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Read more
September 22, 2019

Metagenomic approaches to assess bacteriophages in various environmental niches.

Bacteriophages are ubiquitous and numerous parasites of bacteria and play a critical evolutionary role in virtually every ecosystem, yet our understanding of the extent of the diversity and role of phages remains inadequate for many ecological niches, particularly in cases in which the host is unculturable. During the past 15 years, the emergence of the field of viral metagenomics has drastically enhanced our ability to analyse the so-called viral ‘dark matter’ of the biosphere. Here, we review the evolution of viral metagenomic methodologies, as well as providing an overview of some of the most significant applications and findings in this field of research.

Read more
September 22, 2019

Crosstalk between gut microbiota and Sirtuin-3 in colonic inflammation and tumorigenesis.

Colorectal cancer (CRC) is a disease involving a variety of genetic and environmental factors. Sirtuin-3 (Sirt3) is expressed at a low level in cancer tissues of CRC, but it is unclear how Sirt3 modulates colonic tumorigenesis. In this study, we found that gut microbiota play a central role in the resistance to CRC tumor formation in wild-type (WT) mice through APC (Adenomatous Polyposis Coli)-mutant mouse microbiota transfer via Wnt signaling. We also found that Sirt3-deficient mice were hypersusceptible to colonic inflammation and tumor development through altered intestinal integrity and p38 signaling, respectively. Furthermore, susceptibility to colorectal tumorigenesis was aggravated by initial commensal microbiota deletion via Wnt signaling. Mice with Sirt3-deficient microbiota transfer followed by chemically induced colon tumorigenesis had low Sirt3 expression compared to WT control microbiome transfer, mainly due to a decrease in Escherichia/Shigella, as well as an increase in Lactobacillus reuteri and Lactobacillus taiwanensis. Collectively, our data revealed that Sirt3 is an anti-inflammatory and tumor-suppressing gene that interacts with the gut microbiota during colon tumorigenesis.

Read more
September 22, 2019

Discovery of enzymes for toluene synthesis from anoxic microbial communities.

Microbial toluene biosynthesis was reported in anoxic lake sediments more than three decades ago, but the enzyme catalyzing this biochemically challenging reaction has never been identified. Here we report the toluene-producing enzyme PhdB, a glycyl radical enzyme of bacterial origin that catalyzes phenylacetate decarboxylation, and its cognate activating enzyme PhdA, a radical S-adenosylmethionine enzyme, discovered in two distinct anoxic microbial communities that produce toluene. The unconventional process of enzyme discovery from a complex microbial community (>300,000 genes), rather than from a microbial isolate, involved metagenomics- and metaproteomics-enabled biochemistry, as well as in vitro confirmation of activity with recombinant enzymes. This work expands the known catalytic range of glycyl radical enzymes (only seven reaction types had been characterized previously) and aromatic-hydrocarbon-producing enzymes, and will enable first-time biochemical synthesis of an aromatic fuel hydrocarbon from renewable resources, such as lignocellulosic biomass, rather than from petroleum.

Read more
September 22, 2019

Metagenomic and near full-length 16S rRNA sequence data in support of the phylogenetic analysis of the rumen bacterial community in steers.

Amplicon sequencing utilizing next-generation platforms has significantly transformed how research is conducted, specifically microbial ecology. However, primer and sequencing platform biases can confound or change the way scientists interpret these data. The Pacific Biosciences RSII instrument may also preferentially load smaller fragments, which may also be a function of PCR product exhaustion during sequencing. To further examine theses biases, data is provided from 16S rRNA rumen community analyses. Specifically, data from the relative phylum-level abundances for the ruminal bacterial community are provided to determine between-sample variability. Direct sequencing of metagenomic DNA was conducted to circumvent primer-associated biases in 16S rRNA reads and rarefaction curves were generated to demonstrate adequate coverage of each amplicon. PCR products were also subjected to reduced amplification and pooling to reduce the likelihood of PCR product exhaustion during sequencing on the Pacific Biosciences platform. The taxonomic profiles for the relative phylum-level and genus-level abundance of rumen microbiota as a function of PCR pooling for sequencing on the Pacific Biosciences RSII platform were provided. For more information, see “Evaluation of 16S rRNA amplicon sequencing using two next-generation sequencing technologies for phylogenetic analysis of the rumen bacterial community in steers” P.R. Myer, M. Kim, H.C. Freetly, T.P.L. Smith (2016) [1].

