Critical Reviews in Biotechnology, 11(3):277-295 (1991)

Principles and Biotechnological Applications of Bacterial Ice Nucleation Critical Reviews in Biotechnology Downloaded from informahealthcare.com by University of Notre Dame Australia on 05/07/13 For personal use only.

Argyrios Margaritis and Amarjeet Singh Bassi Department of Chemical and Biochemical Engineering, Faculty of Engineering Science, University of Western Ontario, London, Ontario, Canada, N6A 589

ABSTRACT: Certain aerobic, Gram-negative bacteria, including the epiphytic plant pathogen, Pseudomonas syringae, possess a membrane protein that enables them to nucleate crystallization in supercooled water. Currently, these ice-nucleating (IN) bacteria are being used in snow making and have potential applications in the production and texturing of frozen foods, and as a replacement of silver iodide in cloud seeding. A negative aspect of these IN bacteria is frost damage to plant surfaces. Thus, of the various types of biological ice nucleators, bacteria have been the subject of most research and also appear relevant to the anticipated practical uses. The intent of this review is to explain the identification and ecology of the ice-nucleating bacteria, as well as to discuss aspects of molecular biology related to ice nucleation and consider existing and potential applications of this unique phenomenon. KEY WORDS: ice nucleation, INA bacteria, Pseudomonas syringae, ice-plus bacteria.

1. INTRODUCTION The discovery that certain species of aerobic, Gram-negativebacteria can trigger ice formation in supercooled water has raised interesting new possibilities for applications in the field of ice nucleation. These bacteria (referred to as ice-plus or ice nucleation active bacteria) are highly efficient ice nucleators, being active in temperatures as high as -2.5"C. Thus, they rival silver iodide and other more commonly used ice-nucleating particles. In particular, one species of ice nucleation active (INA) bacteria, namely, Pseudomonas syringae, is an inhabitant of the leaves of many common crops and fruit trees. The yearly frost damage by P. syringae to plants and associated agricultural crops is estimated to be several billion dollars worldwide. At the same time, biochemical engineers and biotechnologists are discovering new beneficial uses for the iceplus bacteria. These include the manufacture of snow, the freeze texturing of different foods, and

new applications in refrigeration in the chemical and bioprocessing industries. What causes these bacteria to act as ice nuclei? Information available at this time reveals that the ice-nucleating phenotype is provided by one structural gene in these bacteria.' The product of this gene expression is a membrane-bound protein. This protein, alone or in combination with membrane phospholipids, may be responsible in imparting ice-nucleating activity to the bacterium. The size, structure, exact location of the protein, and its in vitro isolation are the subjects of intensive research activity. A mutant of the ice-nucleating (IN) bacterium, P . syringae, which lacks the ice nucleation gene, has been prepared by Lindow2s3and othe r ~These . ~ so-called ice-minus bacteria have been tested on strawberry plants in California. The objective in these tests was to investigate whether or not ice-minus P . syringae could displace the more common ice-plus P . syringae populations and, hopefully, prevent frost damage to these

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plants. This release of a recombinant microbe into the environment has been the focus of public debate and c o n t r o v e r ~ y . ~ ~ ~ Several important questions need to be answered before the phenomenon of bacterial ice nucleation can be fully understood.

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1.

2.

3.

What is the make-up of the bacterial icenucleating site? How can the ice nucleation phenotype in the ice-plus bacteria be manipulated? It has been known for some time that environmental and culture conditions do affect this property. However, very little research has been published on environmental factors that affect cell growth in INA. Can ice-minus bacteria efficiently displace ice-plus bacteria from their natural habitat on plant leaves? Growth kinetic studies under batch and continuous culture conditions are needed to answer this question, followed by field testing under actual environmental conditions.

This review makes an attempt to look at various aspects of the bacterial ice nucleation phenomenon and to examine the state of the present research related to the above-mentionedquestions.

II. THE MECHANISMS OF ICE NUCLEATION If ice and pure water are combined at O'C, the two phases will exist in equilibrium, provided heat is neither added nor removed. Removal of heat causes water to convert to ice without the need of nucleation. The system eventually becomes totally solid while maintaining a temperature of 0°C (or very close to this temperature). If, however, ice is initially absent from the system (water only), the solidification follows a very different path because nucleation must precede crystal growth. Upon cooiing, the temperature of this system eventually falls below the freezing point (supercooling) before nucleation and further solidification can proceed. This suggests that nucleation is a necessary step in order to surmount an energy barrier (activation energy) before water converts to ice.

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Nucleation can be defined as a process that generates within a metastable mother phase (e.g., the supercooled state of water) the initial fragments of a more stable phase. These fragments then spontaneously develop into gross fragments and the phase change is then complete. The free energy change during nucleation is given by Equation 1. As shown in Equation 1, the energy barrier that exists for nucleation to occur can be explained on the basis of free energy changes that accompany nucleation:'

where AG = free energy change due to nucleation resulting in the formation of a spherical ice particle of radius, r (kcal); AGp = free energy change per unit volume between liquid water and solid ice during the formation of a spherical ice particle of radius r (kcal/m3); CJ = interfacial energy per unit area between the two phases of water and ice at the given temperature (kcal/m2); and r = radius of a spherical ice particle formed during nucleation (m). The first term on the right is always negative and becomes increasingly so as r increases. This term relates to free energy release as hydrogen bond formation occurs during the association of water molecules into an ordered structure. The second term is always positive as r increases. This term relates to energy required to form the ice-water interface. When r is small, the surface energy term predominates. However, as r increases, the first term becomes more significant and a plot of AG vs. r will pass through a maximums (Figure 1). This maximum represents the free energy of activation for nucleation at the temperature under consideration. A particle with a size corresponding to the maximum AG is said to have a critical radius (r*) because any radius greater or smaller than r* would lead to a decrease in free energy. Particles less than r* will disintegrate, while particles greater than r* will grow. The critical radius is dependent on the degree of supercooling. The magnitude of the critical radius (r*) is influenced by the interfacial free energy per unit area (a),the solid liquid equilibrium temperature (TE), the latent heat of fusion (L), and the degree of supercooling (AT = [T, - TI) where T is the system temperature.

tional to the degree of supercooling, AT, as shown by Equation 3:

t a 83

r*

0:

1 AT

(3)

B. Heterogeneous Nucleation

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I

I

r*

PARTICLE RADIUS-

FIGURE 1. Change in free energy during nucleation (r* = critical radius). (After Fennema, 0. R., Powrie, W. D., and Marth, E.H.,Low TemperaturePreservation of Foods and Living Matter, Marcel Dekker, New York, 1973, 153.)

The above discussion implies a pure water-ice system and the mechanism is called homogeneous nucleation. A. Homogeneous Nucleation Nucleation that takes place in pure systems (i.e., pure ice and water) is referred to as homogeneous nucleation. Only extremely pure water nucleates in this manner, but the probability that pure water will nucleate homogenously at 0°C is zero. As tiny droplets of pure water (less than 1 p,m in diameter) are decreased in temperature, the probability approaches 1 at -41°C. A temperature of - 41°C is usually accepted as the limit to which water can be superc~oled.~ For homogenous nucleation, the critical radius (r*) is given by Equation 2, assuming an incompressible, spherical solid and AH and AS independent of temperature? r* =-2 u TE LAT where r* = critical radius of spherical particle (m); L = latent heat of fusion (kcal/m3); AT = degree of supercooling ("C); and TE = freezing point ("C). Since u, TE and L are constant at constant pressure and do not vary greatly with temperature, then r* becomes inversely propor-

