Proc. Nati. Acad. Sci. USAVol. 85, pp. 1334-1338, March 1988Biochemistry

Size of bacterial ice-nucleation sites measured in situ by radiationinactivation analysis

(Pseudomonas syringae/Erwinia herbicola/kce gene/y radiation)

AREPURA G. GOVINDARAJAN AND STEVEN E. LINDOWDepartment of Plant Pathology, University of California, Berkeley, CA 94720

Communicated by Luis Sequeira, September 28, 1987

ABSTRACT Four bacterial species are known to catalyzeice formation at temperatures just below 00C. To betterunderstand the relationship between the molecular structureof bacterial ice-nucleation site(s) and the quantitative andqualitative features of the ice-nucleation-active phenotype, wedetermined by vrradiation analysis the in situ size of ice-nucleation sites in strains of Pseudomonas syringae and Erwi-nWa herbicola and in Escherchia coli HB101 carrying theplasmid pICE1.1 (containing a 4-kilobase DNA insert from P.syringae that confers ice-nucleation activity). Lyophilized cellsof each bacterial strain were irradiated with a flux of yradiation from 0 to 10.2 Mrad (1 Mrad = 106 J/kg).Differential concentrations of active ice nuclei decreased as afirst-order function of radiation dose in all strains as temper-ature was decreased from - 20C to -14C in PC intervals.Sizes of ice nuclei were calculated from the Vrradiation flux atwhich 37% of initial ice nuclei active within each 10C temper-ature interval remained. The minimum mass of a functionalice nucleus, active only between -r12c and - 130C, was about150 kDa for all strains. The size of ice nuclei increasedlogarithmically with increasing temperature from -12C to-r2C, where the estimated nucleant mass was 19,000 kDa.The ice nucleant in these three bacterial species may representan oligomeric structure, composed at least in part of an icegene product that can self-associate to assume many possiblesizes.

Four species of bacteria are known to act as catalysts for iceformation in aqueous solutions at temperatures only slightlybelow 0C. Several pathovars ofPseudomonas syringae VanHall and some strains of Erwinia herbicola (Lohnis) Dye,Pseudomonas fluorescens biotype G (Migula), and Pseu-domonas viridiflava are the most active sources of biologi-cally derived ice nuclei yet discovered (1-6). These ice-nucleation-active (INA) bacteria are found on plant surfacesand other habitats throughout North America, Europe, andAsia (2, 7, 8). The presence of INA bacteria on leaves andother plant parts prevents water on and within frost-sensitiveplants from supercooling, thus leading to internal ice forma-tion and frost damage (3, 7, 9). It has been hypothesized thatby virtue of their ice-nucleation capability, these bacteriamay play a role in global climatology, serving as atmosphericfreezing nuclei important in precipitation processes (10, 11).

Physiological and biochemical investigations have shownthat the ice-nucleation site of INA bacteria is proteinaceousand preferentially localized in the outer membrane (12).Lipids and other membrane components, while not sufficientto serve as a nucleating site, may be a part of or act tostabilize the active catalytic site (13-15). A cloned 4-kilobaseDNA sequence from the P. syringae ice region confers ice-nucleation activity on Escherichia coli (16). A single large

protein product (ca. 150 kDa) is apparently encoded by theice region of P. syringae (17, 18).

Several workers have employed radiation inactivationanalysis for the estimation of in situ molecular size ofmembrane bound enzymes and other proteins and macro-molecules (19, 20), solubilized proteins (21), and proteinsreconstituted into liposomes (22). This method of size esti-mation is based on classical radiation target theory (23),which relates the loss of biochemical or biological activitywith increasing radiation exposure. The molecular mass ofassayable target molecules is estimated from the rate ofexponential decrease of biological activity with radiationdose absorbed. The advantage of this method of molecularmass estimation is that membrane-bound enzymes or otherproteins need not be solubilized or purified and can bestudied in situ in lyophilized cell preparations or in frozenpure or crude membrane preparations (19, 20). Radiationinactivation analysis is particularly attractive for estimationof membrane-protein size, as it can give information on thetertiary structural organization of the macromolecule fromfunctional studies in situ (20).The structure and organization of bacterial ice-nucleation

