Cold-adapted Organisms Research Articles

Recent Advances in Antifreeze Peptide Preparation: A Review.

Antifreeze agents play a critical role in various fields including tissue engineering, gene therapy, therapeutic protein production, and transplantation. Commonly used antifreeze agents such as DMSO and other organic substances are known to have cytotoxic effects. Antifreeze proteins sourced from cold-adapted organisms offer a promising solution by inhibiting ice crystal formation; however, their effectiveness is hindered by a dynamic ice-shaping (DIS) effect and thermal hysteresis (TH) properties. In response to these limitations, antifreeze peptides (AFPs) have been developed as alternatives to antifreeze proteins, providing similar antifreeze properties without the associated drawbacks. This review explores the methods for acquiring AFPs, with a particular emphasis on chemical synthesis. It aims to offer valuable insights and practical implications to drive the realm of sub-zero storage.

Transposable elements in Drosophila montana from harsh cold environments

BackgroundSubstantial discoveries during the past century have revealed that transposable elements (TEs) can play a crucial role in genome evolution by affecting gene expression and inducing genetic rearrangements, among other molecular and structural effects. Yet, our knowledge on the role of TEs in adaptation to extreme climates is still at its infancy. The availability of long-read sequencing has opened up the possibility to identify and study potential functional effects of TEs with higher precision. In this work, we used Drosophila montana as a model for cold-adapted organisms to study the association between TEs and adaptation to harsh climates.ResultsUsing the PacBio long-read sequencing technique, we de novo identified and manually curated TE sequences in five Drosophila montana genomes from eco-geographically distinct populations. We identified 489 new TE consensus sequences which represented 92% of the total TE consensus in D. montana. Overall, 11–13% of the D. montana genome is occupied by TEs, which as expected are non-randomly distributed across the genome. We identified five potentially active TE families, most of them from the retrotransposon class of TEs. Additionally, we found TEs present in the five analyzed genomes that were located nearby previously identified cold tolerant genes. Some of these TEs contain promoter elements and transcription binding sites. Finally, we detected TEs nearby fixed and polymorphic inversion breakpoints.ConclusionsOur research revealed a significant number of newly identified TE consensus sequences in the genome of D. montana, suggesting that non-model species should be studied to get a comprehensive view of the TE repertoire in Drosophila species and beyond. Genome annotations with the new D. montana library allowed us to identify TEs located nearby cold tolerant genes, and present at high population frequencies, that contain regulatory regions and are thus good candidates to play a role in D. montana cold stress response. Finally, our annotations also allow us to identify for the first time TEs present in the breakpoints of three D. montana inversions.

Rock glacier springs: cool habitats for species on the edge

AbstractUnder climate change, glacier recession and the loss of cold habitats are major threats to aquatic biodiversity. In mountain areas, streams originating from rock glaciers, called “icy seeps”, may represent climate refugia for cold-adapted organisms, given the major persistence of cold waters from these landforms even in unfavourable climates. During late summer 2021, we investigated discharge, turbidity, water chemistry (major ions and trace elements), stable water isotopes (δ18O, δ2H), and macroinvertebrate communities of five rock glacier springs (icy seeps), five glacier springs (glacier springs) and five non-glacial springs (spring brooks) in catchments of the Eastern Italian Alps. In icy seeps, meltwater contribution to runoff (estimated with end-member mixing models) was intermediate between those of the other two spring types. Icy seeps had very cold waters (< 1.5 °C) that were enriched in trace elements, like glacier springs, whereas discharge and turbidity were low, like in spring brooks. Community composition, diversity, and species associations of icy seeps were strongly related to a gradient of chemical harshness (built using trace element concentrations), with less contaminated springs hosting communities like those dwelling in spring brooks. Like glacier springs, those icy seeps with the harshest water chemistry (particularly because of Ni concentrations) and higher meltwater contribution hosted species (e.g., Diamesa steinboecki) that are currently in decline due to glacier loss. This suggests a high conservation value for icy seeps. The protection of these habitats, nowadays overlooked, will be fundamental under the progressive warming and dry-out risk of alpine springs.

Kinetic and dynamical properties of truncated hemoglobins of the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125.

