Ice Nucleation Protein Research Articles

Bioinspired Anti-Icing Hydrogel Enabled by Ice-Nucleating Protein

Ice-nucleating proteins (INPs) are the most effective ice-nucleating agents that play a significant role in preventing freeze injuries in freeze-tolerant organisms. INPs promote ice nucleation in t...

Ice-nucleating proteins are activated by low temperatures to control the structure of interfacial water

Ice-nucleation active (INA) bacteria can promote the growth of ice more effectively than any other known material. Using specialized ice-nucleating proteins (INPs), they obtain nutrients from plants by inducing frost damage and, when airborne in the atmosphere, they drive ice nucleation within clouds, which may affect global precipitation patterns. Despite their evident environmental importance, the molecular mechanisms behind INP-induced freezing have remained largely elusive. We investigate the structural basis for the interactions between water and the ice-nucleating protein InaZ from the INA bacterium Pseudomonas syringae. Using vibrational sum-frequency generation (SFG) and two-dimensional infrared spectroscopy, we demonstrate that the ice-active repeats of InaZ adopt a β-helical structure in solution and at water surfaces. In this configuration, interaction between INPs and water molecules imposes structural ordering on the adjacent water network. The observed order of water increases as the interface is cooled to temperatures close to the melting point of water. Experimental SFG data combined with molecular-dynamics simulations and spectral calculations show that InaZ reorients at lower temperatures. This reorientation can enhance water interactions, and thereby the effectiveness of ice nucleation.

Decorating the surface of Escherichia coli with bacterial lipoproteins: a comparative analysis of different display systems

BackgroundThe display of recombinant proteins on cell surfaces has a plethora of applications including vaccine development, screening of peptide libraries, whole-cell biocatalysts and biosensor development for diagnostic, industrial or environmental purposes. In the last decades, a wide variety of surface display systems have been developed for the exposure of recombinant proteins on the surface of Escherichia coli, such as autotransporters and outer membrane proteins.ResultsIn this study, we assess three approaches for the surface display of a panel of heterologous and homologous mature lipoproteins in E. coli: four from Neisseria meningitidis and four from the host strain that are known to be localised in the inner leaflet of the outer membrane. Constructs were made carrying the sequences coding for eight mature lipoproteins, each fused to the delivery portion of three different systems: the autotransporter adhesin involved in diffuse adherence-I (AIDA-I) from enteropathogenic E. coli, the Lpp’OmpA chimaera and a truncated form of the ice nucleation protein (INP), InaK-NC (N-terminal domain fused with C-terminal one) from Pseudomonas syringae. In contrast to what was observed for the INP constructs, when fused to the AIDA-I or Lpp’OmpA, most of the mature lipoproteins were displayed on the bacterial surface both at 37 and 25 °C as demonstrated by FACS analysis, confocal and transmission electron microscopy.ConclusionsTo our knowledge this is the first study that compares surface display systems using a number of passenger proteins. We have shown that the experimental conditions, including the choice of the carrier protein and the growth temperature, play an important role in the translocation of mature lipoproteins onto the bacterial surface. Despite all the optimization steps performed with the InaK-NC anchor motif, surface exposure of the passenger proteins used in this study was not achieved. For our experimental conditions, Lpp’OmpA chimaera has proved to be an efficient surface display system for the homologous passenger proteins although cell lysis and phenotype heterogeneity were observed. Finally, AIDA-I was found to be the best surface display system for mature lipoproteins (especially heterologous ones) in the E. coli host strain with no inhibition of growth and only limited phenotype heterogeneity.

Gut Escherichia coli expressing Pb2+-adsorption protein reduces lead accumulation in grass carp, Ctenopharyngodon idellus.

