Publications by authors named "Virginia K Walker"

65 Publications

Antibiotic resistance in mucosal bacteria from high Arctic migratory salmonids.

Environ Microbiol Rep 2021 Jun 9. Epub 2021 Jun 9.

College of the Sciences and Mathematics, West Chester University, West Chester, Pennsylvania, USA.

Two related salmonids, Arctic char (Salvelinus alpinus) and lake whitefish (Coregonus clupeaformis) sampled from the high Arctic region of Nunavut, Canada are anadromous fish, migrating annually from the same ice-covered freshwater waterbodies to spend summers in the marine waters of the Arctic Ocean. Microbiota associated with the skin-associated mucus undergo community change coincident with migration, and irrespective of this turnover, antibiotic resistance was detected in mixed bacterial cultures initiated with mucus samples. Although as expected most bacteria were unculturable, however, 5/7 isolates showed susceptibility to a panel of five common antibiotics. The fish were sampled under severe conditions and at remote locations far from human habitation. Regardless, two isolates, 'Carnobacterium maltaromaticum sm-2' and 'Arthrobacter citreus sm', showed multi-resistance to two or more antibiotics including ampicillin and streptomycin indicating multiple resistance genes. It is unknown if these fish bacteria have 'natural' resistance phenotypes or if resistance has been acquired. As result of these observations, we urge long-term monitoring of drug-resistant bacteria in the region and caution the assumption of a lack of drug-resistant organisms even in such extreme environments.
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http://dx.doi.org/10.1111/1758-2229.12975DOI Listing
June 2021

Identification of Arctic Food Fish Species for Anthropogenic Contaminant Testing Using Geography and Genetics.

Foods 2020 Dec 8;9(12). Epub 2020 Dec 8.

Department of Biology, Queen's University, Kingston, ON K7L 3N6, Canada.

The identification of food fish bearing anthropogenic contaminants is one of many priorities for Indigenous peoples living in the Arctic. Mercury (Hg), arsenic (As), and persistent organic pollutants including polychlorinated biphenyls (PCBs) are of concern, and these are reported, in some cases for the first time, for fish sampled in and around King William Island, located in Nunavut, Canada. More than 500 salmonids, comprising Arctic char, lake trout, lake whitefish, and ciscoes, were assayed for contaminants. The studied species are anadromous, migrating to the ocean to feed in the summers and returning to freshwater before sea ice formation in the autumn. Assessments of muscle Hg levels in salmonids from fishing sites on King William Island showed generally higher levels than from mainland sites, with mean concentrations generally below guidelines, except for lake trout. In contrast, mainland fish showed higher means for As, including non-toxic arsenobetaine, than island fish. Lake trout were highest in As and PCB levels, with salmonid PCB congener analysis showing signatures consistent with the legacy of cold-war distant early warning stations. After DNA-profiling, only 4-32 Arctic char single nucleotide polymorphisms were needed for successful population assignment. These results support our objective to demonstrate that genomic tools could facilitate efficient and cost-effective cluster assignment for contaminant analysis during ocean residency. We further suggest that routine pollutant testing during the current period of dramatic climate change would be helpful to safeguard the wellbeing of Inuit who depend on these fish as a staple input to their diet. Moreover, this strategy should be applicable elsewhere.
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http://dx.doi.org/10.3390/foods9121824DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7764770PMC
December 2020

Isolation and Characterization of Ice-Binding Proteins from Higher Plants.

Methods Mol Biol 2020 ;2156:303-332

Department of Biology, Queen's University, Kingston, ON, Canada.

The characterization of ice-binding proteins (IBPs) from plants can involve many techniques, a few of which are presented here. Chief among these methods are tests for ice recrystallization inhibition, an activity characteristic of plant IBPs. Two related procedures are described, both of which can be used to demonstrate and quantify ice-binding activity. First, is the traditional "splat" assay, which can easily be set up using common laboratory equipment, and second, is our modification of this method using superhydrophobic coated sapphire for analysis of multiple samples in tandem. Thermal hysteresis is described as another method for quantifying ice-binding activity, during which ice crystal morphology observations can be used to provide clues about ice-plane binding. Once ice-binding activity has been evaluated, it is necessary to verify IBP identity. We detail two methods for enriching IBPs from complex mixtures using ice-affinity purification, the "ice-finger" and "ice-shell" methods, and we highlight their advantages and limitations for the isolation of plant IBPs. Recombinant IBP expression, necessary for detailed ice-binding analysis, can present challenges. Here, a strategy for recovery of soluble, active protein is described. Lastly, verification of function in planta borrows from standard protocols, but with an additional screen applicable to IBPs. Together, these methods, and a few considerations critical to success, can be used to assist researchers wishing to isolate and characterize IBPs from plants.
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http://dx.doi.org/10.1007/978-1-0716-0660-5_21DOI Listing
March 2021

A practical assessment of nano-phosphate on soybean (Glycine max) growth and microbiome establishment.

Sci Rep 2020 06 4;10(1):9151. Epub 2020 Jun 4.

Department of Biology, Queen's University, Kingston, ON, Canada.

The efficacy of needle-shaped nano-hydroxyapatite (nHA; Ca(PO)(OH)) as a phosphate (Pi) fertilizer was evaluated as well as its impact on soil and soybean (Glycine max) bacterial and fungal communities. Microbial communities were evaluated in soy fertilized with nHA using ITS (internal transcribed spacer) and 16S rRNA high-throughput gene sequencing. Separate greenhouse growth experiments using agriculturally relevant nHA concentrations and application methods were used to assess plant growth and yield compared with no Pi (-P), soluble Pi (+P), and bulk HA controls. Overall, nHA treatments did not show significantly increased growth, biomass, total plant phosphorus concentrations, or yield compared with no Pi controls. Soil and rhizosphere community structures in controls and nHA treatment groups were similar, with minor shifts in the nHA-containing pots comparable to bulk HA controls at equal concentrations. The implementation of nHA in an agriculturally realistic manner and the resulting poor soy growth advises that contrary to some reports under specialized conditions, this nano-fertilizer may not be a viable alternative to traditional Pi fertilizers. If nano-phosphate fertilizers are to achieve their conjectured agricultural potential, alternative nHAs, with differing morphologies, physicochemical properties, and interactions with the soil matrix could be investigated using the evaluative procedures described.
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http://dx.doi.org/10.1038/s41598-020-66005-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7272607PMC
June 2020

Seasonal habitat drives intestinal microbiome composition in anadromous Arctic char (Salvelinus alpinus).

