Omics for understanding microbial functional dynamics

Jansson, Janet; Neufeld, Josh D.; Moran, Mary Ann; Gilbert, Jack A.

doi:10.1111/j.1462-2920.2011.02518.x

Cited by 80 publications

(53 citation statements)

References 17 publications

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“…However, there is still a fundamental lack of methods available for isolating each fraction concurrently for parallel downstream molecular analyses. This method gap is not comparable with the rapid advances made in high-resolution omic-based technologies (Jansson et al, 2012). Indeed, an integrative systems-based strategy, whereby multiple omic-datasets (metagenomic, metatranscriptomic and metaproteomic) are employed holds significant promise for elucidating discrete microbial community dynamics (Muller et al, 2012), such as the linkage of metabolic processes with functional phylotypes (Yu & Zhang, 2012).…”

mentioning

confidence: 92%

A sequential co-extraction method for DNA, RNA and protein recovery from soil for future system-based approaches

Gunnigle

Ramond

Frossard

et al. 2014

Journal of Microbiological Methods

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A co-extraction protocol is presented that sequentially isolates core biopolymer fractions (DNA, RNA, protein) from edaphic microbial communities. In order to confirm compatibility with downstream analyses, bacterial T-RFLP profiles were generated from the DNA-and RNA-derived fractions of an arid-based soil, with metaproteomics undertaken on the corresponding protein fraction. were transferred to sterile 2 ml screw cap tubes (WhiteSci, Gauteng, SA). Ten grams was required for Soil type-A, while only 1 g of soil was required to recover sufficient DNA, RNA and protein from Soil type-B. Thereafter, 0.5 ml of a 10% (w/v) hexadecyltrimethylammonium 3 Sigma) was added to each 2 ml sample tube. Each tube was also supplemented with 0.25 g of zirconia beads (0.1 and 0.5 mm; BioSpec, Bartlesville, OK, USA). Cell lysis was achieved by bead beating, with the aqueous phase pooled and phenol removed by a chloroformisoamyl alcohol (24:1) phase separation (Fig. 1A) Total nucleic acids were precipitated by 30% polyethylene glycol 8000 (Sigma) -1.6M NaCl at 10°C for 2 h with 70% (v/v) ice-cold ethanol wash steps (twice). KeywordsThe organic phase containing the protein was also pooled and retained in a separate tube at 4°C. Residual liquid was removed from the original sample tubes containing the soil and 0.5 ml of fresh 1% sodium dodecyl sulphate (SDS; Merck) extraction buffer (10 mM Tris, 5 mM MgCl 2 ; pH 8), supplemented with protease inhibitor cocktail (10 μl/ml; Sigma) was added (Fig. 1A). A second bead-beating extraction step was applied with subsequent centrifugation and benzonase treatment (250U/μl; Sigma) undertaken on the pooled supernatants to remove any remaining nucleic acids. Thereafter, an additional phenol:chloroform:isoamyl alcohol (25:24:1; pH 8) step was applied to partition the organic phase, which was then added to the organic fraction from the first extraction step (Fig. 1A). Five volumes of 0.1M ammonium acetate in methanol was used to precipitate the protein (-20°C, overnight) with additional washing steps using ice-cold methanol (twice) and acetone (80% v/v; twice). The resulting pellets were resolubilised in 6M guanidine buffer supplemented with 10mM DTT 10mM Tris 5mM CaCL 2 (pH 8) with 1 min of sonication (sonicator bath VWR USC2600).Successful co-extraction of DNA and RNA was confirmed and quantified using a Nanodrop 1000 spectrophotometer (NanoDrop Products, Wilmington, DE, USA) and gel electrophoresis ( Step 1 Initial sample preparationStep 4 T-RFLP communityStep 5 Shotgun label-free metaproteomics 1 2 3 4 5 6 7 8 9 10Choroform:isoamyl alcohol (24:1) stepStep 2 Step 3 Isolation of protein fraction were identified, with 48 RNA-based OTUs recorded (Fig. 1C). These results were consistent with recent studies where commercial DNA extraction kits were employed (Makhalanyane et al. 2013; Stomeo et al., 2013), suggesting that representative phylogenetic information was recovered from oligotrophic soil using this approach. We noted that 21 OTUs were unique for the RNA-derived cDNA samples, confirmin...

