scholarly journals Phylotype-Level Characterization of Complex Communities of Lactobacilli Using a High-Throughput, High-Resolution Phenylalanyl-tRNA Synthetase (pheS) Gene Amplicon Sequencing Approach

2020 ◽  
Vol 87 (1) ◽  
Author(s):  
Shaktheeshwari Silvaraju ◽  
Nandita Menon ◽  
Huan Fan ◽  
Kevin Lim ◽  
Sandra Kittelmann
Keyword(s):  
High Resolution ◽  
Amplicon Sequencing ◽  
Trna Synthetase ◽  
Species Level ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Low Resolution ◽  
Shotgun Metagenomics ◽  
Wide Range

ABSTRACT The lactobacilli identified to date encompass more than 270 closely related species that were recently reclassified into 26 genera. Because of their relevance to industry, there is a need to distinguish between closely related and yet metabolically and regulatory distinct species, e.g., during monitoring of biotechnological processes or screening of samples of unknown composition. Current available methods, such as shotgun metagenomics or rRNA gene-based amplicon sequencing, have significant limitations (high cost, low resolution, etc.). Here, we generated a phylogeny of lactobacilli based on phenylalanyl-tRNA synthetase (pheS) genes and, from it, developed a high-resolution taxonomic framework which allows for comprehensive and confident characterization of the community diversity and structure of lactobacilli at the species level. This framework is based on a total of 445 pheS gene sequences, including sequences of 276 validly described species and subspecies (of a total of 282, including the proposed L. timonensis species and the reproposed L. zeae species; coverage of 98%), and allows differentiation between 265 species-level clades of lactobacilli and the subspecies of L. sakei. The methodology was validated through next-generation sequencing of mock communities. At a sequencing depth of ∼30,000 sequences, the minimum level of detection was approximately 0.02 pg per μl DNA (equaling approximately 10 genome copies per μl template DNA). The pheS approach, along with parallel sequencing of partial 16S rRNA genes, revealed considerable diversity of lactobacilli and distinct community structures across a broad range of samples from different environmental niches. This novel complementary approach may be applicable to industry and academia alike. IMPORTANCE Species formerly classified within the genera Lactobacillus and Pediococcus have been studied extensively at the genomic level. To accommodate their exceptional functional diversity, the over 270 species were recently reclassified into 26 distinct genera. Despite their relevance to both academia and industry, methods that allow detailed exploration of their ecology are still limited by low resolution, high cost, or copy number variations. The approach described here makes use of a single-copy marker gene which outperforms other markers with regard to species-level resolution and availability of reference sequences (98% coverage). The tool was validated against a mock community and used to address diversity of lactobacilli and community structure in various environmental matrices. Such analyses can now be performed at a broader scale to assess and monitor the assembly, structure, and function of communities of lactobacilli at the species level (and, in some cases, even at the subspecies level) across a wide range of academic and commercial applications.

scholarly journals Phylotype-Level Characterization of Complex Lactobacilli Communities Using a High-Throughput, High-Resolution Phenylalanyl-tRNA Synthetase (pheS) Gene Amplicon Sequencing Approach

2020 ◽  
Author(s):  
Shaktheeshwari Silvaraju ◽  
Nandita Menon ◽  
Huan Fan ◽  
Kevin Lim ◽  
Sandra Kittelmann
Keyword(s):  
High Resolution ◽  
Amplicon Sequencing ◽  
Trna Synthetase ◽  
Species Level ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Low Resolution ◽  
Shotgun Metagenomics ◽  
Wide Range

ABSTRACTThe ‘lactobacilli’ to date encompass more than 270 closely related species that were recently re-classified into 26 genera. Because of their relevance to industry, there is a need to distinguish between closely related, yet metabolically and regulatory distinct species, e.g., during monitoring of biotechnological processes or screening of samples of unknown composition. Current available methods, such as shotgun metagenomics or rRNA-based amplicon sequencing have significant limitations (high cost, low resolution, etc.). Here, we generated a lactobacilli phylogeny based on phenylalanyl-tRNA synthetase (pheS) genes and, from it, developed a high-resolution taxonomic framework which allows for comprehensive and confident characterization of lactobacilli community diversity and structure at the species-level. This framework is based on a total of 445 pheS gene sequences, including sequences of 277 validly described species and subspecies (out of a total of 283, coverage of 98%). It allows differentiation between 263 lactobacilli species-level clades out of a total of 273 validly described species (including the proposed species L. timonensis) and a further two subspecies. The methodology was validated through next-generation sequencing of mock communities. At a sequencing depth of ∼30,000 sequences, the minimum level of detection was approximately 0.02 pg per μl DNA (equalling approximately 10 genome copies per µl template DNA). The pheS approach along with parallel sequencing of partial 16S rRNA genes revealed a considerable lactobacilli diversity and distinct community structures across a broad range of samples from different environmental niches. This novel complementary approach may be applicable to industry and academia alike.IMPORTANCESpecies within the former genera Lactobacillus and Pediococcus have been studied extensively at the genomic level. To accommodate for their exceptional functional diversity, the over 270 species were recently re-classified into 26 distinct genera. Despite their relevance to both academia and industry, methods that allow detailed exploration of their ecology are still limited by low resolution, high cost or copy number variations. The approach described here makes use of a single copy marker gene which outperforms other markers with regards to species-level resolution and availability of reference sequences (98% coverage). The tool was validated against a mock community and used to address lactobacilli diversity and community structure in various environmental matrices. Such analyses can now be performed at broader scale to assess and monitor lactobacilli community assembly, structure and function at the species (in some cases even at sub-species) level across a wide range of academic and commercial applications.