Environ Microbiol 2020 08 4;22(8):3112-3125. Epub 2020 Jun 4.

Department of Biology, Queen's University, Kingston, ON, K7L 3N6, Canada.

Intestinal microbial communities from 362 anadromous Arctic char (Salvelinus alpinus) from the high Arctic Kitikmeot region, Nunavut, Canada, were characterized using high-throughput 16S rRNA gene sequencing. The resulting bacterial communities were compared across four seasonal habitats that correspond to different stages of annual migration. Arctic char intestinal communities differed by sampling site, salinity and stages of freshwater residence. Although microbiota from fish sampled in brackish water were broadly consistent with taxa seen in other anadromous salmonids, they were enriched with putative psychrophiles, including the nonluminous gut symbiont Photobacterium iliopiscarium that was detected in >90% of intestinal samples from these waters. Microbiota from freshwater-associated fish were less consistent with results reported for other salmonids, and highly variable, possibly reflecting winter fasting behaviour of these char. We identified microbiota links to age for those fish sampled during the autumn upriver migration, but little impact of the intestinal content and water microbiota on the intestinal community. The strongest driver of intestinal community composition was seasonal habitat, and this finding combined with identification of psychrophiles suggested that water temperature and migratory behaviour are key to understanding the relationship between Arctic char and their symbionts.
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http://dx.doi.org/10.1111/1462-2920.15049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7496496PMC
August 2020

Drosophila as a Model for Developmental Toxicology: Using and Extending the Drosophotoxicology Model.

Methods Mol Biol 2019 ;1965:139-153

Department of Biology, Queen's University, Kingston, ON, Canada.

Fruit flies, Drosophila melanogaster, have been traditionally valued as a simple model system due to their easy and inexpensive culture, their relatively compact genome, and the variety of available genetic tools. However, due to similarities of their neurological and developmental pathways with those of vertebrates, Drosophila also offers advantages for developmental toxicity assays. The ability to distinguish the effects of a toxicant on adult females, males, and the developing offspring adds to the usefulness of this model. Here we describe key techniques to screen chemicals and other potential emerging toxicants such as nanoparticles on adult Drosophila female and male reproductive success. In addition, assessments of relative toxicity can be revealed by viability assays at each developmental stage from the embryo to the pharate, or preemergent, adult.
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http://dx.doi.org/10.1007/978-1-4939-9182-2_10DOI Listing
November 2019

Anadromous Arctic Char Microbiomes: Bioprospecting in the High Arctic.

Front Bioeng Biotechnol 2019 26;7:32. Epub 2019 Feb 26.

Department of Biology, Queen's University, Kingston, ON, Canada.

Northern populations of Arctic char () can be anadromous, migrating annually from the ocean to freshwater lakes and rivers in order to escape sub-zero temperatures. Such seasonal behavior demands that these fish and their associated microbiomes adapt to changes in salinity, temperature, and other environmental challenges. We characterized the microbial community composition of anadromous , netted by Inuit fishermen at freshwater and seawater fishing sites in the high Arctic, both under ice and in open water. Bacterial profiles were generated by DNA extraction and high-throughput sequencing of PCR-amplified 16S ribosomal RNA genes. Results showed that microbial communities on the skin and intestine of Arctic char were statistically different when sampled from freshwater or saline water sites. This association was tested using hierarchical Ward's linkage clustering, showing eight distinct clusters in each of the skin and intestinal microbiomes, with the clusters reflecting sampling location between fresh and saline environments, confirming a salinity-linked turnover. This analysis also provided evidence for a core composition of skin and intestinal bacteria, with the phyla Proteobacteria, Firmicutes, and Cyanobacteria presenting as major phyla within the skin-associated microbiomes. The intestine-associated microbiome was characterized by unidentified genera from families Fusobacteriaceae, Comamonadaceae, Pseudomonadaceae, and Vibrionaceae. The salinity-linked turnover was further tested through ordinations that showed samples grouping based on environment for both skin- and intestine-associated microbiomes. This finding implies that core microbiomes between fresh and saline conditions could be used to assist in regulating optimal fish health in aquaculture practices. Furthermore, identified taxa from known psychrophiles and with nitrogen cycling properties suggest that there is additional potential for biotechnological applications for fish farm and waste management practices.
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http://dx.doi.org/10.3389/fbioe.2019.00032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6399304PMC
February 2019

Ice-Binding Proteins in Plants.

Front Plant Sci 2017 22;8:2153. Epub 2017 Dec 22.

Department of Biomedical and Molecular Sciences, and School of Environmental Studies, Queen's University, Kingston, ON, Canada.

Sub-zero temperatures put plants at risk of damage associated with the formation of ice crystals in the apoplast. Some freeze-tolerant plants mitigate this risk by expressing ice-binding proteins (IBPs), that adsorb to ice crystals and modify their growth. IBPs are found across several biological kingdoms, with their ice-binding activity and function uniquely suited to the lifestyle they have evolved to protect, be it in fishes, insects or plants. While IBPs from freeze-avoidant species significantly depress the freezing point, plant IBPs typically have a reduced ability to lower the freezing temperature. Nevertheless, they have a superior ability to inhibit the recrystallization of formed ice. This latter activity prevents ice crystals from growing larger at temperatures close to melting. Attempts to engineer frost-hardy plants by the controlled transfer of IBPs from freeze-avoiding fish and insects have been largely unsuccessful. In contrast, the expression of recombinant IBP sequences from freeze-tolerant plants significantly reduced electrolyte leakage and enhanced freezing survival in freeze-sensitive plants. These promising results have spurred additional investigations into plant IBP localization and post-translational modifications, as well as a re-evaluation of IBPs as part of the anti-stress and anti-pathogen axis of freeze-tolerant plants. Here we present an overview of plant freezing stress and adaptation mechanisms and discuss the potential utility of IBPs for the generation of freeze-tolerant crops.
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http://dx.doi.org/10.3389/fpls.2017.02153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5744647PMC
December 2017

Microbial Diversity in a Hypersaline Sulfate Lake: A Terrestrial Analog of Ancient Mars.