Due to the low temperature, the Antarctic marine environment is challenging for protein functioning. Cold-adapted organisms have evolved proteins endowed with higher flexibility and lower stability in comparison to their thermophilic homologs, resulting in enhanced reaction rates at low temperatures. The Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) genome is one of the few examples of coexistence of multiple hemoglobin genes encoding, among others, two constitutively transcribed 2/2 hemoglobins (2/2Hbs), also named truncated Hbs (TrHbs), belonging to the Group II (or O), annotated as PSHAa0030 and PSHAa2217. In this work, we describe the ligand binding kinetics and their interrelationship with the dynamical properties of globin Ph-2/2HbO-2217 by combining experimental and computational approaches and implementing a new computational method to retrieve information from molecular dynamic trajectories. We show that our approach allows us to identify docking sites within the protein matrix that are potentially able to transiently accommodate ligands and migration pathways connecting them. Consistently with ligand rebinding studies, our modeling suggests that the distal heme pocket is connected to the solvent through a low energy barrier, while inner cavities play only a minor role in modulating rebinding kinetics.

Frost fighters: unveiling the potential of microbial antifreeze proteins in biotech innovation.

Polar environments pose extreme challenges for life due to low temperatures, limited water, high radiation, and frozen landscapes. Despite these harsh conditions, numerous macro and microorganisms have developed adaptive strategies to reduce the detrimental effects of extreme cold. A primary survival tactic involves avoiding or tolerating intra and extracellular freezing. Many organisms achieve this by maintaining a supercooled state by producing small organic compounds like sugars, glycerol, and amino acids, or through increasing solute concentration. Another approach is the synthesis of ice-binding proteins, specifically antifreeze proteins (AFPs), which hinder ice crystal growth below the melting point. This adaptation is crucial for preventing intracellular ice formation, which could be lethal, and ensuring the presence of liquid water around cells. AFPs have independently evolved in different species, exhibiting distinct thermal hysteresis and ice structuring properties. Beyond their ecological role, AFPs have garnered significant attention in biotechnology for potential applications in the food, agriculture, and pharmaceutical industries. This review aims to offer a thorough insight into the activity and impacts of AFPs on water, examining their significance in cold-adapted organisms, and exploring the diversity of microbial AFPs. Using a meta-analysis from cultivation-based and cultivation-independent data, we evaluate the correlation between AFP-producing microorganisms and cold environments. We also explore small and large-scale biotechnological applications of AFPs, providing a perspective for future research.

Exploring the Frozen Armory: Antiphage Defense Systems in Cold-Adapted Bacteria with a Focus on CRISPR-Cas Systems.

Our understanding of the antiphage defense system arsenal in bacteria is rapidly expanding, but little is known about its occurrence in cold-adapted bacteria. In this study, we aim to shed light on the prevalence and distribution of antiphage defense systems in cold-adapted bacteria, with a focus on CRISPR-Cas systems. Using bioinformatics tools, Prokaryotic Antiviral Defense LOCator (PADLOC) and CRISPRCasTyper, we mapped the presence and diversity of antiphage defense systems in 938 available genomes of cold-adapted bacteria from diverse habitats. We confirmed that CRISPR-Cas systems are less frequent in cold-adapted bacteria, compared to mesophilic and thermophilic species. In contrast, several antiphage defense systems, such as dXTPases and DRTs, appear to be more frequently compared to temperate bacteria. Additionally, our study provides Cas endonuclease candidates with a potential for further development into cold-active CRISPR-Cas genome editing tools. These candidates could have broad applications in research on cold-adapted organisms. Our study provides a first-time map of antiphage defense systems in cold-adapted bacteria and a detailed overview of CRISPR-Cas diversity.

Novel Apoplastic Antifreeze Proteins of Deschampsia antarctica as Enhancer of Common Cell Freezing Media for Cryobanking of Genetic Resources, a Preliminary Study.