Read more
September 22, 2019

The microbiota of freshwater fish and freshwater niches contain omega-3 producing Shewanella species.

Approximately 30 years ago, it was discovered that free-living bacteria isolated from cold ocean depths could produce polyunsaturated fatty acids (PUFA) such as eicosapentaenoic acid (EPA) (20:5n-3) or docosahexaenoic acid (DHA) (22:6n-3), two PUFA essential for human health. Numerous laboratories have also discovered that EPA- and/or DHA-producing bacteria, many of them members of the Shewanella genus, could be isolated from the intestinal tracts of omega-3 fatty acid-rich marine fish. If bacteria contribute omega-3 fatty acids to the host fish in general or if they assist some bacterial species in adaptation to cold, then cold freshwater fish or habitats should also harbor these producers. Thus, we undertook a study to see if these niches also contained omega-3 fatty acid producers. We were successful in isolating and characterizing unique EPA-producing strains of Shewanella from three strictly freshwater native fish species, i.e., lake whitefish (Coregonus clupeaformis), lean lake trout (Salvelinus namaycush), and walleye (Sander vitreus), and from two other freshwater nonnative fish, i.e., coho salmon (Oncorhynchus kisutch) and seeforellen brown trout (Salmo trutta). We were also able to isolate four unique free-living strains of EPA-producing Shewanella from freshwater habitats. Phylogenetic and phenotypic analyses suggest that one producer is clearly a member of the Shewanella morhuae species and another is sister to members of the marine PUFA-producing Shewanella baltica species. However, the remaining isolates have more ambiguous relationships, sharing a common ancestor with non-PUFA-producing Shewanella putrefaciens isolates rather than marine S. baltica isolates despite having a phenotype more consistent with S. baltica strains. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Read more
September 22, 2019

Long-read DNA metabarcoding of ribosomal RNA in the analysis of fungi from aquatic environments.

DNA metabarcoding is widely used to study prokaryotic and eukaryotic microbial diversity. Technological constraints limit most studies to marker lengths below 600 base pairs (bp). Longer sequencing reads of several thousand bp are now possible with third-generation sequencing. Increased marker lengths provide greater taxonomic resolution and allow for phylogenetic methods of classification, but longer reads may be subject to higher rates of sequencing error and chimera formation. In addition, most bioinformatics tools for DNA metabarcoding were designed for short reads and are therefore unsuitable. Here, we used Pacific Biosciences circular consensus sequencing (CCS) to DNA-metabarcode environmental samples using a ca. 4,500 bp marker that included most of the eukaryote SSU and LSU rRNA genes and the complete ITS region. We developed an analysis pipeline that reduced error rates to levels comparable to short-read platforms. Validation using a mock community indicated that our pipeline detected 98% of chimeras de novo. We recovered 947 OTUs from water and sediment samples from a natural lake, 848 of which could be classified to phylum, 397 to genus and 330 to species. By allowing for the simultaneous use of three databases (Unite, SILVA and RDP LSU), long-read DNA metabarcoding provided better taxonomic resolution than any single marker. We foresee the use of long reads enabling the cross-validation of reference sequences and the synthesis of ribosomal rRNA gene databases. The universal nature of the rRNA operon and our recovery of >100 nonfungal OTUs indicate that long-read DNA metabarcoding holds promise for studies of eukaryotic diversity more broadly.© 2018 John Wiley & Sons Ltd.