Lead (Pb2+) is easy to accumulate in fish which become a major source of Pb2+ exposure to humans. In this study, a recombinant Escherichia coli strain expressing Pb2+-specific surface-binding protein anchored by the ice nucleation protein was introduced into grass carp (Ctenopharyngodon idellus) gut to investigate its protective effect against dietary Pb2+ exposure. Pb2+ mostly precipitated on the surface of the engineered strain through Pb2+-specific surface-binding protein, with a maximum adsorption efficiency of 73% and an adsorption capacity of 163μmol/g dry cells. The Pb2+ concentration in engineered bacteria-fed grass carp was reduced significantly, and the residual level of Pb2+ in feces was increased by 76%, compared with the control group. Meantime, the engineered bacteria were able to mitigate the oxidative stress and histological alterations of intestines and dysbiosis of gut microbiota induced by Pb2+exposure. Thus, the engineered bacterium that can effectively reduce Pb2+ residue in grass carp might be a useful tool for decontamintion of lead in aquatic organisms.

Cloning and surface expression of engineered enzyme of organophosphorus hydrolase in E. coli and evaluation of its function

Nowadays, the chemical compounds of organophosphate cause biological contamination due to their presence in the environment and production of metabolites, which endanger the health of humans and organisms. Thus, it is crucial to use a safe method to eliminate the contamination of these compounds. The objective of the present study was cloning and surface expression of the engineered enzyme of organophosphorus hydrolase in E. coli and comparing its function with the non-engineered enzyme. The present study aimed at improving the function of the organophosphorus hydrolase enzyme by expressing this enzyme along with Ice nucleation protein (INP) on the surface of bacterial cells. For this purpose, the OPH enzyme coding sequence was cloned in the pET28a-InaV-N plasmid and sent into E. coli, the strain of BL21 (DE3) and protein expression was evaluated at 37° C after 16 hours of induction. To examine the site of expression of the recombinant protein, cell components (cytoplasm, inner membrane, and outer membrane) were separated and the results were analyzed by using SDS-PAGE. The results revealed that OPH enzyme protein was presented as a protein fusion with a molecular weight of 58 KDa next to the INP anchor on the surface of the outer membrane of the cell. The specific activity of the resulting protein was calculated at 3828.8 U/mg after incubation of this enzyme in the vicinity of the substrate at 37° C. based on the results of the present study, it seems that the expression of engineered enzyme and its presentation on the cell surface is an achievement in using the cell as a degrading agent of organophosphate toxins and eliminating the costs of downstream processes such as purification and refolding of protein.

Ab Initio Determination of Peptide Structures by MicroED.

Structural elucidation of small macromolecules such as peptides has recently been facilitated by a growing number of technological advances to existing crystallographic methods. The emergence of electron micro-diffraction (MicroED) of protein nanocrystals under cryogenic conditions has enabled the interrogation of crystalline peptide assemblies only hundreds of nanometers thick. Collection of atomic or near-atomic resolution data by these methods has permitted the ab initio determination of structures of various amyloid-forming peptides, including segments derived from prions and ice-nucleating proteins. This chapter focuses on the process of ab initio structural determination from nano-scale peptide assemblies and other similar molecules.

Interfacial Water Ordering Is Insufficient to Explain Ice-Nucleating Protein Activity.

Ice-nucleating proteins (INPs) found in bacteria are the most effective ice nucleators known, enabling the crystallization of water at temperatures close to 0 °C. Although their function has been known for decades, the underlying mechanism is still under debate. Here, we show that INPs from Pseudomonas syringae in aqueous solution exhibit a defined solution structure and show no significant conformational changes upon cooling. In contrast, irreversible structural changes are observed upon heating to temperatures exceeding ∼55 °C, leading to a loss of the ice-nucleation activity. Sum-frequency generation (SFG) spectroscopy reveals that active and heat-inactivated INPs impose similar structural ordering of interfacial water molecules upon cooling. Our results demonstrate that increased water ordering is not sufficient to explain INPs' high ice-nucleation activity and confirm that intact three-dimensional protein structures are critical for bacterial ice nucleation, supporting a mechanism that depends on the INPs' supramolecular interactions.

Computationally efficient approach for the identification of ice-binding surfaces and how they bind ice.