scholarly journals Ultra-accurate microbial amplicon sequencing with synthetic long reads

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Benjamin J. Callahan ◽  
Dmitry Grinevich ◽  
Siddhartha Thakur ◽  
Michael A. Balamotis ◽  
Tuval Ben Yehezkel
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
Amplicon Sequencing ◽  
Species Level ◽  
Full Length ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Strain Identification ◽  
Long Reads ◽  
Long Read

Abstract Background Out of the many pathogenic bacterial species that are known, only a fraction are readily identifiable directly from a complex microbial community using standard next generation DNA sequencing. Long-read sequencing offers the potential to identify a wider range of species and to differentiate between strains within a species, but attaining sufficient accuracy in complex metagenomes remains a challenge. Methods Here, we describe and analytically validate LoopSeq, a commercially available synthetic long-read (SLR) sequencing technology that generates highly accurate long reads from standard short reads. Results LoopSeq reads are sufficiently long and accurate to identify microbial genes and species directly from complex samples. LoopSeq perfectly recovered the full diversity of 16S rRNA genes from known strains in a synthetic microbial community. Full-length LoopSeq reads had a per-base error rate of 0.005%, which exceeds the accuracy reported for other long-read sequencing technologies. 18S-ITS and genomic sequencing of fungal and bacterial isolates confirmed that LoopSeq sequencing maintains that accuracy for reads up to 6 kb in length. LoopSeq full-length 16S rRNA reads could accurately classify organisms down to the species level in rinsate from retail meat samples, and could differentiate strains within species identified by the CDC as potential foodborne pathogens. Conclusions The order-of-magnitude improvement in length and accuracy over standard Illumina amplicon sequencing achieved with LoopSeq enables accurate species-level and strain identification from complex- to low-biomass microbiome samples. The ability to generate accurate and long microbiome sequencing reads using standard short read sequencers will accelerate the building of quality microbial sequence databases and removes a significant hurdle on the path to precision microbial genomics.

scholarly journals Molecular and Genomic Characterization of PFAB2: A Non-virulent Bacillus anthracis Strain Isolated from an Indian Hot Spring

2020 ◽  
Vol 20 (7) ◽  
pp. 491-507 ◽  
Author(s):  
Aparna Banerjee ◽  
Vikas K. Somani ◽  
Priyanka Chakraborty ◽  
Rakesh Bhatnagar ◽  
Rajeev K. Varshney ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Culture Media ◽  
Draft Genome ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Taxonomic Resolution ◽  
Thermophilic Bacilli ◽  
Wide Range ◽  
Genomic Characterization

Background: Thermophilic bacilli in both aerobic or facultative anaerobic forms have been isolated for over a hundred years from different mesophilic or thermophilic environments as they are potential source of bioactive secondary metabolites. But the taxonomic resolution in the Bacillus genus at species or at strain level is very challenging for the insufficient divergence of the 16S rRNA genes. One such recurring problem is among Bacillus anthracis, B. cereus and B. thuringiensis. The disease-causing B. anthracis strains have their characteristic virulence factors coded in two wellknown plasmids, namely pXO1 (toxin genes) and pXO2 (capsule genes). Objective: The present study aimed at the molecular and genomic characterization of a recently reported thermophilic and environmental isolate of B. anthracis, strain PFAB2. Methods: We performed comparative genomics between the PFAB2 genome and different strains of B. anthracis, along with closely related B. cereus strains. Results: The pangenomic analysis suggests that the PFAB2 genome harbors no complete prophage genes. Cluster analysis of Bray-Kurtis similarity resemblance matrix revealed that gene content of PFAB2 is more closely related to other environmental strains of B. anthracis. The secretome analysis and the in vitro and in vivo pathogenesis experiments corroborate the avirulent phenotype of this strain. The most probable explanation for this phenotype is the apparent absence of plasmids harboring genes for capsule biosynthesis and toxins secretion in the draft genome. Additional features of PFAB2 are good spore-forming and germinating capabilities and rapid replication ability. Conclusion: The high replication rate in a wide range of temperatures and culture media, the nonpathogenicity, the good spore forming capability and its genomic similarity to the Ames strain together make PFAB2 an interesting model strain for the study of the pathogenic evolution of B. anthracis.

