Menu

Asset Tag: Microbiology

September 22, 2019

Improving eukaryotic genome annotation using single molecule mRNA sequencing.

The advantages of Pacific Biosciences (PacBio) single-molecule real-time (SMRT) technology include long reads, low systematic bias, and high consensus read accuracy. Here we use these attributes to improve on the genome annotation of the parasitic hookworm Ancylostoma ceylanicum using PacBio RNA-Seq.We sequenced 192,888 circular consensus sequences (CCS) derived from cDNAs generated using the CloneTech SMARTer system. These SMARTer-SMRT libraries were normalized and size-selected providing a robust population of expressed structural genes for subsequent genome annotation. We demonstrate PacBio mRNA sequences based genome annotation improvement, compared to genome annotation using conventional sequencing-by-synthesis alone, by identifying 1609 (9.2%) new genes, extended the length of 3965 (26.7%) genes and increased the total genomic exon length by 1.9 Mb (12.4%). Non-coding sequence representation (primarily from UTRs based on dT reverse transcription priming) was particularly improved, increasing in total length by fifteen-fold, by increasing both the length and number of UTR exons. In addition, the UTR data provided by these CCS allowed for the identification of a novel SL2 splice leader sequence for A. ceylanicum and an increase in the number and proportion of functionally annotated genes. RNA-seq data also confirmed some of the newly annotated genes and gene features.Overall, PacBio data has supported a significant improvement in gene annotation in this genome, and is an appealing alternative or complementary technique for genome annotation to the other transcript sequencing technologies.

Read more
September 22, 2019

A single-cell genome for Thiovulum sp.

We determined a significant fraction of the genome sequence of a representative of Thiovulum, the uncultivated genus of colorless sulfur Epsilonproteobacteria, by analyzing the genome sequences of four individual cells collected from phototrophic mats from Elkhorn Slough, California. These cells were isolated utilizing a microfluidic laser-tweezing system, and their genomes were amplified by multiple-displacement amplification prior to sequencing. Thiovulum is a gradient bacterium found at oxic-anoxic marine interfaces and noted for its distinctive morphology and rapid swimming motility. The genomic sequences of the four individual cells were assembled into a composite genome consisting of 221 contigs covering 2.083 Mb including 2,162 genes. This single-cell genome represents a genomic view of the physiological capabilities of isolated Thiovulum cells. Thiovulum is the second-fastest bacterium ever observed, swimming at 615 µm/s, and this genome shows that this rapid swimming motility is a result of a standard flagellar machinery that has been extensively characterized in other bacteria. This suggests that standard flagella are capable of propelling bacterial cells at speeds much faster than typically thought. Analysis of the genome suggests that naturally occurring Thiovulum populations are more diverse than previously recognized and that studies performed in the past probably address a wide range of unrecognized genotypic and phenotypic diversities of Thiovulum. The genome presented in this article provides a basis for future isolation-independent studies of Thiovulum, where single-cell and metagenomic tools can be used to differentiate between different Thiovulum genotypes.

Read more
September 22, 2019

Genomics and host specialization of honey bee and bumble bee gut symbionts.

Gilliamella apicola and Snodgrassella alvi are dominant members of the honey bee (Apis spp.) and bumble bee (Bombus spp.) gut microbiota. We generated complete genomes of the type strains G. apicola wkB1(T) and S. alvi wkB2(T) (isolated from Apis), as well as draft genomes for four other strains from Bombus. G. apicola and S. alvi were found to occupy very different metabolic niches: The former is a saccharolytic fermenter, whereas the latter is an oxidizer of carboxylic acids. Together, they may form a syntrophic network for partitioning of metabolic resources. Both species possessed numerous genes [type 6 secretion systems, repeats in toxin (RTX) toxins, RHS proteins, adhesins, and type IV pili] that likely mediate cell-cell interactions and gut colonization. Variation in these genes could account for the host fidelity of strains observed in previous phylogenetic studies. Here, we also show the first experimental evidence, to our knowledge, for this specificity in vivo: Strains of S. alvi were able to colonize their native bee host but not bees of another genus. Consistent with specific, long-term host association, comparative genomic analysis revealed a deep divergence and little or no gene flow between Apis and Bombus gut symbionts. However, within a host type (Apis or Bombus), we detected signs of horizontal gene transfer between G. apicola and S. alvi, demonstrating the importance of the broader gut community in shaping the evolution of any one member. Our results show that host specificity is likely driven by multiple factors, including direct host-microbe interactions, microbe-microbe interactions, and social transmission.