Recognition and binding of ice by proteins, crystals, and other surfaces is key for their control of the nucleation and growth of ice. Docking is the state-of-the-art computational method to identify ice-binding surfaces (IBS). However, docking methods require a priori knowledge of the ice plane to which the molecules bind and either neglect the competition of ice and water for the IBS or are computationally expensive. Here we present and validate a robust methodology for the identification of the IBS of molecules and crystals that is easy to implement and a hundred times computationally more efficient than the most advanced ice-docking approaches. The methodology is based on biased sampling with an order parameter that drives the formation of ice. We validate the method using all-atom and coarse-grained models of organic crystals and proteins. To our knowledge, this approach is the first to simultaneously identify the ice-binding surface as well as the plane of ice to which it binds, without the use of structure search algorithms. We show that biased simulations even identify surfaces that are too small or too weak to heterogeneously nucleate ice. The biasing simulations can be used to identify of IBS of antifreeze and ice nucleating proteins and to equilibrate ice seeds bound to an IBS for the calculation of heterogeneous ice nucleation rates using classical nucleation theory.

Display of lead-binding proteins on Escherichia coli surface for lead bioremediation.

Cell surface display of heavy metal-binding proteins has been used to enhance the adsorption capacity of heavy metals and the engineered microbial cells can be potentially used for the bioremediation of heavy metals. In this study, the proteins PbrR, PbrR691, and PbrD from the Cupriavidus metallidurans strain CH34 were displayed on the extracellular membrane of Escherichia coli BL21 cells, with the N-domain of ice-nucleation protein as the anchor protein to achieve specific adsorption of lead ions (Pb2+ ) and bioremediation of lead in the soil. The localization of fusion proteins was confirmed by western blot analysis. We investigated the effects of fusion pattern, expression level, heavy metal concentration, and the presence of other heavy metal ions on the adsorption of Pb2+ by these engineered bacteria, and the optimal linker peptide (flexible linker) and inducer concentration (0.5 mM) were obtained. The engineered bacteria showed specific selectivity and strong adsorption capacity for Pb2+ . The maximum Pb2+ adsorption capacity of strains displaying the three proteins (PbrR, PbrR691, and PbrD) were 942.1-, 754.3-, and 864.8-μmol/g cell dry weight, respectively, which was the highest reported to date. The engineered E. coli bacteria were also applied to Pb2+ -contaminated soil and the detoxification effects were observed via the seed germination test and the growth of Nicotiana benthamiana in comparison with the control BL21, which provides the proof-of-concept for in situ remediations of Pb2+ -contaminated water or soil.

Development of a whole-cell biocatalyst for diisobutyl phthalate degradation by functional display of a carboxylesterase on the surface of Escherichia coli

BackgroundPhthalic acid esters (PAEs) are widely used as plasticizers or additives during the industrial manufacturing of plastic products. PAEs have been detected in both aquatic and terrestrial environments due to their overuse. Exposure of PAEs results in human health concerns and environmental pollution. Diisobutyl phthalate is one of the main plasticizers in PAEs. Cell surface display of recombinant proteins has become a powerful tool for biotechnology applications. In this current study, a carboxylesterase was displayed on the surface of Escherichia coli cells, for use as whole-cell biocatalyst in diisobutyl phthalate biodegradation.ResultsA carboxylesterase-encoding gene (carEW) identified from Bacillus sp. K91, was fused to the N-terminal of ice nucleation protein (inpn) anchor from Pseudomonas syringae and gfp gene, and the fused protein was then cloned into pET-28a(+) vector and was expressed in Escherichia coli BL21(DE3) cells. The surface localization of INPN-CarEW/or INPN-CarEW-GFP fusion protein was confirmed by SDS-PAGE, western blot, proteinase accessibility assay, and green fluorescence measurement. The catalytic activity of the constructed E. coli surface-displayed cells was determined. The cell-surface-displayed CarEW displayed optimal temperature of 45 °C and optimal pH of 9.0, using p-NPC2 as substrate. In addition, the whole cell biocatalyst retained ~ 100% and ~ 200% of its original activity per OD600 over a period of 23 days at 45 °C and one month at 4 °C, exhibiting the better stability than free CarEW. Furthermore, approximately 1.5 mg/ml of DiBP was degraded by 10 U of surface-displayed CarEW cells in 120 min.ConclusionsThis work provides a promising strategy of cost-efficient biodegradation of diisobutyl phthalate for environmental bioremediation by displaying CarEW on the surface of E. coli cells. This approach might also provide a reference in treatment of other different kinds of environmental pollutants by displaying the enzyme of interest on the cell surface of a harmless microorganism.