sites with activity at different temperatures are unknown.The Ice phenotype is stable during lyophilization of intactcells and is readily quantified. The frequency with which abacterial cell contains an active ice nucleus is less than 10- 1to 10-7. Thus, few if any cells contain more than one icenucleus. Ice-nucleation sites of these cells are thereforeamenable to size estimations by v-radiation target-size anal-ysis. Here we report the estimated size of the ice-nucleationsites active at different catalytic temperatures in INA bac-teria P. syringae and E. herbicola and in E. coli carrying thecloned P. syringae ice gene.

MATERIALS AND METHODSGrowth and Preparation of Bacterial Cells. The origin and

characteristics of P. syringae strains 31R1 and Cit7, E.herbicola strain 26SR6-2, and E. coli strain HB101 contain-ing the cloned P. syringae ice region on the multicopyplasmid pBR325 (pICE1.1) have been reported (1, 3, 16). Allstrains were grown at 240C for 3 days on mannitol/asparticacid/agar plates [containing mannitol (10 g/liter), L-asparagine (5 g/liter), NaNH4HPO4'4H20 (1.7 g/liter),MgSO4 (0.1 g/liter), NaH2PO4 (7.5 g/liter), KCl (0.75g/liter), and agar (15 g/liter) at pH 6.8] or, when radiationsensitivity of frozen and lyophilized cells was compared, onnutrient agar amended with 2.5% (vol/vol) glycerol. Cellswere removed from the agar surface with a spatula andwashed in 0.1 M potassium phosphate buffer (pH 6.8), andthe cell paste was quickly frozen in liquid nitrogen, lyophi-lized, and stored dry at - 200C. Some cells were alsosuspended in 0.1 M potassium phosphate buffer at a concen-

Abbreviation: INA, ice-nucleation-active.

1334

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Natl. Acad. Sci. USA 85 (1988) 1335

tration of 1010 cells per ml, and 200-Al aliquots were placedin microcentrifuge tubes, frozen quickly in liquid nitrogen,and stored at - 80'C.Sample Preparation and Irradiation. Fifty to 100 mg of

lyophilized bacterial cells were dispensed into 1.5-ml color-less Eppendorf plastic microcentrifuge tubes. The sampleswere dried overnight under vacuum, flushed with nitrogen,and sealed. The microcentrifuge tubes were arranged insidea metallic sample holder enclosed in a Gammacell model 2irradiator held at 22 + 20C. The sample holder was rotatedslowly on a support to 'ensure isodose treatment of thesamples. The 6OCo source, calibrated by Fricke (ferroussulfate) dosimetry (24), generated 0.85 Mrad/hr (1 Mrad =106J/kg) with an energy of 1.25 MeV (1 MeV = 1.6 x 10-13J). A comparison of radiation sensitivity of lyophilized andfrozen-hydrated cells was also performed. Samples of lyoph-ilized and frozen samples prepared as above were placed ininsulated polypropylene cylinders packed with crushed dryice, sealed, and placed in steel sample-exposure containers,which were immersed in a water pool and placed in the pathof a 6'Co y source with a measured flux of 2.4 Mrad/hr.After irradiation, all samples were removed from the cylin-der and packed on dry ice until stored at - 80TC. Duplicatesamples were irradiated with each dose in each experiment,and the ice-nucleus content was measured as describedbelow within 2 days.Measurement of Cumulative and Differential Ice-Nucleus

