Publications by year
In Press
Tennant RK, Jones RT, Brock F, Cook CG, Love J, Turney CSM, Lee R (In Press). A New Flow Cytometry Method Enabling Rapid Purification of Fossil Pollen from Terrestrial Sediments for AMS Radiocarbon Dating. Journal of Quaternary Science
Wagley S, Morcrette H, Kovacs-Simon A, Yang Z, Power A, Tennant R, Love J, Murray N, Titball R, Butler C, et al (In Press). Bacterial dormancy: a subpopulation of viable but non-culturable cells demonstrates better fitness for revival. PLoS Pathogens
Wojcik E, Singleton C, Chapman L, Parker D, Love J (In Press). Plant Biomass as Biofuels. eLS
2023
Porter A, Barber D, Hobbs C, Love J, Power AL, Bakir A, Galloway TS, Lewis C (2023). Uptake of microplastics by marine worms depends on feeding mode and particle shape but not exposure time.
Sci Total Environ,
857(Pt 1).
Abstract:
Uptake of microplastics by marine worms depends on feeding mode and particle shape but not exposure time.
The uptake of microplastics into marine species has been widely documented across trophic levels. Feeding mode is suggested as playing an important role in determining different contamination loads across species, but this theory is poorly supported with empirical evidence. Here we use the two distinct feeding modes of the benthic polychaete, Hediste diversicolor (The Harbour Ragworm) (O.F. Müller, 1776), to test the hypothesis that filter feeding will lead to a greater uptake of microplastic particles than deposit feeding. Worms were exposed to both polyamide microfragments and microfibres in either water (as filter feeders) or sediment (as deposit feeders) for 1 week. No effect of exposure time was found between 1 day and 1 week (p > 0.19) but feeding mode was found to significantly affect the number of microfibres recovered from each worm (p < 0.001). When exposed to microfibers, filter feeding worms took up ≈15,000 % more fibres than deposit feeding worms (p < 0.001), whereas when feeding on microfragments there was no difference between feeding modes. Our data demonstrate that both feeding mode and particle characteristics significantly influence the uptake of microplastics by H. diversicolor. Using imaging flow cytometry, filter feeders were found to take up a broader size range of particles, with significantly more smaller and larger particles than deposit feeders (p < 0.05), commensurate with the range of plastics isolated from the guts of ragworms recovered from the environment. These results demonstrate that biological traits are useful in understanding the uptake of plastics into marine worms and warrant further exploration as a tool for understanding the bioaccessibility of plastics to marine organisms.
Abstract.
Author URL.
2022
Winder JC, Hewlett M, Liu P, Love J (2022). Conversion of Biomass to Chemicals via Electrofermentation of Lactic Acid Bacteria.
Energies,
15(22), 8638-8638.
Abstract:
Conversion of Biomass to Chemicals via Electrofermentation of Lactic Acid Bacteria
Microbial electrosynthesis is the process of supplying electrons to microorganisms to reduce CO2 and yield industrially relevant products. Such systems are limited by their requirement for high currents, resulting in challenges to cell survival. Electrofermentation is an electron-efficient form of microbial electrosynthesis in which a small cathodic or anodic current is provided to a culture to alter the oxidation–reduction potential of the medium and, in turn, alter microbial metabolism. This approach has been successfully utilised to increase yields of diverse products including biogas, butanediol and lactate. Biomass conversion to lactate is frequently facilitated by ensiling plant biomass with homofermentative lactic acid bacteria. Although most commonly used as a preservative in ensiled animal feed, lactate has diverse industrial applications as a precursor for the production of probiotics, biofuels, bioplastics and platform chemicals. Lactate yields by lactic acid bacteria (LAB) are constrained by a number of redox limitations which must be overcome while maintaining profitability and sustainability. To date, electrofermentation has not been scaled past laboratory- or pilot-stage reactions. The increasing ease of genetic modification in a wide range of LAB species may prove key to overcoming some of the pitfalls of electrofermentation at commercial scale. This review explores the history of electrofermentation as a tool for controlling redox balance within bacterial biocatalysts, and the potential for electrofermentation to increase lactate production from low-value plant biomass.
Abstract.
Winder J (2022). Enhancing Lactic Acid Production in Industrial Biocatalysts using Electrofermentation.
Abstract:
Enhancing Lactic Acid Production in Industrial Biocatalysts using Electrofermentation
Lactic acid (LA) is an important industrial chemical precursor. Most LA is produced by homofermentative lactic acid bacteria (LAB). LA production in LAB is constrained by a number of redox limitations. Enhancing LA production for industrial biorefinery applications requires overcoming these limitations while maintaining profitability and sustainability. Microbial electrosynthesis systems can be used to overcome energetic limitations but often require high current densities. Electrofermentation (EF) is an electron-efficient form of microbial electrosynthesis in which a small cathodic or anodic current is provided to a culture to alter the oxidation-reduction potential of the medium and, in turn, alter metabolism. Redox balance is a central driver of LA metabolism, but the potential of EF to enhance LA production has not previously been explored.
Here, EF was tested in pure cultures of two industrial, homofermentative LAB: Lactiplantibacillus plantarum and Pediococcus acidilactici. The approach was tested and optimised using a single-chamber apparatus before moving to a two-chamber H-cell apparatus. In the H-cell, the effect of providing electrons to the culture (cathodic EF) and removing them (anodic EF) was tested in aerobic and anaerobic conditions. The effect of adding the chemical redox mediator Neutral Red (NR) was tested in cathodic treatments. Glucose, lactic acid, acetic acid and ethanol concentrations, growth, and pH of cultures were measured. Imaging flow cytometry was conducted to probe for any potential phenotypic changes during EF. Cathodic EF treatment doubled LA production in anaerobic cultures of L. plantarum. This was unaffected by NR addition, improving the techno-economic feasibility of the platform. In aerobic cultures of P. acidilactici where NR was added, ethanol production was doubled. While the effects of electrical current on overall growth were minimal, some phenotypic effects were observed for P. acidilactici. Overall, in every EF treatment, significant changes to metabolism were observed compared to controls. This study is the first demonstration of bioelectrochemical enhancement and fine-tuning of homolactic LA metabolism in industrial biocatalysts, and if scaled up could contribute significantly to carbon mitigation in the bioeconomy.
Abstract.
Tennant RK, Power AL, Burton SK, Sinclair N, Parker DA, Jones RT, Lee R, Love J (2022). In-situ sequencing reveals the effect of storage on lacustrine sediment microbiome demographics and functionality.
Environmental Microbiome,
17(1).
Abstract:
In-situ sequencing reveals the effect of storage on lacustrine sediment microbiome demographics and functionality
AbstractThe sediment microbiome is a demographically diverse and functionally active biosphere. Ensuring that data acquired from sediment is truly representative of the microbiome is critical to achieving robust analyses. Sample storage and the processing and timing of nucleic acid purification after environmental sample extraction may fundamentally affect the detectable microbial community and thereby significantly alter resultant data. Direct sequencing of environmental samples is increasingly commonplace due to the advent of the portable Oxford Nanopore MinION sequencing device. Here we demonstrate that storing sediment subsamples at − 20 °C or storing the cores at 4 °C for 10 weeks prior to analysis, has a significant effect on the sediment microbiome analysed using sedimentary DNA (sedDNA), especially for Alpha-, Beta- and Deltaproteobacteria species. Furthermore, these significant differences are observed regardless of sediment type. We show that the taxa which are predominantly affected by storage are Proteobacteria, and therefore recommend on-site purifications are performed to ensure an accurate representation of these taxa are observed in the microbiome. Comparisons of sedimentary RNA (sedRNA) analyses, revealed substantial differences between samples purified and sequenced immediately on-site, samples that were frozen before transportation, and cores that were stored at 4 °C prior to analysis. Our data therefore suggest that a more accurate representation of the sediment microbiome demography and functionality may be achieved by environmental sequencing as rapidly as possible to minimise confounding effects of storage.
Abstract.
Love J (2022). Microbial pathways for advanced biofuel production.
Biochem Soc Trans,
50(2), 987-1001.
Abstract:
Microbial pathways for advanced biofuel production.
Decarbonisation of the transport sector is essential to mitigate anthropogenic climate change. Microbial metabolisms are already integral to the production of renewable, sustainable fuels and, building on that foundation, are being re-engineered to generate the advanced biofuels that will maintain mobility of people and goods during the energy transition. This review surveys the range of natural and engineered microbial systems for advanced biofuels production and summarises some of the techno-economic challenges associated with their implementation at industrial scales.
Abstract.
Author URL.
2021
Baugh AV, Howarth TM, West KL, Kerr LEJ, Love J, Parker DA, Fedenko JR, Tennant RK (2021). Draft Genome Sequence of Weissella paramesenteroides STCH-BD1, Isolated from Ensiled Sorghum bicolor.
Microbiology Resource Announcements,
10(7).
Abstract:
Draft Genome Sequence of Weissella paramesenteroides STCH-BD1, Isolated from Ensiled Sorghum bicolor
. Weissella paramesenteroides
. has potential as an industrial biocatalyst due to its ability to produce lactic acid. A novel strain of
. W. paramesenteroides
. was isolated from ensiled sorghum. The genome was sequenced using a hybrid assembly of Oxford Nanopore and Illumina data to produce a 2-Mbp genome and 22-kbp plasmid sequence.
.
Abstract.
West K (2021). Improving Industrial Efficiency of Sustainable Lactic Acid Production.
Abstract:
Improving Industrial Efficiency of Sustainable Lactic Acid Production
Lactic acid (LA) is a valuable, widely used platform chemical with diverse applications in industries such as pharmaceuticals, food storage and textiles. LA has antimicrobial and preservative properties and can also be used a precursor to Poly-Lactic Acid, a biodegradable plastic. Sustainable production of LA is increasingly important and is possible from bacterial fermentation of sugars. The Biodomain business unit at Shell are particularly interested in providing new, sustainable routes to LA production at scale and use a commercial starter culture, MidWestern BioAg Lactic Acid Bacteria (LAB) in pilot fermentation of plant-derived feedstock, which resulted in a range of products, including LA, acetic acid and ethanol, with varying and often unpredictable yields.
This project aimed to understand the basis of that variability by analysing and influencing the microbial content of the fermentation.