Antifreeze proteins (AFPs) are natural biomolecules found in cold-adapted organisms that lower the freezing point of water, allowing survival in icy conditions. These proteins have the potential to improve cryopreservation techniques by enhancing the quality of genetic material postthaw. Deschampsia antarctica, a freezing-tolerant plant, possesses AFPs and is a promising candidate for cryopreservation applications. In this study, we investigated the cryoprotective properties of AFPs from D. antarctica extracts on Atlantic salmon spermatozoa. Apoplastic extracts were used to determine ice recrystallization inhibition (IRI), thermal hysteresis (TH) activities and ice crystal morphology. Spermatozoa were cryopreserved using a standard cryoprotectant medium (C+) and three alternative media supplemented with apoplastic extracts. Flow cytometry was employed to measure plasma membrane integrity (PMI) and mitochondrial membrane potential (MMP) postthaw. Results showed that a low concentration of AFPs (0.05 mg/mL) provided significant IRI activity. Apoplastic extracts from D. antarctica demonstrated a cryoprotective effect on salmon spermatozoa, with PMI comparable to the standard medium. Moreover, samples treated with apoplastic extracts exhibited a higher percentage of cells with high MMP. These findings represent the first and preliminary report that suggests that AFPs derived from apoplastic extracts of D. antarctica have the potential to serve as cryoprotectants and could allow the development of novel freezing media.

Functional aggregation of cell-free proteins enables fungal ice nucleation.

Biological ice nucleation plays a key role in the survival of cold-adapted organisms. Several species of bacteria, fungi, and insects produce ice nucleators (INs) that enable ice formation at temperatures above -10 °C. Bacteria and fungi produce particularly potent INs that can promote water crystallization above -5 °C. Bacterial INs consist of extended protein units that aggregate to achieve superior functionality. Despite decades of research, the nature and identity of fungal INs remain elusive. Here, we combine ice nucleation measurements, physicochemical characterization, numerical modeling, and nucleation theory to shed light on the size and nature of the INs from the fungus Fusarium acuminatum. We find ice-binding and ice-shaping activity of Fusarium IN, suggesting a potential connection between ice growth promotion and inhibition. We demonstrate that fungal INs are composed of small 5.3 kDa protein subunits that assemble into ice-nucleating complexes that can contain more than 100 subunits. Fusarium INs retain high ice-nucleation activity even when only the ~12 kDa fraction of size-excluded proteins are initially present, suggesting robust pathways for their functional aggregation in cell-free aqueous environments. We conclude that the use of small proteins to build large assemblies is a common strategy among organisms to create potent biological INs.

Impact of Global Warming on Kryal Fauna: Thermal Tolerance Response of Diamesa steinboecki (Goetghebuer, 1933; Chironomidae)

The ice fly Diamesa steinboecki Goetghebuer, 1933 (Diptera: Chironomidae: Diamesinae) is exclusive to glacier-fed streams in the East Palaearctic region and is threatened by extinction due to global warming and glacier retreat. To date, no data are available on its thermal tolerance or ability to develop a heat shock response (HSR) or involve other biomarkers when exposed to higher-than-natural temperatures (i.e., >4–6 °C). Our study aimed to investigate the warmth resistance of IV-instar larvae of D. steinboecki in terms of (1) ability to survive heat shock and (2) gene expression of four genes known to be involved in the detoxification/stress response (cytochrome p450 (Cyp450), heat shock protein 70 (hsp70), hsp70 with intron and heat shock protein cognate 70 (hsc70)). Larvae were exposed to short-term shocks for 1 h at increasing temperatures (26, 28, 30, 32, 34, 36, 38, and 40 °C) to estimate the lethal temperature, obtaining high values (LT10 = 38.1 °C, LT50 = 39.2 °C, LT99 = 40.3 °C), suggesting a strong heat resistance up to 38 °C and a very rapid decline in survival thereafter. Moreover, gene expression analysis by real-time PCR was performed on larvae from the control (at 2 °C) and larvae found alive after the previous treatment at 26, 28, 30, 32, 34, 36, and 38 °C. Modulation of the expression was observed only for hsc70 and hsp70 genes. Specifically, hsc70 resulted in constitutive overexpression, even at 26 °C when all larvae were found alive without evidence of suffering. By contrast, hsp70 showed up and downregulation according to the specific temperature, suggesting the activation of an HSR at 28 °C, when some larvae were found alive but suffering (almost paralyzed). The results suggest that, based on LTs, D. steinboecki is more thermally tolerant than other Diamesa species (e.g., D. tonsa) from cold freshwaters, but, as in these, hsp70 and hsc70 are involved in surviving short-term heat shock. This makes the ice fly from the Alps different from Belgica antarctica and other cold-adapted organisms living in extremely cold habitats that, constantly exposed to cold, have lost the ability to develop an HSR. Further research is needed to investigate the response to prolonged exposure to temperatures higher that the natural one, giving new insights into the biological response to climate change of alpine species threatened by extinction.