Concentration Spectra. Cumulative ice-nucleus concentra-tions active at temperatures from 0C to -14C were mea-sured by a droplet-freezing method similar to that describedpreviously (1). Forty 10-,lI droplets from each of seven serial1:10 dilutions of a suspension containing lyophilized cells at1 mg/ml in 0.01 M potassium phosphate buffer (pH 7.0) wereplaced on the surface of a paraffin-coated hollow aluminumblock. Since the most active ice nucleus in a drop willdetermine the temperature at which that drop freezes, onlythe most active ice nucleus in each droplet is detectable.Therefore, many samples from a series of dilutions ofbacterial suspensions are frozen to detect the more numer-ous, but less active, ice nuclei in a population of cells. Theblock was cooled at a rate of 0.20C/min and the temperaturewas recorded continuously with a precision of0.01TC with anembedded digital thermistor thermometer. The true temper-ature of the block was obtained with an accuracy of ± 0.050Cafter applying a correction made by measuring the meltingtemperature of small ice crystals on the surface of the blockafter each experiment. The cumulative number of drops ofeach dilution of bacterial suspension frozen with decreasingtemperature was determined by visual observation. Thecumulative number, N(t), of ice nuclei active at or above agiven temperature t was calculated by a method similar tothat of Vali (25):

N(t) = [ln(l/F)] 10DV

where F is the fraction of droplets unfrozen at temperature t,V is the volume of each droplet used, and D is the number of1:10 serial dilutions to which the original bacterial suspen-sion was subjected.The concentration of ice nuclei active within 10C temper-

ature intervals (differential ice-nucleus concentration) wasdetermined by estimating N(t) at each integral temperaturefrom - 20C to -14C from plots of N(t) vs. t by interpola-tion. The differential ice-nucleus concentration active be-tween temperature t and 10C warmer was obtained as N(t) -N(t + 1). Estimates of differential ice-nucleus concentrationof unirradiated cells were made from samples exposed todifferent doses of y radiation. The logarithm of differentialice-nucleus concentration active within a given 10C temper-ature interval was regressed against dose of y radiation in

Mrad and the differential ice-nucleus concentration for unir-radiated samples was determined by the intercept of suchregression lines with the ordinate.

Target-Size Determination from Ice-Nucleus InactivationData. The logarithm of the number of ice nuclei active within1PC temperature intervals (differential ice-nucleus concen-trations) remaining after exposure to various doses of yradiation was regressed against radiation dosage (in Mrads).The radiation dose that reduced ice-nucleation activitywithin each 1PC temperature interval to 37% of that of theunirradiated sample (D37) was obtained from the slope of theregression line. D3,7 is related to the mass (M) of the targetmolecule active as an ice nucleus at a given temperature:

M = (6.4 x 105)/D37 * st,

where st is a temperature-correction factor that is equal to1.0 for irradiations at temperatures close to 300C (26) andequal to 2.1 at - 850C (27), and where M is given in kDa.

RESULTSCells of P. syringae and other INA bacteria were active ascatalysts for ice formation in supercooled water over a widerange of temperatures (Fig. 1). No change in cumulativeice-nucleus concentration active at a given temperature wasobserved during the lyophilization of P. syringae or of otherINA bacterial cells (data not shown). Cumulative ice-nucleus concentrations increased continuously with decreas-ing temperatures to approximately - 14'C. At temperatureslower than - 14'C, little or no additional ice-nucleationactivity was measured. While some lyophilized P. syringaecells were active in ice nucleation at temperatures as high as- 2.40C, other cells were active as ice nuclei only at muchlower temperatures (Fig. 1). Differential P. syringae ice-nucleus concentrations active within 1PC temperature inter-vals varied widely in the range of -20C to - 14'C (Fig.- 2).Whereas only approximately i0' ice nuclei per mg of lyophi-lized cells were active in the temperature range of - 20C to- 30C, nearly 107 and >108 ice nuclei per mg were active inthe temperature ranges of -30C to - 40C and - 8C- to- 90C, respectively (Fig. 2). Although ice nuclei produced byP. syringae strain Cit7 were active within all temperatureintervals between - 2.5°C and - 140C, differential ice-nu-cleus concentrations dropped to undetectably low levels at

0C,

z00

01

9

8

7

6

5

4

3

2

-2-2

I~ I0 1

-3 -4 -5 -6 -7 -8 -9 -10 -11 -12 -13 -14Tenperature (C)

FIG. 1. Cumulative ice-nucleation activity of Iyophilized cells ofP. syringae strain Cit7 with decreasing temperature as a function offlux of y radiation at 220C. Cumulative ice-nucleus concentrationsfor unirradiated cells (i) and cells irradiated with 0.85 Mrad (A), 1.70Mrad (o), 5.1 Mrad (o), 8.5 Mrad (-), and 10.2 Mrad (o) of yradiation are depicted.