Front Microbiol 2017 26;8:1819. Epub 2017 Sep 26.

Department of Earth, Atmospheric and Planetary Sciences, Massachusetts Institute of Technology, Cambridge, MA, United States.

Life can persist under severe osmotic stress and low water activity in hypersaline environments. On Mars, evidence for the past presence of saline bodies of water is prevalent and resulted in the widespread deposition of sulfate and chloride salts. Here we investigate Spotted Lake (British Columbia, Canada), a hypersaline lake with extreme (>3 M) levels of sulfate salts as an exemplar of the conditions thought to be associated with ancient Mars. We provide the first characterization of microbial structure in Spotted Lake sediments through metagenomic sequencing, and report a bacteria-dominated community with abundant Proteobacteria, Firmicutes, and Bacteroidetes, as well as diverse extremophiles. Microbial abundance and functional comparisons reveal similarities to Ace Lake, a meromictic Antarctic lake with anoxic and sulfidic bottom waters. Our analysis suggests that hypersaline-associated species occupy niches characterized foremost by differential abundance of Archaea, uncharacterized Bacteria, and Cyanobacteria. Potential biosignatures in this environment are discussed, specifically the likelihood of a strong sulfur isotopic fractionation record within the sediments due to the presence of sulfate reducing bacteria. With its high sulfate levels and seasonal freeze-thaw cycles, Spotted Lake is an analog for ancient paleolakes on Mars in which sulfate salt deposits may have offered periodically habitable environments, and could have concentrated and preserved organic materials or their biomarkers over geologic time.
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http://dx.doi.org/10.3389/fmicb.2017.01819DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5623196PMC
September 2017

Impact of food grade and nano-TiO particles on a human intestinal community.

Food Chem Toxicol 2017 Aug 28;106(Pt A):242-249. Epub 2017 May 28.

Department of Biology, Queen's University, Kingston ON K7L 3N6, Canada; Department of Biomedical and Molecular Sciences, School of Environmental Studies, Queen's University, Kingston, ON K7L 3N6, Canada. Electronic address:

Titanium dioxide (TiO) nanoparticles (NPs) are used as an additive (E171 or INS171) in foods such as gum, candy and puddings. To address concerns about the potential hazardous effects of ingested NPs, the toxicity of these food-grade NPs was investigated with a defined model intestinal bacterial community. Each titania preparation (food-grade TiO formulations, E171-1 and E171-6a) was tested at concentrations equivalent to those found in the human intestine after sampling 1-2 pieces of gum or candy (100-250 ppm). At the low concentrations used, neither the TiO food additives nor control TiO NPs had an impact on gas production and only a minor effect on fatty acids profiles (C16:00, C18:00, 15:1 w5c, 18:1 w9c and 18:1 w9c, p < 0.05). DNA profiles and phylogenetic distributions confirmed limited effects on the bacterial community, with a modest decrease in the relative abundance of the dominant Bacteroides ovatus in favor of Clostridium cocleatum (-13% and +14% respectively, p < 0.05). Such minor shifts in the treated consortia suggest that food grade and nano-TiO particles do not have a major effect on human gut microbiota when tested in vitro at relevant low concentrations. However, the cumulative effects of chronic TiO NP ingestion remain to be tested.
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http://dx.doi.org/10.1016/j.fct.2017.05.050DOI Listing
August 2017

Identification of Plant Ice-binding Proteins Through Assessment of Ice-recrystallization Inhibition and Isolation Using Ice-affinity Purification.

J Vis Exp 2017 05 5(123). Epub 2017 May 5.

Department of Biology, Queen's University; Department of Biomedical and Molecular Sciences, Queen's University; School of Environmental Sciences, Queen's University.

Ice-binding proteins (IBPs) belong to a family of stress-induced proteins that are synthesized by certain organisms exposed to subzero temperatures. In plants, freeze damage occurs when extracellular ice crystals grow, resulting in the rupture of plasma membranes and possible cell death. Adsorption of IBPs to ice crystals restricts further growth by a process known as ice-recrystallization inhibition (IRI), thereby reducing cellular damage. IBPs also demonstrate the ability to depress the freezing point of a solution below the equilibrium melting point, a property known as thermal hysteresis (TH) activity. These protective properties have raised interest in the identification of novel IBPs due to their potential use in industrial, medical and agricultural applications. This paper describes the identification of plant IBPs through 1) the induction and extraction of IBPs in plant tissue, 2) the screening of extracts for IRI activity, and 3) the isolation and purification of IBPs. Following the induction of IBPs by low temperature exposure, extracts are tested for IRI activity using a 'splat assay', which allows the observation of ice crystal growth using a standard light microscope. This assay requires a low protein concentration and generates results that are quickly obtained and easily interpreted, providing an initial screen for ice binding activity. IBPs can then be isolated from contaminating proteins by utilizing the property of IBPs to adsorb to ice, through a technique called 'ice-affinity purification'. Using cell lysates collected from plant extracts, an ice hemisphere can be slowly grown on a brass probe. This incorporates IBPs into the crystalline structure of the polycrystalline ice. Requiring no a priori biochemical or structural knowledge of the IBP, this method allows for recovery of active protein. Ice-purified protein fractions can be used for downstream applications including the identification of peptide sequences by mass spectrometry and the biochemical analysis of native proteins.
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http://dx.doi.org/10.3791/55302DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5607887PMC
May 2017

Ice and anti-nucleating activities of an ice-binding protein from the annual grass, Brachypodium distachyon.

Plant Cell Environ 2018 05 18;41(5):983-992. Epub 2017 Feb 18.

Department of Biology, Queen's University, Kingston, Ontario, K7L 3N6, Canada.