Read more
September 22, 2019

Whole genome sequencing of “Faecalibaculum rodentium” ALO17, isolated from C57BL/6J laboratory mouse feces.

Intestinal microorganisms affect host physiology, including ageing. Given the difficulty in controlling for human studies of the gut microbiome, mouse models provide an alternative avenue to study such relationships. In this study, we report on the complete genome of “Faecalibaculum rodentium” ALO17, a bacterium that was isolated from the faeces of a 9-month-old female C57BL/6J mouse. This strain will be utilized in future in vivo studies detailing the relationships between the gut microbiome and ageing.The whole genome sequence of “F. rodentium” ALO17 was obtained using single-molecule, real-time (SMRT) technique on a PacBio instrument. The assembled genome consisted of 2,542,486 base pairs of double-stranded DNA with a GC content of 54.0 % and no plasmids. The genome was predicted to contain 2794 open reading frames, 55 tRNA genes, and 38 rRNA genes. The 16S rRNA gene of ALO17 was 86.9 % similar to that of Allobaculum stercoricanis DSM 13633(T), and the average overall nucleotide identity between strains ALO17 and DSM 13633(T) was 66.8 %. After confirming the phylogenetic relationship between “F. rodentium” ALO17 and A. stercoricanis DSM 13633(T), their whole genome sequences were compared, revealing that “F. rodentium” ALO17 contains more fermentation-related genes than A. stercoricanis DSM 13633(T). Furthermore, “F. rodentium” ALO17 produces higher levels of lactic acid than A. stercoricanis DSM 13633(T) as determined by high-performance liquid chromatography.The availability of the “F. rodentium” ALO17 whole genome sequence will enhance studies concerning the gut microbiota and host physiology, especially when investigating the molecular relationships between gut microbiota and ageing.

Read more
September 22, 2019

Analysis of the gut microbial diversity of dairy cows during peak lactation by PacBio Single-Molecule Real-Time (SMRT) Sequencing.

The gut microbes of dairy cows are strongly associated with their health, but the relationship between milk production and the intestinal microbiota has seldom been studied. Thus, we explored the diversity of the intestinal microbiota during peak lactation of dairy cows.The intestinal microbiota of nine dairy cows at peak lactation was evaluated using the Pacific Biosciences single-molecule real-time (PacBio SMRT) sequencing approach.A total of 32,670 high-quality 16S rRNA gene sequences were obtained, belonging to 12 phyla, 59 families, 107 genera, and 162 species. Firmicutes (83%) were the dominant phylum, while Bacteroides (6.16%) was the dominant genus. All samples showed a high microbial diversity, with numerous genera of short chain fatty acid (SCFA)-producers. The proportion of SCFA producers was relatively high in relation to the identified core intestinal microbiota. Moreover, the predicted functional metagenome was heavily involved in energy metabolism.This study provided novel insights into the link between the dairy cow gut microbiota and milk production.

Read more
September 22, 2019

High-resolution phylogenetic microbial community profiling.

Over the past decade, high-throughput short-read 16S rRNA gene amplicon sequencing has eclipsed clone-dependent long-read Sanger sequencing for microbial community profiling. The transition to new technologies has provided more quantitative information at the expense of taxonomic resolution with implications for inferring metabolic traits in various ecosystems. We applied single-molecule real-time sequencing for microbial community profiling, generating full-length 16S rRNA gene sequences at high throughput, which we propose to name PhyloTags. We benchmarked and validated this approach using a defined microbial community. When further applied to samples from the water column of meromictic Sakinaw Lake, we show that while community structures at the phylum level are comparable between PhyloTags and Illumina V4 16S rRNA gene sequences (iTags), variance increases with community complexity at greater water depths. PhyloTags moreover allowed less ambiguous classification. Last, a platform-independent comparison of PhyloTags and in silico generated partial 16S rRNA gene sequences demonstrated significant differences in community structure and phylogenetic resolution across multiple taxonomic levels, including a severe underestimation in the abundance of specific microbial genera involved in nitrogen and methane cycling across the Lake’s water column. Thus, PhyloTags provide a reliable adjunct or alternative to cost-effective iTags, enabling more accurate phylogenetic resolution of microbial communities and predictions on their metabolic potential.

