functional metagenomics
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Life ◽  
2022 ◽  
Vol 12 (1) ◽  
pp. 85
Author(s):  
Claudia Leoni ◽  
Caterina Manzari ◽  
Hai Tran ◽  
Peter N. Golyshin ◽  
Graziano Pesole ◽  
...  

Amylomaltases are prokaryotic 4-α-glucanotransferases of the GH77 family. Thanks to the ability to modify starch, they constitute a group of enzymes of great interest for biotechnological applications. In this work we report the identification, by means of a functional metagenomics screening of the crystallization waters of the saltern of Margherita di Savoia (Italy), of an amylomaltase gene from the halophilic archaeon Haloquadratum walsbyi, and its expression in Escherichia coli cells. Sequence analysis indicated that the gene has specific insertions yet unknown in homologous genes in prokaryotes, and present only in amylomaltase genes identified in the genomes of other H. walsbyi strains. The gene is not part of any operon involved in the metabolism of maltooligosaccharides or glycogen, as it has been found in bacteria, making it impossible currently to assign a precise role to the encoded enzyme. Sequence analysis of the H. walsbyi amylomaltase and 3D modelling showed a common structure with homologous enzymes characterized in mesophilic and thermophilic bacteria. The recombinant H. walsbyi enzyme showed starch transglycosylation activity over a wide range of NaCl concentrations, with maltotriose as the best acceptor substrate compared to other maltooligosaccharides. This is the first study of an amylomaltase from a halophilic microorganism.


Author(s):  
Ariel Chazan ◽  
Andrey Rozenberg ◽  
Kentaro Mannen ◽  
Takashi Nagata ◽  
Ran Tahan ◽  
...  

2021 ◽  
Vol 12 ◽  
Author(s):  
Paul Tetteh Asare ◽  
Anna Greppi ◽  
Alessia Pennacchia ◽  
Katharina Brenig ◽  
Annelies Geirnaert ◽  
...  

Continuous in vitro fermentation models provide a useful tool for a fast, reproducible, and direct assessment of treatment-related changes in microbiota metabolism and composition independent of the host. In this study, we used the PolyFermS model to mimic the conditions of the chicken cecum and evaluated three nutritive media for in vitro modeling of the chicken cecal microbiota ecology and metabolism. We observed that our model inoculated with immobilized cecal microbiota and fed with a modified Viande Levure medium (mVL-3) reached a high bacterial cell density of up to approximately 10.5 log cells per mL and stable microbiota composition, akin to the host, during 82 days of continuous operation. Relevant bacterial functional groups containing primary fibrolytic (Bacteroides, Bifidobacteriaceae, Ruminococcaceae), glycolytic (Enterococcus), mucolytic (Bacteroides), proteolytic (Bacteroides), and secondary acetate-utilizing butyrate-producing and propionate-producing (Lachnospiraceae) taxa were preserved in vitro. Besides, conserved metabolic and functional Kyoto Encyclopedia of Genes and Genomes pathways were observed between in vitro microbiota and cecal inoculum microbiota as predicted by functional metagenomics analysis. Furthermore, we demonstrated that the continuous inoculation provided by the inoculum reactor generated reproducible metabolic profiles in second-stage reactors comparable to the chicken cecum, allowing for the simultaneous investigation and direct comparison of different treatments with a control. In conclusion, we showed that PolyFermS is a suitable model for mimicking chicken cecal microbiota fermentation allowing ethical and ex vivo screening of environmental factors, such as dietary additives, on chicken cecal fermentation. We report here for the first time a fermentation medium (mVL-3) that closely mimics the substrate conditions in the chicken cecum and supports the growth and metabolic activity of the cecal bacterial akin to the host. Our PolyFermS chicken cecum model is a useful tool to study microbiota functionality and structure ex vivo.


Author(s):  
David Conchouso ◽  
Amani Al-Ma'abadi ◽  
Hayedeh Behzad ◽  
Mohammed Alarawi ◽  
Masahito Hosokawa ◽  
...  

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e11885
Author(s):  
Philippe Bardou ◽  
Sandrine Laguerre ◽  
Sarah Maman Haddad ◽  
Sabrina Legoueix Rodriguez ◽  
Elisabeth Laville ◽  
...  