Plants exposed to sub-zero temperatures face unique challenges that threaten their survival. The growth of ice crystals in the extracellular space can cause cellular dehydration, plasma membrane rupture and eventual cell death. Additionally, some pathogenic bacteria cause tissue damage by initiating ice crystal growth at high sub-zero temperatures through the use of ice-nucleating proteins (INPs), presumably to access nutrients from lysed cells. An annual species of brome grass, Brachypodium distachyon (Bd), produces an ice-binding protein (IBP) that shapes ice with a modest depression of the freezing point (~0.1 °C at 1 mg/mL), but high ice-recrystallization inhibition (IRI) activity, allowing ice crystals to remain small at near melting temperatures. This IBP, known as BdIRI, is unlike other characterized IBPs with a single ice-binding face, as mutational analysis indicates that BdIRI adsorbs to ice on two faces. BdIRI also dramatically attenuates the nucleation of ice by bacterial INPs (up to -2.26 °C). This 'anti-nucleating' activity is significantly higher than previously documented for any IBP.
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http://dx.doi.org/10.1111/pce.12889DOI Listing
May 2018

Knockdown of Ice-Binding Proteins in Brachypodium distachyon Demonstrates Their Role in Freeze Protection.

PLoS One 2016 13;11(12):e0167941. Epub 2016 Dec 13.

Department of Biology, Queen's University, Kingston, ON, Canada.

Sub-zero temperatures pose a major threat to the survival of cold-climate perennials. Some of these freeze-tolerant plants produce ice-binding proteins (IBPs) that offer frost protection by restricting ice crystal growth and preventing expansion-induced lysis of the plasma membranes. Despite the extensive in vitro characterization of such proteins, the importance of IBPs in the freezing stress response has not been investigated. Using the freeze-tolerant grass and model crop, Brachypodium distachyon, we characterized putative IBPs (BdIRIs) and generated the first 'IBP-knockdowns'. Seven IBP sequences were identified and expressed in Escherichia coli, with all of the recombinant proteins demonstrating moderate to high levels of ice-recrystallization inhibition (IRI) activity, low levels of thermal hysteresis (TH) activity (0.03-0.09°C at 1 mg/mL) and apparent adsorption to ice primary prism planes. Following plant cold acclimation, IBPs purified from wild-type B. distachyon cell lysates similarly showed high levels of IRI activity, hexagonal ice-shaping, and low levels of TH activity (0.15°C at 0.5 mg/mL total protein). The transfer of a microRNA construct to wild-type plants resulted in the attenuation of IBP activity. The resulting knockdown mutant plants had reduced ability to restrict ice-crystal growth and a 63% reduction in TH activity. Additionally, all transgenic lines were significantly more vulnerable to electrolyte leakage after freezing to -10°C, showing a 13-22% increase in released ions compared to wild-type. IBP-knockdown lines also demonstrated a significant decrease in viability following freezing to -8°C, with some lines showing only two-thirds the survival seen in control lines. These results underscore the vital role IBPs play in the development of a freeze-tolerant phenotype and suggests that expression of these proteins in frost-susceptible plants could be valuable for the production of more winter-hardy crops.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0167941PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5154533PMC
June 2017

Fate and impact of zero-valent copper nanoparticles on geographically-distinct soils.

Sci Total Environ 2016 Dec 30;573:661-670. Epub 2016 Aug 30.

Department of Natural Sciences, Southern University at New Orleans, New Orleans, LA 70126, USA.

The fate of engineered zero-valent copper nanoparticles (Cu NPs) in soils collected from geographically-distinct regions of the continental United States and incubated under controlled conditions was investigated with respect to NP affinity for soil surfaces and changes in speciation, as well as their impact on bacterial communities. Soil geochemical properties had a great influence on Cu NP migration and transformation. Translocation of Cu NPs was low in soils enriched in organic matter and high in clay and sandy soils. X-ray absorption spectroscopic analysis showed that the highest rates for transformation to Cu ions and adsorption complexes was in acidic soils. Although there was some change in overall bacterial community richness at the level of order in experimental soil, the level of perturbation was evident in side-by-side comparisons of orders using a 50% microbial community change value (MCC). This assessment revealed that generally, Sphingomonas, known for its importance for remediation, and Rhizobiales, symbiotic partners with certain plants appeared susceptible to Cu NPs and their transformation products. The ecological importance of organisms from these orders and its greater vulnerability to Cu NPs suggests need for future targeted studies.
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http://dx.doi.org/10.1016/j.scitotenv.2016.08.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7384298PMC
December 2016

Ice-binding proteins confer freezing tolerance in transgenic Arabidopsis thaliana.

Plant Biotechnol J 2017 01 14;15(1):68-81. Epub 2016 Jul 14.

Department of Biology, Queen's University, Kingston, ON, Canada.

Lolium perenne is a freeze-tolerant perennial ryegrass capable of withstanding temperatures below -13 °C. Ice-binding proteins (IBPs) presumably help prevent damage associated with freezing by restricting the growth of ice crystals in the apoplast. We have investigated the expression, localization and in planta freezing protection capabilities of two L. perenne IBP isoforms, LpIRI2 and LpIRI3, as well as a processed IBP (LpAFP). One of these isoforms, LpIRI2, lacks a conventional signal peptide and was assumed to be a pseudogene. Nevertheless, both LpIRI2 and LpIRI3 transcripts were up-regulated following cold acclimation. LpIRI2 also demonstrated ice-binding activity when produced recombinantly in Escherichia coli. Both the LpIRI3 and LpIRI2 isoforms appeared to accumulate in the apoplast of transgenic Arabidopsis thaliana plants. In contrast, the fully processed isoform, LpAFP, remained intracellular. Transgenic plants expressing either LpIRI2 or LpIRI3 showed reduced ion leakage (12%-39%) after low-temperature treatments, and significantly improved freezing survival, while transgenic LpAFP-expressing lines did not confer substantial subzero protection. Freeze protection was further enhanced by with the introduction of more than one IBP isoform; ion leakage was reduced 26%-35% and 10% of plants survived temperatures as low as -8 °C. Our results demonstrate that apoplastic expression of multiple L. perenne IBP isoforms shows promise for providing protection to crops susceptible to freeze-induced damage.
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http://dx.doi.org/10.1111/pbi.12592DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5253476PMC
January 2017

Structural Basis for the Inhibition of Gas Hydrates by α-Helical Antifreeze Proteins.