Advances in cold-adapted enzymes derived from microorganisms.

Cold-adapted enzymes, produced in cold-adapted organisms, are a class of enzyme with catalytic activity at low temperatures, high temperature sensitivity, and the ability to adapt to cold stimulation. These enzymes are largely derived from animals, plants, and microorganisms in polar areas, mountains, and the deep sea. With the rapid development of modern biotechnology, cold-adapted enzymes have been implemented in human and other animal food production, the protection and restoration of environments, and fundamental biological research, among other areas. Cold-adapted enzymes derived from microorganisms have attracted much attention because of their short production cycles, high yield, and simple separation and purification, compared with cold-adapted enzymes derived from plants and animals. In this review we discuss various types of cold-adapted enzyme from cold-adapted microorganisms, along with associated applications, catalytic mechanisms, and molecular modification methods, to establish foundation for the theoretical research and application of cold-adapted enzymes.

Improving the genetic system for Halorubrum lacusprofundi to allow in-frame deletions.

Halorubrum lacusprofundi is a cold-adapted halophilic archaeon isolated from Deep Lake, Antarctica. Hrr. lacusprofundi is commonly used to study adaptation to cold environments and thereby a potential source for biotechnological products. Additionally, in contrast to other haloarchaeal model organisms, Hrr. lacusprofundi is also susceptible to a range of different viruses and virus-like elements, making it a great model to study virus-host interactions in a cold-adapted organism. A genetic system has previously been reported for Hrr. lacusprofundi; however, it does not allow in-frame deletions and multiple gene knockouts. Here, we report the successful generation of uracil auxotrophic (pyrE2) mutants of two strains of Hrr. lacusprofundi. Subsequently, we attempted to generate knockout mutants using the auxotrophic marker for selection. However, surprisingly, only the combination of the auxotrophic marker and antibiotic selection allowed the timely and clean in-frame deletion of a target gene. Finally, we show that vectors established for the model organism Haloferax volcanii are deployable for genetic manipulation of Hrr. lacusprofundi, allowing the use of the portfolio of genetic tools available for H. volcanii in Hrr. lacusprofundi.

Submilligram Level of Beetle Antifreeze Proteins Minimize Cold-Induced Cell Swelling and Promote Cell Survival.

Hypothermic (cold) preservation is a limiting factor for successful cell and tissue transplantation where cell swelling (edema) usually develops, impairing cell function. University of Wisconsin (UW) solution, a standard cold preservation solution, contains effective components to suppress hypothermia-induced cell swelling. Antifreeze proteins (AFPs) found in many cold-adapted organisms can prevent cold injury of the organisms. Here, the effects of a beetle AFP from Dendroides canadensis (DAFP-1) on pancreatic β-cells preservation were first investigated. As low as 500 µg/mL, DAFP-1 significantly minimized INS-1 cell swelling and subsequent cell death during 4 °C preservation in UW solution for up to three days. However, such significant cytoprotection was not observed by an AFP from Tenebrio molitor (TmAFP), a structural homologue to DAFP-1 but lacking arginine, at the same levels. The cytoprotective effect of DAFP-1 was further validated with the primary β-cells in the isolated rat pancreatic islets in UW solution. The submilligram level supplement of DAFP-1 to UW solution significantly increased the islet mass recovery after three days of cold preservation followed by rewarming. The protective effects of DAFP-1 in UW solution were discussed at a molecular level. The results indicate the potential of DAFP-1 to enhance cell survival during extended cold preservation.