This type of nucleation occurs in impure water when molecules of water aggregate in a crystalline arrangement on nucleating agents such as suspended foreign bodies, surface films, or walls of containers. lo Heterogeneous nucleation is the predominant form of nucleation. It may involve fewer water molecules and a lower activation energy than homogeneous nucleation, but the phenomenon is not well understood. Heterogeneous nucleation results in a decreased degree of supercooling, and it is possible that the nucleating agent may provide a template for ice formation. Studies have been conducted that show that nucleators for ice formation are diverse and widespread. They include substances such as silver iodide ,I1 a-phenazine , I 2 phloroglucinol dihydrate, l 3 metaldehyde,I4 various silicates of clay and mica (kaolinite, covellite, magnetite),I4 steroids (cholesterol),I5 amino acids,'6*17proteins, lectins,'s bacteria,'* and lichen. l 9 MasonI4 has also tabulated the threshold temperature of most inorganic ice nuclei, i.e., the temperature at which the nuclei are most active in ice nucleation. Based on this and other studies, the various ice nuclei may be grouped into specific threshold temperature ranges over which they may be most active. Such a classification is presented in Table 1. It can be seen that INA bacteria are among the most active ice nuclei known. The development of new and better ice-nucleating agents is an ongoing field of research. Recently, a new ice-nucleating aerosol has been developed that is a mixture of AgI and BiI, and provides the closest possible match to the ice lattice." However, the threshold temperature at which this aerosol is active in nucleating ice formation has not been reported. Most inorganic and organic nonviable ice nuclei have fixed threshold temperatures. The advantage of using bacterial ice-nucleating agents is that the threshold temperatures are distributed over a wide range. In addition, bacterial ice nuclei can be obtained

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TABLE 1 Classification of Ice Nuclei and Ice Nucleation Mechanism Based on Temperature Range of Most Activity

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Ice nucleation agent

None Mineral particles of meteoric origin Dust or pollen Crystalline particles, lichen, icenucleating bacteria

Mechanism of Threshold ice nucleation temperature (“C)

Homogeneous - 35 or lower Heterogeneous - 15 or lower

Heterogeneous Heterogeneous

- 10 to - 15 - 2 to - 10

Ref. 9 25

25 14, 18, 19

readily in large numbers by cultivation on inexpensive byproducts and waste materials.

IV. THE ECOLOGICAL AND PHYSIOLOGICAL CHARACTERISTICS OF P. SYRINGA€

111. HISTORICAL BACKGROUND OF BACTERIAL ICE NUCLEATION

Due to its role as the instrumental microbe causing frost damage and its ubiquity in the environment, P . syringae is sufficiently important to summarize its properties here. A plant pathogen that inhabits a large number of plant species, this microorganism was first isolated as a bacterial pathogen of lilac (Syringa vulgaris) in 1902,29and it has since been isolated from several plant species. 30 This bacterium is Gram-negative, rod shaped, and motile (with one or two polar flagella).3’ The microorganism is a facultative aerobe, does not form endospores, and produces fluorescent pigments on media low in Energy yielding pathways in the microorganism are respiratory and not fermentative. P . syringae has 41 pathovars or subgroups; not all pathovars possess ice-nucleating activity. 32 P . syringae is usually differentiated from other pseudomonads by its negative reaction in the oxidase and arginine tests.3’ Another identifying property of this bacterium is the production of a hypersensitive response on Diseases caused by pathovars of P. syringae include foliar blight on snap beans, tomatoes, and wheat; blossom blight on pear trees; and canker formation on olive trees. Its toxic actions are It produces a plant attributed to several toxin called syringomycin ,34 nontoxic to humans,

Ice nucleation initiated by microorganisms was first reported by Schnell and VaW’ when they discovered that decaying tree leaves were an important source of ice-nucleating particles. Later Maki et a1.22 isolated Pseudomonas syringae bacteria from decayed alder leaves. These isolates were found to be highly active in initiating ice formation at temperatures in the range of - 2 to - 5°C (Table 1). Since those findings, several strains of this bacterium and of at least five other bacteria have been identified as efficient ice nucleators. Strains of Erwinia herbicola,IBE . a n a n a ~P, .~f ~z u o r e ~ c e n sP, ~. ~viridifla~a,*~ Xanthomonas compestris ,26 and recombinant Escherichia coli2’ have been shown to possess ice-nucleating activity. The question as to why bacteria possess INA is still open to speculation. The INA phenotype may confer an advantage on bacteria that express it. For example, since these bacteria are epiphytic, ice nucleation and subsequent frost damage to the leaf surface on which these bacteria reside may allow access to nutrients within the leaf tissues. It has also been suggested that the bacteria may participate in rain pre~ipitation.’~*~~ 280

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Warren et a1.42,43 completely sequenced the and also produces lipases and ammonia. All the ice nucleation gene of P . fluorescens and termed plant pathogenic actions caused by this microorit the inaW gene. A partial sequence of the proganism have not been identified. tein of E . herbicola has also been predicted by P . syringae lives on the leaf surface and also nucleotide sequencing of the corresponding invades tissues of the plant. A recent study has genes.44The nucleotide sequence for the ice nufound that the motility of this microorganism concleation gene inaX from the bacterium X . camfers on it epiphytic fitness a d ~ a n t a g eIt. ~has ~ also pestris pv. translucens has also been deterand been found to be present in mined.45The ice nucleation gene sequences from P . syringae is also adapted to living on plants and healthy tissue to which it is n~npathogenic.~~ P . viridzjlava and E. ananas have not yet been reported. The plants may serve as sources of inoculum for The various ice nucleation proteins from the these bacteria. The microorganism can grow rapdifferent INA bacteria have closely related strucidly, with 30- to 150-fold increases in 24 h.29 tures. This has been determined based on two strong pieces of evidence: ( 1 ) the genes transferred to E . coli do not lose the ice nucleation V. MOLECULAR BIOLOGY OF THE ICE phenotype, and (2) antibodies raised against the NUCLEATION SITE inaW protein, i.e., the product of the ice-nucleating gene of P . fluorescens, can recognize A. The Ice Nucleation Genotype and its the inaZ protein from P . syringae after protein Translational Product isolation from an E. coli clone.46 Thus, it has been concluded that the genes are homologous The gene coding for the ice nucleation proand their action is host independent. tein in P . syringae, E. herbicola, and P . fluoWolber and Warren47have described the priroscens has been identified as a small region on mary structure of the bacterial ice nucleation prothe chromosomal DNA of the bacteria. 1*39*40The teins. The majority of the amino acid sequence deletion of this gene completely abolishes ice of each ice-nucleating protein (8 1%) was found nucleation a ~ t i v i t y .The ~ genes of the various to be a complex repeating domain that could be bacteria were purified after cloning and overfurther subdivided into three regions (Figure 2). expression in E. coli. The cloned genes were The first two regions repeat with a high fidelity found to impart ice nucleation activity to the host 2 periodicity of 48 residues. Each of these 48E . ~ o l iand , ~ the ~ level of activity in the transmer units in the fiist two repeating regions conformant was found to correlate with the level of sisted of three 16-mer units with medium fidelity protein expression. A 4.5 kb fragment of DNA repeats. The 16-mer units were themselves comfrom P . syringae and a 5.7 kb fragment from E . posed of two repeating 8-mer units of low fidelity herbicola conferred the INA phenotype on E. repeats, which means that only two positions out coli. of eight were strongly conserved. The third reGreen and Warren4' determined the sepeating section consisted only of 8-mer repeating quence of the ice nucleation gene of P . syringae . units. This gene (termed in&) was found to contain While the majority of the ice nucleation proa single long open reading frame. A striking tein was a central repeating region as described degree of sequence reiteration with a 24 base above, 15% of the sequence was a unique Nperiodicity was observed in this reading frame. terminal domain and 4% formed a C-terminal The consensus repeat sequence noted was domain unique to each protein from the three GCCGGTTATGGCAGCACGCTGACC . The different bacteria. The N-terminal domain was authors found that the predicted translational found to be relatively hydrophobic, which would product contained 1200 amino acids with a moindicate membrane localization while the C-terlecular weight of approximately 120 kDa. Exminal domain was relatively hydrophilic. actly 122 contiguous repeats of the consensus Deletion mutations of the inaZ gene, in which octapeptide Ala-Gly-Tyr-Gly-Ser-Thr-Leu-Thr the remaining sequences were in frame, were could be recognized in it.

