Molecular sizes of lichen ice nucleation sites determined by gamma radiation inactivation analysis

CRYOBIOLOGY

29, 407-413 (1992)

Molecular

Sizes of Lichen Ice Nucleation Gamma Radiation Inactivation

Sites Determined Analysis’

by

THOMAS L. KIEFT’ AND TRACY RUSCETT13 Department

of Biology,

New Mexico

Institute

of Mining and Technology,

Socorro,

New Mexico 87801

It has previously been shown that some species of lichen fungi contain proteinaceous ice nuclei which are active at temperatures as warm as - 2°C. This experiment was undertaken to determine the molecular sizes of ice nuclei in the lichen fungus Rhizopluca chrysoleuca and to compare them to bacterial ice nuclei from Pseudomonas syringae. Gamma radiation inactivation analysis was used to determine molecular weights. Radiation inactivation analysis is based on target theory, which states that the likelihood of a molecule being inactivated by gamma rays increases as its size increases. Three different sources of ice nuclei from the lichen R. chrysoleuca were tested: field-collected lichens, extract of lichen fungus, and a pure culture of the fungus R. chrysoleuca. P. syringae strain Cit7 was used as a source of bacterial ice nuclei. Samples were lyophilized, irradiated with gamma doses ranging from 0 to 10.4 Mrads, and then tested for ice nucleation activity using a droplet-freezing assay. Data for all four types of samples were in rough agreement; sizes of nucleation sites increased logarithmically with increasing temperatures of ice nucleation activity. Molecular weights of nucleation sites active between -3 and -4°C from the bacteria and from the field-collected lichens were approximately 1.0 x lo6 Da. Nuclei from the lichen fungus and in the lichen extract appeared to be slightly smaller but followed the same log-normal pattern with temperature of ice nucleation activity. The data for both the bacterial and lichen ice nuclei are in agreement with ice nucleation theory which states that the size of ice nucleation sites increases logarithmically as the temperature of nucleation increases linearly. This suggests that although some differences exist between bacterial and lichen ice nucleation sites, their molecular sizes are quite similar. o 1992 Academic press, IIIC.

The ability to initiate freezing of supercooled water at relatively warm temperatures has been found in a number of biological systems, including bacteria (17, 19), insects (3, 25), amphibians (22), and lichens (12-14). Bacterial ice nuclei, which have been the most thoroughly investigated of the biological ice nucleating agents, occur in a few species of gram-negative bacteria,

including Pseudomonas syringae, Pseudomonas fluorescens, and Erwinia herbicola.

The bacterial nuclei have been found to be proteins located in the gram-negative outer membrane (18); lipid and carbohydrate components have also been reported (15, 16). Warm-temperature ice nucleation in P. syringae is destroyed by removal of membrane phospholipids (6). Molecular weight measurement by gamma radiation inactivation analysis (7) has demonstrated that the Received August 8, 1991; accepted October 7, 1991. temperature of activity of ice nucleation sites in P. syringae is directly related to the i We thank Steve Lindow and Gabor Vali for helpful discussions. We thank Ron Garcia of the Gamma Irsize of the sites. Bacterial ice nucleation radiation Facility, Sandia National Laboratory for sites which were active at the warmest temtechnical assistance. We also thank Robert Y. Lowrey and Sam Mares of the Department of Energy for use of peratures (-2 to - 3°C) had molecular the Gamma Irradiation Facility. Supported in part by weights of 8000 kDa, whereas nuclei active at the lowest temperatures (- 12 to - 13°C) the Bio-Products Division of Eastman Kodak. ’ To whom correspondence should be addressed. had molecular weights of approximately 3 Present address: Department of Microbiology and 150 kDa. This lower size limit also correImmunology, Louisiana State University Medical sponds to the estimated size for the ice nuCenter, Shreveport, LA 71130. 407 001l-2240/92 $5.00 Copyright Q 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