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.

Freezing from the inside: Ice nucleation in Escherichia coli and Escherichia coli ghosts by inner membrane bound ice nucleation protein InaZ.

Ice nucleation (IN) active bacteria such as Pseudomonas syringae promote the growth of ice crystals more effectively than any material known. Using the specialized ice nucleation protein (INP) InaZ, P. syringae-the well studied epiphytic plant pathogen-attacks plants by frost damage and, likewise fascinating, drives ice nucleation within clouds when airborne in the atmosphere by linkage to the Earth's water cycle. While ice nucleation proteins play a tremendous role for life on the planet, the molecular details of their activity on the bacterial membrane surface are largely unknown. Bacterial ghosts (BGs) derived from Escherichia coli can be used as simplified model systems to study the mode of action of InaZ. In this work, the authors used BGs to study the role of InaZ localization on the luminal side of the bacterial inner membrane. Naturally, P. syringae INPs are displayed on the surface of the outer membrane; so in contrast, the authors engineered an N-terminal truncated form of inaZ lacking the transport sequence for anchoring of InaZ on the outer membrane. This construct was fused to N- and C-terminal inner membrane anchors and expressed in Escherichia coli C41. The IN activity of the corresponding living recombinant E. coli catalyzing interfacial ice formation of supercooled water at high subzero temperatures was tested by a droplet-freezing assay and surface spectroscopy. The median freezing temperature (T50) of the parental living E. coli C41 cells without INP was detected at -20.1 °C and with inner membrane anchored INPs at a T50 value between -7 and -9 °C, demonstrating that the induction of IN from the inside of the bacterium by inner membrane anchored INPs facing the luminal inner membrane side is very similar to IN induced by bacterial INPs located at the outer membrane. Bacterial ghosts derived from these different constructs showed first droplet freezing values between -6 and -8 °C, whereas E. coli C41 BGs alone without carrying inner membrane anchored INPs exhibit a T50 of -18.9 °C. Sum frequency generation spectroscopy showed structural ordered water at the BG/water interface, which increased close to the water melting point. Together, this indicates that the more efficient IN of INP-BGs compared to their living parental strains can be explained by the free access of inner membrane anchored INP constructs to ultrapure water filling the inner space of the BGs.

Electrostatic Interactions Control the Functionality of Bacterial Ice Nucleators.

Bacterial ice-nucleating proteins (INPs) promote heterogeneous ice nucleation more efficiently than any other material. The details of their working mechanism remain elusive, but their high activity has been shown to involve the formation of functional INP aggregates. Here we reveal the importance of electrostatic interactions for the activity of INPs from the bacterium Pseudomonas syringae by combining a high-throughput ice nucleation assay with surface-specific sum-frequency generation spectroscopy. We determined the charge state of nonviable P. syringae as a function of pH by monitoring the degree of alignment of the interfacial water molecules and the corresponding ice nucleation activity. The net charge correlates with the ice nucleation activity of the INP aggregates, which is minimal at the isoelectric point. In contrast, the activity of INP monomers is less affected by pH changes. We conclude that electrostatic interactions play an essential role in the formation of the highly efficient functionally aligned INP aggregates, providing a mechanism for promoting aggregation under conditions of stress that prompt the bacteria to nucleate ice.