Toxicity profiles and protective effects of antifreeze proteins from insect in mammalian models

Antifreeze proteins (AFPs), found in many cold-adapted organisms, can protect them from cold and freezing damages and have thus been considered as additional protectants in current cold tissue preservation solutions that generally include electrolytes, osmotic agents, colloids and antioxidants, to reduce the loss of tissue viability associated with cold-preservation. Due to the lack of toxicity profile studies on AFPs, their inclusion in cold preservation solutions has been a trial-and-error process limiting the development of AFPs’ application in cold preservation. To assess the feasibility of translating the technology of AFPs for mammalian cell cold or cryopreservation, we determined the toxicity profile of two highly active beetle AFPs, DAFP1 and TmAFP, from Dendroides canadensis and Tenebrio molitor in this study. Toxicity was examined on a panel of representative mammalian cell lines including testicular spermatogonial stem cells and Leydig cells, macrophages, and hepatocytes. Treatments with DAFP1 and TmAFP at up to 500 μg/mL for 48 and 72 h were safe in three of the cell lines, except for a 20% decrease in spermatogonia treated with TmAFP. However, both AFPs at 500 μg/mL or below reduced hepatocyte viability by 20–40% at 48 and 72 h. At 1000 μg/mL, DAFP1 and TmAFP reduced viability in most cell lines. While spermatogonia and Leydig cell functions were not affected by 1000 μg/mL DAFP1, this treatment induced inflammatory responses in macrophages. Adding 1000 μg/mL DAFP1 to rat kidneys stored at 4 °C for 48 h protected the tissues from cold-related damage, based on tissue morphology and gene and protein expression of two markers of kidney function. However, DAFP1 and TmAFP did not prevent the adverse effects of cold on kidneys over 72 h. Overall, DAFP1 is less toxic at high dose than TmAFP, and has potential for use in tissue preservation at doses up to 500 μg/mL. However, careful consideration must be taken due to the proinflammatory potential of DAFP1 on macrophages at higher doses and the heighten susceptibility of hepatocytes to both AFPs.

Glacial Expansion or Interglacial Expansion? Contrasting Demographic Models of Four Cold-Adapted Fir Species in North America and East Asia

Understanding and forecasting species’ response to climate change is a critical need for future conservation and management. Two expansion hypotheses, the glacial expansion versus the interglacial expansion, have been proposed to interpret how cold-adapted organisms in the northern hemisphere respond to Quaternary climatic fluctuations. To test these two hypotheses, we originally used two pairs of high-low elevation firs from North America (Abies lasiocarpa and Abies balsamea) and East Asia (Abies chensiensis and Abies nephrolepis). Abies lasiocarpa and Abies chensiensis are widely distributed in high-elevation regions of western North America and central China. Abies balsamea and Abies nephrolepis occur in central North America and northeast China, with much lower elevations. These fir species are typical cold-adapted species and sensitive to climate fluctuations. Here, we integrated the mtDNA and cpDNA polymorphisms involving 44 populations and 585 individuals. Based on phylogeographic analyses, recent historical range expansions were indicated in two high-elevation firs (Abies lasiocarpa and Abies chensiensis) during the last glaciation (43.8–28.4 or 21.9–14.2 kya, 53.1–34.5 or 26.6–17.2 kya). Such glacial expansions in high-elevation firs were further confirmed by the evidence of species distribution modelling, geographic-driven genetic patterns, palynological records, and current distribution patterns. Unlike the north American firs, the SDM models indicated unremarkable expansion or contraction in East Asia firs for its much more stable conditions during different historical periods. Taken together, our findings highly supported that high-elevation firs experienced glacial expansion during the Quaternary climate change in East Asia and North America, as interglacial expansion within low-elevation firs. Under this situation, the critically endangered fir species distributed in high elevation would have no enough higher elevational space to migrate. Facing the increasing global warming, thus we proposed ex-situ conservation of defining conservation units as the most meaningful strategy.

Essential roles of buried phenylalanine in the structural stability of thioredoxin from a psychrophilic Arctic bacterium Sphingomonas sp.