Metagenomic analysis of fermenting sweet sorghum (Sorghum bicolor) biomass showed the LAB inoculum did not dominate the fermentation systems as expected, leading to inefficient processes and unpredictable results. New inocula were investigated, including the use of squeezed liquids from post-ensiled sweet sorghum material, termed “sorghum pressate”. The sorghum pressate proved to be a successful inoculum when added with a carbonate-based pH buffer, with high LA yields (average 102 %) and low variability (± 11 %). Sequencing analysis showed the microbial community of the sorghum pressate was dominated by Weissella paramesenteroides, which was subsequently isolated.
The W. paramesenteroides isolate from post-ensiled sweet sorghum, grows best anaerobically, at 30 °C and pH 6. W. paramesenteroides can metabolise glucose, fructose, and sucrose, which are the three main soluble sugars in sweet sorghum, for growth and efficient LA production. However, when used as a pure strain inoculum for sweet sorghum fermentation, the isolated W. paramesenteroides strain was unproductive, suggesting that a microbial consortium is required for robust fermentation of the biomass.
Abstract.
Scott A, Sueiro Ballesteros L, Bradshaw M, Tsuji C, Power A, Lorriman J, Love J, Paul D, Herman A, Emanueli C, et al (2021). In Vivo Characterization of Endogenous Cardiovascular Extracellular Vesicles in Larval and Adult Zebrafish.
Arterioscler Thromb Vasc Biol,
41(9), 2454-2468.
Abstract:
In Vivo Characterization of Endogenous Cardiovascular Extracellular Vesicles in Larval and Adult Zebrafish.
OBJECTIVE: Extracellular vesicles (EVs) facilitate molecular transport across extracellular space, allowing local and systemic signaling during homeostasis and in disease. Extensive studies have described functional roles for EV populations, including during cardiovascular disease, but the in vivo characterization of endogenously produced EVs is still in its infancy. Because of their genetic tractability and live imaging amenability, zebrafish represent an ideal but under-used model to investigate endogenous EVs. We aimed to establish a transgenic zebrafish model to allow the in vivo identification, tracking, and extraction of endogenous EVs produced by different cell types. APPROACH AND RESULTS: Using a membrane-tethered fluorophore reporter system, we show that EVs can be fluorescently labeled in larval and adult zebrafish and demonstrate that multiple cell types including endothelial cells and cardiomyocytes actively produce EVs in vivo. Cell-type specific EVs can be tracked by high spatiotemporal resolution light-sheet live imaging and modified flow cytometry methods allow these EVs to be further evaluated. Additionally, cryo electron microscopy reveals the full morphological diversity of larval and adult EVs. Importantly, we demonstrate the utility of this model by showing that different cell types exchange EVs in the adult heart and that ischemic injury models dynamically alter EV production. CONCLUSIONS: We describe a powerful in vivo zebrafish model for the investigation of endogenous EVs in all aspects of cardiovascular biology and pathology. A cell membrane fluorophore labeling approach allows cell-type specific tracing of EV origin without bias toward the expression of individual protein markers and will allow detailed future examination of their function.
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Author URL.
Banks AM, Whitfield CJ, Brown SR, Fulton DA, Goodchild SA, Grant C, Love J, Lendrem DW, Fieldsend JE, Howard TP, et al (2021). Key reaction components affect the kinetics and performance robustness of cell-free protein synthesis reactions. Computational and Structural Biotechnology Journal, 20, 218-229.
Langley B (2021). The Anthropogenic Forcing of Coccolithophore Growth.
Abstract:
The Anthropogenic Forcing of Coccolithophore Growth
Global increases in atmospheric CO₂ concentration and temperature are causing changes in ocean chemistry, temperature and circulation, altering light and nutrient regimes. Such changes are expected to impact the coccolithophore community with organisms responding through phenotypic and physiological plasticity, genetic change and/or dispersal to more hospitable habitats. Understanding how the coccolithophore community responds to climate variability is vital for predicting ecosystem functioning and the fate of the global carbon cycle under a changing climate. Despite these urgent concerns, the coccolithophore response to recent global climate change remains poorly understood. Fossil coccospheres, the external calcite structure produced by the excretion of interlocking plates by coccolithophores, can provide a rare window into cell size in the past and an archive of the response of coccolithophores to environmental change. In turn, coccolithophore cell size has the potential to impose fundamental constraints on ecological and biogeochemical processes. In this thesis, I develop novel techniques combining imaging flow cytometry and cross-polarised light (ISX+PL) to rapidly and reliably visually isolate and quantify the morphological characteristics of coccospheres from marine sediment to enable cell size reconstructions. This method overcomes the constraints of labour-intensive manual microscopy allowing rapid identification and analysis of coccospheres within sediment. By employing ISX+PL, coccolithophore community cell size and community size structure of a subpolar North Atlantic community is reconstructed from ~1750 to ~2014 C.E. to investigate how modern coccolithophores may be responding to recent climate change. Results suggest average community cell size and community size structure was insensitive to increases in CO₂ concentration and SST since the mid-1900s. Stability in the species assemblage may be attributed to the low magnitude of environmental change and broad tolerances of the dominant species of the subpolar North Atlantic. The results imply environmental changes in the subpolar North Atlantic are not yet considerable enough to impact fundamental traits that affect the biogeochemical and ecological functioning of the coccolithophore ecosystem. In regions where climate change is resulting in higher local environmental variability and in communities that have a broader cell size composition with a subordinate community of larger cells, changes in cell size may be observed and should be a focus for future investigations.
Abstract.
Power AL, Barber DG, Groenhof SRM, Wagley S, Liu P, Parker DA, Love J (2021). The Application of Imaging Flow Cytometry for Characterisation and Quantification of Bacterial Phenotypes.
FRONTIERS IN CELLULAR AND INFECTION MICROBIOLOGY,
11 Author URL.
2020
Langley B, Halloran PR, Power A, Rickaby REM, Chana P, Diver P, Thornalley D, Hacker C, Love J (2020). A new method for isolating and analysing coccospheres within sediment.
Scientific Reports,
10(1).
Abstract:
A new method for isolating and analysing coccospheres within sediment
AbstractSize is a fundamental cellular trait that is important in determining phytoplankton physiological and ecological processes. Fossil coccospheres, the external calcite structure produced by the excretion of interlocking plates by the phytoplankton coccolithophores, can provide a rare window into cell size in the past. Coccospheres are delicate however and are therefore poorly preserved in sediment. We demonstrate a novel technique combining imaging flow cytometry and cross-polarised light (ISX+PL) to rapidly and reliably visually isolate and quantify the morphological characteristics of coccospheres from marine sediment by exploiting their unique optical and morphological properties. Imaging flow cytometry combines the morphological information provided by microscopy with high sample numbers associated with flow cytometry. High throughput imaging overcomes the constraints of labour-intensive manual microscopy and allows statistically robust analysis of morphological features and coccosphere concentration despite low coccosphere concentrations in sediments. Applying this technique to the fine-fraction of sediments, hundreds of coccospheres can be visually isolated quickly with minimal sample preparation. This approach has the potential to enable rapid processing of down-core sediment records and/or high spatial coverage from surface sediments and may prove valuable in investigating the interplay between climate change and coccolithophore physiological/ecological response.
Abstract.
Wagley S, Morcrette H, Kovacs-Simon A, Yang ZR, Power A, Tennant RK, Love J, Murray N, Titball RW, Butler CS, et al (2020). Bacterial dormancy: a subpopulation of viable but non-culturable cells demonstrates better fitness for revival.
Morcrette H, Kovacs-Simon A, Tennant RK, Love J, Wagley S, Yang ZR, Studholme DJ, Soyer OS, Champion OL, Butler CS, et al (2020). Campylobacter jejuni 11168H Exposed to Penicillin Forms Persister Cells and Cells with Altered Redox Protein Activity.
Frontiers in Cellular and Infection Microbiology,
10Abstract:
Campylobacter jejuni 11168H Exposed to Penicillin Forms Persister Cells and Cells with Altered Redox Protein Activity
The formation of persister cells is one mechanism by which bacteria can survive exposure to environmental stresses. We show that Campylobacter jejuni 11168H forms persister cells at a frequency of 10−3 after exposure to 100 × MIC of penicillin G for 24 h. Staining the cell population with a redox sensitive fluorescent dye revealed that penicillin G treatment resulted in the appearance of a population of cells with increased fluorescence. We present evidence, to show this could be a consequence of increased redox protein activity in, or associated with, the electron transport chain. These data suggest that a population of penicillin G treated C. jejuni cells could undergo a remodeling of the electron transport chain in order to moderate membrane hyperpolarization and intracellular alkalization; thus reducing the antibiotic efficacy and potentially assisting in persister cell formation.
Abstract.
Whitfield CJ, Banks AM, Dura G, Love J, Fieldsend JE, Goodchild SA, Fulton DA, Howard TP (2020). Cell-free protein synthesis in hydrogel materials.
Chem Commun (Camb),
56(52), 7108-7111.
Abstract:
Cell-free protein synthesis in hydrogel materials.
We report a method for embedding cell-free protein synthesis reactions in macro-scale hydrogel materials without a free liquid phase. This paper focuses on methods of preparation for a variety of hydrogels and an investigation of the impact that the hydrogel material has on cell-free protein synthesis.
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Author URL.
McDermott C (2020). Development of an Ensiling Process for Industrial Lactic Acid Production using Beta vulgaris as a Candidate Feedstock.
Abstract:
Development of an Ensiling Process for Industrial Lactic Acid Production using Beta vulgaris as a Candidate Feedstock
Lactic acid has a wide range in uses in the food, pharmaceutical and cosmetic industries, and can be used in the formation of Polylactic acid (PLA), a biodegradable plastic. Currently, there is rising interest in renewable lactic acid production from biomass.
The aim was to develop an economically feasible ensiling process for lactic acid production, using Beta vulgaris as the chosen feedstock. To determine optimal ensiling conditions, the use of buffer treatments, nutrient additives, and incubation temperature and duration were investigated. Two optimal buffer treatments that were established: 7.5% w/v CaCO3 and 0.75% w/v Ca(OH)2 and 6% w/v Mg(OH)2 produced the highest lactic acid yields, with the lowest variability. Ensiling with addition of yeast extract and soybean meal significantly improved lactic acid production, with yeast extract increasing lactic acid titres by a maximum of 67.5%, and soybean meal increasing titres by a maximum of 63.3%. This shows the importance of a nutrient additive in this process to provide the lactic acid bacteria with a nitrogenous supplement. It was also discovered that the optimal buffer treatments had differing optimal ensiling temperatures and duration. The optimal ensiling temperature for 7.5% w/v CaCO3 was 45 °C and the optimal duration was 14 days. For the 0.75% w/v Ca(OH)2 and 6% w/v Mg(OH)2 treatment, the optimal ensiling temperature was 30 °C and the optimal duration was 8 days.