Read more
September 22, 2019

Different next generation sequencing platforms produce different microbial profiles and diversity in cystic fibrosis sputum.

Cystic fibrosis (CF) is an autosomal recessive disease characterized by recurrent lung infections. Studies of the lung microbiome have shown an association between decreasing diversity and progressive disease. 454 pyrosequencing has frequently been used to study the lung microbiome in CF, but will no longer be supported. We sought to identify the benefits and drawbacks of using two state-of-the-art next generation sequencing (NGS) platforms, MiSeq and PacBio RSII, to characterize the CF lung microbiome. Each has its advantages and limitations.Twelve samples of extracted bacterial DNA were sequenced on both MiSeq and PacBio NGS platforms. DNA was amplified for the V4 region of the 16S rRNA gene and libraries were sequenced on the MiSeq sequencing platform, while the full 16S rRNA gene was sequenced on the PacBio RSII sequencing platform. Raw FASTQ files generated by the MiSeq and PacBio platforms were processed in mothur v1.35.1.There was extreme discordance in alpha-diversity of the CF lung microbiome when using the two platforms. Because of its depth of coverage, sequencing of the 16S rRNA V4 gene region using MiSeq allowed for the observation of many more operational taxonomic units (OTUs) and higher Chao1 and Shannon indices than the PacBio RSII. Interestingly, several patients in our cohort had Escherichia, an unusual pathogen in CF. Also, likely because of its coverage of the complete 16S rRNA gene, only PacBio RSII was able to identify Burkholderia, an important CF pathogen.When comparing microbiome diversity in clinical samples from CF patients using 16S sequences, MiSeq and PacBio NGS platforms may generate different results in microbial community composition and structure. It may be necessary to use different platforms when trying to correctly identify dominant pathogens versus measuring alpha-diversity estimates, and it would be important to use the same platform for comparisons to minimize errors in interpretation. Copyright © 2016 Elsevier B.V. All rights reserved.

Read more
September 22, 2019

Soil bacterial communities are shaped by temporal and environmental filtering: evidence from a long-term chronosequence.

Soil microbial communities are abundant, hyper-diverse and mediate global biogeochemical cycles, but we do not yet understand the processes mediating their assembly. Current hypothetical frameworks suggest temporal (e.g. dispersal limitation) and environmental (e.g. soil pH) filters shape microbial community composition; however, there is limited empirical evidence supporting this framework in the hyper-diverse soil environment, particularly at large spatial (i.e. regional to continental) and temporal (i.e. 100 to 1000 years) scales. Here, we present evidence from a long-term chronosequence (4000 years) that temporal and environmental filters do indeed shape soil bacterial community composition. Furthermore, nearly 20 years of environmental monitoring allowed us to control for potentially confounding environmental variation. Soil bacterial communities were phylogenetically distinct across the chronosequence. We determined that temporal and environmental factors accounted for significant portions of bacterial phylogenetic structure using distance-based linear models. Environmental factors together accounted for the majority of phylogenetic structure, namely, soil temperature (19%), pH (17%) and litter carbon:nitrogen (C:N; 17%). However, of all individual factors, time since deglaciation accounted for the greatest proportion of bacterial phylogenetic structure (20%). Taken together, our results provide empirical evidence that temporal and environmental filters act together to structure soil bacterial communities across large spatial and long-term temporal scales. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

Read more
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.

Read more
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.

Read more
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.

Read more

Subscribe for blog updates:

Talk with an expert

If you have a question, need to check the status of an order, or are interested in purchasing an instrument, we're here to help.