281

8-MERS -16-MERS 48-MERS 4 8-BIERS 8-MERS

N H -

UNIQUE

2

-16-MERS

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8

8

16

8

8

16

-

8-MERS

8

- UNIQUE - COOH

8

16

second order of periodiaity

M Third order of periodioity

FIGURE 2.

A simplified representation of the primary structure of the ice nucleation protein. The top shows that the protein consists of N-unique and C-unique terminals and a central repeating region. The first two sections of the central repeating region have three orders of periodicity as depicted below the cartoon of the entire sequence: a primary repetitive sequence of 8 amino acid residues, superimposed by 16-mer repeats and 48-mer repeats. (Adapted from Wolber, P. and Warren, G. J., TBS, 14, 179, 1989.)

used to probe the function of the ice nucleation Removal of the N-terminal unique domain caused the ice nucleation activity of the P. syringae bacteria to decrease with threshold temperatures lower than - 5°C. Removal of the Cterminal unique domain caused a total loss of ice nucleation activity, while successive deletions in the repeating domain caused a corresponding decrease in ice nucleation temperatures. The secondary structure of the protein is thought to be a P-pleated sheet, punctuated by 5 to 6 turns per 48 amino acid sequence. The repeated unit is hydrophilic and particularly rich in serine and threonine. Based on this information, Wolber and Wanen have presented structural models of the ice nucleation protein (INP) and suggest that the INP may mimic the ice lattice49 (Figure 3). The INP may provide a template for ice formation. Structural characterization is still too incomplete to supply an adequate working model, due undoubtedly to the complexity and size of the nuclei.

282

B. Additional Factors Involved in Ice Nucleation Kozloff et al .50 have presented evidence that a lipid, phosphatidylinositol (PI), may be a component of the ice nucleation site. They originally hypothesized the involvement of the PI molecule based on the observation that another carbohydrate, mesoinositol, has a steric arrangement of hydroxyl groups topotactic with that in ice. PI was shown to cause hemagglutination of the lectins that had previously been shown to inhibit ice nucleation. It is therefore possible that PI may be the target of these lectins. Govindarajan and L i n d o ~ ~also ' * showed ~~ a relationship existing between loss of membrane lipids and corresponding loss in ice nucleation activity. These authors found that the delipidation of partially purified outer membranes of the bacteria by various delipidating agents led to a significant loss in INA. They suggest further that a hydrophobic environment produced either by lip-

A

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B

n

FIGURE 3. Structural models in which the INA protein displays a symmetry retated to that of Ice. Drawings are not to scale. (A) symmetry of ice illustrated by the spatial arrangement of 12 water molecules (left), the symmetry of this structure (center), and the way this symmetry becomes extended as more water is added (right); (6) triangular model of protein; a 48-residue repeat is shown at left and its symmetry is demonstrated at center and right; (C)antiparallel double helix model; a 48-residue repeat of each of the intertwined chains is shown at left; their individual, combined, and extended symmetries are demonstrated at center.4e

ids or certain detergent micelles is required for proper assembly and organization of an oligomeric ice protein complex. Another possible hypothesis is to consider that the PI molecule is responsible in anchoring the protein to the membrane. Lows3 has reviewed this aspect in terms of the general functional role of glycosylphosphatfdylinositol (GPI)as a dynamic and versatile anchor for cell surface proteins.

C. Localization of Ice Nucleation Activity Sprang and L i n d ~ whave ~ ~ shown that ice nuclei were present in cell membranes and found that 80% of the ice nuclei were associated with the outer membrane fraction. Phelps et al.55 showed that growing cultures of ice-nucleating E. herbicolu shed ice nuclei into the medium. Evidence indicated that the nuclei shed were associated with membrane vesicles shed under the same conditions as the nuclei. The shedding of

the membrane vesicles also occurred in ice-minus strains of E. herbicolu. Lindow et al.56 have quantified the ice nucleation activity in different sub-cellular fractions of P. syringue and E. coli containing the ice nucleation gene. For both the microorganisms, the ice nuclei were found to be localized in cell envelopes. Ice nucleation activity was found in Triton-X 100 insoluble membrane fragments as well as in slowly sedimenting high-density membrane fragments. The outer membranes of these Gram-negative bacteria had nearly all the ice nucleation activity associated with them. No ice nucleation was detected in any soluble cell fractions.

D. Measurement of Size of MembraneBound Ice Nuclei To circumvent the necessity of extraction and purification, Govindarajan and LindowS7 em-

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ployed gamma radiation target analysis to measure the in situ size of ice nucleation sites in strains of P . syringue, E. herbicolu, and rDNA E. coli cell containing the ice gene. The minimal size of a functional ice nucleus active below - 10°C was about 1.5 X lo5 Da for all strains. The size of the ice nuclei increased logarithmically, with activity at increasing nucleation temperatures from - 12°C to - 2°C where the estimated molecular weight was 1.9 X lo7Da (Figure 4). The authors concluded that the ice-nucleating site may be an oligomenc structure that can selfassociate to assume many possible sizes.

hydrophilic centers may exist on a relatively hydrophobic site. Such theoretical considerations have been applied by the authors to ice nucleation by silver iodide and silver bromide. M U Z U ~ O ~ ~ has used conformal energy calculations to theoretically predict the conformation of the ice nucleation protein. Burke and Lindowm have used heterogeneous ice nucleation theory to calculate the ice nucleation probability of bacterial ice nuclei at various supercooling temperatures, assuming various surface properties, shapes, and sizes of heterogeneous nucleating particles. These were then compared with experimentally measured sizes of the bacterial ice nucleators of P . syringae and other INA bacteria. Good fit was obtained for particles (spheres, cylinders, and disks) that had surface tension identical to that of ice.

E. Theoretical Considerations A theoretical basis of the ice nucleation phenomenon has been proposed by Thangaraj et al.58 They suggest that the nucleation capability of an ice-nucleating site depends on lattice match and symmetry, water solubility, size of the nucleating site, polarization ability, hydrophobicity, surface charge, and the number and strength of surface sites capable of absorbing water molecules. Thus,

F. Isolation of the Ice Nucleation Protein No studies have yet been published that report the utilization of the isolated ice nucleation protein in its native form. The relationship be-

7

6.5

6

5.5

5

4.5

'

1

I

1

I

1

1

1

I

I

I

-2 -3 - 4 - 5 - 6 -7 - 8 -9 -10 -11 -12 -13 -14

Temperature (C) FIGURE 4. Molecular mass of ice nucleation sites of different strains of ice nucleation active bacteria as a function of temperature (T) of expression of ice nucleation activity estimated by sensitivity to gamma radiation: f . syringae strain 31R1 (m), f .syfingae strain cit7 (o),f.herbicola strain 26SR62 (q, and E. coli strain HB101 (*).57

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tween ice nucleation activity and cell viability also needs to be clearly established. The protein fractions have been isolated and characterized as described by Deninger et al. ,& Wolber et al. ,61 and others.62

VI. ENVIRONMENTAL MANIPULATION OF BACTERIAL ICE NUCLEATION ACTIVITY A systematic perusal of the literature revealed that despite the potential commercial importance of Pseudomonas syringae as an efficient ice nucleation agent, very little research has been published on the environmental factors that affect its cell growth and INA. The INA expressed by bacteria is a function of the strain genotype, pH, temperature, composition of the growth medium, and aeration. Bacteria grown on a solid medium may express levels of INA different from those cultured in the liquid broth. The kinetics of INA development during P. syringae cell growth in bioreactors is under investigation in our laboratories.'j3 Bacterial INA has been found to be growth a s ~ o c i a t e dThe . ~ ~measurement of ice nucleation activity is described below, and the effects of several environmental parameters are considered.