The earth harbors trillions of bacterial species adapted to very diverse ecosystems thanks to specific metabolic function acquisition. Most of the genes responsible for these functions belong to uncultured bacteria and are still to be discovered. Functional metagenomics based on activity screening is a classical way to retrieve these genes from microbiomes. This approach is based on the insertion of large metagenomic DNA fragments into a vector and transformation of a host to express heterologous genes. Metagenomic libraries are then screened for activities of interest, and the metagenomic DNA inserts of active clones are extracted to be sequenced and analysed to identify genes that are responsible for the detected activity. Hundreds of metagenomics sequences found using this strategy have already been published in public databases. Here we present the MINTIA software package enabling biologists to easily generate and analyze large metagenomic sequence sets, retrieved after activity-based screening. It filters reads, performs assembly, removes cloning vector, annotates open reading frames and generates user friendly reports as well as files ready for submission to international sequence repositories. The software package can be downloaded from https://github.com/Bios4Biol/MINTIA.


2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Léa Chuzel ◽  
Samantha L. Fossa ◽  
Madison L. Boisvert ◽  
Samanta Cajic ◽  
René Hennig ◽  
...  

Abstract Background Sulfate modification of N-glycans is important for several biological functions such as clearance of pituitary hormones or immunoregulation. Yet, the prevalence of this N-glycan modification and its functions remain largely unexplored. Characterization of N-glycans bearing sulfate modifications is hampered in part by a lack of enzymes that enable site-specific detection of N-glycan sulfation. In this study, we used functional metagenomic screening to identify enzymes that act upon sulfated N-acetylglucosamine (GlcNAc). Using multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (xCGE-LIF) -based glycoanalysis we proved their ability to act upon GlcNAc-6-SO4 on N-glycans. Results Our screen identified a sugar-specific sulfatase that specifically removes sulfate from GlcNAc-6-SO4 when it is in a terminal position on an N-glycan. Additionally, in the absence of calcium, this sulfatase binds to the sulfated glycan but does not remove the sulfate group, suggesting it could be used for selective isolation of sulfated N-glycans. Further, we describe isolation of a sulfate-dependent hexosaminidase that removes intact GlcNAc-6-SO4 (but not asulfated GlcNAc) from a terminal position on N-glycans. Finally, the use of these enzymes to detect the presence of sulfated N-glycans by xCGE-LIF is demonstrated. Conclusion The present study demonstrates the feasibility of using functional metagenomic screening combined with glycoanalytics to discover enzymes that act upon chemical modifications of glycans. The discovered enzymes represent new specificities that can help resolve the presence of GlcNAc-6-SO4 in N-glycan structural analyses.


Author(s):  
Che Fai Alex Wong ◽  
Liisa van Vliet ◽  
Swapnil Vilas Bhujbal ◽  
Chengzhi Guo ◽  
Marit Sletmoen ◽  
...  

2021 ◽  
Author(s):  
David Conchouso ◽  
Amani Al-Ma’abadi ◽  
Hayedeh Behzad ◽  
Mohammed Alarawi ◽  
Masahito Hosokawa ◽  
...  

<p>Droplet microfluidics techniques have shown promising results to study single-cells at high throughput. However, their adoption in laboratories studying “-omics” sciences is still irrelevant because of the field’s complex and multidisciplinary nature. To facilitate their use, here we provide engineering details and organized protocols for integrating three droplet-based microfluidic technologies into the metagenomic pipeline to enable functional screening of bioproducts at high throughput. First, a device encapsulating single-cells in droplets at a rate of ~ 250 Hz is described considering droplet size and cell growth. Then, we expand on previously reported fluorescent activated droplet sorting (FADS) systems to integrate the use of 4 independent fluorescence-exciting lasers (e.g., 405, 488, 561, 637 nm) in a single platform to make it compatible with different fluorescence-emitting biosensors. For this sorter, both hardware and software are provided and optimized for effortlessly sorting droplets at 60 Hz. Then, a passive droplet merger was also integrated into our method to enable adding new reagents to already made droplets at a rate of 200 Hz. Finally, we provide an optimized recipe for manufacturing these chips using silicon dry-etching tools. Because of the overall integration and the technical details presented here, our approach allows biologists to quickly use microfluidic technologies and achieve both single-cell resolution and high-throughput (> 50,000 cells/day) capabilities to mining and bioprospecting metagenomic data.</p>


2021 ◽  
Author(s):  
David Conchouso ◽  
Amani Al-Ma’abadi ◽  
Hayedeh Behzad ◽  
Mohammed Alarawi ◽  
Masahito Hosokawa ◽  
...  