Biophys J 2015 Oct;109(8):1698-705

Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada; Department of Biology, Queen's University, Kingston, Ontario, Canada. Electronic address:

Kinetic hydrate inhibitors (KHIs) are used commercially to inhibit gas hydrate formation and growth in pipelines. However, improvement of these polymers has been constrained by the lack of verified molecular models. Since antifreeze proteins (AFPs) act as KHIs, we have used their solved x-ray crystallographic structures in molecular modeling to explore gas hydrate inhibition. The internal clathrate water network of the fish AFP Maxi, which extends to the protein's outer surface, is remarkably similar to the {100} planes of structure type II (sII) gas hydrate. The crystal structure of this water web has facilitated the construction of in silico models for Maxi and type I AFP binding to sII hydrates. Here, we have substantiated our models with experimental evidence of Maxi binding to the tetrahydrofuran sII model hydrate. Both in silico and experimental evidence support the absorbance-inhibition mechanism proposed for KHI binding to gas hydrates. Based on the Maxi crystal structure we suggest that the inhibitor adsorbs to the gas hydrate lattice through the same anchored clathrate water mechanism used to bind ice. These results will facilitate the rational design of a next generation of effective green KHIs for the petroleum industry to ensure safe and efficient hydrocarbon flow.
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http://dx.doi.org/10.1016/j.bpj.2015.08.041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4624156PMC
October 2015

Vegetation-associated impacts on arctic tundra bacterial and microeukaryotic communities.

Appl Environ Microbiol 2015 Jan 31;81(2):492-501. Epub 2014 Oct 31.

Department of Biology, Queen's University, Kingston, Ontario, Canada

The Arctic is experiencing rapid vegetation changes, such as shrub and tree line expansion, due to climate warming, as well as increased wetland variability due to hydrological changes associated with permafrost thawing. These changes are of global concern because changes in vegetation may increase tundra soil biogeochemical processes that would significantly enhance atmospheric CO2 concentrations. Predicting the latter will at least partly depend on knowing the structure, functional activities, and distributions of soil microbes among the vegetation types across Arctic landscapes. Here we investigated the bacterial and microeukaryotic community structures in soils from the four principal low Arctic tundra vegetation types: wet sedge, birch hummock, tall birch, and dry heath. Sequencing of rRNA gene fragments indicated that the wet sedge and tall birch communities differed significantly from each other and from those associated with the other two dominant vegetation types. Distinct microbial communities were associated with soil pH, ammonium concentration, carbon/nitrogen (C/N) ratio, and moisture content. In soils with similar moisture contents and pHs (excluding wet sedge), bacterial, fungal, and total eukaryotic communities were correlated with the ammonium concentration, dissolved organic nitrogen (DON) content, and C/N ratio. Operational taxonomic unit (OTU) richness, Faith's phylogenetic diversity, and the Shannon species-level index (H') were generally lower in the tall birch soil than in soil from the other vegetation types, with pH being strongly correlated with bacterial richness and Faith's phylogenetic diversity. Together, these results suggest that Arctic soil feedback responses to climate change will be vegetation specific not just because of distinctive substrates and environmental characteristics but also, potentially, because of inherent differences in microbial community structure.
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http://dx.doi.org/10.1128/AEM.03229-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4277566PMC
January 2015

Juvenile hormone-receptor complex acts on mcm4 and mcm7 to promote polyploidy and vitellogenesis in the migratory locust.

PLoS Genet 2014 Oct 23;10(10):e1004702. Epub 2014 Oct 23.

State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, China.

Juvenile hormone (JH), a sesquiterpenoid produced by the corpora allata, coordinates insect growth, metamorphosis, and reproduction. While JH action for the repression of larval metamorphosis has been well studied, the molecular basis of JH in promoting adult reproduction has not been fully elucidated. Methoprene-tolerant (Met), the JH receptor, has been recently shown to mediate JH action during metamorphosis as well as in vitellogenesis, but again, the precise mechanism underlying the latter has been lacking. We have now demonstrated using Met RNAi to phenocopy a JH-deprived condition in migratory locusts, that JH stimulates DNA replication and increases ploidy in preparation for vitellogenesis. Mcm4 and Mcm7, two genes in the DNA replication pathway were expressed in the presence of JH and Met. Depletion of Mcm4 or Mcm7 inhibited de novo DNA synthesis and polyploidization, and resulted in the substantial reduction of vitellogenin mRNA levels as well as severely impaired oocyte maturation and ovarian growth. By using luciferase reporter and electrophoretic mobility shift assays, we have shown that Met directly regulates the transcription of Mcm4 and Mcm7 by binding to upstream consensus sequences with E-box or E-box-like motifs. Our work suggests that the JH-receptor complex acts on Mcm4 and Mcm7 to regulate DNA replication and polyploidy for vitellogenesis and oocyte maturation.
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http://dx.doi.org/10.1371/journal.pgen.1004702DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4207617PMC
October 2014

Perturbation of bacterial ice nucleation activity by a grass antifreeze protein.

Biochem Biophys Res Commun 2014 Sep 1;452(3):636-41. Epub 2014 Sep 1.

Department of Biology, Queen's University, Kingston, ON K7L 3N6, Canada. Electronic address:

Certain plant-associating bacteria produce ice nucleation proteins (INPs) which allow the crystallization of water at high subzero temperatures. Many of these microbes are considered plant pathogens since the formed ice can damage tissues, allowing access to nutrients. Intriguingly, certain plants that host these bacteria synthesize antifreeze proteins (AFPs). Once freezing has occurred, plant AFPs likely function to inhibit the growth of large damaging ice crystals. However, we postulated that such AFPs might also serve as defensive mechanisms against bacterial-mediated ice nucleation. Recombinant AFP derived from the perennial ryegrass Lolium perenne (LpAFP) was combined with INP preparations originating from the grass epiphyte, Pseudomonas syringae. The presence of INPs had no effect on AFP activity, including thermal hysteresis and ice recrystallization inhibition. Strikingly, the ice nucleation point of the INP was depressed up to 1.9°C in the presence of LpAFP, but a recombinant fish AFP did not lower the INP-imposed freezing point. Assays with mutant LpAFPs and the visualization of bacterially-displayed fluorescent plant AFP suggest that INP and LpAFP can interact. Thus, we postulate that in addition to controlling ice growth, plant AFPs may also function as a defensive strategy against the damaging effects of ice-nucleating bacteria.
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http://dx.doi.org/10.1016/j.bbrc.2014.08.138DOI Listing
September 2014

Expression and localization of an ice nucleating protein from a soil bacterium, Pseudomonas borealis.