Biochemistry: Govindarajan and Lindow

1336 Biochemistry: Govindarajan and Lindow

z

0).3

8.5

8

7.5

7

6.5

6

5.5

5

4.5-2 -3 -4 -5 -6 -7 -8 -9 -10 -11 -12 -13

Temperature (C)

00)E

z

0)

01

-14 -15

FIG. 2. Differential concentration of ice nuclei produced by P.syringae strain Cit7 active within different 1PC temperature inter-vals. Ice-nucleus concentrations of unirradiated lyophilized cellsthat were active within integral temperatures from - 2TC to - 13TCwere estimated from cumulative ice-nucleus concentrations, N(t),active at each integral temperature, t, obtained from Fig. 1 as N(t) -N(t + 1). Differential ice-nucleus concentration within the temper-ature range t + 1 to t is depicted as a function of t.

temperatures lower than -14C (Fig. 2). A similar pattern ofdifferential ice-nucleus concentrations in P. syringae strain31RI, E. herbicola 26SR6-2, and E. coli HB1O1(pICE1.1)over these temperature intervals was observed (data notshown).

Ice-nucleation sites active at the highest temperatureswere more susceptible to inactivation by y radiation thanthose ice nuclei active only at lower temperatures. Cumula-tive ice-nucleus concentrations of lyophilized cells of P.syringae strain Cit7 exposed to differing amounts of yradiation at 220C exhibited a family of nonparallel curveswhen plotted against decreasing temperature (Fig. 1). Cumu-lative ice-nucleus concentrations active at - 50C or higherwere reduced by a factor of ca. 100 and 105 after exposure ofcells to 0.85 Mrad and 1.7 Mrad, respectively, while ice-nucleus concentrations active at temperatures of - 10'C andhigher were reduced only by a factor of 1.5 and 3.0,respectively. The temperature threshold of detectable icenucleation decreased from - 2.50C to - 7.3YC for untreatedcells and cells exposed to 10.2 Mrad of y radiation, respec-tively. Cumulative ice-nucleus concentrations of all fourINA bacterial strains exposed to differing amounts of 'yradiation displayed qualitatively similar families of nonparal-lel distributions as shown in Fig. 1 when plotted againstdecreasing temperatures (data not shown).

P. syringae ice nuclei active within different 1PC temper-ature intervals each decreased in number logarithmically butat different rates as a function of increasing y-radiationdosage. Significant linear relationships were observed whenthe logarithms of differential ice-nucleus concentrations oflyophilized cells remaining after y irradiation at 220C wereregressed against the dose of y radiation (Fig. 3). Rates ofinactivation of ice nuclei with y-radiation dose were highestfor ice nuclei active at the highest temperatures (-3C to- 40C) and lowest for ice nuclei active only at low temper-atures (- 12'C to - 130C). Similar log-linear relationshipsbetween differential ice-nucleus concentrations active withindifferent temperature ranges and y-radiation doses wereobtained for all four INA bacterial strains. The value of D37was readily obtained by linear regression analysis for icenuclei active within all 1PC temperature intervals, sincehighly significant log-linear relationships between differen-tial ice-nucleus concentrations and y-radiation doses wereobserved over all temperature intervals measured and for allINA bacterial strains.

8

7

6

5

4

3

2

0o 1 2 3 4 5 6 7 8 9 10

Gamma Radiation (Mrad)

FIG. 3. Exponential loss of P. syringae strain Cit7 ice nucleiactive at different temperature intervals as a function of flux of yradiation. Lyophilized cells were irradiated at 220C with vY radiationand differential ice-nucleus concentrations as a function of decreas-ing temperature were determined as in Fig. 2. The logarithm of theconcentration of ice nuclei active between - 12'C and - 130C (m),- 80C and - 90C (o), - 50C and - 60C (e), and - 30C and - 40C (o)is depicted as a function of increasing y-radiation flux to which thecells were exposed.