Sugar beet ensiling at the optimal conditions, including a selected buffer treatment and nutrient addition results in high lactic acid titres of up to 159.4 g l-1 and a yield of 81.1% (maximum theoretical conversion of hexose sugars to lactic acid) and is therefore a successful ensiling process for lactic acid production. However, further work should be carried out to improve the cost-effectiveness of the process.
Abstract.
Fogwill CJ, Turney CSM, Menviel L, Baker A, Weber ME, Ellis B, Thomas ZA, Golledge NR, Etheridge D, Rubino M, et al (2020). Southern Ocean carbon sink enhanced by sea-ice feedbacks at the Antarctic Cold Reversal.
Nature Geoscience,
13(7), 489-497.
Abstract:
Southern Ocean carbon sink enhanced by sea-ice feedbacks at the Antarctic Cold Reversal
The Southern Ocean occupies 14% of the Earth’s surface and plays a fundamental role in the global carbon cycle and climate. It provides a direct connection to the deep ocean carbon reservoir through biogeochemical processes that include surface primary productivity, remineralization at depth and the upwelling of carbon-rich water masses. However, the role of these different processes in modulating past and future air–sea carbon flux remains poorly understood. A key period in this regard is the Antarctic Cold Reversal (ACR, 14.6–12.7 kyr bp), when mid- to high-latitude Southern Hemisphere cooling coincided with a sustained plateau in the global deglacial increase in atmospheric CO2. Here we reconstruct high-latitude Southern Ocean surface productivity from marine-derived aerosols captured in a highly resolved horizontal ice core. Our multiproxy reconstruction reveals a sustained signal of enhanced marine productivity across the ACR. Transient climate modelling indicates this period coincided with maximum seasonal variability in sea-ice extent, implying that sea-ice biological feedbacks enhanced CO2 sequestration and created a substantial regional marine carbon sink, which contributed to the plateau in CO2 during the ACR. Our results highlight the role Antarctic sea ice plays in controlling global CO2, and demonstrate the need to incorporate such feedbacks into climate–carbon models.
Abstract.
Nicholls J (2020). The Development of Molecular Approaches for Investigating the Temperature Response of Soil Carbon Fluxes.
Abstract:
The Development of Molecular Approaches for Investigating the Temperature Response of Soil Carbon Fluxes
Soil microbial respiration and methanogenesis are key sources of atmospheric carbon. Conflicting evidence exists as to how these processes acclimate to temperature fluxes. Thermal acclimation of temperate soils might result in downregulation of microbial soil respiration rates and, therefore, has the potential to mitigate atmospheric soil carbon losses predicted due to global warming. The extent of thermal acclimation and the mechanisms behind it (substrate depletion, genetic adaption, physiological changes, or microbial community composition) remain unclear. We investigated the temperature response of soil microbial respiration by warming (37 °C and 45 °C) and cooling (15 °C) a soil sample over a 14-week period. Soil respiration rates acclimated to temperature after 8 weeks and metagenomic analysis of the acclimated soil microbiomes revealed an increase in the relative abundance of Proteobacteria, Actinobacteria and Chlamydiae phyla.
Additionally, the investigation of the physiological temperature response of an important environmental methanogen led to novel insights into methanogenic acclimation. Methanosarcina barkeri incubated at optimum (37 °C), cool (15 °C) and warm (45 °C) temperatures for 4 weeks displayed thermal acclimation of methanogenesis at the end of the incubation period. Transcriptomic analysis of acclimated cultures identified potential molecular signatures of methanogenic acclimation, primarily the apparent preference for utilizing the methyl-reducing methanogenic pathway within acclimated cultures.
Understanding the acclimation responses of soil carbon fluxes will increase the predictive power of climate change models and enable more accurate predictions of the carbon-sequestration potential of natural carbon offsetting projects. This study demonstrates the importance of including the temperature responses of soil microbial communities and individual soil organisms in predicting the loss of carbon from soils due to climate change.
Abstract.
Welsh C, Pike L, Elliott J, Bailey J, Quintin-Baxendale R, Billington J, Matousek A, Matthews C, Dumitrescu D, Murphy JF, et al (2020). Why is it so hard to enact responsible change?: Scientists need to work more closely with other social groups to implement sustainable innovation.
EMBO Rep,
21(4).
Abstract:
Why is it so hard to enact responsible change?: Scientists need to work more closely with other social groups to implement sustainable innovation.
Science is key to developing sustainable products and solutions. But scientists also need to work more with governments, industry and society to help implement those solutions.
Abstract.
Author URL.
2019
Tennant RK, Ayine M, Power A, Gilman J, Hewlett M, James P, Singleton C, Parker D, Love J (2019). A Hybrid Sequencing Approach Completes the Genome Sequence of Thermoanaerobacter ethanolicus JW 200. Microbiology Resource Announcements, 8(3).
Singleton C, Gilman J, Rollit J, Zhang K, Parker DA, Love J (2019). A design of experiments approach for the rapid formulation of a chemically defined medium for metabolic profiling of industrially important microbes.
PLoS One,
14(6).
Abstract:
A design of experiments approach for the rapid formulation of a chemically defined medium for metabolic profiling of industrially important microbes.
Geobacillus thermoglucosidans DSM2542 is an industrially important microbe, however the complex nutritional requirements of Geobacilli confound metabolic engineering efforts. Previous studies have utilised semi-defined media recipes that contain complex, undefined, biologically derived nutrients which have unknown ingredients that cannot be quantified during metabolic profiling. This study used design of experiments to investigate how individual nutrients and interactions between these nutrients contribute to growth. A mathematically derived defined medium has been formulated that has been shown to robustly support growth of G. thermoglucosidans in two different environmental conditions (96-well plate and shake flask) and with a variety of lignocellulose-based carbohydrates. This enabled the catabolism of industrially relevant carbohydrates to be investigated.
Abstract.
Author URL.
Whitfield CJ, Banks AM, Dura G, Love J, Fieldsend JE, Goodchild SA, Fulton DA, Howard TP (2019). Cell-free genetic devices confer autonomic and adaptive properties to hydrogels.
Reid T (2019). Developing synthetic consortia for industrial biofuel production from lignocellulose.
Abstract:
Developing synthetic consortia for industrial biofuel production from lignocellulose
This report describes work initiated at the Shell Biodomain Houston to evaluate candidate species for use as consolidated bioprocessing (CBP) hosts for conversion of lignocellulose (LC) to fuel. A systematic literature review regarding desirable characteristics for LC CBP hosts identified eight potential species: Bacillus subtilis 168, Cellulomonas fimi, Clostridium cellulolyticum, Clostridium thermocellum, Escherichia coli, Lactobacillus plantarum, Thermoanaerobacterium saccharolyticum and Thermobifida fusca. Commercially available API CH 50 kits were shown to be a rapid convenient method of testing the ability of all the species to ferment a wide range of carbohydrates. Analysis of batches cultured in the most important LC-derived sugars (D-glucose, cellobiose, D-xylose, L-arabinose) for biomass, ethanol, acetate and lactate production prompted T. fusca to be discarded as a potential host. This highlighted the high efficiency to which L. plantarum could achieve high product (lactic acid) yields. Physiological characterisation by imaging flow cytometry (IFC) established that it is possible to separate populations of different bacteria for the purpose of future sorting of complex consortia. Based on all these results a two-member microbial consortium was designed in which the biomass-to-model fuel product (lactate) pathway was shared among two species in co-culture with each species being specialised for specific processes i.e. C. fimi as the cellulolytic, and L. plantarum as the biofuel-producing specialist. For population monitoring of consortia IFC technology was utilised to establish a microbial demographic of C. fimi and L. plantarum separately and co-cultured together. Subsequently, a novel method to determine species abundance in synthetic consortia was developed allowing high-throughput testing. This could be used in industry for rapid process optimisation of synthetic consortia, and as an online monitoring and management system to detect consortium population-balance in industrial fermentations.
Abstract.
Reid T (2019). Developing synthetic consortia for industrial biofuel production from lignocellulose.
Abstract:
Developing synthetic consortia for industrial biofuel production from lignocellulose
This report describes work initiated at the Shell Biodomain Houston to evaluate candidate species for use as consolidated bioprocessing (CBP) hosts for conversion of lignocellulose (LC) to fuel. A systematic literature review regarding desirable characteristics for LC CBP hosts identified eight potential species: Bacillus subtilis 168, Cellulomonas fimi, Clostridium cellulolyticum, Clostridium thermocellum, Escherichia coli, Lactobacillus plantarum, Thermoanaerobacterium saccharolyticum and Thermobifida fusca. Commercially available API CH 50 kits were shown to be a rapid convenient method of testing the ability of all the species to ferment a wide range of carbohydrates. Analysis of batches cultured in the most important LC-derived sugars (D-glucose, cellobiose, D-xylose, L-arabinose) for biomass, ethanol, acetate and lactate production prompted T. fusca to be discarded as a potential host. This highlighted the high efficiency to which L. plantarum could achieve high product (lactic acid) yields. Physiological characterisation by imaging flow cytometry (IFC) established that it is possible to separate populations of different bacteria for the purpose of future sorting of complex consortia. Based on all these results a two-member microbial consortium was designed in which the biomass-to-model fuel product (lactate) pathway was shared among two species in co-culture with each species being specialised for specific processes i.e. C. fimi as the cellulolytic, and L. plantarum as the biofuel-producing specialist. For population monitoring of consortia IFC technology was utilised to establish a microbial demographic of C. fimi and L. plantarum separately and co-cultured together. Subsequently, a novel method to determine species abundance in synthetic consortia was developed allowing high-throughput testing. This could be used in industry for rapid process optimisation of synthetic consortia, and as an online monitoring and management system to detect consortium population-balance in industrial fermentations.
Abstract.
Zhang K, Wells P, Liang Y, Love J, Parker DA, Botella C (2019). Effect of diluted hydrolysate as yeast propagation medium on ethanol production.
Bioresour Technol,
271, 1-8.
Abstract:
Effect of diluted hydrolysate as yeast propagation medium on ethanol production.