scholarly journals Coordination of microbe–host homeostasis by crosstalk with plant innate immunity

Nature Plants ◽  
2021 ◽  
Author(s):  
Ka-Wai Ma ◽  
Yulong Niu ◽  
Yong Jia ◽  
Jana Ordon ◽  
Charles Copeland ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Amplicon Sequencing ◽  
Host Susceptibility ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Root Growth Inhibition ◽  
Natural Soil ◽  
Molecular Pattern ◽  
Immune Related Genes ◽  
Molecular Patterns

AbstractPlants grown in natural soil are colonized by phylogenetically structured communities of microbes known as the microbiota. Individual microbes can activate microbe-associated molecular pattern (MAMP)-triggered immunity (MTI), which limits pathogen proliferation but curtails plant growth, a phenomenon known as the growth–defence trade-off. Here, we report that, in monoassociations, 41% (62 out of 151) of taxonomically diverse root bacterial commensals suppress Arabidopsis thaliana root growth inhibition (RGI) triggered by immune-stimulating MAMPs or damage-associated molecular patterns. Amplicon sequencing of bacterial 16S rRNA genes reveals that immune activation alters the profile of synthetic communities (SynComs) comprising RGI-non-suppressive strains, whereas the presence of RGI-suppressive strains attenuates this effect. Root colonization by SynComs with different complexities and RGI-suppressive activities alters the expression of 174 core host genes, with functions related to root development and nutrient transport. Furthermore, RGI-suppressive SynComs specifically downregulate a subset of immune-related genes. Precolonization of plants with RGI-suppressive SynComs, or mutation of one commensal-downregulated transcription factor, MYB15, renders the plants more susceptible to opportunistic Pseudomonas pathogens. Our results suggest that RGI-non-suppressive and RGI-suppressive root commensals modulate host susceptibility to pathogens by either eliciting or dampening MTI responses, respectively. This interplay buffers the plant immune system against pathogen perturbation and defence-associated growth inhibition, ultimately leading to commensal–host homeostasis.

scholarly journals Microdiversity and phylogeographic diversification of bacterioplankton in pelagic freshwater systems revealed through long-read amplicon sequencing

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Yusuke Okazaki ◽  
Shohei Fujinaga ◽  
Michaela M. Salcher ◽  
Cristiana Callieri ◽  
Atsushi Tanaka ◽  
...  
Keyword(s):  
16S Rrna ◽  
Regional Scale ◽  
Scale Up ◽  
Amplicon Sequencing ◽  
Freshwater Ecosystems ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Metagenomic Sequencing ◽  
Long Read

Abstract Background Freshwater ecosystems are inhabited by members of cosmopolitan bacterioplankton lineages despite the disconnected nature of these habitats. The lineages are delineated based on > 97% 16S rRNA gene sequence similarity, but their intra-lineage microdiversity and phylogeography, which are key to understanding the eco-evolutional processes behind their ubiquity, remain unresolved. Here, we applied long-read amplicon sequencing targeting nearly full-length 16S rRNA genes and the adjacent ribosomal internal transcribed spacer sequences to reveal the intra-lineage diversities of pelagic bacterioplankton assemblages in 11 deep freshwater lakes in Japan and Europe. Results Our single nucleotide-resolved analysis, which was validated using shotgun metagenomic sequencing, uncovered 7–101 amplicon sequence variants for each of the 11 predominant bacterial lineages and demonstrated sympatric, allopatric, and temporal microdiversities that could not be resolved through conventional approaches. Clusters of samples with similar intra-lineage population compositions were identified, which consistently supported genetic isolation between Japan and Europe. At a regional scale (up to hundreds of kilometers), dispersal between lakes was unlikely to be a limiting factor, and environmental factors or genetic drift were potential determinants of population composition. The extent of microdiversification varied among lineages, suggesting that highly diversified lineages (e.g., Iluma-A2 and acI-A1) achieve their ubiquity by containing a consortium of genotypes specific to each habitat, while less diversified lineages (e.g., CL500-11) may be ubiquitous due to a small number of widespread genotypes. The lowest extent of intra-lineage diversification was observed among the dominant hypolimnion-specific lineage (CL500-11), suggesting that their dispersal among lakes is not limited despite the hypolimnion being a more isolated habitat than the epilimnion. Conclusions Our novel approach complemented the limited resolution of short-read amplicon sequencing and limited sensitivity of the metagenome assembly-based approach, and highlighted the complex ecological processes underlying the ubiquity of freshwater bacterioplankton lineages. To fully exploit the performance of the method, its relatively low read throughput is the major bottleneck to be overcome in the future.