A. Measurement of Ice Nucleation Activity The droplet freezing assay (ValP4 and L i n d o ~ is ~ a~ rapid ) and quantitative measure of the ice nucleation activity of INA bacteria. In this technique, 30 to 40 drops of suitably diluted ice-nucleating bacterial cultures of small volumes (I0pl) are placed on a hydrophobic surface (paraffin-coated aluminium foil boats). The aluminium foil boats are placed in a temperature-controlled environment, for example, floating on the surface of a refrigerated bath containing chilled refrigerant (ethanol or a 5050 ethylene glycol and water mixture) or on a thermoelectric (Peilter) cold plate. The temperature is gradually lowered at a constant rate and the fraction of droplets that freeze at each temperature is noted. From this

information a droplet freezing spectrum is obtained. This is a plot of the fraction of droplets frozen at a given temperature vs. the temperature. The fraction of droplets freezing follows the Poisson distribution and can be converted to the number of ice nuclei per milliliter active at all temperatures warmer than and including the temperature of measurement. This quantitative measure of ice nucleation activity is termed the cumulative nucleus frequency (CNF) and is given by Equation 4:

(4) where CNF = cumulative nucleus frequency (cumulative number of ice nuclei/ml of droplet volume); f = the fraction of droplets frozen; and V = the volume of an individual droplet (ml). If the cell concentration is known, then the specific ice nucleation frequency can be obtained by dividing the CNF/ml by the cell number or gram dry weight per milliliter. This specific ice nucleation activity (i.e., ice nucleation activity per cell) can then be used to compare bacterial samples produced under different environmental growth conditions. Table 2 lists several variables that may affect the freezing of droplets containing ice nuclei (and, hence, the CNF) as the temperature of a sample is lowered. Variables of primary importance in

TABLE 2 List of All Possible Variables That Can Affect the Freezing of Droplets in the Bacterial Ice Nucleation Assay Temperature of drop Volume of drop Numbers of ice nuclei present in the drop - this depends on: Concentration of cells Ratio of ice nuclei per cell Distribution of ice nuclei in the original cell sample Time for which drop is held at a given temperature Heat transfer characteristics of the cooling surface pH of drop Separation of the bacterial cells from the growth medium by centrifugation and washing

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this list are the temperature, volume of the drop, and the concentration of cells in the drop. No studies, except perhaps the original study by Vali and S t a n ~ b u r y ,have ~ ~ appeared on how other variables in Table 2 affect INA measurements with this assay. It has been observed in our laboratory that the time for which the drops are held at a given temperature (or cooling rate of the refrigerated bath) and the pH of the droplets can affect the reproducibility of results.63

The fact that increasing the cell concentration decreases supercooling can be explained on the basis of chemical bonding principles. More cells (hence more ice nuclei) are required at warmer temperatures in order to increase the probability that at least one ice nucleation site is active in causing the necessary rearrangement of water molecules from a disordered liquid state into the ordered state of an ice lattice.

C. pH

6. Cell Concentration Maki et a1.22have studied the effect of cell concentration on ice nucleation activity at various freezing temperatures (Figure 5 ) . They found that increasing the cell concentration in the droplets resulted in ice nucleation at warmer temperatures; conversely, dilution of the cell cultures increased supercooling. A cell concentration of lo6 per ml or higher was needed to initiate ice nucleation at temperatures between -2" and -5°C. Also, as is evident in Figure 5, at high cell concentrations (> lo7 cells per ml), the ice nucleation activity becomes independent of the number of cells. Similar results have been reported by Obata et a1.% and Stewert and Bear.67

The pH for growth of ice-nucleating bacteria is in the range of 5.0 to 9.0. The optimum pH for cell growth is 7.0. For the expression of high levels of ice nucleation activity in P. syringae cultures, an optimum pH range of 5.5 to 6.7 has been identified by Hendricks et a1.68However, Kozloff et d.'' found that there was no change in ice nucleation activity of either P. syringae or E. herbicolu as the pH was changed from 5.0 to 9.2. When the pH fell below 5.0, the INA decreased. Obata et al.69also found no effect of pH on the INA of P. fluorescens. Observations by Kozloff et al.'* and Obata et al.69 were limited to reporting the warmest nucleation temperatures in large populations of bacteria; cumulative nucleus frequencies were not given. Thus, their observations that pH does not affect INA may be suspect because such a technique (reporting only warmest ice nucleation temperature) has associated with it a large error due to the heterogeneous populations of ice nuclei present in any given cell sample.

D. Growth Temperature

0

-4

-8

-12

-16

-20

-24

Heterogeneous nucleotion ternperoture(C)

FIGURE 5. Effect of cell concentration on the ice nucleation activity of P. syringae C-9. (From Maki, L. R., Galyan, E. L., Chang-Chien, M., and Caldwell, D. R., Appl. Microbiol., 28(3),456, 1974.)

286

While Pseudomonas species can grow in the range of 4°C to 30°C, the optimum temperature of cell growth is 20°C. The effects of lower growth temperature on INA have not been reported in literature. Obata et al.69investigated INA of cells grown above the optimum temperature of growth of P. fluorescens. They found a significant decrease in INA for such Cells grown at 20"c and then conditioned for several hours at 4°C have been found to show increased levels of INA.~~

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E. Medium Composition While P . syringae can utilize a large number of carbon and nitrogen sources, the development of ice nucleation activity varies with the growth medium used. Two patents have appeared recently on composition of growth media for the production of cells with high ice-nucleating activity. Lynn et al.71 proposed a medium with glucose as a carbon source and an a-ketoglutarate yielding amino acid as the nitrogen source. The medium also contained phosphate and trace amounts of iron and zinc. HendrickP proposed a simpler medium comprised of mannitol, yeast extract, and magnesium sulfate. The effects of varying carbon and nitrogen sources on the INA of P . fluorescens have been studied by Obata et al.69Glucose, glycerol, and citric acid yielded cells with high INA. Malt extract and sodium nitrate were found to be poor substances for growth, while ammonium salts produced cells with high INA. Luria broth and nutrient broth have been found to be poor media for expression of ice nucleation. Phenol derivatives as carbon sources do not yield ice-nucleating cells. However, polyethylene sorbiton fatty acids and Tween 20 increased ice nucleation activity. Mineral composition has been found to affect ice nucleation activity. Two or more minerals may act synergistically in enhancing INA.

F. Additional Environmental Factors Several chemicals have been found to decrease INA. n-Octylbenzyldimethyl ammonium iodide (0.1, 1, 10, 100 ppm) decreased the INA of E . ananas. This chemical was more effective at 15°C than at 5°C.” The reasons for this are not clear. An ascorbic acid-copper complex de.~~ have creased INA of P . f l u o r e ~ c e n sLectins been found to inhibit bacterial INA.’* The INA is also decreased with borate compounds and sulfhydryl agents. l 8 Delipidation has been shown to decrease INA, which lends evidence to the theory that lipids participate in the ice nucleation Studies by O’Brien and L i n d o ~indicate ~~ that the ice nucleation activity of individual bacterial strains resident on plant surfaces is a func-

tion of complex interacting factors such as strain genotype, host plant species, humidity, light, etc. The INA measurements of cells grown in culture were found to be lower than on plant surfaces. Lind~w also ~ ~found a lack of correlation between INA of bacteria grown in the laboratory and on plant surfaces. INA seems to be preserved if bacterial are concentrated, frozen in liquid nitrogen, lyophilized, and either stored at -20°C or beINA also seems to be retained by dead cells, provided no lysis takes place.76

VII. FROST CONTROL WITH ICE-MINUS BACTERIA

A. Causes of Frost Damage Most plants supercool below the freezing points of the water solution within their tissues. Total avoidance of freezing by supercooling may provide some protection (to about - 4°C) for tender horticultural crops like corn and beans that have little resistance to freezing. In these crops, there is little resistance to the spread of ice through the plasmalemma into the cytosol, and lethal intracellular freezing occurs close to the nucleation temperature. Most temperate zone crops that show freezing resistance freeze extracellularly. Though extracellular freezing causes the withdrawal of water from within the cell and cell dehydration, ice does not penetrate the cell. When the temperature improves, the cells regain water and survive. Cell dehydration may, however, cause injury by cellular deformation and membrane rupture. Intracellular freezing is usually lethal, and the primary reason for cell injury or death. Deep supercooling is one mechanism of avoiding freeze damage. Repeated freezing injury also makes the plant susceptible to attack by microorganisms.