Highly significant linear relationships were observed forall four INA bacterial strains tested when the logarithm ofthe molecular mass of ice-nucleating structures active withindifferent 10C temperature intervals was regressed against thetemperature at which ice-nucleation activity was expressed(Fig. 4). The significant linear relationships y = - 0.189x +7.48, r = -0.99 (P < 0.01); y = -0.248x + 7.699, r =-0.946 (P < 0.01); y = -0.173x + 7.06, r = 0.816 (P <0.05); and y = -0.186x + 7.42, r = -0.928 (P < 0.01)between the logarithm of the molecular mass of ice-nucleation sites (y) and the temperature of ice nucleation (x)was found for lyophilized cells of P. syringae strain Cit7, P.syringae strain 31R1, E. herbicola strain 26SR6-2, and E.coli HB1O1(pICE1.1), respectively, irradiated at 22TC. In allfour strains of INA bacteria the molecular mass of theice-nucleating structure increased log-linearly with increas-ing nucleation temperature (Fig. 4). There was no significantdifference in the estimated in situ molecular mass of ice-nucleation sites active at a given temperature amoqg thebacterial strains within the range of nucleation temperatures

6.5

1e 6

5 5.5

0-J 5

4.5-2 -3 -4 -5 -6 -7 -8 -9 -10 -11 -12 -13 -14

Temperatue (C)

FIG. 4. Molecular mass (Da) of ice-nucleating structures ofvarious INA bacterial strains active within different 1PC temperatureintervals estimated by sensitivity to y radiation at 220C. Thelogarithm of the size of ice-nucleation sites for P. syringae strain31R1 (i), P. syringae strain Cit7 (o), E. herbicola strain 26SR6-2 (a),and E. coli HB101(pICE1.1) (e) active between temperatures + Iand t is depicted as a function of t.

I- I

Proc. Natl. Acad. Sci. USA 85 (1988)

-. .

Proc. Natl. Acad. Sci. USA 85 (1988) 1337

7

0

N

0)

6.5

6

5.5-7

Temperature (C)

-8 -9 -10

FIG. 5. Molecular mass (Da) of ice-nucleating structures of P.syringae strain Cit7 active within different 1PC temperature intervalsestimated by sensitivity of lyophilized cells (o) or frozen hydratedcells (n) to y radiation at - 850C. The logarithm of the size of icenucleants active between temperature t + 1 and t is depicted as afunction of t.

tested (Fig. 4) as indicated by a covariance analysis of theseregression lines. The smallest ice-nucleating structures mea-

sured, active in the temperature range - 12'C to - 13'C,were approximately 150 kDa for all INA bacteria tested. Thelargest ice-nucleation sites measured, active in the temper-ature range - 2C to -3C, had masses of approximately8000 kDa for every INA bacterial strain tested.The size of structures active in ice nucleation within a

given 1C temperature interval estimated from frozen-hydrated samples and lyophilized cells did not differ signif-icantly (Fig. 5). The dosage of y radiation required to reducethe ice-nucleation activity of samples held at - 85TC by 64%within any 1PC temperature interval was about twice that ofsamples irradiated at 22TC (data not shown). After applyinga temperature-correction factor of 2.1 for D37 in estimationsof nucleant mass from measurements made at - 85TC for icenuclei active within each 1PC temperature interval, the sizesof nucleating structures active within each temperatureinterval did not differ appreciably either from estimatesmade from lyophilized samples irradiated at 22TC (Fig. 4) orbetween frozen and lyophilized samples irradiated at - 850C(Fig. 5).

DISCUSSION

Inactivation of biological and/or biochemical activity ofmacromolecules by radiation exposure directly relates mo-lecular structure with function and is the only means cur-

rently available for analyzing sizes of macromolecular com-ponents in functional biological membranes (20, 22, 28-34).Estimated molecular masses of macromolecules by radiationinactivation have varied from estimates made by conven-tional biochemical methods by an average of only 14% (26,29). This method appeared particularly suited for analysis ofbacterial ice-nucleation sites because it estimates the mass ofthe functional unit that is the minimal structure or assemblyof structures required for ice-nucleation catalytic activity(28). In addition, since the expression of ice-nucleationactivity is quantitatively related to the presence and compo-sition of other outer membrane components, studies ofice-nucleant structure currently can be done only in situ.

