CASE STUDIES

Transforming our ability to understand and manage our environment.

Examples of the Genomics work supported by the Liverpool and Sheffield nodes of the NERC Biomolecular Analysis Facility (the predecessor to NEOF) from 2019.

Examples of Proteomics work supported by the Centre for Proteomics Research, University of Liverpool.

Examples of Metabolomics work supported by the Centre for Proteomics Research, University of Liverpool.

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Environmental monitoring of SARS-CoV-2 (2021)

NEOF led the design of sequencing assays and generation data and analysis.

(Hilary et al 2021 Water Research 200, 117214
Brown et al 2021 Summary for SAGE)

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In support of the UK national response to COVID-19, NEOF prioritised work to use our skills in eDNA to detect and characterise coronavirus in wastewater. Working with the University of Bangor, we were able to demonstrate that SARS-CoV-2 could be genome-sequenced from wastewater taken from treatment plants, and were able to characterise its genetic diversity through the first wave of the pandemic. Having demonstrated the potential of the method, we then worked with UK Health Security Agency (UKHSA) Monitoring Health Protection Programme to deliver national surveillance of variants in wastewater for more than 1000 samples per week. This initial work by NEOF led to a major consortium of sequencing laboratories supported by funding from UKHSA. Highlights have included a rapid report to the CMO of the alpha (“Kent”) variant present in samples from London through November and December 2020, and an outbreak of an E484K mutation in Bristol used as a case study in a paper to SAGE. 

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Bumblebee colony density on farmland (2021)

NEOF supported training student in molecular methods and analysis within the visitor facility.
(Timberlake et al, 2021 JAE 1016-16)
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NEOF provided support and training to a PhD student project in the NEOF Visitor Facility to understand how the landscape affects the density of bumblebees, which are an important pollinator providing an essential ecosystem function. Molecular genotyping was used to estimate colony density on farmland and how this varied with nectar supply and garden cover. These results discovered the importance of the late spring as a bottleneck for resources, and highlighted the role that environmental stewardship programmes of farmland could have in increasing the density of bumblebee colonies, for example by modifying mowing regimes to delay flowering at field edges. This provides an example of how omics methods can provide an environmental solution within a systems approach. This work involved both international partners and conservation bodies, and received media interest, including in the farming press.

Timberlake et al. (2021) Bumblebee colony density on farmland is influenced by late-summer nectar supply and garden cover J Appl Ecol 58, 1006–1016.

Photo credit: Francis Franklin

Gradients in richness and turnover of a forest passerine’s diet prior to breeding: A mixed model approach applied to faecal metabarcoding data (2020)

(Shutt et al. 2020 Molecular Ecology, 29, 1199–1213). 
NBAF supported metabarcoding and bioinformatic analysis.
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Generalist insectivorous bird species may feed on a range of different prey items, though quantifying variation in prey type and feeding preferences remains a substantial challenge. In this study, Shutt et al. used faecal DNA metabarcoding to assess how diet of blue tits varies across a transect of 39 sites in Scotland. They showed that most prey items are rare, and that diet composition shows substantial change through the feeding season. This study provides useful insights into temperate foodwebs, and shows how DNA metabarcoding can provide a robust framework for estimating how diet composition changes in both space and time.
Photo credit: Rob Knell

Meiotic drive reduces egg-to-adult viability in stalk-eyed flies (2019)