Cryobiology 2014 Aug 12;69(1):110-8. Epub 2014 Jun 12.

Department of Biology, Queen's University, Kingston, ON K7L 3N6, Canada. Electronic address:

An ice nucleating protein (INP) coding region with 66% sequence identity to the INP of Pseudomonas syringae was previously cloned from P. borealis, a plant beneficial soil bacterium. Ice nucleating activity (INA) in the P. borealis DL7 strain was highest after transfer of cultures to temperatures just above freezing. The corresponding INP coding sequence (inaPb or ina) was used to construct recombinant plasmids, with recombinant expression visualized using a green fluorescent protein marker (gfp encoding GFP). Although the P. borealis strain was originally isolated by ice-affinity, bacterial cultures with membrane-associated INP-GFP did not adsorb to pre-formed ice. Employment of a shuttle vector allowed expression of ina-gfp in both Escherichia coli and Pseudomonas cells. At 27 °C, diffuse fluorescence appeared throughout the cells and was associated with low INA. However, after transfer of cultures to 4 °C, the protein localized to the poles coincident with high INA. Transformants with truncated INP sequences ligated to either gfp, or an antifreeze protein-gfp fusion showed that the repetitive ice-nucleation domain was not necessary for localization. Such localization is consistent with the flanking residues of the INP associating with a temperature-dependent secretion apparatus. A polar location would facilitate INP-INP interactions resulting in the formation of larger aggregates, serving to increase INA. Expression of INPs by P. borealis could function as an efficient atmospheric dispersal mechanism for these soil bacteria, which are less likely to use these proteins for nutrient procurement, as has been suggested for P. syringae.
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http://dx.doi.org/10.1016/j.cryobiol.2014.06.001DOI Listing
August 2014

The effect of silver nanoparticles on seasonal change in arctic tundra bacterial and fungal assemblages.

PLoS One 2014 13;9(6):e99953. Epub 2014 Jun 13.

Department of Biology, Queen's University, Kingston, Ontario, Canada; School of Environmental Studies, Queen's University, Kingston, Ontario, Canada.

The impact of silver nanoparticles (NPs) and microparticles (MPs) on bacterial and fungal assemblages was studied in soils collected from a low arctic site. Two different concentrations (0.066% and 6.6%) of Ag NPs and Ag MPs were tested in microcosms that were exposed to temperatures mimicking a winter to summer transition. Toxicity was monitored by differential respiration, phospholipid fatty acid analysis, polymerase chain reaction-denaturing gradient gel electrophoresis and DNA sequencing. Notwithstanding the effect of Ag MPs, nanosilver had an obvious, additional impact on the microbial community, underscoring the importance of particle size in toxicity. This impact was evidenced by levels of differential respiration in 0.066% Ag NP-treated soil that were only half that of control soils, a decrease in signature bacterial fatty acids, and changes in both richness and evenness in bacterial and fungal DNA sequence assemblages. Prominent after Ag NP-treatment were Hypocreales fungi, which increased to 70%, from only 1% of fungal sequences under control conditions. Genera within this Order known for their antioxidant properties (Cordyceps/Isaria) dominated the fungal assemblage after NP addition. In contrast, sequences attributed to the nitrogen-fixing Rhizobiales bacteria appeared vulnerable to Ag NP-mediated toxicity. This combination of physiological, biochemical and molecular studies clearly demonstrate that Ag NPs can severely disrupt the natural seasonal progression of tundra assemblages.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0099953PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4057283PMC
May 2015

Isolation and characterization of ice-binding proteins from higher plants.

Methods Mol Biol 2014 ;1166:255-77

Department of Biomedical and Molecular Sciences, Queen's University, Kingston, ON, Canada, K7L 3N6.

The characterization of ice-binding proteins from plants can involve many techniques, only a few of which are presented here. Chief among these methods are tests for ice recrystallization inhibition activity. Two distinct procedures are described; neither is normally used for precise quantitative assays. Thermal hysteresis assays are used for quantitative studies but are also useful for ice crystal morphologies, which are important for the understanding of ice-plane binding. Once the sequence of interest is cloned, recombinant expression, necessary to verify ice-binding protein identity can present challenges, and a strategy for recovery of soluble, active protein is described. Lastly, verification of function in planta borrows from standard protocols, but with an additional screen applicable to ice-binding proteins. Here we have attempted to assist researchers wishing to isolate and characterize ice-binding proteins from plants with a few methods critical to success.
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http://dx.doi.org/10.1007/978-1-4939-0844-8_19DOI Listing
January 2015

Towards the selection of a produced water enrichment for biological gas hydrate inhibitors.

Environ Sci Pollut Res Int 2014 Sep 13;21(17):10254-61. Epub 2014 May 13.

Department of Biology, Queen's University, Kingston, Ontario, K7L 3N6, Canada,

Economic concerns associated with the recovery of non-conventional hydrocarbon reserves include unexpected ice as well as ice-like gas hydrate formation. Antifreeze proteins (AFPs) inhibit ice growth, and experiments with fish, plant, and insect AFPs have shown promise of effective gas hydrate inhibition in lab-scale experiments. If produced on an industrial scale, AFPs could provide a more environmentally friendly alternative to kinetic inhibitors, but a large-scale production of these AFPs is not currently feasible. We believe that these difficulties could be surmounted by the production of microbial AFPs, but to date, only a few such proteins have been identified and purified, and none of these are associated with hydrocarbon reserves. Here, we have used ice-affinity and freeze-thaw stress to select microbes derived from oil and gas formation water, or produced water, as a source of anaerobic microbial communities. Ice-affinity successfully incorporated anaerobic bacteria under aerobic conditions, and the mixed culture had ice-associating properties. Under these conditions, ice-affinity selection does not result in cultivatable isolates, but similar, cultivable microbes were obtained following freeze-thaw selection under anaerobic conditions. Since these mixed cultures inhibited the growth of ice crystals, they also have the potential to inhibit hydrate growth. Overall, freeze-thaw selection provides a promising first step towards the isolation of microbes capable of the inhibition of ice and gas hydrate growth, for possible application for energy exploration and recovery at high-latitudes and in-deep, cold waters.
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http://dx.doi.org/10.1007/s11356-014-2912-xDOI Listing
September 2014

Monitoring the developmental impact of copper and silver nanoparticle exposure in Drosophila and their microbiomes.

