16s-rrna.net Metagenome Sequencing, 16s Sequencing, 18s Sequencing, and Fungi Sequencing with MRDNA

1. Bioengineered. 2015;6(2):89-98. doi: 10.1080/21655979.2015.1018493.

 

Inhibition of the growth of Bacillus subtilis DSM10 by a newly discovered

antibacterial protein from the soil metagenome.

 

O'Mahony MM(1), Henneberger R, Selvin J, Kennedy J, Doohan F, Marchesi JR, Dobson

AD.

 

Author information:

(1)a School of Microbiology and Marine Biotechnology Center; Environmental

Research Institute; University College Cork ; Cork , Ireland.

 

A functional metagenomics based approach exploiting the microbiota of suppressive

soils from an organic field site has succeeded in the identification of a clone

with the ability to inhibit the growth of Bacillus subtilis DSM10. Sequencing of

the fosmid identified a putative β-lactamase-like gene abgT. Transposon

mutagenesis of the abgT gene resulted in a loss in ability to inhibit the growth

of B. subtilis DSM10. Further analysis of the deduced amino acid sequence of AbgT

revealed moderate homology to esterases, suggesting that the protein may possess

hydrolytic activity. Weak lipolytic activity was detected; however the clone did

not appear to produce any β-lactamase activity. Phylogenetic analysis revealed

the protein is a member of the family VIII group of lipase/esterases and clusters

with a number of proteins of metagenomic origin. The abgT gene was sub-cloned

into a protein expression vector and when introduced into the abgT transposon

mutant clones restored the ability of the clones to inhibit the growth of B.

subtilis DSM10, clearly indicating that the abgT gene is involved in the

antibacterial activity. While the precise role of this protein has yet to fully

elucidated, it may be involved in the generation of free fatty acid with

antibacterial properties. Thus functional metagenomic approaches continue to

provide a significant resource for the discovery of novel functional proteins and

it is clear that hydrolytic enzymes, such as AbgT, may be a potential source for

the development of future antimicrobial therapies.

 

DOI: 10.1080/21655979.2015.1018493

PMCID: PMC4601227

PMID: 25692994  [PubMed - indexed for MEDLINE]

 

 

2. Microbiologyopen. 2013 Oct;2(5):717-24. doi: 10.1002/mbo3.110. Epub 2013 Jul 25.

 

Characterization of mineral phosphate solubilization traits from a barley

rhizosphere soil functional metagenome.

 

Chhabra S(1), Brazil D, Morrissey J, Burke JI, O'Gara F, N Dowling D.

 

Author information:

(1)Department of Science and Health, Institute of Technology Carlow, Carlow,

Ireland.

 

Mineral phosphate solubilization (MPS) microorganisms are important for their

provision of orthophosphate anions for plant growth promotion activity in soil.

In this study, we applied a functional metagenomic approach to identify this

trait directly from the microbiome in barley rhizosphere soil that had not

received P fertilizer over a 15-year period. A fosmid system was used to clone

the metagenome of which 18,000 clones (~666 Mb of DNA) was screened for MPS.

Functional assays and High Performance Liquid Chromatography analysis recognized

gluconic acid production and MPS activity in the range 24.8-77.1 mmol/L and

27.6-38.16 μg/mL, respectively, when screened in an Escherichia coli host (at

frequency of one MPS-positive clone hit per 114 Mb DNA tested). The MPS clones

(with average insert size of ~37 kb) were analysed by 454 Roche sequencing and

annotated. A number of genes/operons with homology to Phosphorous (P) uptake,

regulatory and solubilization mechanisms were identified, linking the MPS

function to the uncultivated microbiome present in barley rhizosphere soil.

 

© 2013 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

 

DOI: 10.1002/mbo3.110

PMCID: PMC3831634

PMID: 23894099  [PubMed - indexed for MEDLINE]

 

 

3. J Biotechnol. 2015 May 10;201:60-8. doi: 10.1016/j.jbiotec.2014.09.010. Epub 2014

Sep 18.

 

Successful heterologous expression of a novel chitinase identified by sequence

analyses of the metagenome from a chitin-enriched soil sample.

 

Stöveken J(1), Singh R(1), Kolkenbrock S(2), Zakrzewski M(3), Wibberg D(3),

Eikmeyer FG(3), Pühler A(3), Schlüter A(3), Moerschbacher BM(4).

