Cold requirement for maximal activity of the bacterial ice nucleation protein INAZ in transgenic plants

The bacterial ice nucleation gene inaZ confers production of ice nuclei when transferred into transgenic plants. Conditioning of the transformed plant tissue at temperatures near 0°C greatly increased the ice nucleation activity in plants, and maximum ice nucleation activity was achieved only after low-temperature conditioning for about 48 h. Although the transgenic plants contain similar amounts of inaZ mRNA at both normal and low temperatures, low temperatures are required for accumulation of INAZ protein. We propose that the stability of the INAZ protein and thus ice nucleation activity in the transgenic plants is enhanced by low-temperature conditioning.     

Deletion mutagenesis of the ice nucleation gene from Pseudomonas syringae S203

Summary The ice nucleation gene inaZ, from Pseudomonas syringae S203, was manipulated to produce a series of defined rearrangements in its coding sequence without changing the reading frame. The effects of these mutations on the ice nucleation phenotype were determined in a heterologous host, Escherichia coli K12. Deletions which disrupted the periodicity of 16 codons, in a repetitive region of inaZ, caused the frequencies of ice nuclei in the bacterial population to be significantly depressed; the nuclei with thresholds at warmer temperatures were most affected. In contrast, when the periodicity was left intact, deletions and duplications in the same region had only slight effects on nucleation activity. Deletions removing part or all of one of the nonrepetitive regions (that encoding the amino-terminal domain of the InaZ protein) did not abolish nucleation activity, but caused it to be limited to cooler threshold temperatures. In contrast, the non-repetitive carboxy-terminal domain of the InaZ protein was shown to be essential for ice nucleation at all temperatures. The differential requirements (for periodicity, and for the amino-terminus) in forming nuclei with different thresholds may be significant for understanding what determines the threshold temperature of an ice nucleus.     

Conserved repetition in the ice nucleation gene inaX from Xanthomonas campestris pv. translucens

Summary The nucleotide sequence was determined for the bacterial ice nucleation gene, inaX, from Xanthomonas campestris pathovar translucens X56S. Comparison of the nucleotide sequence of inaX to the previously characterized ice nucleation genes, inaZ from Pseudomonas syringae S203, inaW from Pseudomonas fluorescens MS1650, and iceE from Erwinia herbicola M1 revealed a 65.8%, 67.8%, and 68.8% homology, respectively. Within the internal, repetitive domain of the translated product of inaX are 153 consecutive octapeptide repeat units.     

Clustering of ice nucleation protein correlates with ice nucleation activity

Antibodies raised against a synthetic peptide specifically detect ice nucleation proteins from Pseudomonas species in Western blots. In immunofluorescent staining of whole bacteria, the antibodies reveal the protein in clusters, as indicated by patches of intense fluorescence in Escherichia coli cells heterologously expressing Pseudomonas ice nucleation genes. The abundance, size, and brightness of the clusters vary considerably from cell to cell. Their varying sizes may explain the variability in activity of bacterial ice nuclei. Growth at lower temperatures produces more ice nuclei, and gives brighter and more frequent patches, than growth at 37 degrees C. The observed clustering may thus reflect formation of functional ice nucleation sites in vivo. The presence of ice nucleation protein in clusters is also correlated with alterations in cell morphology.     

Bacterial ice nucleation: a factor in frost injury to plants

Heterogeneous ice nuclei are necessary, and the common epiphytic ice nucleation active (INA) bacteria Pseudomonas syringae van Hall and Erwinia herbicola (Löhnis) Dye are sufficient to incite frost injury to sensitive plants at −5°C. The ice nucleation activity of the bacteria occurs at the same temperatures at which frost injury to sensitive plants occurs in nature. Bacterial ice nucleation on leaves can be detected at about −2°C, whereas the leaves themselves, i.e. without INA bacteria, contain nuclei active only at much lower temperatures. The temperature at which injury to plants occurs is predictable on the basis of the ice nucleation activity of leaf discs, which in turn depends on the number and ice nucleation activity of their resident bacteria. Bacterial isolates which are able to incite injury to corn at −5°C are always active as ice nuclei at −5°C. INA bacteria incited frost injury to all of the species of sensitive plants tested.     