Thioredoxin (Trx), a small redox protein, exhibits thermal stability at high temperatures regardless of its origin, including psychrophiles. Trxs have a common structure consisting of the central β-sheet flanked by an aliphatic cluster on one side and an aromatic cluster on the other side. Although the roles of aromatic amino acids in the folding and stability of proteins have been studied extensively, the contributions of aromatic residues to the stability and function of Trx, particularly Trxs from cold-adapted organisms, have not been fully elucidated. This study examined the roles of aromatic amino acids in the aromatic cluster of a Trx from the psychrophilic Arctic bacterium Sphingomonas sp. PAMC 26621 (SpTrx). The aromatic cluster of SpTrx was comprised of W11, F26, F69, and F80, in which F26 at the β2 terminus was buried inside. The substitution of tyrosine for F26 changed the SpTrx conformation substantially compared to that of F69 and F80. Further biochemical and spectroscopic investigations on F26 showed that the F26Y, F26W, and F26A mutants resulted in structural instability of SpTrx in both urea- and temperature-induced unfolding and lower insulin reduction activities. The Trx reductase (SpTR) showed lower catalytic efficiencies against F26 mutants compared to the wild-type SpTrx. These results suggest that buried F26 is essential for maintaining the active-site conformation of SpTrx as an oxidoreductase and its structural stability for interactions with SpTR at colder temperatures.

Use of NanoSIMS to Identify the Lower Limits of Metabolic Activity and Growth by Serratia liquefaciens Exposed to Sub-Zero Temperatures.

Serratia liquefaciens is a cold-adapted facultative anaerobic astrobiology model organism with the ability to grow at a Martian atmospheric pressure of 7 hPa. Currently there is a lack of data on its limits of growth and metabolic activity at sub-zero temperatures found in potential habitable regions on Mars. Growth curves and nano-scale secondary ion mass spectrometry (NanoSIMS) were used to characterize the growth and metabolic threshold for S. liquefaciens ATCC 27,592 grown at and below 0 °C. Cells were incubated in Spizizen medium containing three stable isotopes substituting their unlabeled counterparts; i.e., 13C-glucose, (15NH4)2SO4, and H218O; at 0, −1.5, −3, −5, −10, or −15 °C. The isotopic ratios of 13C/12C, 15N/14N, and 18O/16O and their corresponding fractions were determined for 240 cells. NanoSIMS results revealed that with decreasing temperature the cellular amounts of labeled ions decreased indicating slower metabolic rates for isotope uptake and incorporation. Metabolism was significantly reduced at −1.5 and −3 °C, almost halted at −5 °C, and shut-down completely at or below −10 °C. While growth was observed at 0 °C after 5 days, samples incubated at −1.5 and −3 °C exhibited significantly slower growth rates until growth was detected at 70 days. In contrast, cell densities decreased by at least half an order of magnitude over 70 days in cultures incubated at ≤ −5 °C. Results suggest that S. liquefaciens, if transported to Mars, might be able to metabolize and grow in shallow sub-surface niches at temperatures above −5 °C and might survive—but not grow—at temperatures below −5 °C.

Dynamic cross correlation analysis of Thermus thermophilus alkaline phosphatase and determinants of thermostability

BackgroundUnderstanding the determinants of protein thermostability is very important both from the theoretical and applied perspective. One emerging view in thermostable enzymes seems to indicate that a salt bridge/charged residue network plays a fundamental role in their thermostability. MethodsThe structure of alkaline phosphatase (AP) from Thermus thermophilus HB8 was solved by X-ray crystallography at 2.1 Å resolution. The obtained structure was further analyzed by molecular dynamics studies at different temperatures (303 K, 333 K and 363 K) and compared to homologous proteins from the cold-adapted organisms Shewanella sp. and Vibrio strain G15–21. To analyze differences in measures of dynamic variation, several data reduction techniques like principal component analysis (PCA), residue interaction network (RIN) analysis and rotamer analysis were used. Using hierarchical clustering, the obtained results were combined to determine residues showing high degree dynamical variations due to temperature jumps. Furthermore, dynamic cross correlation (DCC) analysis was carried out to characterize networks of charged residues. ResultsTop clustered residues showed a higher propensity for thermostabilizing mutations, indicating evolutionary pressure acting on thermophilic organisms. The description of rotamer distributions by Gini coefficients and Kullback–Leibler (KL) divergence both revealed significant correlations with temperature. DCC analysis revealed a significant trend to de-correlation of the movement of charged residues at higher temperatures. SignificanceThe de-correlation of charged residues detected in Thermus thermophilus AP, highlights the importance of dynamic electrostatic network interactions for the thermostability of this enzyme.