If ice-nucleation sites active within a unique 1C temper-ature interval are similarly sized structures, a simple loga-rithmic loss of differential ice-nucleus content with increas-ing y-radiation dosage was expected, based on theory andempirical studies (20). The sizes of ice-nucleation sites active

within a given MC temperature interval appear to be rela-tively discrete. However, the size of sites active at temper-atures differing by more than 3YC differ greatly in size (Fig.4). A complex curve of loss of ice nuclei with increasingy-radiation dose (described only by two or more exponentialfunctions) was observed when concentrations of ice nucleiactive over large (>40C) temperature intervals were used inthese studies (data not shown). Such complex decay curveswere expected from radiation target theory if ice nucleationsites active within this larger temperature range were com-posed of different-sized structures. These observations cor-roborate data (Fig. 4) that indicate that ice-nucleation sitesactive at different temperatures differ greatly in size. Whilea single hit with y radiation appears sufficient to reduce theice-nucleation temperature of an ice-nucleating structure atleast MC (Fig. 3), such irradiated structures may retain someability to nucleate ice at substantially lower temperatures.However, because the differential concentration of ice nu-clei generally increased with decreasing temperatures (Fig.2), the contribution of partially inactivated higher tempera-ture ice nuclei to lower temperature nuclei would be quan-titatively very low.The minimum functional size of ice-nucleating structures,

which may differ from the size of individual components ofthe structure or the actual catalytic site, is obtained fromanalysis of inactivation of ice nuclei by y radiation. Theice-nucleating site may occupy only a fraction of the struc-tural unit that is disrupted by irradiation. y rays causerandom primary ionizations, generating about 1500 kcal/mol(26) in the target molecule. The dissipation of this energywithin the target molecule damages the structure of themolecule with concomitant loss of biological function (26).In many enzymes, which consist of several molecular sub-units conjointly required for expression of activity, theestimated molecular sizes correspond to the sum of the massof each of the components (20, 22, 26). The use of lyophi-lized samples in radiation studies of membrane-bound pro-teins has occasionally been reported to yield artifactuallylarge target sizes, presumably due to spatial changes inmembrane structure during drying that might lead to aggre-gation (35). No difference in sizes of ice-nucleating struc-tures estimated from irradiated lyophilized and frozen-hydrated samples was observed (Fig. 5), indicating that thelarge size of ice nucleants (Figs. 4 and 5) is not due toartificial aggregation due to sample preparation. The resultsof Fig. 3 are consistent with the presence of proteinaceousoligomers of different sizes that are composed of tightlycoupled components (19, 28). The large differences in sizesof ice-nucleating structures active at different temperaturesand the large size of ice-nucleation sites active at tempera-tures approaching 00C (Figs. 4 and 5) are both consistentwith a homologous or heterologous oligomer of smallermacromolecular constituents. The 150-kDa P. syringae icegene product (17) may constitute such a constituent. Theamino acid sequence of a P. syringae Ice protein predictedfrom DNA sequence analysis of a cloned ice gene is largelycomposed of a tandem repeat of a 16 amino acid consensussequence (18). Such a structure either individually or inmultimers could act as a site for the orientation of watermolecules, acting as an ice nucleant. The smallest icenucleants measured, active between - 12'C and - 13TC, hadmasses of approximately 150 kDa (Fig. 4). The minimum sizeof functional subunits of ice-nucleation sites, detected at- 12'C to - 13'C, was approximately 150 kDa and possiblyrepresents the expression of ice nucleation by single P.syringae Ice proteins. The largest ice-nucleation sites mea-sured, active between - 2°C and - 3°C, possibly representan oligomer of 53 such subunits. Ice nucleation by P.syringae and other INA bacteria studied is expressed ingreatest frequency at temperatures lower than - 70C (Fig. 2).