Sci Total Environ 2014 Jul 22;487:822-9. Epub 2014 Jan 22.

Department of Biology, Queen's University, Kingston, Ontario K7L 3N6, Canada; School of Environmental Studies, Queen's University, Kingston, Ontario K7L 3N6, Canada. Electronic address:

There is concern that waste waters containing manufactured metal nanoparticles (NPs) originating from consumer goods, will find their way into streams and larger water bodies. Aquatic invertebrates could be vulnerable to such pollution, and here we have used fruit flies, Drosophila melanogaster, as a model invertebrate, to test for the effect of NPs on fitness. Both copper NP and microparticle (MP)-containing medium slowed development, reduced adult longevity and decreased sperm competition. In contrast, ingestion of silver resulted in a significant reduction in developmental success only if the metal particles were nanosized. Ag NP-treatments resulted in reduced developmental success as assessed by larval and pupal survival as well as larval climbing ability, but there was no impact of silver on adult longevity and little effect on reproductive success. However, Cu NPs generally appeared to be no more toxic to this invertebrate model than the bulk counterpart. The impact of silver ingestion in larvae was further investigated by 454 pyrosequencing of the 16S rRNA genes of the midgut flora. There was a striking reduction in the diversity of the gut microbiota of Ag NP-treated larvae with a rise in the predominance of Lactobacillus brevis and a decrease in Acetobacter compared to control or Ag MP-treatment groups. Importantly, these experiments show that perturbation of the microbial assemblage within a metazoan model may contribute to Ag NP-mediated toxicity. These observations have implications for impact assessments of nanoparticles as emerging contaminants.
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http://dx.doi.org/10.1016/j.scitotenv.2013.12.129DOI Listing
July 2014

Ice recrystallization inhibition mediated by a nuclear-expressed and -secreted recombinant ice-binding protein in the microalga Chlamydomonas reinhardtii.

Appl Microbiol Biotechnol 2013 Nov 15;97(22):9763-72. Epub 2013 Sep 15.

Center for Biotechnology (CeBiTec), Faculty of Biology, Bielefeld University, Universitätsstrasse 27, 33615, Bielefeld, Germany.

A Lolium perenne ice-binding protein (LpIBP) demonstrates superior ice recrystallization inhibition (IRI) activity and has proposed applications in cryopreservation, food texturing, as well as in being a "green" gas hydrate inhibitor. Recombinant production of LpIBP has been previously conducted in bacterial and yeast systems for studies of protein characterization, but large-scale applications have been hitherto limited due to high production costs. In this work, a codon-optimized LpIBP was recombinantly expressed and secreted in a novel one-step vector system from the nuclear genome of the green microalga Chlamydomonas reinhardtii. Both mixotrophic and photoautotrophic growth regimes supported LpIBP expression, indicating the feasibility of low-cost production using minimal medium, carbon dioxide, and light energy as input. In addition, multiple growth and bioproduct extraction cycles were performed by repetitive batch cultivation trials, demonstrating the potential for semi-continuous production and biomass harvesting. Concentrations of recombinant protein reached in this proof of concept approach were sufficient to demonstrate IRI activity in culture media without additional purification or concentration, with activity further verified by thermal hysteresis and morphology assays. The incorporation of the recombinant LpIBP into a model gas hydrate offers the promise that algal production may eventually find application as a "green" hydrate inhibitor.
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http://dx.doi.org/10.1007/s00253-013-5226-xDOI Listing
November 2013

The effect of freeze-thaw conditions on arctic soil bacterial communities.

Biology (Basel) 2013 Feb 28;2(1):356-77. Epub 2013 Feb 28.

Department of Biology, Queen's University, Kingston Ontario, K7L 3N6, Canada.

Climate change is already altering the landscape at high latitudes. Permafrost is thawing, the growing season is starting earlier, and, as a result, certain regions in the Arctic may be subjected to an increased incidence of freeze-thaw events. The potential release of carbon and nutrients from soil microbial cells that have been lysed by freeze-thaw transitions could have significant impacts on the overall carbon balance of arctic ecosystems, and therefore on atmospheric CO2 concentrations. However, the impact of repeated freezing and thawing with the consequent growth and recrystallization of ice on microbial communities is still not well understood. Soil samples from three distinct sites, representing Canadian geographical low arctic, mid-arctic and high arctic soils were collected from Daring Lake, Alexandra Fjord and Cambridge Bay sampling sites, respectively. Laboratory-based experiments subjected the soils to multiple freeze-thaw cycles for 14 days based on field observations (0 °C to -10 °C for 12 h and -10 °C to 0 °C for 12 h) and the impact on the communities was assessed by phospholipid fatty acid (PLFA) methyl ester analysis and 16S ribosomal RNA gene sequencing. Both data sets indicated differences in composition and relative abundance between the three sites, as expected. However, there was also a strong variation within the two high latitude sites in the effects of the freeze-thaw treatment on individual PLFA and 16S-based phylotypes. These site-based heterogeneities suggest that the impact of climate change on soil microbial communities may not be predictable a priori; minor differential susceptibilities to freeze-thaw stress could lead to a "butterfly effect" as described by chaos theory, resulting in subsequent substantive differences in microbial assemblages. This perspectives article suggests that this is an unwelcome finding since it will make future predictions for the impact of on-going climate change on soil microbial communities in arctic regions all but impossible.
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http://dx.doi.org/10.3390/biology2010356DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4009868PMC
February 2013

Assessing the impact of copper and zinc oxide nanoparticles on soil: a field study.