(Finnegan et al. 2019 Proceedings of the Royal Society London B 286, 20191414).
NBAF supported microsatellite genotyping and data analysis.
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Several species exhibit the phenomenon of Sex-Ratio (SR) meiotic drive, a selfish genetic element located on the X-chromosome that causes dysfunction of Y-bearing sperm, and one that has the potential to be exploited in the control of harmful insect vectors such as some mosquitoes. SR is transmitted to up to 100% of offspring, causing extreme sex-ratio bias. SR in several species is found in a stable polymorphism, suggesting that there must be strong frequency-dependent selection resisting its spread. This study investigated the effect of SR on female and male egg-to-adult viability in the Malaysian stalk-eyed fly, Teleopsis dalmanni. SR meiotic drive in this species is old, and appears to be broadly stable at a moderate (ca 20%) frequency. Large-scale controlled crosses were used to estimate the strength of selection acting against SR in female and male carriers. SR was found to reduce the egg-to-adult viability of both sexes. In females, homozygous females experienced greater reduction in viability and the deleterious effects of SR were additive. The male deficit in viability was not different from that in homozygous females. The study contributed to our understanding of how these reductions in egg-to-adult survival maintain the SR polymorphism in this species.

Individual variation in early-life telomere length and survival in a wild mammal (2019)

(van Lieshout et al. 2019, Molecular Ecology 28, 4152–4165).
NBAF supported telomere length analysis using quantitative PCR methods
.
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Individual variation in survival probability due to differential responses to early-life environmental conditions is important in the evolution of life histories and senescence. A biomarker allowing quantification of such individual variation, and that links early-life environmental conditions with survival by providing a measure of the conditions that have been experienced, is telomere length. This study examined telomere dynamics in 24 cohorts of European badgers (Meles meles). It found a complex cross-sectional relationship between telomere length and age, with no apparent loss over the first 29 months, but with both decreases and increases in telomere length at older ages. Overall, there was low within-individual consistency in telomere length over individual lifetimes. Unexpectedly, some individuals showed reliable increases in telomere length. There was evidence that early-life telomere length predicts lifespan due to an effect on survival to adulthood (≥1 year old), but not on adult survival probability. These results showed that the relationship between early-life telomere length and lifespan was driven by conditions in early-life, where early-life telomere length varied strongly among cohorts. Our data provide evidence for associations between early-life telomere length and individual life history, and highlight the dynamics of telomere length across individual lifetimes due to individuals experiencing different early-life environments.

Parallel evolution of complex centipede venoms revealed by comparative proteotranscriptomic analyses (2019)

(Jenner et al. 2019, Molecular Biology and Evolution 36, 2748–2763).
NBAF supported transcriptome sequencing
.
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The evolution of venom is an ancient adaptation to the predatory lifestyle of centipedes. Until now, venom evolution has been studied in only one of the five orders of centipede. Using transcriptome sequencing for non-model species, Jenner et al. were able to uncover a striking diversity of venom components comprising 93 phylogenetically distinct protein and peptide families. They were able to uncover the processes of gene duplication leading to this diversity and found that the majority, 67, of these families were found in single orders. Integration of transcriptomes with proteomic analysis was central to their approach in assigning function to gene families.
Photo credit: Colin Kinear

Divergent national-scale trends of microbial and animal biodiversity revealed across diverse temperate soil ecosystems (2019)

(George et al. 2019 Nature Communications 10, 1107).
NBAF supported metabarcoding.
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Biodiversity within soil accounts for around 25% of global biodiversity, yet the large-scale distribution patterns of this biodiversity are very poorly understood. Here, George et al. used rapid, molecular approaches to quantity this diversity across UK landscapes in a major survey of 436 sites. They showed that animal microbial diversity followed markedly different patterns; animal biodiversity was driven by intensive land use, whereas microbial biodiversity was driven by soil properties. This benchmark survey of UK soil, using genomic methods, provides a solid framework to understand and value this important component of UK biodiversity.

Lack of long-term acclimation in Antarctic encrusting species suggests vulnerability to warming (2019)

(Clark et al. 2019 Nature Communications, 10, 3383).
NBAF supported RNA-seq and metabarcoding.
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Polar marine invertebrates are predicted to experience major population declines due to warming oceans. Understanding how individual species’ respond to temperature changes in an experimental setting may allow us to predict wider responses in ocean systems. Clark et al. investigated the responses of spatially dominant calcified marine worms in artificial heating experiments using RNA-Seq and expression profiling. They contrasted these results with co-occurring prokaryotic bacteria communities investigated with amplicon sequencing. Marine worms showed significant stress responses in warmed environments, with this response contrasting to bacteria that showed no difference in community composition with temperature. These results show different ecosystem changes in response to a changing climate.
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Proteomics case study: Targeting specific proteins or peptides 

Aim: To identify sex peptides from Drosophila subobscura accessory gland samples. 