Biochemistry: Govindarajan and Lindow

1338 Biochemistry: Govindarajan and Lindow

More than 1000 ice-nucleation sites active between - 80Cand - 90C for every ice nucleus active between - 20C and-3C are found in P. syringae cells (Fig. 2). Large oligo-mers, providing ice-nucleation sites active at temperatureshigher than - 70C, may be more unstable or less likely toform than the smaller oligomers or monomers that expressice-nucleation activity at colder temperatures. Evidence fortemperature sensitivity of higher temperature (-750C) icenuclei above 270C has been reported (36) and suggests thatinstability of large ice nuclei may be a factor. A less likelyexplanation of the apparent large size of ice-nucleation sitesincludes a dispersed collection of monomeric structures inthe membrane that are all required for expression. Since thelogarithm of the size of ice-nucleating structures was acontinuous linear function of'the temperature of ice cataly-sis, and since the individual components of an active sitemay be relatively small, a much larger number of possiblesizes of ice-nucleation sites than were arbitrarily estimatedhere are possible.

It is significant that the sizes of ice-nucleation sites in P.syringae and in E. coli containing the P. syringae ice genewere nearly identical (Fig. 4). These two bacterial speciesexhibit many biochemical and physiological differences. Thespecificity of ice nucleation appears to be determined by thecloned ice gene and is expressed in quite different biologicalenvironments. The cloned ice gene of P. syringae appears toproduce a gene product directly involved in ice nucleation.

It is unlikely that any nonproteinaceous molecules in thevicinity of the ice-nucleation site were estimated by y-radiation inactivation. The mass of nonproteinaceous com-ponents such as oligosaccharides and lipids that may berequired for nucleation activity (13) does not generallycontribute' to the estimation of target size obtained byradiation inactivation analysis, because energy is not effi-ciently transferred to noncovalently bound molecules orthose bound via amino acid side chains (37-39).

Classical theories predict that logarithmically increasingsizes of insoluble homogeneous particles function as icenuclei at linearly increasing temperatures (40-42). The na-ture of heterogeneous ice-nucleation sites is unknown, but isgenerally presumed to involve the orientation of a largenumber of water molecules by hydrogen bonding or hydro-phobic exclusion forming a regular array of water moleculesresembling an ice-like lattice (40). Uncertainties in the sur-face interface parameter and the geometry of nucleation sitesmake exact predictions of the size of such sites active at agiven temperature difficult (42). If the surface parameter isassumed to be very ice-like, then the predicted diameter ofnucleation sites active at -5C is about 160 A (42). Such alarge site could be formed from a proteinaceous templatewith an estimated mass of 2500 kDa (Fig. 4). The resultspresented here generally agree with the predicted size ofnucleation sites. The results also give insight into the possi-ble structural assembly of Ice proteins into multimericice-nucleation sites.

We thank Dr. Richard M. Lemmon of Lawrence Berkeley Labo-ratory and Dr. David Trombino of the Lawrence Livermore Na-tional Laboratory, respectively, for providing access to 6Co gener-ators and assistance in their operation, and to Dr. J. Loper forhelpful suggestions on the manuscript. This work was supported byGrant 84-CRCR-1-1393 from the U.S. Department of Agriculture.

1. Lindow, S. E., Amy, D. C. & Upper, C. D. (1978) Phytopa-thology 68, 523-527.

2. Lindow, S. E., Arny, D. C. & Upper, C. D. (1978) Appl.Environ. Microbiol. 36, 831-838.

3. Amy, D. C., Lindow, S. E. & Upper, C. D. (1976) Nature(London) 262, 282-284.

4. Maki, L. R. & Willoughby, K. J. (1978) J. Appl. Meteorol. 17,1049-1053.

5. Maki, L. R., Golyon, E. L., Chang-Chien, M. & Caldwell,D. R. (1974) Appl. Microbiol. 28, 456-460.

6. Paulin, J. P. & Luisetti, J. (1978) in Proceedings of the FourthInternational Conference on Plant Pathogenic Bacteria (Sta-tion de Pathologie Vdgdtale et Phytobacteriologie, Gibert-Clarey, Tours, France), Vol. 2, pp. 403-431.