The high catalytic rate of the cold-active Vibrio alkaline phosphatase requires a hydrogen bonding network involving a large interface loop.

The role of surface loops in mediating communication through residue networks is still a relatively poorly understood part in the study of cold adaptation of enzymes, especially in terms of their quaternary interactions. Alkaline phosphatase (AP) from the psychrophilic marine bacterium Vibriosplendidus (VAP) is characterized by an analogous large surface loop in each monomer, referred to as the large loop, that hovers over the active site of the other monomer. It presumably has a role in the high catalytic efficiency of VAP which accompanies its extremely low thermal stability. Here, we designed several different variants of VAP with the aim of removing intersubunit interactions at the dimer interface. Breaking the intersubunit contacts from one residue in particular (Arg336) reduced the temperature stability of the catalytically potent conformation and caused a 40% drop in catalytic rate. The high catalytic rates of enzymes from cold-adapted organisms are often associated with increased dynamic flexibility. Comparison of the relative B-factors of the R336L crystal structure to that of the wild-type confirmed surface flexibility was increased in a loop on the opposite monomer, but not in the large loop. The increase in flexibility resulted in a reduced catalytic rate. The large loop increases the area of the interface between the subunits through its contacts and may facilitate an alternating structural cycle demanded by a half-of-sites reaction mechanism through stronger ties, as the dimer oscillates between high affinity (active) or low phosphoryl group affinity (inactive).

The role of hydro-environmental factors in Mayfly (Ephemeroptera, Insecta) community structure: Identifying threshold responses.

Freshwater organisms are threatened by changes in stream flow and water temperature regimes due to global climate change and anthropogenic activities. Threats include the disappearance of narrow‐tolerance species and loss of favorable thermal conditions for cold‐adapted organisms. Mayflies are an abundant and diverse indicator of river health that performs important functional roles. The relative importance of key hydro‐environmental factors such as water temperature and flow volumes in structuring these communities has rarely been explored in the tropical regions of Africa. Here, we investigate the response of mayfly species diversity to these factors in the Luvuvhu catchment, a strategic water source area in the arid northeastern region of South Africa. Mayfly larvae were sampled monthly in stones‐in‐current biotopes across 23 sites over a one‐year period. The relationship between these environmental drivers and mayfly diversity was modeled using linear mixed effects models (LMMs) and a model‐based multivariate approach. Threshold Indicator Taxa Analysis (TITAN) was used to model the response of mayfly species to important gradients and identify thresholds of change. Site‐specific characteristic were the most important predictor of mayfly diversity, and there was considerable variation over time, with mayfly diversity peaking during winter. Along this, gradient temperature was the best predictor of assemblage structure, with five out of six reliable indicator species being cold‐adapted, and a community threshold response at 19°C. Results support laboratory‐based thresholds of temperature for mayfly species survival and development, extending empirical evidence to include field‐based observations. Increased global (climate change) and local (riparian vegetation removal, impoundments) changes are predicted to have negative impacts on mayfly diversity and ultimately on ecosystem function.

Inhibition of Bacterial Ice Nucleators Is Not an Intrinsic Property of Antifreeze Proteins.

Cold-adapted organisms use antifreeze proteins (AFPs) or ice-nucleating proteins (INPs) for the survival in freezing habitats. AFPs have been reported to be able to inhibit the activity of INPs, a property that would be of great physiological relevance. The generality of this effect is not understood, and for the few known examples of INP inhibition by AFPs, the molecular mechanisms remain unclear. Here, we report a comprehensive evaluation of the effects of five different AFPs on the activity of bacterial ice nucleators using a high-throughput ice nucleation assay. We find that bacterial INPs are inhibited by certain AFPs, while others show no effect. Thus, the ability to inhibit the activity of INPs is not an intrinsic property of AFPs, and the interactions of INPs and different AFPs proceed through protein-specific rather than universal molecular mechanisms.