Cell surface-expression of the phosphate-binding protein PstS: System development, characterization, and evaluation for phosphorus removal and recovery

Simultaneous overabundance and scarcity of inorganic phosphate (Pi) is a critical issue driving the development of innovative water/wastewater treatment technologies that not only facilitate Pi removal to prevent eutrophication, but also recover Pi for agricultural reuse. Here, a cell-surface expressed high-affinity phosphate binding protein (PstS) system was developed, and its Pi capture and release potential was evaluated. E. coli was genetically modified to express PstS on its outer membrane using the ice nucleation protein (INP) as an anchoring motif. Verification of protein expression and localization were performed utilizing SDS-polyacrylamide gel electrophoresis (SDS-PAGE), western blot, and outer membrane separation analyses. Cell surface characterization was investigated through acid-base titration, X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared spectroscopy (FTIR). These tests provided information on the macromolecular structure and composition of the bacteria surface as well as the proton-exchange properties of the surface functional groups (i.e., pKa values). Phosphate desorption and adsorption batch experiments were conducted to evaluate the effects of temperature, pH, and ionic strength on phosphate capture and release. The PstS surface-displayed cells demonstrated greater potential to release and capture phosphate compared to non-modified cells. Higher temperatures up to 40°C, basic pH conditions (pH = 10.5), and higher ionic strength up to 1.0 mol/L KCl promoted 20%–50% higher phosphate release.

Ice Binding Proteins: Diverse Biological Roles and Applications in Different Types of Industry.

More than 80% of Earth’s surface is exposed periodically or continuously to temperatures below 5 °C. Organisms that can live in these areas are called psychrophilic or psychrotolerant. They have evolved many adaptations that allow them to survive low temperatures. One of the most interesting modifications is production of specific substances that prevent living organisms from freezing. Psychrophiles can synthesize special peptides and proteins that modulate the growth of ice crystals and are generally called ice binding proteins (IBPs). Among them, antifreeze proteins (AFPs) inhibit the formation of large ice grains inside the cells that may damage cellular organelles or cause cell death. AFPs, with their unique properties of thermal hysteresis (TH) and ice recrystallization inhibition (IRI), have become one of the promising tools in industrial applications like cryobiology, food storage, and others. Attention of the industry was also caught by another group of IBPs exhibiting a different activity—ice-nucleating proteins (INPs). This review summarizes the current state of art and possible utilizations of the large group of IBPs.

Antifreeze proteins: effective adaptations of organisms for low temperature survival

Adaptation to low temperature presents many challenges. On a cellular level, structural integrity and metabolism are profoundly affected in cold temperatures, and at freezing temperatures, ice crystal formation must be prevented or controlled for an organism to survive. Antifreeze proteins (AFP) and glycoproteins (AFGP) are protective molecules expressed by organisms to effectively cope with these challenges. These proteins are extremely effective at protecting tissues from chilling and freezing damage, and are active at low concentrations compared to other cryoprotectants, such as glycerol. Many organisms can prevent ice crystal formation in their tissues by supercooling, where the freezing point of cellular fluid is decreased below the normal freezing point of water. Those that can tolerate freezing use ice nucleating proteins (INP) and AFPs to restrict the growth and coalescence of ice crystals. Studies on the evolution of AFPs and AFGPs in polar species reveal that the genes arose from a variety of mechanisms - including lateral gene transfer, gene duplication and convergent evolution - in response to climate change. AFPs directly bind to ice crystal faces, slowing or preventing crystal growth and coalescence. They also act to reduce membrane permeability by interacting with bilayer lipids, thereby preventing leakage during chilling. Practical applications of these molecules include tissue preservation, cold tolerance in crops and food animals, frozen food storage, and freeze-proof surface coatings.

Enzymatic Synthesis of Nucleoside Triphosphates and Deoxynucleoside Triphosphates by Surface-Displayed Kinases.