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cleation protein monomer of P. syringae (24). A similar ice nucleation protein of 180 kDa has been identified in a Pseudomonas fluorescent (2). Larger nucleation sites are thought to result from aggregation of protein monomers (7, 8, 24). These findings also conform to ice nucleation theory, which states that only large molecules can serve as templates for the ordering of liquid water molecules into crystalline lattices (ice) at warm temperatures (4, 20). Biological ice nucleation activity has been found in a number of lichens; the warmest nucleation so far observed has been in R. chrysoleuca (12, 13). Warmtemperature lichen ice nucleation sites are produced by the fungal component of the lichen symbiosis (12, 13). The lichen ice nuclei share characteristics with the bacterial ice nuclei in that they are proteinaceous (14) and they are active at temperatures as warm as -2°C (12). However, they differ from bacterial ice nuclei in being more temperature-stable, active over a wider pH range, and active in the absence of membrane lipids (14). The selective advantage conferred by ice nucleation activity to lichens has not been determined; however, ice nucleation may enhance moisture uptake from the atmosphere (12, 14). This study was conducted to compare the molecular sizes of lichen and bacterial ice nucleation sites and to determine the sizes of these sites in relation to the temperatures at which they initiate freezing of supercooled water. The gamma radiation method of molecular size determination is based on radiation target theory and has been used for many membrane proteins, including bacterial ice nuclei (5, 7, 9-11, 21). It was selected over more traditional methods of molecular weight determination because lichen ice nuclei have not yet been isolated and they are produced by the lichen fungi in low amounts relative to other fungal proteins. The radiation inactivation method also has the advantage of measuring molecular sizes of macromolecules in situ, i.e., in

RUSCETTI

the cellular form in which they occur in nature. Ice nuclei are particularly well suited to gamma irradiation inactivation analysis because their activity can be easily quantilied. MATERIALS

AND

METHODS

Lichens. Three different sources of R. chrysoleuca lichen nuclei were prepared for

gamma radiation analysis: (a) a homogenate of field-collected lichens, (b) a crude extract of field-collected lichens, and (c) an axenic culture of the lichen fungus R. chrysoleuca. Lichen samples were collected on May 24, 1989, from the surface of rhyolytic rocks on South Baldy Mountain in the Magdalena Mountains of central New Mexico. They were sealed in plastic bags and stored at - 20°C prior to analysis. Fieldcollected lichens were crushed and homogenized in a mortar and pestle and crude extracts were prepared from a portion of this lichen material by the method of Kieft and Ruscetti (14). Crushed lichen material was added to distilled water to make a slurry which was then sonicated on ice (5-7 min in 30-s bursts at 70 W output) and centrifuged (27,000g for 10 min). The supernatant was decanted and stored at - 20°C. An axenic culture of R. chrysoleuca, provided by Vernon Ahmadjian of Clark University, was cultured at 17°C in Lilly and Bamett’s medium containing 10 g glucose, 2.0 g proline, 1.0 g KH,PO,, 0.5 g MgSO, . H,O, 0.2 mg Fe(NO& .9H,O, 0.2 mg ZnSO,, =4H,O, 100 kg thiamine, and 5 mg biotin 1-l H,O (1). All three types of lichen material were frozen at -50°C in a shell freezer (Labconco, Kansas City, MO) and then lyophilized to remove all water prior to irradiation. Bacteria. An ice nucleating bacterium, P. syringae strain Cit7, provided by Dr. Steven Lindow, University of California, was cultured at 20°C on tryptic soy agar (Baltimore Biological Laboratories, Baltimore, MD) for 5 days. Bacterial cells were

SIZES OF LICHEN

ICE NUCLEATION

scraped from the agar surface with a spatula, frozen at -50°C and lyophilized. Gamma irradiation analysis. Aliquots of lyophilized samples were sealed in screwcap polycarbonate cryotubes (12 x 60 mm). Amounts per cryotube were approximately 35 mg for the homogenized lichen, 10 mg for the lichen extract, 3 mg for the lichen fungus, and 15 mg for the bacteria. Cryotubes were purged with N, before sealing. Gamma irradiation was carried out at the gamma irradiation facility of Sandia National Laboratory. Sealed lyophilized samples were transported on dry ice to this facility. Gamma irradiation took place at 22°C. The gamma source was approximately 125 kCi of 6oCo in rods which surrounded the irradiation chamber. The flux of radiation was 38.6 krad min- ‘. Samples were exposed for lengths of time ranging from 0 to 270 min, resulting in gamma irradiation doses of 0 to 10.4 Mrads. The dosage was monitored by thermoluminescence dosimetry using CaF,. All samples, including the unirradiated controls, were held at 22°C for the same time period (270 min) regardless of the radiation dose. After irradiation, samples were transported to New Mexico Tech on dry ice and stored at -70°C prior to analysis of ice nucleation activity. Ice nucleation analysis. Ice nucleation activity was quantified by the dropletfreezing assay of Vali (23). Lichen material or bacteria were suspended in deionizeddistilled H,O. Serial lo-fold dilutions of these suspensions were made in deionizeddistilled H,O. Each dilution was placed in twenty lo-p1 droplets on the surface of a paraffin-coated aluminum cold plate. The temperature of the cold plate was lowered at a rate of 0.3”C min- ’ while the number of drops frozen was recorded. The cumulative number of nuclei (N) active at a particular temperature (Z’) was calculated from the equation (23) N(T) = [--ln(1 - f)]l(V