B. Economic Considerations Brown and B l a ~ k b u r n ,Q ~ ~ a m m e ,and ~~ A n d r e w ~have ~ ~ reviewed the impact of freezing temperatures on crop production in Canada. Early spring frosts often damage the buds and shoots of crops like alfalfa and winter wheat when the

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temperature drops to -2°C or lower after these crops begin regrowth in spring. Late spring or early summer frosts sometimes damage seedlings or transplants of tender annuals and blossoms of perennial fruit crops. Late summer and autumn frosts are a threat to the development period of crops such as canola, tobacco, soyabeans, corn, tomatoes, potatoes, peppers, and other tender horticultural crops. The economic impact of freezing and frost damage on spring grains, corn, oilseeds, and vegetables in Saskatchewan, Canada, ranged from $11 million to $13 million in 1974, 1979, and 1982. The freeze in 1982 on August 28 caused losses of $13.4 million in cereal production in Saskatchewan and $1.6 million in Manitoba. The major oilseed crops in Canada, soyabeans, canola, and sunflower, are susceptible to frost damage. Seedlings that have not undergone hardening die at - 3°C to - 4°C. The economic impact of frost damage in the U.S. has been estimated to be over a billion dollars per year.8oIce-nucleating bacteria have been identified recently in frost damage to wheat, oats, barley, canola, and mustard.81

Application of water to plants -Water can be applied to plants directly or by the use of sprinklers. The limitation is that a heavy buildup of ice may occur, causing wilting and damage to plant tissues. Application of fogs, smoke, and chemical foam - These techniques are used generally in areas where frost is infrequent or unseasonal. Chemicals - Bactericides, copper-containing fungicides or antibiotics can be sprayed on plant surfaces, either to destroy the INA bacterial or limit their ice nucleation activity. Fish antifreeze glycoproteins (FAGPs) Such proteins have been used to inhibit bacterial ice nucleators. FAGPs from the Antarctic fish Dissostichus rnawsoni inhibited the ice nucleation activity of membrane vesicles from Erwinia herbicolaS2This inhibition effect showed saturation at high concentration of FAGP and conformed to a simple binding reaction between the FAGP and the nucleation center. Antagonistic bacteria - The displacement of the INA bacteria with other nonpathogenic species of bacteria is of increasing interest. Cody et aLS3have studied a number of species that can reduce the populations of ice-plus bacteria on plant surfaces.

C. Frost Control Several methods may be used to avoid frost damage. These include: Site selection - Low-temperature damage in horticultural crops can be avoided by selective planting on sites with a low risk of water injury and spring frosts. Genetic improvement - More frost-resistant and frost-hardy plants can be produced. Protective covers - Plants are often covered by earth, straw, or other mulching materials to prevent winter injury. Plastic cloches filled with water are also used. Protective paints and wraps have been used to protect trees. Greenhouses and cold frames provide a controlled environment for sensitive and young plants. Wind machines and heaters - These are used to mix cold air with warm air. The use of such systems is limited to small areas and, with increasing energy costs, is becoming prohibitive. Also, such systems can be used only on a shortterm or emergency basis.

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D. Application of Ice-minus Bacteria The idea behind frost control by ice-minus bacteria is to displace the ice-plus populations in their natural habitat on plants with ice-minus bacteria populations. Since the two types of bacteria are exactly identical except for ice-nucleating ability, the problems of host specificity and adaptation to plant surface do not arise. The ice-minus bacteria are sprayed on plant surfaces before ice-plus populations develop, and grow to a sufficient number to prevent the latter from colonizing the plant surface. Thus, this seems to be a simple and effective means of frost control. Four experiments conducted independently by Advanced Genetic Sciences (AGS) and by Dr. S. Lindow of University of California at Berkeley were carried out to evaluate the dispersal, environmental fate, colonization potential, and competition between ice-plus and ice-minus strains.

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The experiments were performed under actual field conditions with prior approval from the U.S. Environmental Protection Agency. AGS carried out field testing of the ice-minus bacteria in small field trials on 2500 strawberry plants. The results were found to be encouraging and larger field trials were then conducted on 17,400 plants in 1988.84Lindow's tests used potato plants to compare the competitive abilities of ice-minus and ice-plusP. syringae strains. The data on all these tests are a~ailable.~' It was found that blossoms and fruits treated with the ice-minus bacteria had a significantly reduced probability of freezing between - 2.5"Cand - 4.0"C. Lindemann and Suslows6 carried out controlled laboratory tests in order to determine the probability of environmental risk associated with the release of ice-minus P. syringae and P . fzuorescens deletion mutant strains into the environment for frost control applications. The survival of the ice-minus strains in the soil and on the roots of actively growing strawberry plants was first considered. Three weeks after inoculation, populations of the ice-minus strains declined to undetectable levels, while the ice-plus strains on control soil and plants decreased only tenfold. P. syringae and P. jhorescens ice-minus strains were found to grow rapidly on strawberry, almond, pear, cherry, and blackberry blossoms (populations greater than lo7 cfu per blossom). Established populations of ice-plus cells, however, inhibited the ice-minus strains. A number of other variables were also investigated by the authors. These included dissemination during spray inoculation, dissemination by insects, and effects of freezing and thawing on survival of ice-minus strains in vivo. Based on these studies, the authors concluded that the ice-minus deletion mutants expressed no competitive advantage over the ice-plus strain and exhibited only a limited survival capability in the environment.

VIII. APPLICATIONS OF ICE-PLUS BACTERIA

A. Food Industry Applications While ice-minus bacteria are being tested in frost control, researchers are considering alter-

native uses for ice-plus bacteria elsewhere. Watanabe and AraP7 have studied the application of ice-plus Erwinia ananus in the efficient freeze drying of foods. The use of these bacteria to raise supercooling temperatures led to savings in refrigeration cost, shortening of freezing times, and efficient production. Arai and Watanabe23studied the freeze texturing of food materials using ice-plus E. ananans. The bacterial cells, when added to isotropic aqueous dispersions or hydrogels of proteins and polysaccaharides, converted the bulk water into directional ice crystals at subzero temperatures not lower than - 5°C with anisotropically textured products. Raw egg white, bovine blood, soyabean curd, milk curd, and aqueous dispersions or slurries of soyabean protein isolate were tested successfully. The use of ice nuclei in the food industry requires that they be robust and environmentally safe. They must also be nontoxic, nonpathogenic, and palatable. These criteria are best met by preparations in which the ice nuclei have been extracted and purified away from their bacterial source. Cell-free preparations are also likely to be accepted by national regulatory agencies. This involves the development of high levels of ice nucleation protein in the bacteria, then purification and concentration in an active form. Such a development has yet to reach a commercial scale.

6. The Manufacture of Artificial Snow Since its invention in the early 1950s, artificial snow production has been enhancing ski conditions at a growing number of ski areas, and is considered a necessity for many. Most small ski facilities operate 10 to 30 snow guns (nozzles), and larger ski areas may operate hundreds of them. The snow guns are usually run all night, and handle between 10 and 100 gallons per minute of water, along with several hundred standard cubic feet of compressed air. The process of snow making is very energy intensive and even a small improvement in efficiency can translate into considerable savings in energy costs. The snow making process is essentially a spray freezing operation. Water is mixed with compressed air and forced through an internal

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mixing nozzle (snow gun), from which it expands into the ambient air, causing it to atomize and cool simultaneously. Under typical snow making conditions, the cooling provided by the free jet is small compared with the ambient cooling. The role of the compressed air is mainly to atomize the water droplets. The engineering considerations involved in a typical snow making process have been outlined by Liao and Ng.88 Several variables can affect the process. These include water temperature and pressure, flow rate, droplet diameter, ice nuclei present, and nucleation temperature. Chen and KevorkiannYhave identified certain requirements for successful artificial snow manufacture: I.