Inhibition of bacterial ice nucleation by polyglycerol polymers

The simple linear polymer polyglycerol (PGL) was found to apparently bind and inhibit the ice nucleating activity of proteins from the ice nucleating bacterium Pseudomonas syringae. PGL of molecular mass 750 Da was added to a solution consisting of 1 ppm freeze-dried P. syringae 31A in water. Differential ice nucleator spectra were determined by measuring the distribution of freezing temperatures in a population of 98 drops of 1 microL volume. The mean freezing temperature was lowered from -6.8 degrees C (control) to -8.0,-9.4,-12.5, and -13.4 degrees C for 0.001, 0.01, 0.1, and 1% w/w PGL concentrations, respectively (SE < 0.2 degrees C). PGL was found to be an ineffective inhibitor of seven defined organic ice nucleating agents, whereas the general ice nucleation inhibitor polyvinyl alcohol (PVA) was found to be effective against five of the seven. The activity of PGL therefore seems to be specific against bacterial ice nucleating protein. PGL alone was an ineffective inhibitor of ice nucleation in small volumes of environmental or laboratory water samples, suggesting that the numerical majority of ice nucleating contaminants in nature may be of nonbacterial origin. However, PGL was more effective than PVA at suppressing initial ice nucleation events in large volumes, suggesting a ubiquitous sparse background of bacterial ice nucleating proteins with high nucleation efficiency. The combination of PGL and PVA was particularly effective for reducing ice formation in solutions used for cryopreservation by vitrification.     

Supercooling and nucleation of ice in single cells

An emulsion droplet formation procedure was employed to isolate yeast cells and, in separate experiments, human red blood cells, one from another in individual droplets, and to segregate extraneous materials catalyzing the formation of ice. Emulsification succeeded in isolating the cells and permitted the observation of the supercooling of droplets containing cells whereby each droplet was observed to nucleate ice at a temperature that depended only upon the components of the droplet. The droplet formation procedures were characterized. It was shown that the surface coatings and the carrier fluids used in the preparation of the emulsions did not act as ice nucleators. It was, in this manner, possible to study the nucleation of ice brought about by supercooling and homogeneous nucleation in the volume of the droplet or by the catalysis of nucleation on or in the cells contained in the droplets. It was shown that yeast cells and red blood cells could each be supercooled to about ?40 °C in short-term experiments. The results also revealed that yeast cells did not store for infinite times at temperatures above the observed upper limit of homogeneous nucleation. The yeast cells died at rates that were exponential functions of time at ?20, ?22.5, ?25, ?29 and ?33 °C. The temperature dependence of the death rate did not correspond to a process with a normal Arrhenius activation energy. The temperature dependence did, however, suggest a potentiated heterogeneous catalysis of ice resulting in the death of the yeast cells.     

Frost injury and heterogeneous ice nucleation in leaves of tuber-bearing solanum species : ice nucleation activity of external source of nucleants

The heterogeneous ice nucleation characteristics and frost injury in supercooled leaves upon ice formation were studied in nonhardened and cold-hardened species and crosses of tuber-bearing Solanum. The ice nucleation activity of the leaves was low at temperatures just below 0°C and further decreased as a result of cold acclimation. In the absence of supercooling, the nonhardened and cold-hardened leaves tolerated extracellular freezing between −3.5° and −8.5°C. However, if ice initiation in the supercooled leaves occurred at any temperature below −2.6°C, the leaves were lethally injured.

To prevent supercooling in these leaves, various nucleants were tested for their ice nucleating ability. One% aqueous suspensions of fluorophlogopite and acetoacetanilide were found to be effective in ice nucleation of the Solanum leaves above −1°C. They had threshold temperatures of −0.7° and −0.8°C, respectively, for freezing in distilled H₂O. Although freezing could be initiated in the Solanum leaves above −1°C with both the nucleants, 1% aqueous fluorophlogopite suspension showed overall higher ice nucleation activity than acetoacetanilide and was nontoxic to the leaves. The cold-hardened leaves survived between −2.5° and −6.5° using 1% aqueous fluorophlogopite suspension as a nucleant. The killing temperatures in the cold-hardened leaves were similar to those determined using ice as a nucleant. However, in the nonhardened leaves, use of fluorophlogopite as a nucleant resulted in lethal injury at higher temperatures than those estimated using ice as a nucleant.