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

PacBio sequencing and its applications.

Single-molecule, real-time sequencing developed by Pacific BioSciences offers longer read lengths than the second-generation sequencing (SGS) technologies, making it well-suited for unsolved problems in genome, transcriptome, and epigenetics research. The highly-contiguous de novo assemblies using PacBio sequencing can close gaps in current reference assemblies and characterize structural variation (SV) in personal genomes. With longer reads, we can sequence through extended repetitive regions and detect mutations, many of which are associated with diseases. Moreover, PacBio transcriptome sequencing is advantageous for the identification of gene isoforms and facilitates reliable discoveries of novel genes and novel isoforms of annotated genes, due to its ability to sequence full-length transcripts or fragments with significant lengths. Additionally, PacBio’s sequencing technique provides information that is useful for the direct detection of base modifications, such as methylation. In addition to using PacBio sequencing alone, many hybrid sequencing strategies have been developed to make use of more accurate short reads in conjunction with PacBio long reads. In general, hybrid sequencing strategies are more affordable and scalable especially for small-size laboratories than using PacBio Sequencing alone. The advent of PacBio sequencing has made available much information that could not be obtained via SGS alone. Copyright © 2015 The Authors. Production and hosting by Elsevier Ltd.. All rights reserved.

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

Periodic pattern of genetic and fitness diversity during evolution of an artificial cell-like system.

Genetic and phenotypic diversity are the basis of evolution. Despite their importance, however, little is known about how they change over the course of evolution. In this study, we analyzed the dynamics of the adaptive evolution of a simple evolvable artificial cell-like system using single-molecule real-time sequencing technology that reads an entire single artificial genome. We found that the genomic RNA population increases in fitness intermittently, correlating with a periodic pattern of genetic and fitness diversity produced by repeated diversification and domination. In the diversification phase, a genomic RNA population spreads within a genetic space by accumulating mutations until mutants with higher fitness are generated, resulting in an increase in fitness diversity. In the domination phase, the mutants with higher fitness dominate, decreasing both the fitness and genetic diversity. This study reveals the dynamic nature of genetic and fitness diversity during adaptive evolution and demonstrates the utility of a simplified artificial cell-like system to study evolution at an unprecedented resolution. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Read more
September 22, 2019

Great differences in performance and outcome of high-throughput sequencing data analysis platforms for fungal metabarcoding.

Along with recent developments in high-throughput sequencing (HTS) technologies and thus fast accumulation of HTS data, there has been a growing need and interest for developing tools for HTS data processing and communication. In particular, a number of bioinformatics tools have been designed for analysing metabarcoding data, each with specific features, assumptions and outputs. To evaluate the potential effect of the application of different bioinformatics workflow on the results, we compared the performance of different analysis platforms on two contrasting high-throughput sequencing data sets. Our analysis revealed that the computation time, quality of error filtering and hence output of specific bioinformatics process largely depends on the platform used. Our results show that none of the bioinformatics workflows appears to perfectly filter out the accumulated errors and generate Operational Taxonomic Units, although PipeCraft, LotuS and PIPITS perform better than QIIME2 and Galaxy for the tested fungal amplicon dataset. We conclude that the output of each platform requires manual validation of the OTUs by examining the taxonomy assignment values.

Read more
September 22, 2019

Soil microclimate changes affect soil fungal communities in a Mediterranean pine forest.