Yeast propagation using 50% diluted hydrolysate in water was utilized for the fermentation of hydrolysate derived from pre-treated ensiled sweet sorghum. The purpose was to condition the yeast to the inhibitors generated during the ensiling of sweet sorghum. The conditioned seed cultures exhibited similar fermentation performance and superior kinetics than the inoculum prepared in YPD medium. Furthermore, the conditioned yeast showed increased tolerance to the increased levels of these inhibitors, including ethanol, acetic and lactic acids, demonstrating an effective way to increase the robustness of yeast fermentation for ethanol production.
Abstract.
Author URL.
Brain C (2019). Establishment of an Ensiling System for the Production of Lactic Acid from Sorghum bicolor (Sweet Sorghum) Biomass.
Abstract:
Establishment of an Ensiling System for the Production of Lactic Acid from Sorghum bicolor (Sweet Sorghum) Biomass
Lactic acid is an economically important organic acid that has a wide range of applications in industry, including in the manufacture of sustainable and biodegradable plastic alternative poly-lactic acid (PLA). Transition towards a bio-based economy requires increased production of lactic acid and other industrially important chemicals from biomass. Lignocellulosic biomass is a preferred feedstock for production, as it is inexpensive and abundant, however the recalcitrant nature of lignocellulose makes its utilisation challenging. Therefore, development of a consolidated bioprocessing (CBP) system for efficient conversion of lignocellulosic biomass to value-added products is required.
Lactic acid is currently produced via fermentation of expensive carbohydrate-based feedstocks by lactic acid bacteria (LAB). Application of an ensiling system may overcome problems associated with current fermentation methods and enable conversion of lignocellulosic biomass to lactic acid via CBP. Ensiling is commonly used for the preservation of freshly harvested crops for animal feed, including sweet sorghum, which contains a high concentration of soluble sugar and lignocellulose. In this study, a series of lab-scale ensiling experiments were performed, in which the effects of various conditions and additives on ensiling product yields from sweet sorghum were evaluated. Addition of CaCO3 was identified as an effective method of pH control, resulting in a 2.8 x increase in lactic acid production by endogenous LAB. Inoculation with two different species of LAB (Lactobacillus plantarum and Pediococcus acidilactici) and addition of a commercial cellulose enzyme package were demonstrated to have minimal influence on lactic acid yield compared to ensiling temperature and time. An ensiling temperature of 45 °C resulted in high lactic acid selectivity and potential degradation of some lignocellulose. However, due to variability between ensiling experiments, further investigation is required before optimal ensiling temperature and time can be confirmed for maximum production of lactic acid from sweet sorghum.
Abstract.
Gachanja N (2019). Evaluation of Beta vulgaris as a Candidate Feedstock for Industrial Lactic Acid Production.
Abstract:
Evaluation of Beta vulgaris as a Candidate Feedstock for Industrial Lactic Acid Production
An increase in global demands for fuel from renewable sources has accelerated research into alternative energy sources that are economical and can reduce greenhouse gases. These renewable fuels must also not cause further challenges by competing for agricultural land use with food sources. Therefore, lignocellulosic feedstock, which are cheap and abundant non-food materials are an emerging solution. Concurrently, there is also rising interest in sustainable and renewable based chemicals. Lactic acid is valuable in many industries and is one such chemical. In addition to use as a preservative and emulsifying agent, lactic acid is also a precursor for poly-lactic acid, a biodegradable and biocompatible polymer. Microbial fermentation with Beta vulgaris (sugar beet) in a low-technology process such as ensiling has the potential to provide high titers of ethanol for use as a biofuel and lactic acid for industry.
In this project, the utilisation of seven carbohydrates, sucrose, glucose, fructose, xylose, arabinose, cellobiose and D-galacturonic acid by several lactic acid bacteria that were considered potential inoculants for sugar beet ensiling was assessed. This was used to determine the optimal microbial consortia for use as an inoculant to produce high lactic acid yields. A laboratory sugar beet ensiling protocol was also developed and various factors including shredding sugar beet hypocotyls prior to ensiling, incubation between 32-37 °C, 5-7% w/v CaCO3 and 5% w/v sodium chloride determined to produce 20-25 mmol g-1 lactic acid. Ensiling with Saccharomyces cerevisiae, acid and the antibiotic Lactrol resulted in high ethanol selectivity and yields. Lastly, metagenomic analysis of sugar beet samples prior and post ensiling determined the endogenous microbial community and changes to relative distributions during ensiling. Analysis showed Carnobacterium was the most abundant genus and most likely responsible for lactic acid production and sugar beet samples prior to ensiling had greater bacterial diversity than post-ensiled samples. Performing ensiling at optimal conditions with selected additive resulted in high titers of the desired product and reduced by-product formation demonstrating Beta vulgaris can be successfully ensiled.
Abstract.
Tennant RK, Holzer B, Love J, Tchilian E, White HN (2019). Higher levels of B-cell mutation in the early germinal centres of an inefficient secondary antibody response to a variant influenza haemagglutinin.
Immunology,
157(1), 86-91.
Abstract:
Higher levels of B-cell mutation in the early germinal centres of an inefficient secondary antibody response to a variant influenza haemagglutinin.
Designing improved vaccines against mutable viruses such as dengue and influenza would be helped by a better understanding of how the B-cell memory compartment responds to variant antigens. Towards this we have recently shown, after secondary immunization of mice with a widely variant dengue virus envelope protein with only 63% amino acid identity, that IgM+ memory B cells with few mutations supported an efficient secondary germinal centre (GC) and serum response, superior to a primary response to the same protein. Here, further investigation of memory responses to variant proteins, using more closely related influenza virus haemagglutinins (HA) that were 82% identical, produced a variant-induced boost response in the GC dominated by highly mutated B cells that failed, not efficiently improving serum avidity even in the presence of extra adjuvant, and that was worse than a primary response. This supports a hypothesis that over a certain level of antigenic differences, cross-reactive memory B-cell populations have reduced competency for affinity maturation. Combined with our previous observations, these findings also provide new parameters of success and failure in antibody memory responses.
Abstract.
Author URL.
Tennant RK, Lux T, Sambles C, Kuhn N, Petticrew E, Oldfield R, Parker D, Hatton J, Moore K, Lee R, et al (2019). Palaeogenomics of the Hydrocarbon Producing Microalga Botryococcus braunii. Scientific Reports, 9
Gilman J, Singleton C, Tennant RK, James P, Howard TP, Lux T, Parker DA, Love J (2019). Rapid, Heuristic Discovery and Design of Promoter Collections in Non-Model Microbes for Industrial Applications.
ACS Synth Biol,
8(5), 1175-1186.
Abstract:
Rapid, Heuristic Discovery and Design of Promoter Collections in Non-Model Microbes for Industrial Applications.
Well-characterized promoter collections for synthetic biology applications are not always available in industrially relevant hosts. We developed a broadly applicable method for promoter identification in atypical microbial hosts that requires no a priori understanding of cis-regulatory element structure. This novel approach combines bioinformatic filtering with rapid empirical characterization to expand the promoter toolkit and uses machine learning to improve the understanding of the relationship between DNA sequence and function. Here, we apply the method in Geobacillus thermoglucosidasius, a thermophilic organism with high potential as a synthetic biology chassis for industrial applications. Bioinformatic screening of G. kaustophilus, G. stearothermophilus, G. thermodenitrificans, and G. thermoglucosidasius resulted in the identification of 636 100 bp putative promoters, encompassing the genome-wide design space and lacking known transcription factor binding sites. Eighty of these sequences were characterized in vivo, and activities covered a 2-log range of predictable expression levels. Seven sequences were shown to function consistently regardless of the downstream coding sequence. Partition modeling identified sequence positions upstream of the canonical -35 and -10 consensus motifs that were predicted to strongly influence regulatory activity in Geobacillus, and artificial neural network and partial least squares regression models were derived to assess if there were a simple, forward, quantitative method for in silico prediction of promoter function. However, the models were insufficiently general to predict pre hoc promoter activity in vivo, most probably as a result of the relatively small size of the training data set compared to the size of the modeled design space.
Abstract.
Author URL.
2018
Brown SR, Staff M, Lee R, Love J, Parker DA, Aves SJ, Howard TP (2018). Design of Experiments Methodology to Build a Multifactorial Statistical Model Describing the Metabolic Interactions of Alcohol Dehydrogenase Isozymes in the Ethanol Biosynthetic Pathway of the Yeast Saccharomyces cerevisiae.
ACS Synth Biol,
7(7), 1676-1684.
Abstract:
Design of Experiments Methodology to Build a Multifactorial Statistical Model Describing the Metabolic Interactions of Alcohol Dehydrogenase Isozymes in the Ethanol Biosynthetic Pathway of the Yeast Saccharomyces cerevisiae.
Multifactorial approaches can quickly and efficiently model complex, interacting natural or engineered biological systems in a way that traditional one-factor-at-a-time experimentation can fail to do. We applied a Design of Experiments (DOE) approach to model ethanol biosynthesis in yeast, which is well-understood and genetically tractable, yet complex. Six alcohol dehydrogenase (ADH) isozymes catalyze ethanol synthesis, differing in their transcriptional and post-translational regulation, subcellular localization, and enzyme kinetics. We generated a combinatorial library of all ADH gene deletions and measured the impact of gene deletion(s) and environmental context on ethanol production of a subset of this library. The data were used to build a statistical model that described known behaviors of ADH isozymes and identified novel interactions. Importantly, the model described features of ADH metabolic behavior without explicit a priori knowledge. The method is therefore highly suited to understanding and optimizing metabolic pathways in less well-understood systems.
Abstract.
Author URL.
Murray AJ, Love J, Redwood MD, Orozco RL, Tennant RK, Woodhall F, Goodridge F, Macaskie LE (2018). Enhancement of Photosynthetic Productivity by Quantum Dots Application. In Stavrou VN (Ed) Nonmagnetic and Magnetic Quantum Dots, IntechOpen.
Tennant RK, Holzer B, Love J, Tchilian E, White HN (2018). Highly mutated memory cells drive an inefficient secondary antibody response to a variant protein.