     

Low-temperature conditioning of the ice nucleation active bacterium,Erwinia herbicola

Rapid “low-temperature conditioning” and “solute conditioning” of the ice nucleation active bacterium Erwinia herbicola No. 26 are described. Conditioning is the process by which the ability to initiate ice at high temperatures is gained in these bacteria. The cumulative ice nucleator concentration, N[T], was used to measure the number of ice nucleators present in the bacterial systems. N[T] was determined at temperatures from −2 ° to −10 °C and was measured under varying conditioning temperature, time, and solute regimes. Values of N[T] increased rapidly on cooling samples from 30 to 5 °C. The optimum low temperature for conditioning was 5 °C. The conditioning process followed first-order reaction kinetics and time constants (1/rate constant) were between 43 and 62 min at 5 °C. Individual ice nucleators were isolated in droplets and were stable for at least 2 hr. Low-temperature conditioning did not occur when protein synthesis was inhibited by eliminating amino acids in the low-temperature conditioning media or by using the protein synthesis inhibitors chloramphenicol and streptomycin. Analysis of low-temperature conditioning, using heterogeneous ice nucleation theory predicted that ice nucleators are large and have diameters ranging from 80 Å (active at −8 °C) to 300 Å (active at −3 °C). In conclusion, it was predicted that conditioning resulted from growth of the nucleator from about 80 to 300 Å, from a change in the surface properties of 300 Å nucleator making it more similar to ice, or from a combination of these.     

Expression of a bacterial ice nucleation gene in plants

We have introduced an ice nucleation gene (inaZ) from Pseudomonas syringae pv. syringae into Nicotiana tabacum, a freezing-sensitive species, and Solanum commersonii, a freezing-tolerant species. Transformants of both species showed increased ice nucleation activity over untransformed controls. The concentration of ice nuclei detected at −10.5°C in 15 different primary transformants of S. commersonii varied by over 1000-fold, and the most active transformant contained over 100 ice nuclei/mg of tissue. The temperature of the warmest freezing event in plant samples of small mass was increased from approximately −12°C in the untransformed controls to −4°C in inaZ-expressing transformants. The threshold nucleation temperature of samples from transformed plants did not increase appreciably with the mass of the sample. The most abundant protein detected in transgenic plants using immunological probes specific to the inaZ protein exhibited a higher mobility on sodium dodecyl sulfate polyacrylamide gels than the inaZ protein from bacterial sources. However, some protein with a similar mobility to the inaZ protein could be detected. Although the warmest ice nucleation temperature detected in transgenic plants is lower than that conferred by this gene in P. syringae (−2°C), our results demonstrate that the ice nucleation gene of P. syringae can be expressed in plant cells to produce functional ice nuclei.     

Nonlinear relationship between concentration and activity of a bacterial ice nucleation protein

The expression level of an ice nucleation gene (inaZ) was varied in Escherichia coli to observe the relationship between activity and gene product. The ice nucleation activity increased as the 2nd to 3rd power of the membrane concentration of the inaZ gene product, implying that molecules of InaZ protein interact cooperatively in groups of two to three at the rate-limiting step of ice nucleus assembly. The 2nd to 3rd power relationship was independent of the threshold temperature at which ice nucleation was measured and was consistent over a 500-fold range of protein concentration. Such a relationship indicates that the same rate-limiting step must be common to the formation of ice nuclei displaying all the various threshold temperatures within a bacterial population. Observations of Pseudomonas syringae, expressing the inaZ gene at various levels, were consistent with a similar relationship and hence a similar mechanism of ice nucleus assembly in Pseudomonas.     

Ice nucleation temperature of individual leaves in relation to population sizes of ice nucleation active bacteria and frost injury

Ice nucleation temperatures of individual leaves were determined by a tube nucleation test. With this assay, a direct quantitative relationship was obtained between the temperatures at which ice nucleation occurred on individual oat (Avena sativa L.) leaves and the population sizes of ice nucleation active (INA) bacteria present on those leaves. In the absence of INA bacteria, nucleation of supercooled growth-chamber grown oat leaves did not occur until temperatures were below approximately −5°C. Both nucleation temperature and population size of INA bacteria were determined on the same individual, field-grown oat leaves. Leaves with higher ice nucleation temperatures harbored larger populations of INA bacteria than did leaves with lower nucleation temperatures. Log₁₀ mean populations of INA bacteria per leaf were 5.14 and 3.51 for leaves with nucleation temperatures of −2.5°C and −3.0°C, respectively. Nucleation frequencies (the ratio of ice nuclei to viable cells) of INA bacteria on leaves were lognormally distributed. Strains from two very different collections of Pseudomonas syringae and one of Erwinia herbicola were cultured on nutrient glycerol agar and tested for nucleation frequency at −5°C. Nucleation frequencies of these bacterial strains were also lognormally distributed within each of the three sets. The tube nucleation test was used to determine the frequency with which individual leaves in an oat canopy harbored large populations of INA bacteria throughout the growing season. This test also predicted relative frost hazard to tomato (Lycopersicon esculentum Mill) plants.     