Soil microclimate is a potentially important regulator of the composition of plant-associated fungal communities in climates with significant drought periods. Here, we investigated the spatio-temporal dynamics of soil fungal communities in a Mediterranean Pinus pinaster forest in relation to soil moisture and temperature. Fungal communities in 336 soil samples collected monthly over 1 year from 28 long-term experimental plots were assessed by PacBio sequencing of ITS2 amplicons. Total fungal biomass was estimated by analysing ergosterol. Community changes were analysed in the context of functional traits. Soil fungal biomass was lowest during summer and late winter and highest during autumn, concurrent with a greater relative abundance of mycorrhizal species. Intra-annual spatio-temporal changes in community composition correlated significantly with soil moisture and temperature. Mycorrhizal fungi were less affected by summer drought than free-living fungi. In particular, mycorrhizal species of the short-distance exploration type increased in relative abundance under dry conditions, whereas species of the long-distance exploration type were more abundant under wetter conditions. Our observations demonstrate a potential for compositional and functional shifts in fungal communities in response to changing climatic conditions. Free-living fungi and mycorrhizal species with extensive mycelia may be negatively affected by increasing drought periods in Mediterranean forest ecosystems.© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

Read more
September 22, 2019

A manganese superoxide dismutase (MnSOD) from red lip mullet, Liza haematocheila: Evaluation of molecular structure, immune response, and antioxidant function.

Manganese superoxide dismutase (MnSOD) is a nuclear-encoded antioxidant metalloenzyme. The main function of this enzyme is to dismutase the toxic superoxide anion (O2-) into less toxic hydrogen peroxide (H2O2) and oxygen (O2). Structural analysis of mullet MnSOD (MuMnSOD) was performed using different bioinformatics tools. Pairwise alignment revealed that the protein sequence matched to that derived from Larimichthys crocea with a 95.2% sequence identity. Phylogenetic tree analysis showed that the MuMnSOD was included in the category of teleosts. Multiple sequence alignment showed that a SOD Fe-N domain, SOD Fe-C domain, and Mn/Fe SOD signature were highly conserved among the other examined MnSOD orthologs. Quantitative real-time PCR showed that the highest MuMnSOD mRNA expression level was in blood cells. The highest expression level of MuMnSOD was observed in response to treatment with both Lactococcus garvieae and lipopolysaccharide (LPS) at 6?h post treatment in the head kidney and blood. Potential ROS-scavenging ability of the purified recombinant protein (rMuMnSOD) was examined by the xanthine oxidase assay (XOD assay). The optimum temperature and pH for XOD activity were found to be 25?°C and pH 7, respectively. Relative XOD activity was significantly increased with the dose of rMuMnSOD, revealing its dose dependency. Activity of rMuMnSOD was inhibited by potassium cyanide (KCN) and N-N’-diethyl-dithiocarbamate (DDC). Moreover, expression of MuMnSOD resulted in considerable growth retardation of both gram-positive and gram-negative bacteria. Results of the current study suggest that MuMnSOD acts as an antioxidant enzyme and participates in the immune response in mullet. Copyright © 2018 Elsevier Ltd. All rights reserved.

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

High-resolution community profiling of arbuscular mycorrhizal fungi.

Community analyses of arbuscular mycorrhizal fungi (AMF) using ribosomal small subunit (SSU) or internal transcribed spacer (ITS) DNA sequences often suffer from low resolution or coverage. We developed a novel sequencing based approach for a highly resolving and specific profiling of AMF communities. We took advantage of previously established AMF-specific PCR primers that amplify a c. 1.5-kb long fragment covering parts of SSU, ITS and parts of the large ribosomal subunit (LSU), and we sequenced the resulting amplicons with single molecule real-time (SMRT) sequencing. The method was applicable to soil and root samples, detected all major AMF families and successfully discriminated closely related AMF species, which would not be discernible using SSU sequences. In inoculation tests we could trace the introduced AMF inoculum at the molecular level. One of the introduced strains almost replaced the local strain(s), revealing that AMF inoculation can have a profound impact on the native community. The methodology presented offers researchers a powerful new tool for AMF community analysis because it unifies improved specificity and enhanced resolution, whereas the drawback of medium sequencing throughput appears of lesser importance for low-diversity groups such as AMF.© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Read more
September 22, 2019

Human and rhesus macaque KIR haplotypes defined by their transcriptomes.