 

Author information:

(1)Department of Plant Biology and Biotechnology, Westphalian Wilhelm's

University Münster, Schlossplatz 8, 48143 Münster, Germany. (2)evocatal GmbH,

Alfred-Nobel-Strasse 10, 40789 Monheim am Rhein, Germany. (3)Center for

Biotechnology, CeBiTec, Bielefeld University, Universitätsstraße 27, 33615

Bielefeld, Germany. (4)Department of Plant Biology and Biotechnology, Westphalian

Wilhelm's University Münster, Schlossplatz 8, 48143 Münster, Germany. Electronic

address: moersch@uni-muenster.de.

 

Chitin and its derivative chitosan are abundant natural polysaccharides with many

potential industrial applications. Metagenomic analysis of chitin-enriched soil

samples using the Roche Genome Sequencer FLX platform led to the identification

of several novel genes for chitin and chitosan modifying enzymes (CCMEs) which

may be used to produce novel chitosans. The sequencing approach yielded 2,281,090

reads with an average length of 378 bp amounting to a total sequence information

of approximately 851 Mb. Assembly of the obtained sequences comprised 699,710

reads representing 30.68% of all reads. A total of 6625 contigs larger than 500

bp containing 16,289 predicted genes are included in the assembly. Taxonomic

profiling of the indigenous microbial community by applying the software CARMA

revealed that 96.1% of the reads were of bacterial origin including 17% assigned

to the family Xanthomonadaceae. Several putative genes encoding CCMEs were

identified by comparison against the GenBank database, inclusive a full-length

chitinase gene which was codon optimized for Escherichia coli and heterologously

synthesized as a Strep-tagged protein in E. coli Rosetta 2 using the pET vector

system. Approximately 5mg of the novel active chitinase was purified as

demonstrated by dot assay analysis using glycol chitin as a substrate. Next

generation metagenomic sequencing, thus, emerges as a new and powerful tool for

the identification of potentially novel biocatalysts of biotechnological value.

 

Copyright © 2014 Elsevier B.V. All rights reserved.

 

DOI: 10.1016/j.jbiotec.2014.09.010

PMID: 25240439  [PubMed - indexed for MEDLINE]

 

 

4. PLoS One. 2016 Aug 30;11(8):e0161979. doi: 10.1371/journal.pone.0161979.

eCollection 2016.

 

Quantifying the Sensitivity of Soil Microbial Communities to Silver Sulfide

Nanoparticles Using Metagenome Sequencing.

 

Doolette CL(1), Gupta VV(2), Lu Y(3), Payne JL(4), Batstone DJ(3), Kirby JK(5),

Navarro DA(1,)(5), McLaughlin MJ(1,)(5).

 

Author information:

(1)School of Agriculture Food and Wine, The University of Adelaide, Adelaide,

Australia. (2)CSIRO Agriculture, Functional Microbial Ecology, Adelaide,

Australia. (3)Advanced Water Management Centre (AWMC), The University of

Queensland, St. Lucia, Australia. (4)School of Natural and Built Environments,

University of South Australia, Adelaide, Australia. (5)CSIRO Land and Water,

Environmental Contaminant Mitigation and Technologies Research Program, Adelaide,

Australia.

 

Soils are a sink for sulfidised-silver nanoparticles (Ag2S-NPs), yet there are

limited ecotoxicity data for their effects on microbial communities. Conventional

toxicity tests typically target a single test species or function, which does not

reflect the broader community response. Using a combination of quantitative PCR,

16S rRNA amplicon sequencing and species sensitivity distribution (SSD) methods,

we have developed a new approach to calculate silver-based NP toxicity thresholds

(HCx, hazardous concentrations) that are protective of specific members

(operational taxonomic units, OTUs) of the soil microbial community. At the HC20

(80% of species protected), soil OTUs were significantly less sensitive to

Ag2S-NPs compared to AgNPs and Ag+ (5.9, 1.4 and 1.4 mg Ag kg-1, respectively).

However at more conservative HC values, there were no significant differences.

These trends in OTU responses matched with those seen in a specific microbial

function (rate of nitrification) and amoA-bacteria gene abundance. This study

provides a novel molecular-based framework for quantifying the effect of a

toxicant on whole soil microbial communities while still determining sensitive

genera/species. Methods and results described here provide a benchmark for

microbial community ecotoxicological studies and we recommend that future

revisions of Soil Quality Guidelines for AgNPs and other such toxicants consider

this approach.

 

DOI: 10.1371/journal.pone.0161979

PMCID: PMC5004803

PMID: 27575719  [PubMed - in process]

 

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