PLoS One 2012 8;7(8):e42663. Epub 2012 Aug 8.

Department of Biology, Dowling College, Oakdale, New York, United States of America.

It is not known if the annual production of tonnes of industrial nanoparticles (NPs) has the potential to impact terrestrial microbial communities, which are so necessary for ecosystem functioning. Here, we have examined the consequences of adding zero valent copper and zinc oxide NPs to soil in pots that were then maintained under field conditions. The fate of these NPs, as well as changes in the microbial communities, was monitored over 162 days. Both NP types traveled through the soil matrix, albeit at differential rates, with Cu NPs retained in the soil matrix at a higher rate compared to ZnO NPs. Leaching of Cu and Zn ions from the parent NPs was also observed as a function of time. Analysis of microbial communities using culture-dependent and independent methods clearly indicated that Cu and ZnO NPs altered the microbial community structure. In particular, two orders of organisms found in rhizosphere, Flavobacteriales and Sphingomonadales, appeared to be particularly susceptible to the presence of NPs. Together, the migration of NPs through soil matrix and the ability of these potential pollutants to influence the composition of microbial community in this field study, cannot help but raise some environmental concerns.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0042663PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3414451PMC
February 2013

Cross-tolerance between osmotic and freeze-thaw stress in microbial assemblages from temperate lakes.

FEMS Microbiol Ecol 2012 Nov 13;82(2):405-15. Epub 2012 Jun 13.

Department of Biology, Queen's University, Kingston, ON, Canada.

Osmotic stress can accompany increases in solute concentrations because of freezing or high-salt environments. Consequently, microorganisms from environments with a high-osmotic potential may exhibit cross-tolerance to freeze stress. To test this hypothesis, enrichments derived from the sediment and water of temperate lakes with a range of salt concentrations were subjected to multiple freeze-thaw cycles. Surviving isolates were identified and metagenomes were sampled prior to and following selection. Enrichments from alkali lakes were typically the most freeze-thaw resistant with only 100-fold losses in cell viability, and those from freshwater lakes were most susceptible, with cell numbers reduced at least 100,000-fold. Metagenomic analysis suggested that selection reduced assemblage diversity more in freshwater samples than in those from saline lakes. Survivors included known psychro-, halo- and alkali-tolerant bacteria. Characterization of freeze-thaw-resistant isolates from brine and alkali lakes showed that few isolates had ice-associating activities such as antifreeze or ice nucleation properties. However, all brine- and alkali-derived isolates had high intracellular levels of osmolytes and/or appeared more likely to form biofilms. Conversely, these phenotypes were infrequent amongst the freshwater-derived isolates. These observations are consistent with microbial cross-tolerance between osmotic and freeze-thaw stresses.
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http://dx.doi.org/10.1111/j.1574-6941.2012.01404.xDOI Listing
November 2012

Prospecting for ice association: characterization of freeze-thaw selected enrichment cultures from latitudinally distant soils.

Can J Microbiol 2012 Apr 21;58(4):402-12. Epub 2012 Mar 21.

Department of Biology, Queen's University, Kingston, ON K7L 3N6, Canada.

Freeze-thaw stress has previously been shown to alter soil community structure and function. We sought to further investigate this stress on enriched microbial consortia with the aim of identifying microbes with ice-associating adaptations that facilitate survival. Enrichments were established to obtain culturable psychrotolerant microbes from soil samples from the latitudinal extremes of the Canadian Shield plateau. The resulting consortia were subjected to consecutive freeze-thaw cycles, and survivors were putatively identified by their 16S rRNA gene sequences. Even though the northerly site was exposed to longer, colder winters and large spring-time temperature fluctuations, the selective regime similarly affected both enriched consortia. Quantitative PCR and metagenomic sequencing were used to determine the frequency of a subset of the resistant microbes in the original enrichments. The metagenomes showed 22 initial genera, only 6 survived and these were not dominant prior to selection. When survivors were assayed for ice recrystallization inhibition and ice nucleation activities, over 60% had at least one of these properties. These phenotypes were not more prevalent in the northern enrichment, indicating that regarding these adaptations, the enrichment strategy yielded seemingly functionally similar consortia from each site.
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http://dx.doi.org/10.1139/w2012-010DOI Listing
April 2012

Antifreeze protein from freeze-tolerant grass has a beta-roll fold with an irregularly structured ice-binding site.

J Mol Biol 2012 Mar 28;416(5):713-24. Epub 2012 Jan 28.

Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada K7L 3N6.

The grass Lolium perenne produces an ice-binding protein (LpIBP) that helps this perennial tolerate freezing by inhibiting the recrystallization of ice. Ice-binding proteins (IBPs) are also produced by freeze-avoiding organisms to halt the growth of ice and are better known as antifreeze proteins (AFPs). To examine the structural basis for the different roles of these two IBP types, we have solved the first crystal structure of a plant IBP. The 118-residue LpIBP folds as a novel left-handed beta-roll with eight 14- or 15-residue coils and is stabilized by a small hydrophobic core and two internal Asn ladders. The ice-binding site (IBS) is formed by a flat beta-sheet on one surface of the beta-roll. We show that LpIBP binds to both the basal and primary-prism planes of ice, which is the hallmark of hyperactive AFPs. However, the antifreeze activity of LpIBP is less than 10% of that measured for those hyperactive AFPs with convergently evolved beta-solenoid structures. Whereas these hyperactive AFPs have two rows of aligned Thr residues on their IBS, the equivalent arrays in LpIBP are populated by a mixture of Thr, Ser and Val with several side-chain conformations. Substitution of Ser or Val for Thr on the IBS of a hyperactive AFP reduced its antifreeze activity. LpIBP may have evolved an IBS that has low antifreeze activity to avoid damage from rapid ice growth that occurs when temperatures exceed the capacity of AFPs to block ice growth while retaining the ability to inhibit ice recrystallization.
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http://dx.doi.org/10.1016/j.jmb.2012.01.032DOI Listing
March 2012