Samples: 3 x 10 excised excretory glands from D. subobscura. 

Results: There is no protein database for D. sub in UniProt, However, we were able to identify a suitable database by contacting other re-searchers who work with this organism. In addition, by pre-calculating the expected masses of trypsin cleavage products from the specific sex peptides of interest, we were able to perform target-ed analysis of these peptides to get a positive identification. 

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Proteomics case study: Species identification 

Aim: Rapid identification of species and sex, based on pattern recognition of mass spectra, for closely related species that are notoriously diffi-cult to identify by eye. 

Samples: 150-170 whole mosquito samples of each of D. bifasciata, D. pseudoobscura, D. suboscura, D. melanogaster and D. simulans were used to build a species model; 400 male and 400 female mos-quitoes across the 5 species were used to build the sex determina-tion model. 

Results: 95 % accuracy in species determination, and 99 % accuracy in sex determination. 

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Proteomics case study: Impact of viral infection on host proteome 

Aim: To quantify changes to the Asian tiger mosquito proteome when ex-posed to virus. 

Samples: 3 replicates of 14 conditions. 

Results: By using TMT isobaric labelling, more than 5000 proteins were quantified including 11 out of 12 virus proteins. Proteins were iden-tified from a genome database. 

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Proteomics case study: Secretome proteome of pseudomonas aeruginosa. 

Aim: To identify the difference in multi-drug resistance strain verse the wild type. 

Samples: 5 replicates of 2 pseudomonas aeruginosa strains. 

Results: Proteins were concentrated from 10 mL to 100 μL by strataclean beads. Digestion was conducted from straight from the strataclean beads. Over 1000 proteins were identified in each sample. Sam-ples were processed for label free quantification. 

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Proteomics case study: Identification of phosphorylation targets for a phosphatase in human cell line. 

Aim: To determine which proteins are phosphorylated and the down stream effects of phosphorylation. 

Samples: 4 replicates of 4 clonal cell lines. 

Results: Samples were labelled with TMT isobaric labelling. The sample was fractionated into 12 fractions and then each used to deter mine i) global protein changes and ii) phosphopeptide changes. Around 6000 proteins and in excess of 10,000 phosphopeptides were identified, with the vast majority being quantified across all 16 samples.

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Metabolomics case study: Using metabolomics to understand how environmental bacteria respond to pharmaceutical exposure

Mol. BioSyst. 2016, 12, 1367. https://doi.org/10.1039/c5mb00889a

PLoS ONE 2016 11: e0156509. https://doi.org/10.1371/journal.pone.0156509

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It has been known for decades that human pharmaceuticals are present in wastewater treatment plants and pollute rivers and estuaries at levels that affect aquatic organisms (see PNAS paper).  What isn’t know is how microbial communities respond to exposure to APIs (active pharmaceutical ingredients) and this is likely to be at the phenotypic level where the microorganism responds to this stress.  In addition, bacteria and fungi may transform APIs into other substances that could also affect both the planktonic and benthic communities.

In a paper published in Molecular BioSystems we used a combination of metabolic fingerprinting using Fourier transform infrared (FT-IR) spectroscopy with metabolic profiling using gas chromatography-mass spectrometry (GC-MS) to investigate phenotypic changes in an environmental isolate of Pseudomonas putida.  Initially this bacterium was exposed to six APIs (acetaminophen, atenolol, diclofenac, ibuprofen, mefenamic acid, propranolol) at levels that did not affect the growth profile of the organism.  Focussing on propranolol (a β-blocker used to treat hypertension) we found significant changes in carbon metabolism and in particular alterations in the concentration of energy related metabolites, including ATP.  In addition, we found profound changes in the saturation levels of cardiolipins which may be related to the synthesis and activation of solvent extrusion pumps which would eliminate propranolol from inside these bacterial cells.