Impacts of switching tillage to no-tillage and vice versa on soil structure, enzyme activities, and prokaryotic community profiles in Argentinean semi-arid soils

2021 ◽  
Author(s):  
L A Gabbarini ◽  
E Figuerola ◽  
J P Frene ◽  
N B Robledo ◽  
F M Ibarbalz ◽  
...  
Keyword(s):  
Community Structure ◽  
Soil Structure ◽  
Management Practices ◽  
Soil Health ◽  
Amplicon Sequencing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Prokaryotic Community ◽  
C Cycle

Abstract The effects of tillage on soil structure, physiology, and microbiota structure were studied in a long-term field experiment, with side-to-side plots, established to compare effects of conventional tillage (CT) vs. no-till (NT) agriculture. After 27 years, part of the field under CT was switched to NT and vice versa. Soil texture, soil enzymatic profiles, and the prokaryotic community structure (16S rRNA genes amplicon sequencing) were analysed at two soil depths (0–5, 5–10 cm) in samples taken 6, 18, and 30 months after switching tillage practices. Soil enzymatic activities were higher in NT than CT, and enzymatic profiles responded to the changes much earlier than the overall prokaryotic community structure. Beta diversity measurements of the prokaryotic community indicated that the levels of stratification observed in long-term NT soils were already recovered in the new NT soils thirty months after switching from CT to NT. Bacteria and Archaea OTUs, which responded to NT were associated with coarse soil fraction, SOC and C cycle enzymes while CT responders were related to fine soil fractions and S cycle enzymes. This study showed the potential of managing the soil prokaryotic community and soil health through changes in agricultural management practices.

scholarly journals High resolution species detection: accurate long read eDNA metabarcoding of North Sea fish using Oxford Nanopore sequencing

2021 ◽  
Author(s):  
Karlijn Doorenspleet ◽  
Lara Jansen ◽  
Saskia Oosterbroek ◽  
Oscar Bos ◽  
Pauline Kamermans ◽  
...  
Keyword(s):  
High Resolution ◽  
North Sea ◽  
Fish Species ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Nanopore Sequencing ◽  
Nature Restoration ◽  
Oxford Nanopore ◽  
Field Samples ◽  
Long Read

To monitor the effect of nature restoration projects in North Sea ecosystems, accurate and intensive biodiversity assessments are vital. DNA based techniques and especially environmental DNA (eDNA) metabarcoding from seawater is becoming a powerful monitoring tool. However, current approaches are based on genetic target regions of <500 nucleotides, which offer limited taxonomic resolution. This study aims to develop and validate a long read nanopore sequencing method for eDNA that enables improved identification of fish species. We designed a universal primer pair targeting a 2kb region covering the 12S and 16S rRNA genes of fish mitochondria. eDNA was amplified and sequenced using the Oxford Nanopore MiniON. Sequence data was processed using the new pipeline Decona, and accurate consensus identities of above 99.9% were retrieved. The primer set efficiency was tested with eDNA from a 3.000.000 L zoo aquarium with 31 species of bony fish and elasmobranchs. Over 55% of the species present were identified on species level and over 75% on genus level. Next, our long read eDNA metabarcoding approach was applied to North Sea eDNA field samples collected at ship wreck sites, the Gemini Offshore Wind Farm, the Borkum Reef Grounds and a bare sand bottom. Here, location specific fish and vertebrate communities were obtained. Incomplete reference databases still form a major bottleneck in further developing high resolution long read metabarcoding. Yet, the method has great potential for rapid and accurate fish species monitoring in marine field studies.

scholarly journals Transfer of a Multiclass Method for over 60 Antibiotics in Food from High Resolution to Low Resolution Mass Spectrometry