x

D),

[II

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wheref = the proportion of drops frozen, V = the volume of each droplet, and D = the factor by which the material was diluted. The cumulative number of nuclei per gram of material vs temperature of activity (ice nucleation spectrum) was plotted. A frequency distribution of nuclei active at various 1” intervals of temperature was also calculated for each nucleus preparation. For example, the nuclei which were active at -4°C c T < - 3°C were summed and labeled as the -4°C interval; the same procedure was used for each 1” temperature interval from - 4°C through - 9°C. Calculation of nucleation site molecular weights. The log of the number of nuclei

active within each 1” temperature interval was plotted against the radiation dose. According to radiation target theory, the steeper the negative slope of this inactivation curve, the larger the target site. The molecular weights of the nucleation sites were calculated using the equation (11) M = 6.4 x lO?D,,,

PI

where M = the molecular weight of the target site in daltons and D,, = the dose in Mrad which results in 37% of the molecules remaining active. A linear regression was calculated for the irradiation inactivation curve of log nuclei gg ’ vs radiation dose for each of the 1” temperature intervals. Using these linear regressions, D,, was interpolated as the dose resulting in 37% of the Y-intercept value for nuclei per grams. Equation [2] was then used to solve for the molecular weight of the nucleation sites in each 1” temperature interval. RESULTS

Each of the preparations showed high concentrations of warm-temperature ice nuclei (Fig. 1). The warmest temperature of ice nucleation activity was observed in the P. syringae cells (-2.5”(Z). The warmest ice nucleation activities detected in the lichen preparations ranged from - 3.3 to - 4.1”C. The highest concentrations per

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FIG. 1. Ice nucleation spectra (i.e., cumulative concentration of ice nuclei as temperature decreases) for unirradiated (0) P. syringae strain Cit7 bacterial cells, (A) field-collected homogenized R. chrysoleuca lichen, (A) cells of the lichen fungus R. chrysoleuca cultured axe&ally, and (0) a crude extract of the lichen R. chrysoleuca.

gram of material were found in the fieldcollected lichen and in the lichen extract. The lowest concentration of nuclei among the four preparations was found in the axenic culture of the lichen fungus. The bacterial spectrum for P. syringae Cit7 was similar to those previously reported (7). The frequency distribution of the lichen homogenate ice nuclei (Fig. 2) showed a single mode, unlike the bimodal distribution commonly observed in P. syringae (7) and also observed in P. syringae Cit7 in this experiment . Ice nucleation activity was sensitive to gamma irradiation in all four sample types. Typical values for the concentrations of ice

RUSCETTI

nuclei vs gamma radiation dose are shown for the lichen homogenate for three temperature intervals in Fig. 3. The negative slopes of concentrations vs dose increased in steepness with increasing temperatures of ice nucleation activity. The nucleation sites which were active at the warmest temperatures were thus the most likely to be inactivated by gamma irradiation and thus had the greatest size. The molecular sizes of the nucleation sites for each of the sample types showed a log-normal pattern (Fig. 4) with the warmest temperature nuclei having the largest molecular size. The molecular size of the nucleation sites increased logarithmically as the temperature of ice nucleation activity increased linearly. This conforms IL) the pattern previously reported for P. syringae Cit7 (7). Regression equations for these lognormal relationships were y = 0.170x + 6.65 (r2 = 0.869) for P. syringue, y = 0.177x + 6.49 (2 = 0.442) for the lichen homogenate, y = 0.187x + 6.41 (r2 = 0.941) for the lichen fungus, and y = 0.132~ + 5.93 (3 = 0.417) for the lichen extract, where x = temperature (“C) and y = log molecular weight (Da). One can conclude from these regressions that the ice nuclei measured in the bacteria were about 45%

Dose -14

-12

-10

-6

Temperature

-6

-4

-2

(‘C)

FIG. 2. Frequency distribution of ice nuclei in 1°C increments of the temperatures of activity for the unirradiated field-collected homogenized sample of the lichen R. chrysoleuca. Each temperature interval (7) contains all of the ice nuclei which initiated freezing of water within the interval ZT to T + 1°C.

(MRad)

FIG. 3. Concentration of ice nuclei as a function of

gamma radiation dose for three temperature intervals (- 3, - 4 and - 5°C) for the field-collected homogenized lichen R. chrysoleuca. For each temperature interval, the slope of the regression line was used to determine D,,, the dose which diminished the concentration of active nuclei to 37% of the original unirradiated nucleus concentration (the Y-intercept). 0, -4°C; 0, -5°C; and A, -6°C.