Power A, Tennant RK, Jones RT, Tang Y, Du J, Worsley AT, Love J (2018). Monitoring Impacts of Urbanisation and Industrialisation on Air Quality in the Anthropocene Using Urban Pond Sediments. Frontiers in Earth Science, 6
Burton BR, Tennant RK, Love J, Titball RW, Wraith DC, White HN (2018). Variant proteins stimulate more IgM+ GC B-cells revealing a mechanism of cross-reactive recognition by antibody memory. eLife
2017
Bryant JA, Love J (2017). Biofuels. In (Ed) Biofuels and Bioenergy, 1-19.
McCalmont J, Clifton-Brown J, Hastings A (2017). Development of Miscanthus as a Bioenergy Crop. In Love J, Bryant J (Eds.)
Biofuels and Bioenergy, John Wiley & Sons.
Abstract:
Development of Miscanthus as a Bioenergy Crop
Abstract.
Shi H, Love J, Hu W (2017). Editorial: Melatonin in Plants.
FRONTIERS IN PLANT SCIENCE,
8 Author URL.
Sambles C, Middelhaufe S, Soanes D, Kolak D, Lux T, Moore K, Matoušková P, Parker D, Lee R, Love J, et al (2017). Genome sequence of the oleaginous yeast Rhodotorula toruloides strain CGMCC 2.1609. Genomics Data, 13, 1-2.
Tennant RK, Sambles CM, Diffey GE, Moore KA, Love J (2017). Metagenomic Analysis of Silage. Journal of Visualized Experiments, 119, e54936-e54936.
Tennant RK, Sambles CM, Diffey GE, Moore KA, Love J (2017). Metagenomic Analysis of Silage. Journal of Visualized Experiments(119).
Sambles C, Moore K, Lux TM, Jones K, Littlejohn GR, Gouveia JD, Aves SJ, Studholme DJ, Lee R, Love J, et al (2017). Metagenomic analysis of the complex microbial consortium associated with cultures of the oil-rich alga<i>Botryococcus braunii</i>. MicrobiologyOpen, 6(4), e00482-e00482.
Love J, Bryant JA (2017). Postscript. In (Ed) Biofuels and Bioenergy, 285-287.
Cook C, Dayananda C, Tennant RK, Love J (2017). Third‐Generation Biofuels from the Microalga, Botryococcus braunii. In Love J, Bryant J (Eds.) Biofuels and Bioenergy, Wiley.
2016
Jones KJ, Moore K, Sambles C, Love J, Studholme DJ, Aves SJ (2016). Draft Genome Sequences of Achromobacter piechaudii GCS2, Agrobacterium sp. Strain SUL3, Microbacterium sp. Strain GCS4, Shinella sp. Strain GWS1, and Shinella sp. Strain SUS2 Isolated from Consortium with the Hydrocarbon-Producing Alga Botryococcus braunii.
Genome Announc,
4(1).
Abstract:
Draft Genome Sequences of Achromobacter piechaudii GCS2, Agrobacterium sp. Strain SUL3, Microbacterium sp. Strain GCS4, Shinella sp. Strain GWS1, and Shinella sp. Strain SUS2 Isolated from Consortium with the Hydrocarbon-Producing Alga Botryococcus braunii.
A variety of bacteria associate with the hydrocarbon-producing microalga Botryococcus braunii, some of which may influence its growth. We report here the genome sequences for Achromobacter piechaudii GCS2, Agrobacterium sp. strain SUL3, Microbacterium sp. strain GCS4, and Shinella sp. strains GWS1 and SUS2, isolated from a laboratory culture of B. braunii, race B, strain Guadeloupe.
Abstract.
Author URL.
Johns AMB, Love J, Aves SJ (2016). Four Inducible Promoters for Controlled Gene Expression in the Oleaginous Yeast Rhodotorula toruloides.
Front Microbiol,
7Abstract:
Four Inducible Promoters for Controlled Gene Expression in the Oleaginous Yeast Rhodotorula toruloides.
Rhodotorula (Rhodosporidium) toruloides is an oleaginous yeast with great biotechnological potential, capable of accumulating lipid up to 70% of its dry biomass, and of carotenoid biosynthesis. However, few molecular genetic tools are available for manipulation of this basidiomycete yeast and its high genomic GC content can make routine cloning difficult. We have developed plasmid vectors for transformation of R. toruloides which include elements for Saccharomyces cerevisiae in-yeast assembly; this method is robust to the assembly of GC-rich DNA and of large plasmids. Using such vectors we screened for controllable promoters, and identified inducible promoters from the genes NAR1, ICL1, CTR3, and MET16. These four promoters have independent induction/repression conditions and exhibit different levels and rates of induction in R. toruloides, making them appropriate for controllable transgene expression in different experimental situations. Nested deletions were used to identify regulatory regions in the four promoters, and to delimit the minimal inducible promoters, which are as small as 200 bp for the NAR1 promoter. The NAR1 promoter shows very tight regulation under repressed conditions as determined both by an EGFP reporter gene and by conditional rescue of a leu2 mutant. These new tools facilitate molecular genetic manipulation and controllable gene expression in R. toruloides.
Abstract.
Author URL.
Love J, Graham SW, Irwin JA, Ashton PA, Bretagnolle F, Abbott RJ (2016). Self-pollination, style length development and seed set in self-compatible Asteraceae: evidence from Senecio vulgaris L.
Plant Ecology and Diversity,
9(4), 371-379.
Abstract:
Self-pollination, style length development and seed set in self-compatible Asteraceae: evidence from Senecio vulgaris L.
Background: Variation in style length has been reported in Senecio vulgaris and has been associated with outcrossing rate. Aims: to determine if (i) long styles lack germinated pollen on stigmas left to self-pollinate, (ii) successful self-pollination causes styles to stop elongating and shrink in length and (iii) seed set increases with the amount of pollen deposited on stigmas. Methods: Determined germinated self-pollen on stigmas of long and short styles after auto-self-pollination; scored style length over 48 h in self-pollinated and non-pollinated florets; recorded seed set after placing different amounts of pollen on stigmas. Results: Most long-styled florets had zero or low amounts of germinated pollen on stigmas in contrast to most short-styled florets. Styles initially elongated to the same length in self-pollinated and non-pollinated florets, then shrank in length in self-pollinated florets while continuing to elongate in non-pollinated florets. Seed set increased with number of pollen grains deposited on stigmas. Conclusions: Successful self-pollen deposition and/or germination on stigmas of S. vulgaris are indicated by presence of short styles, whereas the opposite is indicated by presence of long styles in florets left to self-pollinate. Self-pollination causes styles to shrink after initially elongating. Seed set is dependent on the amount of pollen deposited on stigmas.
Abstract.
Gilman J, Love J (2016). Synthetic promoter design for new microbial chassis.
Biochem Soc Trans,
44(3), 731-737.
Abstract:
Synthetic promoter design for new microbial chassis.
The judicious choice of promoter to drive gene expression remains one of the most important considerations for synthetic biology applications. Constitutive promoter sequences isolated from nature are often used in laboratory settings or small-scale commercial production streams, but unconventional microbial chassis for new synthetic biology applications require well-characterized, robust and orthogonal promoters. This review provides an overview of the opportunities and challenges for synthetic promoter discovery and design, including molecular methodologies, such as saturation mutagenesis of flanking regions and mutagenesis by error-prone PCR, as well as the less familiar use of computational and statistical analyses for de novo promoter design.
Abstract.
Author URL.
2015
Hutt H, Everson R, Grant M, Love J, Littlejohn G (2015). How clumpy is my image?: Scoring in crowdsourced annotation tasks.
Soft Computing,
19(6), 1541-1552.
Abstract:
How clumpy is my image?: Scoring in crowdsourced annotation tasks
The use of citizen science to obtain annotations from multiple annotators has been shown to be an effective method for annotating datasets in which computational methods alone are not feasible. The way in which the annotations are obtained is an important consideration which affects the quality of the resulting consensus annotation. In this paper, we examine three separate approaches to obtaining consensus scores for instances rather than merely binary classifications. To obtain a consensus score, annotators were asked to make annotations in one of three paradigms: classification, scoring and ranking. A web-based citizen science experiment is described which implements the three approaches as crowdsourced annotation tasks. The tasks are evaluated in relation to the accuracy and agreement among the participants using both simulated and real-world data from the experiment. The results show a clear difference in performance between the three tasks, with the ranking task obtaining the highest accuracy and agreement among the participants. We show how a simple evolutionary optimiser may be used to improve the performance by reweighting the importance of annotators.
Abstract.
Littlejohn GR, Mansfield JC, Parker D, Lind R, Perfect S, Seymour M, Smirnoff N, Love J, Moger J (2015). In vivo chemical and structural analysis of plant cuticular waxes using stimulated Raman scattering microscopy.
Plant Physiol,
168(1), 18-28.
Abstract:
In vivo chemical and structural analysis of plant cuticular waxes using stimulated Raman scattering microscopy.
The cuticle is a ubiquitous, predominantly waxy layer on the aerial parts of higher plants that fulfils a number of essential physiological roles, including regulating evapotranspiration, light reflection, and heat tolerance, control of development, and providing an essential barrier between the organism and environmental agents such as chemicals or some pathogens. The structure and composition of the cuticle are closely associated but are typically investigated separately using a combination of structural imaging and biochemical analysis of extracted waxes. Recently, techniques that combine stain-free imaging and biochemical analysis, including Fourier transform infrared spectroscopy microscopy and coherent anti-Stokes Raman spectroscopy microscopy, have been used to investigate the cuticle, but the detection sensitivity is severely limited by the background signals from plant pigments. We present a new method for label-free, in vivo structural and biochemical analysis of plant cuticles based on stimulated Raman scattering (SRS) microscopy. As a proof of principle, we used SRS microscopy to analyze the cuticles from a variety of plants at different times in development. We demonstrate that the SRS virtually eliminates the background interference compared with coherent anti-Stokes Raman spectroscopy imaging and results in label-free, chemically specific confocal images of cuticle architecture with simultaneous characterization of cuticle composition. This innovative use of the SRS spectroscopy may find applications in agrochemical research and development or in studies of wax deposition during leaf development and, as such, represents an important step in the study of higher plant cuticles.
Abstract.
Author URL.
2014
Littlejohn GR, Mansfield JC, Christmas JT, Witterick E, Fricker MD, Grant MR, Smirnoff N, Everson RM, Moger J, Love J, et al (2014). An update: improvements in imaging perfluorocarbon-mounted plant leaves with implications for studies of plant pathology, physiology, development and cell biology.