 

We went onto conduct an in-depth metabolomics study focussing on the efflux pumps involved in solvent extrusion which was published in PLoS ONE.  In this study we included a series of efflux pump knockout mutants of P. putida DOT-T1E to assess the effect of this transporter on propranolol and metabolism.

 

These studies have opened up the exciting prospect of understanding how microbial communities adapt when exposed to toxic materials.

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Metabolomics case study: Metabolic Profiling of Geobacter sulfurreducens during Industrial Bioprocess Scale-Up

Appl. Environ. Micro. 2015 81: 3288-3298. https://doi.org/10.1128/AEM.00294-15

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Geobacter sulfurreducens is an important bacterium as it is capable of global recycling of metals and therefore is important for bioremediation applications.  In addition, this organism produces biogenic magnetite nanoparticles (BMNPs) which may have industrial application.  However, during industrial scale-up of this BMNP production bioprocess we found an extended lag phase of 24 h for cells grown in 5-litre bioreactors compared to only 6 h those grown in 100 mL bottles.

In a paper published in Applied and Environmental Microbiology we investigated the phenotypic changes associated with the extended lag-phase during scale up of this bioprocess scale up using gas chromatography-mass spectrometry (GC-MS) for metabolic profiling.

 

GC-MS analysis showed that there were several changes in metabolites during scaleup and when relevant metabolites were overlaid onto a metabolic network of G. sulfurreducens we found that there was a limited availability of oxaloacetate and nicotinamide which would seem to be the main metabolic bottlenecks.  Metabolite feeding experiments found that nicotinamide supplementation (1 mM) significantly reduced the lag phase in 5 L bioreactors from 24 h to 6 h, therefore resulting in a more rapid industrial bioprocess.

 

This study illustrates the exciting prospect of using metabolomics to enhance industrial scaleup of environmentally relevant microbial systems.

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Metabolomics case study: Metabolism in Action: Developing stable isotope probing using Raman and infrared imaging for understanding metabolic flux at the single cell level

Anal. Chem. 2021, 93, 6, 3082–3088; https://doi.org/10.1021/acs.analchem.0c03967

Analyst 2021; 146, 1734-1746; https://doi.org/10.1039/D0AN02319A

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Complex microbial communities are found throughout ecosystems on the planet and these play essential functions that support animal and aquatic life.

Despite the vital role that bacteria play, linking these microbes with specific metabolic functions is difficult, since microbial communities consist of numerous and phylogenetically diverse microbes.  This is compounded by the microscopic size of these microorganism (1-2 µm).

There is therefore a need to develop metabolomics solutions that generate images that are based on the biochemistry of the sample under analysis, and that provide information at micron and sub-micrometre scales.  With the advent of spontaneous Raman and variants that can be tuned to specific vibrations (viz SRS and CARS), these, along with optical-photothermal infrared (O-PTIR) microscopy open up the tantalising opportunity to follow metabolism in microbial communities.

Crossfeed

 

In a recent series of papers published in Analytical Chemistry and Analyst we have shown that the cellular uptake of stable isotope-labelled compounds by bacteria can be probed at the single-cell level using both Raman and infrared spectroscopies.  This is due to the ability of these imaging techniques to monitor chemical vibrations that are affected by the incorporation of “heavy” atoms by cells and thus can be used to understand microbial systems.  In this series of experiments, we have shown that it is possible to measure incorporation of 13C and 15N substrates, follow the dynamics of the rate of incorporation, as well as employing deuterated water to assess cross feeding in microbial communities.

This research has opened up the exciting prospect that these enabling chemical imaging technologies will guide the identification of primary substrate consumers in complex microbial communities in situ.  This we believe represents a key step towards the characterisation of novel genes, enzymes and metabolic flux analysis in microbial consortia.