SIZES OF LICHEN ICE NUCLEATION

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tured on tryptic soy agar for 5 days at 20°C whereas Govindarajan and Lindow grew their cells on a mannitol/aspartic acid agar for 3 days at 24°C. Variations in growth conditions are known to intluence the expression of ice nucleation activity in bacteria; e.g., colder incubations favor 4-1 I ! I I greater production of warm-temperature -10 -9 -8 -7 -6 -5 -4 -3 Temperature (“C) ice nuclei (17). Culture conditions may also FIG. 4. Molecular masses (Da) of the ice nucleation influence the efficiency with which the nusites in (0) P. syringae strain Cit7 bacterial cells, (A) clei function. Differences in conformation field-collected homogenized R. chrysoleuca lichen, of ice nucleation proteins and cell mem(A) cells of the lichen fungus R. chrysoleuca cultured axenically, and (0) a crude extract of the lichen R. branes may account for the inequalities in chrysoleuca. The molecular masses were determined molecular weights between the two experifor each 1°C temperature interval. ments. Differences in growth conditions and also larger than the homogenized field-collected in preparation of the material prior to lichen nuclei over the full range of temper- gamma irradiation may also account for atures of ice nucleation activity. The actual some of the slight disparities observed in masses determined for the nuclei from P. nucleation sites among the three different syringae Cit7 and from the homogenized, types of R. chrysoleuca nuclei. The regresfield-collected R. chrysoleuca (each active sion for the ice nuclei indicated that the between - 3 and - 4°C) were 1.6 x lo6 Da smallest masses among the four types of and 1.0 x lo6 Da, respectively. sample preparations were found in the lichen extract. It may be that the extract conDISCUSSION tains nuclei which have little if any associDespite the known differences in ice nu- ated cellular material. They could then be clei between lichens and bacteria (12, 14), effective ice nuclei with lower susceptibilthe sizes of the nuclei in both types of or- ity to radiation inactivation than the nucleganisms are similar for any given tempera- ation sites in the other sample preparations. ture range. The lichen ice nuclei thus con- The fact that the lichen homogenate and the form to ice nucleation theory which states crude lichen extract each contained apthat warm-temperature ice nucleation re- proximately the same concentration of nuquires a relatively large ice nucleation site, clei per gram of material indicates that the i.e., a molecule with a large radius of cur- extraction procedure is not an efficient one. vature (4, 20). A large proportion of the nuclei remains asAlthough the data reported here on the sociated with the particulate material which bacterial ice nuclei agree with the general is removed as the centrifugation pellet pattern of molecular size vs temperature of (Kieft and Ruscetti, unpublished data). The activity previously reported by Govindara- relatively low concentrations of ice nuclei jan and Lindow (7), the actual masses mea- observed in the lichen fungus (mycobiont) sured in this study were smaller by a factor are consistent with values previously meaof approximately three. The bacteria in our sured (13). The lower concentration of nuexperiment also differed in having a higher clei in the axenic mycobiont culture may be concentration of nuclei per g of cells. These explained by two factors: the mycobiont differences may be a result of different cul- was not isolated from the same fieldture conditions used for P. syringae Cit7 in collected lichen, and it is impossible to culour laboratory. Our bacterial cells were cul- ture the mycobiont under conditions which

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are identical to in situ conditions in the lichen symbiosis. In situ conditions appear to favor greater production of ice nuclei. Conditions for optimizing expression of ice nucleation activity in the lichen mycobionts have yet to be determined; however, lower incubation temperatures appear to promote ice nucleation activity. The agreement in masses of ice nucleation sites between the lichen fungus and the homogenized, fieldcollected lichen suggests that the in situ conformations of the nucleation sites are similar in the two sample preparations. The log-normal relationship between molecular size and temperature of ice nucleation activity is consistent with a model for the nucleation sites in which small proteinaceous subunits which are active only at low temperature aggregate to form large, warm-temperature ice nuclei. Such a scheme has been proposed for the bacterial ice nuclei (7,8,24). The sensitivity of lichen ice nuclei to agents which destroy tertiary and quaternary protein structure in proteins (14) also supports this model. However, much more research is needed to test the theory in lichens. Although ice nucleation activity presumably arose separately in such diverse taxonomic groups as bacteria, fungi, insects, and amphibians, convergent evolution may have created structurally similar ice nucleation sites. Certain features may be required for effective warm-temperature ice nucleation, regardless of the cellular milieu or antecedent molecules. These required features might include proteins with repeating sequences of hydrophilic amino acids (8, 24) and aggregation of protein subunits to make large, warm-temperature ice nuclei. Detailed structural analysis of the lichen nucleation sites, as well as those in other eucaryotes, is needed to test this hypothesis. The data presented here provide further support for existing ideas regarding the nature of biological ice nucleation activity, particularly regarding the molecular sizes of ice nucleation sites.

RUSCETTI REFERENCES

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