2. 3.

The water spray must have droplets of cerrain diameter (220 to 700 pm). The water droplets must be seeded with ice nuclei. The droplets should have sufficient residence time in the ambient air to be able to convert to snow.

Recognizing the key role of ice nuclei, Eastman Kodak in collaboration with AGS has patented a product called Snomax (freeze-dried P. syringue) used in artificial snow manufacture.w This product is used for ski slopes and skating rinks and where snow is used as a construction material, e.g., in the Arctic. Kocak et a1.% have patented a process for the manufacture of snow. A schematic diagram is shown in Figure 6. Pure

P. syringue culture is ultra-filtered to form a slurry that is then dipped in liquid nitrogen to form pellets. The pellets are freeze dried and bagged. The product is marketed as Snomax snow inducer. Each pack contains 300 g of material that dissolves in 400 1 of water. The solution is then injected into the snow making water supply. The environmental effects of such snow have reportedly been investigated by the authors, but the results of such investigations have not been described.

C. Other Current and Potential Applications A patent has recently appeared on the application of bacterial ice nuclei in immunoas~ays.~' The use of the INA bacteria in improving the efficiency in air conditioning and refrigeration systems during thermal storage has also been con~ i d e r e dThe . ~ ~ nucleation of ice in such systems at higher temperatures increases system efficiency and reduces energy consumption. This becomes increasingly important with expanding volume and larger loads. A novel and important application that has been developed is the BIND (bacterial ice nucleation assay) test.92This assay is used as a rapid test for food poisoning bacteria. For application to testing for Salmonella, first a Salmonella-specific bacteriophage is incorporated with the gene coding for the ice nucleation protein. The bac-

AIR

D I P P I N EI I N LIQUIC N IT ROGEN

t I

PACK1 NG

FREEZE DRY ING

SNOW f NDUCI NG BACTERIA FIGURE 6.

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Schematic diagram for production of snow-inducing bacteria.80

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warmer temperatures is slower and purer ice crystals are produced.

teriophage reagent is then added to test samples of food that may contain Salmonella, together with a green fluorescent dye. Any Salmonella present are infected with the bacteriophage. The test samples are then incubated at 37°C to allow the formation of the ice nucleation protein. The test samples are chilled to - 5°C. If the bacteria are present, the samples freeze and the green dye becomes dark red and nonfluorescent. Samples that lack the bacteria and control samples without the bacteriophage do not freeze and remain bright green and fluorescent. The total time for the assay is 2 to 6 h compared with currently available Salmonella tests that take 2 to 5 d. Freeze concentration is used in the chemical processing industry as a process for concentrating dilute streams by refrigeration techniques.93 Currently, it is being utilized in treating hazardous wastes, concentrating fruit juices, freezing food, and purifying organic chemicals. These are potential areas where bacterial ice nuclei may be successfully applied.94A flow chart of this potential application is presented in Figure 7. By using bacteria, supercooling to very low temperatures can be prevented and energy costs can be reduced. Supercooling to low temperatures leads to rapid freezing and formation of impure ice crystals that trap the solute. Ice formation at

BACTERIAL I C E L

r

D I L U T E FEED STREAM

V

IX. FUTURE DIRECTIONS Bacterial ice nucleation research is focused in three different directions: (1) the production of efficient frost controllers; (2) the determination of the structure, location, and composition of the ice nucleation site; and (3) the production of efficient ice-nucleating bacteria for various applications, many of which are yet to be discovered. While agricultural scientists are concerned with the first approach, biochemists and genetic engineers are exploring the second area. The third issue may be of interest to biochemical engineers, physicists, and meteorologists. Thus, the various pieces of this puzzle of bacterial ice nucleation are being slowly assembled as more and more knowledge becomes available.

ACKNOWLEDGMENTS This research was supported by the Natural Sciences and Engineering Research Council of Canada through Operating Grant OGP4388 awarded to Dr. A. Margaritis.

CONCENTRATE NUCLEI

FREEZING

4

FILTRATION

MELTING OF I C E CRYSTALS

PURE WATER FIGURE 7. Flow chart for the potential application of bacterialice nuclei in freeze concentration.

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1. Orser, C . , Staskawicz, B. J., Loper, J . ,

Panopoulos, N. J., Dahlbeck, D., Lindow, S. E., and Schroth, M. N., Cloning of genes involved in bacterial ice nucleation and fluorescent pigmenusiderophore production, in Molecular Genetics of Bacterial Plant Interaction, F’iihler, A., Ed., SpringerVerlag, New York, 1983, 353. 2. Lindow, S. E., Biological control of frost injury to tomato with non-ice nucleation active epiphytic bacteria, in Proc. 4th Int. Conf. Plant Pathol., Abstr. 3 . Lindow, S. E., Ecology of Pseudomonas syringae relevant to the field use of ice-minus deletion mutants constructed in vitro for plant frost control, in Engineering Organisms in the Environment: Scientific Issues, Halvorson, H. O . , F’ramer, D., and Rogul, M., Eds., American Society for Microbiology, Washington, D.C., 1985, 23. 4. Warren, G. J., Lindemann, J., Suslow, T. V., and Green, R. L., Ice nucleation deficient bacteria as frost protection agents, in Biotechnology in Agricultural Chemistry, LeBaron, H. M., Mumma, R . O., Honeycutt, R. C., and Duesing, J. H., Eds., American Chemical Society, Washington, D.C., 1987, 215. 5. Van Brunt, J., Environmental release: a portrait of opinion and opposition, Biotechnology, 5(6), 558, 1987. 6. Ice minus tests finally take place, Biotechnol. News, 7(11), 2, 1987. 7. Lamer, V. K., Nucleation in phase transitions, Ind. Eng. Chem., 44(6), 1270, 1952. 8. Fennema, 0.R., Powrie, W. D., and Marth, E. H., Low Temperature Preservation of Foods and Living Matter, Marcel Dekker, New York, 1973, 153. 9. Fletcher, N. H., in The Chemical Physics of Ice, Cambridge Univesity Press, 1970, 85. 10. Turnbull, D. and Vonnegut, B., Nucleation catalysits, I d . Eng. Chem., 44(6), 1292, 1952. 1 1 . Fukuta, N. and Mason, B. J., Epitaxial growth of ice on organic crystals, J . Phys. Chem. Soiids, 715, 1963. 12. Head, R. B., Icenucleation by a-Phenazine, Nature, 196, 736, 1962. 13. Garten, V. A. and Head, R. B., A theoretical basis of ice nucleation by organic crystals, Nature, 205, 160, 1965. 14. Mason, B. J., The growth of snow crystals, Sci. Amer., 204(1), 120, 1961. 15. Head, R. B., Steroids as ice nucleators, Nature, 191, 1058, 1961. 16. Power, B. A. and Power, R. F., Some amino acids as ice nucleators, Nature, 194, 1170, 17. Barthakur, N. and Maybank, J., Anomalous behaviour of some amino acids as ice nucleators, Nature, 200, 866, 1963.