Front Plant Sci,
5Abstract:
An update: improvements in imaging perfluorocarbon-mounted plant leaves with implications for studies of plant pathology, physiology, development and cell biology.
Plant leaves are optically complex, which makes them difficult to image by light microscopy. Careful sample preparation is therefore required to enable researchers to maximize the information gained from advances in fluorescent protein labeling, cell dyes and innovations in microscope technologies and techniques. We have previously shown that mounting leaves in the non-toxic, non-fluorescent perfluorocarbon (PFC), perfluorodecalin (PFD) enhances the optical properties of the leaf with minimal impact on physiology. Here, we assess the use of the PFCs, PFD, and perfluoroperhydrophenanthrene (PP11) for in vivo plant leaf imaging using four advanced modes of microscopy: laser scanning confocal microscopy (LSCM), two-photon fluorescence microscopy, second harmonic generation microscopy, and stimulated Raman scattering (SRS) microscopy. For every mode of imaging tested, we observed an improved signal when leaves were mounted in PFD or in PP11, compared to mounting the samples in water. Using an image analysis technique based on autocorrelation to quantitatively assess LSCM image deterioration with depth, we show that PP11 outperformed PFD as a mounting medium by enabling the acquisition of clearer images deeper into the tissue. In addition, we show that SRS microscopy can be used to image PFCs directly in the mesophyll and thereby easily delimit the "negative space" within a leaf, which may have important implications for studies of leaf development. Direct comparison of on and off resonance SRS micrographs show that PFCs do not to form intracellular aggregates in live plants. We conclude that the application of PFCs as mounting media substantially increases advanced microscopy image quality of living mesophyll and leaf vascular bundle cells.
Abstract.
Author URL.
Lux TM, Lee R, Love J (2014). Genome-wide phylogenetic analysis of the pathogenic potential of Vibrio furnissii.
Front Microbiol,
5Abstract:
Genome-wide phylogenetic analysis of the pathogenic potential of Vibrio furnissii.
We recently reported the genome sequence of a free-living strain of Vibrio furnissii (NCTC 11218) harvested from an estuarine environment. V. furnissii is a widespread, free-living proteobacterium and emerging pathogen that can cause acute gastroenteritis in humans and lethal zoonoses in aquatic invertebrates, including farmed crustaceans and molluscs. Here we present the analyses to assess the potential pathogenic impact of V. furnissii. We compared the complete genome of V. furnissii with 8 other emerging and pathogenic Vibrio species. We selected and analyzed more deeply 10 genomic regions based upon unique or common features, and used 3 of these regions to construct a phylogenetic tree. Thus, we positioned V. furnissii more accurately than before and revealed a closer relationship between V. furnissii and V. cholerae than previously thought. However, V. furnissii lacks several important features normally associated with virulence in the human pathogens V. cholera and V. vulnificus. A striking feature of the V. furnissii genome is the hugely increased Super Integron, compared to the other Vibrio. Analyses of predicted genomic islands resulted in the discovery of a protein sequence that is present only in Vibrio associated with diseases in aquatic animals. We also discovered evidence of high levels horizontal gene transfer in V. furnissii. V. furnissii seems therefore to have a dynamic and fluid genome that could quickly adapt to environmental perturbation or increase its pathogenicity. Taken together, these analyses confirm the potential of V. furnissii as an emerging marine and possible human pathogen, especially in the developing, tropical, coastal regions that are most at risk from climate change.
Abstract.
Author URL.
2013
Reid EL, Weynberg KD, Love J, Isupov MN, Littlechild JA, Wilson WH, Kelly SL, Lamb DC, Allen MJ (2013). Functional and structural characterisation of a viral cytochrome b5.
FEBS Lett,
587(22), 3633-3639.
Abstract:
Functional and structural characterisation of a viral cytochrome b5.
Cytochrome b5 is a ubiquitous electron transport protein. The sequenced viral OtV-2 genome, which infects Ostreococcus tauri, was predicted to encode a putative cytochrome b5 enzyme. Using purified OtV-2 cytochrome b5 we confirm this protein has identical spectral properties to purified human cytochrome b5 and additionally that the viral enzyme can substitute for yeast cytochrome b5 in yeast cytochrome P450 51 mediated sterol 14α-demethylation. The crystal structure of the OtV-2 cytochrome b5 enzyme reveals a single domain, comprising four β sheets, four α helices and a haem moiety, which is similar to that found in larger eukaryotic cytochrome proteins. As a product of a horizontal gene transfer event involving a subdomain of the host fumarate reductase-like protein, OtV-2 cytochrome b5 appears to have diverged in function and is likely to have evolved an entirely new role for the virus during infection. Indeed, lacking a hydrophobic C-terminal anchor, OtV-2 encodes the first cytosolic cytochrome b5 characterised. The lack of requirement for membrane attachment (in contrast to all other microsomal cytochrome b5s) may be a reflection of the small size of the host cell, further emphasizes the unique nature of this virus gene product and draws attention to the potential importance of cytochrome b5 metabolic activity at the extremes of cellular scale.
Abstract.
Author URL.
Hutt H, Everson R, Grant M, Love J, Littlejohn G (2013). How clumpy is my image? Evaluating crowdsourced annotation tasks.
2013 13th UK Workshop on Computational Intelligence, UKCI 2013, 136-143.
Abstract:
How clumpy is my image? Evaluating crowdsourced annotation tasks
The use of citizen science to obtain annotations from multiple annotators has been shown to be an effective method for annotating datasets in which computational methods alone are not feasible. The way in which the annotations are obtained is an important consideration which affects the quality of the resulting consensus estimates. In this paper, we examine three separate approaches to obtaining scores for instances rather than merely classifications. To obtain a consensus score annotators were asked to make annotations in one of three paradigms: classification, scoring and ranking. A web-based citizen science experiment is described which implements the three approaches as crowdsourced annotation tasks. The tasks are evaluated in relation to the accuracy and agreement among the participants using both simulated and real-world data from the experiment. The results show a clear difference in performance between the three tasks, with the ranking task obtaining the highest accuracy and agreement among the participants. We show how a simple evolutionary optimiser may be used to improve the performance by reweighting the importance of annotators. © 2013 IEEE.
Abstract.
Howard TP, Middelhaufe S, Moore K, Edner C, Kolak DM, Taylor GN, Parker DA, Lee R, Smirnoff N, Aves SJ, et al (2013). Synthesis of customized petroleum-replica fuel molecules by targeted modification of free fatty acid pools in Escherichia coli.
Proc Natl Acad Sci USA,
110, 7636-7641.
Abstract:
Synthesis of customized petroleum-replica fuel molecules by targeted modification of free fatty acid pools in Escherichia coli
Biofuels are the most immediate, practical solution for mitigating dependence on fossil hydrocarbons, but current biofuels (alcohols and biodiesels) require significant downstream processing and are not fully compatible with modern, mass-market internal combustion engines. Rather, the ideal biofuels are structurally and chemically identical to the fossil fuels they seek to replace (i.e. aliphatic n- and iso-alkanes and -alkenes of various chain lengths). Here we report on production of such petroleum-replica hydrocarbons in Escherichia coli. The activity of the fatty acid (FA) reductase complex from Photorhabdus luminescens was coupled with aldehyde decarbonylase from Nostoc punctiforme to use free FAs as substrates for alkane biosynthesis. This combination of genes enabled rational alterations to hydrocarbon chain length (Cn) and the production of branched alkanes through upstream genetic and exogenous manipulations of the FA pool. Genetic components for targeted manipulation of the FA pool included expression of a thioesterase from Cinnamomum camphora (camphor) to alter alkane Cn and expression of the branched-chain α-keto acid dehydrogenase complex and β-keto acyl-acyl carrier protein synthase III from Bacillus subtilis to synthesize branched (iso-) alkanes. Rather than simply reconstituting existing metabolic routes to alkane production found in nature, these results demonstrate the ability to design and implement artificial molecular pathways for the production of renewable, industrially relevant fuel molecules.
Abstract.
Author URL.
2012
Love J, Tennant RK, Jones RT, Lee R (2012). A new flow cytometry enabling rapid purification of diatoms from silica-rich lacustrine sediments.
Journal of Paleolimnology,
49(2), 305-309.
Abstract:
A new flow cytometry enabling rapid purification of diatoms from silica-rich lacustrine sediments.
Sub-fossil diatoms represent an important archive of past environmental change in both terrestrial and marine settings. In recent years the isotopic analysis of diatom frustules has developed into an important area of paleo-environmental research. The extraction and concentration of diatoms from marine and lake sediments has proven difficult, particularly in silica rich samples. Here we present a new method for the rapid extraction of diatom frustules from lacustrine sediment using flow cytometry. This new technique produces samples suitable for geochemical and isotope-based research with a high degree of purity over a much shorter time frame than existing techniques, hours rather than days.
Abstract.
Littlejohn GR, Love J (2012). A simple method for imaging Arabidopsis leaves using perfluorodecalin as an infiltrative imaging medium.
Journal of visualized experiments : JoVE(59).
Abstract:
A simple method for imaging Arabidopsis leaves using perfluorodecalin as an infiltrative imaging medium.
The problem of acquiring high-resolution images deep into biological samples is widely acknowledged. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm -1 ) is lower than that of water (72 dynes cm -1 ) and also below the limit (25-30 dyne cm -1 ) for stomatal penetration, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO 2 and O 2 , which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation, surgery, artificial blood, oxygenation of growth media, and studies of ice crystal formation in plants. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.
Abstract.
2011
Debieux CM, Dridge EJ, Mueller CM, Splatt P, Paszkiewicz K, Knight I, Florance H, Love J, Titball RW, Lewis RJ, et al (2011). A bacterial process for selenium nanosphere assembly.
Proc Natl Acad Sci U S A,
108(33), 13480-13485.
Abstract:
A bacterial process for selenium nanosphere assembly.