The killer-cell Ig-like receptors (KIRs) play a central role in the immune recognition in infection, pregnancy, and transplantation through their interactions with MHC class I molecules. KIR genes display abundant copy number variation as well as high levels of polymorphism. As a result, it is challenging to characterize this structurally dynamic region. KIR haplotypes have been analyzed in different species using conventional characterization methods, such as Sanger sequencing and Roche/454 pyrosequencing. However, these methods are time-consuming and often failed to define complete haplotypes, or do not reach allele-level resolution. In addition, most analyses were performed on genomic DNA, and thus were lacking substantial information about transcription and its corresponding modifications. In this paper, we present a single-molecule real-time sequencing approach, using Pacific Biosciences Sequel platform to characterize the KIR transcriptomes in human and rhesus macaque (Macaca mulatta) families. This high-resolution approach allowed the identification of novel Mamu-KIR alleles, the extension of reported allele sequences, and the determination of human and macaque KIR haplotypes. In addition, multiple recombinant KIR genes were discovered, all located on contracted haplotypes, which were likely the result of chromosomal rearrangements. The relatively high number of contracted haplotypes discovered might be indicative of selection on small KIR repertoires and/or novel fusion gene products. This next-generation method provides an improved high-resolution characterization of the KIR cluster in humans and macaques, which eventually may aid in a better understanding and interpretation of KIR allele-associated diseases, as well as the immune response in transplantation and reproduction. Copyright © 2018 by The American Association of Immunologists, Inc.

Read more
September 22, 2019

Below-ground organic matter accumulation along a boreal forest fertility gradient relates to guild interaction within fungal communities.

Plant-soil interactions link ecosystem fertility and organic matter accumulation below ground. Soil microorganisms play a central role as mediators of these interactions, but mechanistic understanding is still largely lacking. Correlative data from a coniferous forest ecosystem support the hypothesis that interactions between fungal guilds play a central role in regulating organic matter accumulation in relation to fertility. With increasing ecosystem fertility, the proportion of saprotrophic basidiomycetes increased in deeper organic layers, at the expense of ectomycorrhizal fungal species. Saprotrophs correlated positively with the activity of oxidative enzymes, which in turn favoured organic matter turnover and nitrogen recycling to plants. Combined, our findings are consistent with a fungus-mediated feedback loop, which results in a negative correlation between ecosystem fertility and below-ground carbon storage. These findings call for a shift in focus from plant litter traits to fungal traits in explaining organic matter dynamics and ecosystem fertility in boreal forests.© 2017 John Wiley & Sons Ltd/CNRS.

Read more
September 22, 2019

Order of removal of conventional and nonconventional introns from nuclear transcripts of Euglena gracilis.

Nuclear genes of euglenids and marine diplonemids harbor atypical, nonconventional introns which are not observed in the genomes of other eukaryotes. Nonconventional introns do not have the conserved borders characteristic for spliceosomal introns or the sequence complementary to U1 snRNA at the 5′ end. They form a stable secondary structure bringing together both exon/intron junctions, nevertheless, this conformation does not resemble the form of self-splicing or tRNA introns. In the genes studied so far, frequent nonconventional introns insertions at new positions have been observed, whereas conventional introns have been either found at the conserved positions, or simply lost. In this work, we examined the order of intron removal from Euglena gracilis transcripts of the tubA and gapC genes, which contain two types of introns: nonconventional and spliceosomal. The relative order of intron excision was compared for pairs of introns belonging to different types. Furthermore, intermediate products of splicing were analyzed using the PacBio Next Generation Sequencing system. The analysis led to the main conclusion that nonconventional introns are removed in a rapid way but later than spliceosomal introns. Moreover, the observed accumulation of transcripts with conventional introns removed and nonconventional present may suggest the existence of a time gap between the two types of splicing.

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.