292

18. Kozloff, L. M., Schofield, M. A., and Lute, M., Ice nucleation activity of Pseudomonas syringae and Envinia herbicofa, J . Bacteriol., 153(1), 222, 1983. 19. Kieft, T. L., Ice nucleation activity in lichen, Appl. Environ. Microbiol., 54(7), 1678, 1988. 20. Scott, P. T., Altered and enhanced iodargyrite with bismuth thioiodide for use in precipitation enhancement operations, Gov. Rep. Announce. Index (U.S.) 88(3), 1988. 21. Schnell, R. C. and Vali, G., Atmospheric ice nuclei from decomposing vegetation, Nature, 236, 163, 1973. 22. Maki, L. R., Galyan, E. L., Chang-Chien, M., and Caldwell, D. R., Appl. Microbiol., 28(3), 456, 1974. 23, Arai, S. and Watanabe, M., Freeze texturing of food materals by ice nucleation with the bacterium Envinia ananas, Agric. Biol. Chem., 50(1), 169, 1986. 24. Maki, L. R. and Willoughby, K. J., Bacteria as biogenic sources of freezing nucleu, J. Appl. Meteorol., 17, 1049, 1978. 25. Lindow, S . E., The role of bacterial ice nucleation in frost injury to plants, Annu. Rev. Phytopathol., 241, 363, 1983. 26. Kim, H. K., Orser, C., Lindow, S. E., andsands, D. C., Xanthomonas campestris pv. transluscens strains active in ice nucleation, Plant Dis., 71(1 l), 994, 1987. 27. Orser, C., Staskawicz, B. J., Panopoulus, N. J., Dahlbeck, D., and Lindow, S. E., Cloning and expression of bacterial ice nucleation genes in E. coli, J. Bacteriol., 164(1), 359. 1985. 28. Sands, D. C., Scharen, A. L., Carpenter, E., Caple, J., and Snider, J. R., A hypothetical bioprecipitation cycle involving ice-nucleation and dew condensing bacteria, plants and rainfall, in Proc. 6th Int. Congr. PlantPathol., Civerolo, E. L., Collmer, A., Davis, R. E., and Gillaspie, A. G., Eds., Martinus Nijhoff, The Hague, 1987. 29. Hiram, S. S., Ecology and physiology of Pseudomonas syringae, Biotechnology, 3( 12), 1073, 1987. 30. Hirano, S. S., Nordheim, E. V., Amy, D. C., and Upper, C. D., Lognormal distributions of epiphytic bacterial populations on leaf surfaces, Appl. Environ. Microbiol., 44(3), 605, 1982. 3 1. Breed, R. S., Murray, E. G., and Hitchens, A. P., U s . , Bergey’s Manual of Determinative Bacteriology, 6th ed., Williams & Wilkins, Baltimore, 1948, 189. 32. Dye,D. W.,Bradbury, J. F., Goto, M., Hayward, A. C., Lelliot, R. A., and Schroth, M. N., International standards for naming pathovars of phytopathogenic bacteria and a list of pathovar names and pathotype strains, Rev. Plant Pathol., 59(4), 153, 1980. 33. Atkinson, M. M. and Bakur, J. C., Association of

Critical Reviews in Biotechnology Downloaded from informahealthcare.com by University of Notre Dame Australia on 05/07/13 For personal use only.

host plasma membrane K+/H+ exchange with multiplication of Pseudomonas syringae pv. syringae in Phaseolus vulgaris, Phytopathology, 77(9), 1273, 1987. 34. Seemiiller, E. and Arnold, M., Pathogenicity, syringomycin production and other characteristics of pseudomonad strains isolated from deciduous fruit trees, in Proc. 4th Int. Conf. Plant Path. Bacteria (Station de Pathologie, Eds.), 703. 35. Haefele, D. M. and Lindow, S. E., Flagellar motility confers epiphytic fitness advantages upon Pseudomonas syringae, Appl. Environ. Microbiol., 53(lo), 2528, 1987. 36, Lindemann, J., Constandine, H. A., Barchet, W. R., and Upper, C. D., Plants as sources of airborne bacteria including ice nucleation bacteria, Appl. Environ. Microbiol., 44, 1059, 1982. 37. Neegaard, P., Seeds, Vols. I and 11, Macmillan, London, 1977. 38. Lindow, S. E., Amy, D. C., and Upper, C. D., Distribution of ice nucleation active bacteria on plants in nature, Appl. Environ. Microbiol., 36, 831, 1978. 39. Orser, C. S., Lotstein, R., Lahue, E., Willis, D. K., and Panopoulos, N. J., Structural and functional analysis of Pseudomonas syringae pv. syringae ice region and construction of ice deletion mutants, Phytopathology, 74, (Abstr.), 798, 1984. 40.Cornto, L. V., Wolber, P. K., and Warren, G. J., Ice nucleation activity of Pseudomonas jluorescens: mutagenesis, complementation analysis and identification of a gene product, EMBO J., 5, 231, 1986. 41. Green, R. L. and Warren, G. J., Physical and functional repetition in a bacterial ice nucleation gene, Nature, 317, 645, 1985. 42. Warren, G. J., Corotto, L., and Wobler, P., Nucl. Acids Res., 14, 8047, 1986. 43. Warren, G., Wolber, P., and Green, R., Functional significance of oligonucleotide repeats in a bacterial ice nucleation gene, in Proc. 6th Int. Conf. Plant Pathol. Bacteria, Civerolo, E . L . , Collmer, A., Davis, R. E., and Gillaspie,A. G., U s . , Martinius Nijhoff, The Hague, 1987, 1013. 44. Phelps, P., The Expression of Ice Nucleation Activity in Bacteria and the Structure of the Ice Nucleation Site, Ph.D. thesis, University of Colorado, Boulder, 1987. 45. Zhao, J. L. and Orser, C. S., Conserved repetition in the ice nucleation gene inaX from Xanthomonas campestris pv. translucens, Mol. Gen. Genet., 223(1), 163, 1990. 46. Deninger, C. A., MueUer, G. D., and Wolber, P. K., Immunological characterization of ice nucleation proteins from Pseudomonas syringae, Pseudomonasjluorescens. and Erwinia herbicola, J. Bacteriol., 170(2), 669, 1988. 47. Wolber, P. and Warren, G. J., Bacterial ice nucleation proteins, T I M , 14, 179, 1989. 48. Green, R. L., Corotto, L. V., and Warren, G. J.,

Deletion mutagenesis of the ice nucleation gene from Pseudomonas syringae S203, Mol. Gen. Genet., 215(1), 165, 1988. 49. Wolber, P. K. and Warren, G. J., Structural modelling of the ice nucleation protein of Pseudomonas syringae, Biophys. J . , 49 (Abstr.), 293a, 1986. 50. Kozloff, L. M., Lute, M., and Westway, D., Phosphatidylinositol as a component of the ice nucleating site of pseudomonas syringae and Erwinia herbicola, Science, 226, 845, 1984. 51. Govindarajan, A. G. and Lindow, S. E., Phospholipid requirements for expression of ice nuclei in bacterial membranes in vitro, Plant Physiol., 75 (Abstr.), 143, 1984. 52. Govindarajan, A. G. and Lindow, S. E., Phospholipid requirements for expression of ice nuclei in bacterial membranes in vitro, J. Biol. Chern., 263(9), 9333, 1988. 53. Low, M. G., glycosyl-phosphatidylinositol:a versatile anchor for cell surface proteins, FASEB J., 3, 1600, 1989. 54. Sprang, M. L. and Lindow, S. E., Subcellular localization and partial characterization of ice nucleation activity of Pseudomonas syringae and Erwinia herbicola, Phytopathology, 71 (Abstr.), 256, 1981. 55. Phelps, P., Giddings, T. H., Prochoda, M., and Fall, R., Release of cell free ice nuclei by Erwinia herbicola, J. Bacteriol., 167, 496, 1986. 56. Lindow, S. E., Lahue, E., Govindarajan, A. G., Panapoulos, N. J., and Gies, D., Localization of ice nucleation activity and the iceC gene product in Pseudomonas syringae and Escherichia coli, Mol. Plant Microb. Interact., 2(5), 262, 1989. 57. Govindarajan, A. G. and Lindow, S. E., Size of bacterial ice nucleation sites measured in situ by radiation inactivation analysis, Proc. Natl. Acad. Sci. U.S.A., 85, 1334, 1988. 58. ThangaraJ,K., Palanisamy, M., Gobinathan, R., and Ramasamy, P., Substrate foreign atoms and ice nucleation activity, J. Colloid. tntegace Sci., 126(2), 463, 1988. 59. Mimno, H., Prediction of the conformation of ice nucleation protein by conformal energy calculations, Proteins Struct. Function Genet., 5(1), 47, 1989. 60.Burke, M. J. and Lindow, S. E., Surface properties and size of the ice nucleation site in ice nucleation active bacteria: theoretical considerations, Cryobiology, 26, 80, 1990. 61. Wolber, P. K., Deininger, C. A., Southworth, M. W., Vandeker-chove, J., Montagu, M. V., and Warren, G. J., Identification and purification of a bacterial ice nucleation protein, Proc. Natl. Acad. Sci. U.S.A., 83, 7256, 1986. 62. Duman, J. G., Morris, J. P., and Castellino, F. J., Purification and composition of the ice nucleation protein from queens of the hornet Vespula masculata, J. Comp. Physiol. Biol., 154, 79, 1984. 63. Bassi, A. S. and Margaritis, A,, Kinetics of growth