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mentioning

confidence: 92%

A sequential co-extraction method for DNA, RNA and protein recovery from soil for future system-based approaches

Gunnigle

Ramond

Frossard

et al. 2014

Journal of Microbiological Methods

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“…In addition, he discovered the importance of microbial root inhabitants for plant growth and health. Since that time, much has been learned about microorganism and plant host interactions, especially in recent years by means of new next generation sequencing (NGS) techniques, "omics"-technologies, and microscopic methods [2][3][4][5]. The rhizosphere is of central importance not only for plant nutrition, health, and quality, but also for microorganismdriven carbon sequestration, ecosystem functioning, and nutrient cycling in terrestrial ecosystems.…”

Section: The Plant As Meta-organismmentioning

confidence: 99%

Next-Generation Bio-Products Sowing the Seeds of Success for Sustainable Agriculture

et al. 2013

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Abstract:Plants have recently been recognized as meta-organisms due to a close symbiotic relationship with their microbiome. Comparable to humans and other eukaryotic hosts, plants also harbor a "second genome" that fulfills important host functions. These advances were driven by both "omics"-technologies guided by next-generation sequencing and microscopic insights. Additionally, these new results influence applied fields such as biocontrol and stress protection in agriculture, and new tools may impact (i) the detection of new bio-resources for biocontrol and plant growth promotion, (ii) the optimization of fermentation and formulation processes for biologicals, (iii) stabilization of the biocontrol effect under field conditions, and (iv) risk assessment studies for biotechnological applications. Examples are presented and discussed for the fields mentioned above, and next-generation bio-products were found as a sustainable alternative for agriculture.

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“…Even an individual scientist may deal with cases from various biological domains in her overall research. While most of the phenomena studied by systems biologists are processes taking place within single organisms or even single cells (Helms, 2008), there is also research pertaining to the interaction of microorganisms or the distribution and transfer of genes across whole microbial communities (e.g., using data of metagenomics projects), so that systems biology can have connections to ecology (Crawford et al, 2005;Jansson et al, 2012). Studies on (changes in) gene regulatory networks are germane to evolutionary biology (Davidson, 2006;Davidson & Erwin, 2006;Linksvayer et al, 2012;O'Malley & Soyer, 2012).…”

Section: Systems Biologymentioning

confidence: 99%

Systems biology and the integration of mechanistic explanation and mathematical explanation

Brigandt

2013

Studies in History and Philosophy of Science Part C: Studies in

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The paper discusses how systems biology is working toward complex accounts that integrate explanation in terms of mechanisms and explanation by mathematical models-which some philosophers have viewed as rival models of explanation. Systems biology is an integrative approach, and it strongly relies on mathematical modeling. Philosophical accounts of mechanisms capture integrative in the sense of multilevel and multifield explanations, yet accounts of mechanistic explanation (as the analysis of a whole in terms of its structural parts and their qualitative interactions) have failed to address how a mathematical model could contribute to such explanations. I discuss how mathematical equations can be explanatorily relevant. Several cases from systems biology are discussed to illustrate the interplay between mechanistic research and mathematical modeling, and I point to questions about qualitative phenomena (rather than the explanation of quantitative details), where quantitative models are still indispensable to the explanation. Systems biology shows that a broader philosophical conception of mechanisms is needed, which takes into account functional-dynamical aspects, interaction in complex networks with feedback loops, system-wide functional properties such as distributed functionality and robustness, and a mechanism's ability to respond to perturbations (beyond its actual operation). I offer general conclusions for philosophical accounts of explanation.

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Omics for understanding microbial functional dynamics

Cited by 80 publications

References 17 publications

A sequential co-extraction method for DNA, RNA and protein recovery from soil for future system-based approaches

A sequential co-extraction method for DNA, RNA and protein recovery from soil for future system-based approaches

Next-Generation Bio-Products Sowing the Seeds of Success for Sustainable Agriculture

Systems biology and the integration of mechanistic explanation and mathematical explanation

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