During selenate respiration by Thauera selenatis, the reduction of selenate results in the formation of intracellular selenium (Se) deposits that are ultimately secreted as Se nanospheres of approximately 150 nm in diameter. We report that the Se nanospheres are associated with a protein of approximately 95 kDa. Subsequent experiments to investigate the expression and secretion profile of this protein have demonstrated that it is up-regulated and secreted in response to increasing selenite concentrations. The protein was purified from Se nanospheres, and peptide fragments from a tryptic digest were used to identify the gene in the draft T. selenatis genome. A matched open reading frame was located, encoding a protein with a calculated mass of 94.5 kDa. N-terminal sequence analysis of the mature protein revealed no cleavable signal peptide, suggesting that the protein is exported directly from the cytoplasm. The protein has been called Se factor a (SefA), and homologues of known function have not been reported previously. The sefA gene was cloned and expressed in Escherichia coli, and the recombinant His-tagged SefA purified. In vivo experiments demonstrate that SefA forms larger (approximately 300 nm) Se nanospheres in E. coli when treated with selenite, and these are retained within the cell. In vitro assays demonstrate that the formation of Se nanospheres upon the reduction of selenite by glutathione are stabilized by the presence of SefA. The role of SefA in selenium nanosphere assembly has potential for exploitation in bionanomaterial fabrication.
Abstract.
Author URL.
Reid EL, Worthy CA, Probert I, Ali ST, Love J, Napier J, Littlechild JA, Somerfield PJ, Allen MJ (2011). Coccolithophores: functional biodiversity, enzymes and bioprospecting.
Mar Drugs,
9(4), 586-602.
Abstract:
Coccolithophores: functional biodiversity, enzymes and bioprospecting.
Emiliania huxleyi is a single celled, marine phytoplankton with global distribution. As a key species for global biogeochemical cycling, a variety of strains have been amassed in various culture collections. Using a library consisting of 52 strains of E. huxleyi and an 'in house' enzyme screening program, we have assessed the functional biodiversity within this species of fundamental importance to global biogeochemical cycling, whilst at the same time determining their potential for exploitation in biocatalytic applications. Here, we describe the screening of E. huxleyi strains, as well as a coccolithovirus infected strain, for commercially relevant biocatalytic enzymes such as acid/alkali phosphodiesterase, acid/alkali phosphomonoesterase, EC1.1.1-type dehydrogenase, EC1.3.1-type dehydrogenase and carboxylesterase.
Abstract.
Author URL.
Lux TM, Lee R, Love J (2011). Complete genome sequence of a free-living Vibrio furnissii sp. nov. strain (NCTC 11218).
J Bacteriol,
193(6), 1487-1488.
Author URL.
2010
Love J (2010). Conference report: Society for general microbiology bioenergy fuel sources symposium. Biofuels, 1(1), 15-17.
Littlejohn GR, Gouveia JD, Edner C, Smirnoff N, Love J (2010). Perfluorodecalin enhances in vivo confocal microscopy resolution of Arabidopsis thaliana mesophyll.
New Phytol,
186(4), 1018-1025.
Abstract:
Perfluorodecalin enhances in vivo confocal microscopy resolution of Arabidopsis thaliana mesophyll.
*Air spaces in the leaf mesophyll generate deleterious optical effects that compromise confocal microscopy. Leaves were mounted in the nontoxic, nonfluorescent perfluorocarbon, perfluorodecalin (PFD), and optical enhancement and physiological effect were assessed using confocal microscopy and chlorophyll fluorescence. Mounting leaves of Arabidopsis thaliana in PFD significantly improved the optical qualities of the leaf, thereby enabling high-resolution laser scanning confocal imaging over twofold deeper into the mesophyll, compared with using water. Incubation in PFD had less physiological impact on the mounted specimen than water. We conclude that the application of PFD as a mounting medium substantially increases confocal image resolution of living mesophyll and vascular bundle cells, with minimal physiological impact.
Abstract.
2009
Dodd AN, Gardner MJ, Hotta CT, Hubbard KE, Dalchau N, Robertson FC, Love J, Sanders D, Webb AAR (2009). Response to comment on "the Arabidopsis circadian clock incorporates a cADPR-based feedback loop".
Science,
326(5950).
Abstract:
Response to comment on "the Arabidopsis circadian clock incorporates a cADPR-based feedback loop"
Xu et al. were unable to measure circadian oscillations of cyclic adenosine diphosphate ribose (cADPR). Their experiments showing very low concentrations of cADPR lack appropriate controls, which suggests that technical limitations might explain their negative result. Xu et al. also report that chemically induced ADP ribosyl cyclase did not alter clock function, which exactly replicates our findings.
Abstract.
2008
Murphy AJ, Kemp F, Love J (2008). Surface plasmon resonance characterization of calspermin-calmodulin binding kinetics.
Anal Biochem,
376(1), 61-72.
Abstract:
Surface plasmon resonance characterization of calspermin-calmodulin binding kinetics.
We cloned, expressed, and purified a chimeric fusion between a soluble green fluorescent protein (smGFP) and the calmodulin binding protein calspermin. We have shown that the fusion protein, labeled smGN, has a K(i) in the calmodulin-dependent cyclic nucleotide phosphodiesterase activity assay of 1.97 nM, i.e. 3800 times smaller than that of the commonly used calmodulin inhibitor W7. Association and dissociation rate constants (k(a) and k(d)) and the dissociation equilibrium constant (K(D)) of smGN for calmodulin were determined using surface plasmon resonance (SPR). The k(a)=1.24 x 10(6)M(-1)s(-1), the k(d)=5.49 x 10(-3)s(-1), and the K(D)=4.42 x 10(-9)M. We also found that the GFP moiety was important for successfully binding calspermin to the surface of the CM5 flow cell at a sufficiently high concentration for SPR, and that this procedure may be used for SPR analysis of other acidic polypeptides, whose pI< or =4. To determine whether smGN might also bind to other calmodulin-like proteins in a heterologous system, we purified proteins from a plant total cell extract or a plant total protein extract by affinity chromatography against smGN. The purified proteins were identified as calmodulins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and liquid chromatography-tandem mass spectrometry, indicating a high level of specificity. We conclude that the high affinity and specific binding between smGN and calmodulin make it an easily localized recombinant alternative to chemical calmodulin inhibitors.
Abstract.
Author URL.
2007
Xu X, Hotta CT, Dodd AN, Love J, Sharrock R, Lee YW, Xie Q, Johnson CH, Webb AAR (2007). Distinct Light and Clock Modulation of Cytosolic Free Ca2+ Oscillations and Rhythmic<i>CHLOROPHYLL A/B BINDING PROTEIN2</i>Promoter Activity in<i>Arabidopsis</i>.
The Plant Cell,
19(11), 3474-3490.
Abstract:
Distinct Light and Clock Modulation of Cytosolic Free Ca2+ Oscillations and RhythmicCHLOROPHYLL A/B BINDING PROTEIN2Promoter Activity inArabidopsis
AbstractPlants have circadian oscillations in the concentration of cytosolic free calcium ([Ca2+]cyt). To dissect the circadian Ca2+-signaling network, we monitored circadian [Ca2+]cyt oscillations under various light/dark conditions (including different spectra) in Arabidopsis thaliana wild type and photoreceptor and circadian clock mutants. Both red and blue light regulate circadian oscillations of [Ca2+]cyt. Red light signaling is mediated by PHYTOCHROME B (PHYB). Blue light signaling occurs through the redundant action of CRYPTOCHROME1 (CRY1) and CRY2. Blue light also increases the basal level of [Ca2+]cyt, and this response requires PHYB, CRY1, and CRY2. Light input into the oscillator controlling [Ca2+]cyt rhythms is gated by EARLY FLOWERING3. Signals generated in the dark also regulate the circadian behavior of [Ca2+]cyt. Oscillations of [Ca2+]cyt and CHLOROPHYLL A/B BINDING PROTEIN2 (CAB2) promoter activity are dependent on the rhythmic expression of LATE ELONGATED HYPOCOTYL and CIRCADIAN CLOCK-ASSOCIATED1, but [Ca2+]cyt and CAB2 promoter activity are uncoupled in the timing of cab1 (toc1-1) mutant but not in toc1-2. We suggest that the circadian oscillations of [Ca2+]cyt and CAB2 promoter activity are regulated by distinct oscillators with similar components that are used in a different manner and that these oscillators may be located in different cell types in Arabidopsis.
Abstract.
Love J, Dodd AN, Gardner MJ, Webb ARR (2007). The Arabidopsis Circadian Clock Incorporates a cADPR-Based Feedback Loop .
2005
Akesson A, Persson S, Love J, Boss WF, Widell S, Sommarin M (2005). Overexpression of the Ca2+-binding protein calreticulin in the endoplasmic reticulum improves growth of tobacco cell suspensions (Nicotiana tabacum) in high-Ca2+ medium. Physiologia Plantarum, 123(1), 92-99.
Love, J. Webb, A.R.R. (2005). The plant clock shows its metal: Circadian regulation of cytosolic free Ca2+. Trends in Plant Science, 10(1), 15-21.
2004
Love J, Dodd AN, Webb ARR (2004). Circadian and diurnal calcium oscillations encode photoperiodic information in Arabidopsis. The Plant Cell, 16(4), 956-966.
2003
Stevenson-Paulik J, Love J, Boss WF (2003). Differential regulation of two Arabidopsis type III phosphatidylinositol 4-kinase isoforms. A regulatory role for the pleckstrin homology domain.
Plant Physiol,
132(2), 1053-1064.
Abstract:
Differential regulation of two Arabidopsis type III phosphatidylinositol 4-kinase isoforms. A regulatory role for the pleckstrin homology domain.
Here, we compare the regulation and localization of the Arabidopsis type III phosphatidylinositol (PtdIns) 4-kinases, AtPI4Kalpha1 and AtPI4Kbeta1, in Spodoptera frugiperda (Sf9) insect cells. We also explore the role of the pleckstrin homology (PH) domain in regulating AtPI4Kalpha1. Recombinant kinase activity was found to be differentially sensitive to PtdIns-4-phosphate (PtdIns4P), the product of the reaction. The specific activity of AtPI4Kalpha1 was inhibited 70% by 0.5 mm PtdIns4P. The effect of PtdIns4P was not simply due to charge because AtPI4Kalpha1 activity was stimulated approximately 50% by equal concentrations of the other negatively charged lipids, PtdIns3P, phosphatidic acid, and phosphatidyl-serine. Furthermore, inhibition of AtPI4Kalpha1 by PtdIns4P could be alleviated by adding recombinant AtPI4Kalpha1 PH domain, which selectively binds to PtdIns4P (Stevenson et al. 1998). In contrast, the specific activity of AtPI4Kbeta1, which does not have a PH domain, was stimulated 2-fold by PtdIns4P but not other negatively charged lipids. Visualization of green fluorescent protein fusion proteins in insect cells revealed that AtPI4Kalpha1 was associated primarily with membranes in the perinuclear region, whereas AtPI4Kbeta1 was in the cytosol and associated with small vesicles throughout the cytoplasm. Expression of AtPI4Kalpha1 without the PH domain in the insect cells compromised PtdIns 4-kinase activity and caused mislocalization of the kinase. The green fluorescent protein-PH domain alone was associated with intracellular membranes and the plasma membrane. In vitro, the PH domain appeared to be necessary for association of AtPI4Kalpha1 with fine actin filaments. These studies support the idea that the Arabidopsis type III PtdIns 4-kinases are responsible for distinct phosphoinositide pools.