293

Critical Reviews in Biotechnology Downloaded from informahealthcare.com by University of Notre Dame Australia on 05/07/13 For personal use only.

and ice nucleation activity of Pseudomonas syringae, University of Western Ontario, London, Canada, in preparation. 64. Vali, G., quantitative evaluation of experimental results on the heterogeneous freezing nucleation of supercooled liquids, J. Atmos. Sci., 28, 402, 1971. 65. Valie, G. and Stansbury, E. J., Time dependent characteristics of the heterogeneous nucleation of ice, Can. J. Phys., 44,477, 1966. 66. Obata, H., Nakai, T., Tanashita, J., and Tokuyama, T., Identification of an ice nucleating bacterium and its ice nucleation properties, J . Fermentation Bioeng., 67(3), 143, 1989. 67. Stewert, W. E., Jr. and Bear, L. L., Concentration effects of ice nucleation active bacteria on the water nucleation temperature, in Proc. 23rd Intersoc. Energy Convers. Eng. Conf., Vol. 2, Goswami, D. Y., Ed., American Society of Mechanical Engineers, New York, 1988, 147. 68. Hendricks, D. M., Orrego, S. A., and Ward, P. J., Production of microorganisms with high ice nucleating activity, European Patent 0261624, 1988. 69. Obata, H., Saeki, Y., Tanishita, J., and Tokiyama, T., Ice nucleating activity of Pseudornonas fluorescens, J . Fermentation Technol., 65(6), 693, 1987. 70. Rogers, J. S., Stall, R. E., and Burke, M. J., Low temperature conditioning of the ice nucleation active bacterium Erwinia herbicola, Cryobiology, 24, 270, 1987. 71. Lynn, S. Y. and Noto, G. D., Fermentation of Microorganisms Having High Ice Nucleating Activity Using a Defined Medium, European Patent EP 0272669, 1988. 72. Watanabe, M., Watabe, S., and Soichi, A., Interaction of an anti-nucleating chemical and an ice nucleating bacterium: a case study with n-octylbenzyldimethyl ammonium salt and Erwinia ananas, Agric. Biol. Chem., 52(8), 1869, 1988. 73. Obata, H., Mariguchi, Y., and Tokiyama, T., Reactive species responsible for inactivating the ice nucleating activity of Pseudomonasfluorescens KUIN1 by an ascorbic acid-copper (11) ion system, Agric. Biol. Chem., 52(8), 1869, 1988. 74. O’Brien, R. D. and Lindow, S. E., Effect of plant species and environmental conditions on ice nucleation activity of Pseudomonas syringae on leaves, Appl. Environ. Microbiol., 54(9), 228 1, 1988. 75. Lmdow, S, E., Lack of correlation of in vitro antibiosis with antagonism of ice nucleation active bacteria on leaf surfaces by non-ice nucleation active bacteria, Phyropathology, 78(4), 444, 1988. 76. Suslow, T. V., Lindow, S. E., and Dix, H., Enhancement of snow production by ice nucleation active bacteria in air:water systems, in Proc. 6th Int. Conf. Plant Path., Bacteria, Civerolo, E. L.,Collmer, A., Davis, R. E., and Gillaspie, A. G., Eds., Martinus Nijhoff, The Hague, 1987,

294

77. Brown, D. M. and Blackburn, W. J., Impacts of freezing temperatures on crop production in Canada, Can. J. Plant Sci., 67, 1167, 1987. 78. Quamme, H. A., Low temperature stress in Canadian horticultural production - an overview, Can. J. Plant Sci., 67, 1135, 1987. 79. Andrews, C. J., Low temperature stress in field and forage crop production -an overview, Can. J. Plant Sci., 67, 1121, 1987. 80. White, G. F. and Haas, J. E., Assessment ofResearch on Natural Hazards, MIT Press, Cambridge, 1975, 304. 81. Gusta, L. V. and O’Connor, B. J., Frost tolerance of wheat, oats, barley, canola, mustard and the role of ice nucleating bacteria, Can. .I. Plant Sci., 67, 1155, 1987. 82. Porody-Morreale, A., Murphy, K. P., DiCera, E., Fall,R., DeVries, A. L., and Gill,S. J., Inhibition of bacterial ice nucleators by fish anti-freeze glycoproteins, Nature, 333, 782, 1988. 83. Cody, Y. S., Gross, D. C., Proebsting, E. L., Jr., and Spotts, R. A., Suppression of ice nucleation active Pseudomonas syringae by antagonistic bacteria in fruit tree orchards and evaluations of frost control, Phytopathology, 77(7), 1036, 1987. 84. Field tests conducted, Biofechnol. News, 8(1), 1988. 85. U.S. Environmental Protection Agency Experimental Use Permit Documtents, Ref. nos. 55269-EUP-2, 54306-EUD-1, and 54306-EUP-2, 1988. 86. Lindemann, J. and Suslow, T. V., Characteristics relevant to the question of environmental fate of genetically engineered INA deletion mutant strain of Pseudomonas, in Proc. 6th Int. Con! Plant Path. Bacteria, Civerolo, E. L., Collmer, A., Davis, R. E., and Gillaspie, A. G., Eds., Martinus Nijhoff, The Hague, 1987. 87. Watanabe, M. and Arai, S., Freezing of water in the presence of the ice nucleation active bacterium Erwinia ananas and its application for the efficient freeze drying of food, Agric. Biol. Chem., 51(2), 557, 1987. 88. Liao, J. C. and Ng, K. C., Effect of ice nucleators on snow making and spray freezing. Ind. Eng. Chem. Res., 29, 361, 1990. 89. Chen, J. and Kevorkian, V., Heat and mass transfer in making snow, Id.Eng. Chem. Process Design Dev., 10(1), 75, 1971. 90.Kocak, R. and Van Gemert, H., Man-made snow: biotechnology assisting the snow making industry, J. Biotechnol., 2(1), 37, 1988. 91. Warren, G. J. and Wolber, P. K., Ice Nucleation Fluorescence Immunoassay Kits Using Bacterial Ice Nucleation Proteins, U.S. patent 4,784,943, 1989. 92. Worthy, W., Bacterial assay exploits ice nucleation research, Chem. Eng. News, 23. 93. Chowdhury, J., CPI warms up to freeze concentration, Chem. Eng. News, 24.

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94. Ryder, J. M., Biogenic Ice Nucleation in the Freez-

ing of Fish, Ph.D thesis, University of Rhode Island, Kingston, 1987.

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Principles and biotechnological applications of bacterial ice nucleation.

Certain aerobic, Gram-negative bacteria, including the epiphytic plant pathogen, Pseudomonas syringae, possess a membrane protein that enables them to...
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