<p>Droplet microfluidics techniques have shown promising results to study single-cells at high throughput. However, their adoption in laboratories studying “-omics” sciences is still irrelevant because of the field’s complex and multidisciplinary nature. To facilitate their use, here we provide engineering details and organized protocols for integrating three droplet-based microfluidic technologies into the metagenomic pipeline to enable functional screening of bioproducts at high throughput. First, a device encapsulating single-cells in droplets at a rate of ~ 250 Hz is described considering droplet size and cell growth. Then, we expand on previously reported fluorescent activated droplet sorting (FADS) systems to integrate the use of 4 independent fluorescence-exciting lasers (e.g., 405, 488, 561, 637 nm) in a single platform to make it compatible with different fluorescence-emitting biosensors. For this sorter, both hardware and software are provided and optimized for effortlessly sorting droplets at 60 Hz. Then, a passive droplet merger was also integrated into our method to enable adding new reagents to already made droplets at a rate of 200 Hz. Finally, we provide an optimized recipe for manufacturing these chips using silicon dry-etching tools. Because of the overall integration and the technical details presented here, our approach allows biologists to quickly use microfluidic technologies and achieve both single-cell resolution and high-throughput (> 50,000 cells/day) capabilities to mining and bioprospecting metagenomic data.</p>


mBio ◽  
2021 ◽  
Vol 12 (3) ◽  
Author(s):  
Kimberley V. Sukhum ◽  
Rhiannon C. Vargas ◽  
Manish Boolchandani ◽  
Alaric W. D’Souza ◽  
Sanket Patel ◽  
...  

ABSTRACT In agricultural settings, microbes and antimicrobial resistance genes (ARGs) have the potential to be transferred across diverse environments and ecosystems. The consequences of these microbial transfers are unclear and understudied. On dairy farms, the storage of cow manure in manure pits and subsequent application to field soil as a fertilizer may facilitate the spread of the mammalian gut microbiome and its associated ARGs to the environment. To determine the extent of both taxonomic and resistance similarity during these transitions, we collected fresh manure, manure from pits, and field soil across 15 different dairy farms for three consecutive seasons. We used a combination of shotgun metagenomic sequencing and functional metagenomics to quantitatively interrogate taxonomic and ARG compositional variation on farms. We found that as the microbiome transitions from fresh dairy cow manure to manure pits, microbial taxonomic compositions and resistance profiles experience distinct restructuring, including decreases in alpha diversity and shifts in specific ARG abundances that potentially correspond to fresh manure going from a gut-structured community to an environment-structured community. Further, we did not find evidence of shared microbial community or a transfer of ARGs between manure and field soil microbiomes. Our results suggest that fresh manure experiences a compositional change in manure pits during storage and that the storage of manure in manure pits does not result in a depletion of ARGs. We did not find evidence of taxonomic or ARG restructuring of soil microbiota with the application of manure to field soils, as soil communities remained resilient to manure-induced perturbation. IMPORTANCE The addition of dairy cow manure—stored in manure pits—to field soil has the potential to introduce not only organic nutrients but also mammalian microbial communities and antimicrobial resistance genes (ARGs) to soil communities. Using shotgun sequencing paired with functional metagenomics, we showed that microbial community composition changed between fresh manure and manure pit samples with a decrease in gut-associated pathobionts, while ARG abundance and diversity remained high. However, field soil communities were distinct from those in manure in both microbial taxonomic and ARG composition. These results broaden our understanding of the transfer of microbial communities in agricultural settings and suggest that field soil microbial communities are resilient against the deposition of ARGs or microbial communities from manure.


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