Abstract.
Author URL.
Stevenson-Paulik JM, Love J, Boss WF (2003). Erratum: Differential Regulation of Two Arabidopsis Type III Phosphatidylinositol 4-Kinase Isoforms. A Regulatory Role for the Pleckstrin Homology Domain (Plant Physiology (2003) 132 (1053-1064)). Plant Physiology, 133(2).
2002
Love J, Allen GC, Gatz C, Thompson WF (2002). Differential Top10 promoter regulation by six tetracycline analogues in plant cells. Journal of Experimental Botany, 53(376), 1871-1877.
Perera IY, Love J, Heilmann I, Thompson WF, Boss WF (2002). Up-regulation of phosphoinositide metabolism in tobacco cells constitutively expressing the human type I inositol polyphosphate 5-phosphatase.
Plant Physiol,
129(4), 1795-1806.
Abstract:
Up-regulation of phosphoinositide metabolism in tobacco cells constitutively expressing the human type I inositol polyphosphate 5-phosphatase.
To evaluate the impact of suppressing inositol 1,4,5-trisphosphate (InsP(3)) in plants, tobacco (Nicotiana tabacum) cells were transformed with the human type I inositol polyphosphate 5-phosphatase (InsP 5-ptase), an enzyme which specifically hydrolyzes InsP(3). The transgenic cell lines showed a 12- to 25-fold increase in InsP 5-ptase activity in vitro and a 60% to 80% reduction in basal InsP(3) compared with wild-type cells. Stimulation with Mas-7, a synthetic analog of the wasp venom peptide mastoparan, resulted in an approximately 2-fold increase in InsP(3) in both wild-type and transgenic cells. However, even with stimulation, InsP(3) levels in the transgenic cells did not reach wild-type basal values, suggesting that InsP(3) signaling is compromised. Analysis of whole-cell lipids indicated that phosphatidylinositol 4,5-bisphosphate (PtdInsP(2)), the lipid precursor of InsP(3), was greatly reduced in the transgenic cells. In vitro assays of enzymes involved in PtdInsP(2) metabolism showed that the activity of the PtdInsP(2)-hydrolyzing enzyme phospholipase C was not significantly altered in the transgenic cells. In contrast, the activity of the plasma membrane PtdInsP 5 kinase was increased by approximately 3-fold in the transgenic cells. In vivo labeling studies revealed a greater incorporation of (32)P into PtdInsP(2) in the transgenic cells compared with the wild type, indicating that the rate of PtdInsP(2) synthesis was increased. These studies show that the constitutive expression of the human type I InsP 5-ptase in tobacco cells leads to an up-regulation of the phosphoinositide pathway and highlight the importance of PtdInsP(2) synthesis as a regulatory step in this system.
Abstract.
Author URL.
Persson S, Love J, Tsou P-L, Robertson D, Thompson WF, Boss WF (2002). When a day makes a difference. Interpreting data from endoplasmic reticulum-targeted green fluorescent protein fusions in cells grown in suspension culture.
Plant Physiol,
128(2), 341-344.
Author URL.
2001
Persson S, Wyatt SE, Love J, Thompson WF, Robertson D, Boss WF (2001). The Ca(2+) status of the endoplasmic reticulum is altered by induction of calreticulin expression in transgenic plants.
Plant Physiol,
126(3), 1092-1104.
Abstract:
The Ca(2+) status of the endoplasmic reticulum is altered by induction of calreticulin expression in transgenic plants.
To investigate the endoplasmic reticulum (ER) Ca(2+) stores in plant cells, we generated tobacco (Nicotiana tabacum; NT1) suspension cells and Arabidopsis plants with altered levels of calreticulin (CRT), an ER-localized Ca(2+)-binding protein. NT1 cells and Arabidopsis plants were transformed with a maize (Zea mays) CRT gene in both sense and antisense orientations under the control of an Arabidopsis heat shock promoter. ER-enriched membrane fractions from NT1 cells were used to examine how altered expression of CRT affects Ca(2+) uptake and release. We found that a 2.5-fold increase in CRT led to a 2-fold increase in ATP-dependent (45)Ca(2+) accumulation in the ER-enriched fraction compared with heat-shocked wild-type controls. Furthermore, after treatment with the Ca(2+) ionophore ionomycin, ER microsomes from NT1 cells overproducing CRT showed a 2-fold increase in the amount of (45)Ca(2+) released, and a 2- to 3-fold increase in the amount of (45)Ca(2+) retained compared with wild type. These data indicate that altering the production of CRT affects the ER Ca(2+) pool. In addition, CRT transgenic Arabidopsis plants were used to determine if altered CRT levels had any physiological effects. We found that the level of CRT in heat shock-induced CRT transgenic plants correlated positively with the retention of chlorophyll when the plants were transferred from Ca(2+)-containing medium to Ca(2+)-depleted medium. Together these data are consistent with the hypothesis that increasing CRT in the ER increases the ER Ca(2+) stores and thereby enhances the survival of plants grown in low Ca(2+) medium.
Abstract.
Author URL.
2000
Love J, Scott AC, Thompson WF (2000). Stringent control of transgene expression in Arabidopsis thaliana using the Top10 promoter system.
Plant Journal,
21(6), 579-588.
Abstract:
Stringent control of transgene expression in Arabidopsis thaliana using the Top10 promoter system
We show that the tightly regulated tetracycline-sensitive Top10 promoter system (Weinmann et al. Plant J. 1994, 5, 559-569) is functional in Arabidopsis thaliana. A pure breeding A. thaliana line (JL-tTA/8) was generated which expressed a chimeric fusion of the tetracycline repressor and the activation domain of Herpes simplex virus (tTA), from a single transgenic locus. Plants from this line were crossed with transgenics carrying the ER-targeted green fluorescent protein coding sequence (mGFP5) under control of the Top10 promoter sequence. Progeny from this cross displayed ER-targeted GFP fluorescence throughout the plant, indicating that the tTA-Top10 promoter interaction was functional in A. thaliana. GFP expression was repressed by 100 ng ml-1 tetracycline, an order of magnitude lower than the concentration used previously to repress expression in Nicotiana tabacum. Moreover, the level of GFP expression was controlled by varying the concentration of tetracycline in the medium, allowing a titred regulation of transgenic activity that was previously unavailable in A. thaliana. The kinetics of GFP activity were determined following de-repression of the Top10::mGFP5 transgene, with a visible ER-targeted GFP signal appearing from 24 to 48 h after de-repression.
Abstract.
1998
Moutinho A, Love J, Trewavas AJ, Malhó R (1998). Distribution of calmodulin protein and mRNA in growing pollen tubes.
Sexual Plant Reproduction,
11(3), 131-139.
Abstract:
Distribution of calmodulin protein and mRNA in growing pollen tubes
Pollen tube growth is a vital process for angiosperm fertilisation and is dependent on the presence of a tip-focused gradient of cytosolic free calcium ([Ca2+](c)). In order to clarify some of the target molecules which convey the Ca2+ signal information, we investigated calmodulin distribution during tube growth. Fluorescently labelled calmodulin was pressure microinjected into pollen tubes and its distribution monitored by confocal microscopy. Calmodulin distributes evenly throughout the cell, but some of its binding sites form a V-shaped collar behind the apical region. This specific association dissipates upon growth arrest, and suggests an interaction of calmodulin with cytoskeletal-bound target proteins. The distribution of calmodulin mRNA was also analysed by microinjection of fluorescently labelled mRNA. No specific pattern was observed, with an even localisation in the body of tube and a lower concentration in the cell apex. Studies with localised application of inhibitors/activators indicate that calmodulin plays a crucial role in tip elongation but does not direct tube orientation.
Abstract.
1997
Love J, Brownlee C, Trewavas AJ (1997). Ca2+ and Calmodulin Dynamics during Photopolarization in Fucus serratus Zygotes.
Plant Physiol,
115(1), 249-261.
Abstract:
Ca2+ and Calmodulin Dynamics during Photopolarization in Fucus serratus Zygotes.
The role of Ca2+ in zygote polarization in fucoid algae (Fucus, Ascophyllum, and Pelvetia species) zygote polarization is controversial. Using a local source of Fucus serratus, we established that zygotes form a polar axis relative to unilateral light (photopolarization) between 8 and 14 h after fertilization (AF), and become committed to this polarity at approximately 15 to 18 h AF. We investigated the role of Ca2+, calmodulin, and actin during photopolarization by simultaneously exposing F. serratus zygotes to polarizing light and various inhibitors. Neither removal of Ca2+ from the culture medium or high concentrations of EGTA and LaCl3 had any effect on photopolarization. Bepridil, 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester, nifedipine, and verapamil, all of which block intracellular Ca2 release, reduced photopolarization from 75 to 30%. The calmodulin antagonists N-(6-aminohexyl)-5-chloro-L-naphthalenesulfonamide and trifluoperazine inhibited photopolarization in all zygotes, whereas N-(6-aminohexyl)-L-naphthalenesulfonamide had no effect. Cytochalasin B, cytochalasin D, and latrunculin B, all of which inhibit actin polymerization, had no effect on photopolarization, but arrested polar axis fixation. The role of calmodulin during polarization was investigated further. Calmodulin mRNA from the closely related brown alga Macrocystis pyrifera was cloned and the protein was expressed in bacteria. Photopolarization was enhanced following microinjections of this recombinant calmodulin into developing zygotes. Confocal imaging of fluorescein isothiocyanate-labeled recombinant calmodulin in photopolarized zygotes showed a homogenous signal distribution at 13 h AF, which localized to the presumptive rhizoid site at 15 h AF.
Abstract.
Author URL.
1995
LOVE J, TREWAVAS AJ (1995). CALMODULIN INVOLVEMENT IN POLARIZATION OF FUCUS-SERRATUS ZYGOTES.
Author URL.