The role of ice nucleation active bacteria in supercooling of citrus tissues

The frost sensitivity of Citrus sinensis in relation to the presence of biogenic ice nuclei was studied. In commercially managed citrus groves the ice nucleation active (INA) bacterium Pseudomonas syringae reached 6 × 10⁴ colony forming units (CFU) leaf⁻¹, a population sufficiently high to catalyze ice formation. However, a transient loss of bacterial nucleation activity was noticeable at subzero field temperatures, followed by resumption as temperatures rose. This loss was apparently due to a temporary transition of INA to ice nucleation inactive (INI) bacteria. Field application of Bordeaux mixture, copper hydroxide, streptomycin, and 2-hydroxypropylmethanethiolsulfonate (HPMTS), resulted in reduction of INA bacterial populations to detectability (≤ 10² CFU leaf⁻¹) limits. However, the corresponding reduction in ice nucleation events in treated samples as compared to controls at nucleation temperature ≥−3°C was not as dramatic. It ranged from approximately 7% in samples treated with the bactericide HPMTS, to 35% in samples treated with chemicals possessing combined bactericidal - fungicidal action (coppers). Since a quantitative relationship exists between ice nucleation events on individual leaves and the INA bacterial populations harbored by these leaves, these results suggest the co-existence of a bacterial and a proteinaceous, yet non-bacterial ice nucleating source in citrus, both active at ≥−3°C.     

Factors affecting ice nucleation in plant tissues

Factors affecting the ice nucleation temperature of plants and plant tissues were examined. The mass of a sample had a marked effect on ice nucleation temperature. Small tissue samples supercooled to −10°C and were not accurate predictors of the nucleation temperature of intact plants in either laboratory or field experiments. This effect was not unique to plant tissues and was observed in autoclaved and control soil samples. Ice nucleation temperatures of bean, corn, cotton, and soybean seedlings were influenced by the length of subzero exposure, presence of ice nucleation active bacteria, and leaf surface wetness. The number of factors influencing ice nucleation temperature suggested that predicting the freezing behavior of plants in the field will be complex.     

Components of ice nucleation structures of bacteria

Nonprotein components attached to the known protein product of the inaZ gene of Pseudomonas syringae have been identified and shown to be necessary for the most efficient ice nucleation of supercooled H2O. Previous studies have shown that cultures of Ina+ bacteria have cells with three major classes of ice-nucleating structures with readily differentiated activities. Further, some cells in the culture have nucleating activities intermediate between those of the different classes and presumably have structures that are biosynthetic intermediates between those of the different classes. Since these structures cannot be readily isolated and analyzed, their components have been identified by the use of specific enzymes or chemical probes, by direct incorporation of labeled precursors, and by stimulation of the formation of specific classes of freezing structures by selective additions to the growth medium. From these preliminary studies it appears that the most active ice nucleation structure (class A) contains the ice nucleation protein linked to phosphatidylinositol and mannose, probably as a complex mannan, and possibly glucosamine. These nonprotein components are characteristic of those used to anchor external proteins to cell membranes of eucaryotic cells and suggest that a similar but not identical anchoring mechanism is required for efficient ice nucleation structure. The class B structure has been found to contain protein presumably linked to the mannan and glucosamine moieties but definitely not to the phosphatidylinositol. The class C structure, which has the poorest ice nucleation activity, appears to be the ice nucleation protein linked to a few mannose residues and to be partially imbedded in the outer cell membrane.     