Molecules ◽  
2019 ◽  
Vol 24 (16) ◽  
pp. 2935 ◽  
Author(s):  
Giusepponi ◽  
Paoletti ◽  
Barola ◽  
Moretti ◽  
Saluti ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
High Resolution ◽  
Development Process ◽  
Tandem Mass ◽  
Laboratory Studies ◽  
Low Resolution ◽  
Method Transfer ◽  
Wide Range ◽  
Microbial Residues

A multiclass method has been developed to screen and confirm a wide range of anti-microbial residues in muscle and milk, and validated using liquid-chromatography coupled to (low-resolution, LR) tandem mass spectrometry (LC-QqQ). Over sixty antibiotics, belonging to ten distinct families, were included in the method scope. The development process was rapidly concluded as a result of two previously implemented methods. This consisted of identical sample treatments, followed by liquid chromatography, and coupled with high-resolution (HR) mass spectrometry (LC-Q-Orbitrap). The validation study was performed in the range between 10–1500 μg·kg−1 for muscles and 2–333 μg·kg−1 for milk. The main performance characteristics were estimated and, then, compared to those previously obtained with HR technique. The validity of the method transfer was ascertained also through inter-laboratory studies.

Phenotypic and molecular characterization of chickpea rhizobia isolated from different areas of Tunisia

2007 ◽  
Vol 53 (3) ◽  
pp. 427-434 ◽  
Author(s):  
Boulbaba L’taief ◽  
Bouaziz Sifi ◽  
Maher Gtari ◽  
Mainassara Zaman-Allah ◽  
Mokhtar Lachaâl
Keyword(s):  
Rflp Analysis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Phenotypic Characteristics ◽  
Cicer Arietinum L ◽  
Mesorhizobium Ciceri ◽  
Length Polymorphisms ◽  
Reference Strains ◽  
Tolerance To Salinity

Several phenotypic markers were used in this study to determine the biodiversity of rhizobial strains nodulating Cicer arietinum L. in various areas of Tunisia. They include symbiotic traits, the use of 21 biochemical substrates, and tolerance to salinity and pH. In addition, restriction fragment length polymorphisms (RFLPs) of PCR-amplified 16S rDNA were compared with those of reference strains. Numeric analysis of the phenotypic characteristics showed that the 48 strains studied fell into three distinct groups. This heterogeneity was highly supported by the RFLP analysis of 16S rRNA genes, and two ribotypes were identified. Chickpea rhizobia isolated from Tunisian soils are both phenotypically and genetically diverse. Results showed that 40 and 8 isolates were assigned, respectively, to Mesorhizobium ciceri and Mesorhizobium mediterraneum .

scholarly journals Evaluating seasonal hydrological extremes in mesoscale (pre-)Alpine basins at coarse 0.5° and fine hyperresolution

2019 ◽  
Vol 23 (3) ◽  
pp. 1593-1609 ◽  
Author(s):  
Joost Buitink ◽  
Remko Uijlenhoet ◽  
Adriaan J. Teuling
Keyword(s):  
High Resolution ◽  
Land Cover ◽  
Swiss Alps ◽  
Hydrological Response ◽  
Low Resolution ◽  
Continental Scale ◽  
Wide Range ◽  
Resolution Model ◽  
High Resolution Model ◽  
Basin Response

Abstract. Hydrological models are being applied for impact assessment across a wide range of resolutions. In this study, we quantify the effect of model resolution on the simulated hydrological response in five mesoscale basins in the Swiss Alps using the distributed hydrological model Spatial Processes in Hydrology (SPHY). We introduce a new metric to compare a range of values resulting from a distributed model with a single value: the density-weighted distance (DWD). Model simulations are performed at two different spatial resolutions, matching common practices in hydrology: 500 m × 500 m matching regional-scale models, and 40 km × 40 km matching global-scale modeling. We investigate both the intra-basin response in seasonal streamflow and evapotranspiration from the high-resolution model and the difference induced by the two different spatial resolutions, with a focus on four seasonal extremes, selected based on temperature and precipitation. Results from the high-resolution model show that the intra-basin response covers a surprisingly large range of anomalies and show that it is not uncommon to have both extreme positive and negative flux anomalies occurring simultaneously within a catchment. The intra-basin response was grouped by land cover, where different dominant runoff-generating processes are driving the differences between these groups. The low-resolution model failed to capture the diverse and contrasting response from the high-resolution model, since neither the complex topography nor land cover classes were properly represented. DWD values show that, locally, the hydrological response simulated with a high-resolution model can be a lot more extreme than a low-resolution model might indicate, which has important implications for global or continental scale assessments carried out at coarse grids of 0.5∘×0.5∘ or 0.25∘×0.25∘ resolution.

Sign in / Sign up

Export Citation Format

Share Document