Nucleoside triphosphates and deoxynucleoside triphosphates are important biochemical molecules. In this study, recombinant Escherichia coli that could display nucleotide kinases (INP-N-NMKases) and acetate kinase (INP-N-ACKase) on the cell surface were constructed by fusing an enzyme (NMKase/ACKase) to the N-terminus of ice nucleation protein (INP-N). By using intact recombinant bacteria cells as a catalyst coupled with an ACKase-catalyzed adenosine-5'-triphosphate (ATP) regeneration system, nucleoside triphosphates (NTPs) and deoxynucleoside triphosphates (dNTPs) could be synthesized efficiently. In a reaction system with 5mmol/l substrate, the conversion rates of cytidine-5'-triphosphate (CTP) and deoxycytidine-5'-triphosphate (dCTP) were 96% and 93%, respectively, the conversion rate of ATP and deoxyadenosine-5'-triphosphate (dATP) was 96%, the conversion rate of deoxythymidine-5'-triphosphate (dTTP) was 91%, and the conversion rate of uridine-5'-triphosphate (UTP) was 80%. There was no obvious degradation. At 37°C, the stability of the surface-displayed fusion protein, especially in the presence of the substrate, was significantly improved. Each whole cell could be reused more than 8 times.

Complete Genome Sequence of Pseudomonas sp. Strain KUIN-1, a Model Strain for Studies on the Production of Cell-Free Ice Nucleation Proteins

Pseudomonas sp. (formerly Pseudomonas fluorescens) strain KUIN-1 is an ice-nucleating bacterium that was isolated from the leaves of field beans (Phaseolus vulgaris L.). This microorganism can release cell-free ice nucleation proteins and shows cold shock-induced freezing tolerance. Here, we report the 6,028,589-bp complete genome sequence of Pseudomonas sp. KUIN-1.

A comparative evaluation of freezing criteria and molecular characterization of epiphytic ice-nucleating (Ice+) and non-ice-nucleating (Ice−) Pseudomonas syringae and Pseudomonas fluorescens

The ice-nucleation-active bacteria that produce ice-nucleating proteins (INPs), especially Pseudomonas species, may play a critical potential role in frost injury. The ice-minus variant of bacteria is a mutant, lacking the genes responsible for the production of INPs. The aim of the project was the isolation of relevant bacteria. Pseudomonas-like bacteria were screened from 66 leaves samples and were identified by biochemical as well as molecular methods with rpoD, 16S rDNA-flu and hrpl primers. The ice nucleation activity (INA) of isolates was performed using the drop-freezing method. The existence of the ina gene in isolates was investigated by specific inaZ and inaW primers. The effect of various temperatures, salt concentration and pH on growth and INA were investigated. Finally, the pathogenicity and freezing of INA+ and INA− bacteria on tomatoes and oranges were estimated. In this study, 57 isolates were identified as Pseudomonas including five P. syringae strains and seven P. fluorescens strains. The freezing profiles revealed that P. fluorescens strain 22A as INA− bacteria and three strains of P. syringae 18A, 52A and 52B are also recommended as strong INA+ strains. Most of the P. syringae isolates were active at −2 to −8 °C (type I and II), whereas P. fluorescens strains had an efficient activity at −8 to −12 °C (type III). Unlike the selected P. syringae bacteria, P. fluorescens strains did not show signs of frost injury on tomatoes and oranges.

Ice Nucleation Properties of Ice-binding Proteins from Snow Fleas.

Ice-binding proteins (IBPs) are found in many organisms, such as fish and hexapods, plants, and bacteria that need to cope with low temperatures. Ice nucleation and thermal hysteresis are two attributes of IBPs. While ice nucleation is promoted by large proteins, known as ice nucleating proteins, the smaller IBPs, referred to as antifreeze proteins (AFPs), inhibit the growth of ice crystals by up to several degrees below the melting point, resulting in a thermal hysteresis (TH) gap between melting and ice growth. Recently, we showed that the nucleation capacity of two types of IBPs corresponds to their size, in agreement with classical nucleation theory. Here, we expand this finding to additional IBPs that we isolated from snow fleas (the arthropod Collembola), collected in northern Israel. Chemical analyses using circular dichroism and Fourier-transform infrared spectroscopy data suggest that these IBPs have a similar structure to a previously reported snow flea antifreeze protein. Further experiments reveal that the ice-shell purified proteins have hyperactive antifreeze properties, as determined by nanoliter osmometry, and also exhibit low ice-nucleation activity in accordance with their size.