Effects of solution composition on the theoretical prediction of ice nucleation kinetics and thermodynamics

Predictions by various mathematical models of intracellular ice formation (proposed by Mazur, Pitt, Toner, and Karlsson, respectively) were compared to the known thermodynamic and kinetic behavior of ice formation in supercooled aqueous systems. The older models (Mazur, Pitt, and Toner) significantly underestimated the magnitude of colligative nonequilibrium freezing point depression in response to increased concentration of solutes, such as salts or cryoprotectants. Furthermore, kinetics predicted using phenomenological models (by Mazur and Pitt) exhibited implausible temperature-dependence, with the probability of intracellular ice formation being allowed to increase even at temperatures below the glass transition point. The Toner model, on the other hand, produced invalid results at temperatures below −48 °C. The Karlsson model was the only model that consistently yielded realistic predictions over a wide range of temperatures and solute concentrations, especially in the presence of cryoprotectant additives. To facilitate adoption of the Karlsson model of intracellular ice nucleation, the complete set of model equations has been collected and described in detail.     

Active control of the nucleation temperature enhances freezing survival of multipotent mesenchymal stromal cells

Cryopreservation is a technique that has been extensively used for storage of multipotent mesenchymal stromal cells (MSCs) in regenerative medicine. Therefore, improving current cryopreservation procedures in terms of increasing cell viability and functionality is important. In this study, we optimized the cryopreservation protocol of MSCs derived from the common marmoset Callithrix jacchus (cj), which can be used as a non-human primate model in various pathological and transplantation studies and have a great potential for regenerative medicine. We have investigated the effect of the active control of the nucleation temperature using induced nucleation at a broad range of temperatures and two different dimethylsulfoxide concentrations (Me₂SO, 5% (v/v) and 10%, (v/v)) to evaluate the overall effect on the viability, metabolic activity and recovery of cells after thawing. Survival rate and metabolic activity displayed an optimum when ice formation was induced at −10 °C. Cryomicroscopy studies indicated differences in ice crystal morphologies as well as differences in intracellular ice formation with different nucleation temperatures. High subzero nucleation temperatures resulted in larger extracellular ice crystals and cellular dehydration, whereas low subzero nucleation temperatures resulted in smaller ice crystals and intracellular ice formation.     

Purification and composition of an ice nucleating protein from queens of the hornet,Vespula maculata

Summary Hemolymph ice nucleating factors are found in many freeze tolerant insects. These factors function to initiate ice nucleation in the extracellular fluid at fairly high subzero temperatures thereby minimizing the possibility of lethal intracellular ice formation.An ice nucleating protein was purified from the hymolymph of pupal bald faced hornets,Vespula maculata. This is the first ice nucleating protein to be purified. The protein has a molecular weight of 74,000, as determined by SDS-PAGE, and is quite hydrophilic. Glutamate and/or glutamine accounts for 20% of the amino acid residues. It is likely that the hydrophilic nature of the protein is involved in the ability of the protein to function as an ice nucleator.     

<Emphasis Type="Italic">Polytrichum commune</Emphasis> spores nucleate ice and associated microorganisms increase the temperature of ice nucleation activity onset

Moss spores disperse via wind and have been found previously in precipitation and air samples. Their presence in the atmosphere led to this study’s examining the potential of moss spores to contribute to ice nucleation, a process necessary for ice formation in clouds prior to precipitation. Ice nucleation assays were conducted using Polytrichum commune spores that were either associated with natural assemblages of microbes or extracted aseptically from capsules and subsequently confirmed to be free of culturable microbes. Liquid suspensions of capsule spores and non-sterile spores nucleated ice at temperatures as high as ?12 and ?7 °C, respectively. When capsule and non-sterile spores were heated at 95 °C for 10 min, which killed all culturable microbes on non-sterile spores, both nucleated ice from ?10 to ?13 °C. An additional non-sterile spore sample collected from partially opened capsules in a forested ecosystem (ID, USA) nucleated ice at temperatures as high as ?7 °C, similar to non-sterile P. commune spores. This is the first set of results to indicate that P. commune spores themselves are capable of nucleating ice at temperatures higher than many abiological particles such as mineral dust (≤?15 °C) and that natural assemblages of microbes can increase their ice nucleation efficiency. Future studies aimed at determining the abundance of moss spores in the atmosphere and the identity of ice-nucleating microbes associated with them will provide further insights into the ability of moss spores to impact precipitation patterns.     

Perturbation of bacterial ice nucleation activity by a grass antifreeze protein

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.