1. Bioengineered. 2015;6(2):89-98. doi: 10.1080/21655979.2015.1018493.
O'Mahony MM(1), Henneberger R, Selvin J, Kennedy J, Doohan F, Marchesi JR, Dobson
(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.
PMID: 25692994 [PubMed - indexed for MEDLINE]
2. Microbiologyopen. 2013 Oct;2(5):717-24. doi: 10.1002/mbo3.110. Epub 2013 Jul 25.
Chhabra S(1), Brazil D, Morrissey J, Burke JI, O'Gara F, N Dowling D.
(1)Department of Science and Health, Institute of Technology Carlow, Carlow,
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.
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
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).
(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
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.
PMID: 25240439 [PubMed - indexed for MEDLINE]
4. PLoS One. 2016 Aug 30;11(8):e0161979. doi: 10.1371/journal.pone.0161979.
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).
(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,
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
PMID: 27575719 [PubMed - in process]