7. Lindow, S. E. (1983) Annu. Rev. Phytopathol. 21, 363-384.8. Lindow, S. E. (1982) in Plant Cold Hardiness, eds. Li, P. H. &

Sahai, A. (Academic, New York), pp. 395-416.9. Lindow, S. E., Amy, D. C. & Upper, C. D. (1982) Plant

Physiol. 70, 1090-1093.10. Lindemann, J., Constantinidou, H. A., Barchet, W. R. &

Upper, C. D. (1982) Appl. Environ. Microbiol. 43, 159-271.11. Vali, G., Christensen, M., Fresh, R. W., Galyon, E. L., Maki,

L. R. & Schnell, R. (1976) J. Atmos. Sci. 33, 1565-1570.12. Sprang, M. L. & Lindow, S. E. (1981) Phytopathology 71, 890

(abstr.).13. Govindarajan, A. G. & Lindow, S. E. (1984) Plant Physiol. 75,

43 (abstr.).14. Kozloff, L. M., Schofield, M. A. & Lute, M. (1983) J. Bacte-

riol. 153, 222-231.15. Kozloff, L. M., Lute, M. & Westaway, D. (1984) Science 226,

845-846.16. Orser, C. S., Staskawicz, B. J., Panopoulos, N. J., Dahlbeck,

D. & Lindow, S. E. (1985) J. Bacteriol. 164, 359-366.17. Govindarajan, A. G. & Lindow, S. E. (1985) Phytopathology

75, 1380 (abstr.).18. Green, R. L. & Warren, G. J. (1985) Nature (London) 317,

645-648.19. Kepner, G. R. & Macey, R. I. (1968) Biochim. Biophys. Acta

163, 188-203.20. Kempner, E. S. & Schlegel, W. (1979) Anal. Biochem. 92, 2-10.21. Noel, H., Beauregard, G., Potier, M., Bleau, G., Chapdelaine,

A. & Roberts, K. D. (1983) Biochim. Biophys. Acta 758,88-90.

22. Goldkom, T., Rimon, G., Kempner, E. S. & Kaback, H. R.(1984) Proc. Natl. Acad. Sci. USA 81, 1021-1025.

23. Lea, D. (1946) Actions of Radiations on Living Cells (Cam-bridge Univ. Press, London), p. 69.

24. Spinks, J. W. T. & Woods, R. J. (1964) An Introduction toRadiation Chemistry (Wiley, New York), pp. 105-112.

25. Vali, G. (1971) J. Atmos. Sci. 28, 402-406.26. Kempner, E. S. & Miller, J. H. (1983) Science 222, 586-589.27. Kempner, E. S. & Haigler, H. T. (1982) J. Biol. Chem. 257,

13297-13299.28. Schlegel, W., Kempner, E. S. & Rodbell, M. (1979) J. Biol.

Chem. 254, 5168-5176.29. Kempner, E. S., Miller, J. H., Schlegel, W. & Hearon, J. Z.

(1980) J. Biol. Chem. 255, 6826-6831.30. Nielsen, T. B., Lad, P. M., Preston, M. S., Kempner, E.,

Schlegel, W. & Rodbell, M. (1981) Proc. Natl. Acad. Sci. USA78, 722-726.

31. Saccomani, G., Sachs, G., Cuppoletti, J. & Jung, C. Y. (1981)J. Biol. Chem. 256, 7727-7729.

32. Powell, W. F. & Pollard, E. C. (1955) Radiat. Res. 2, 109-118.33. Hutchinson, F., Preston, A. & Vogel, B. (1957) Radiat. Res. 7,

465-472.34. Jagger, J. & Wilson, D. (1955) Radiat. Res. 3, 127-134.35. Angelides, K. J., Nutter, T. J., Elmer, L. W. & Kempner,

E. S. (1985) J. Biol. Chem. 260, 3431-3439.36. Lindow, S. E. (1983) Phytopathology 73, 809 (abstr.).37. Lad, P. M., Preston, M. S., Welton, A. F., Nielsen, T. B. &

Rodbell, M. (1978) Biochim. Biophys. Acta 551, 368-381.38. Schwartz, A. L., Ster, C. J. & Kempner, E. S. (1984) J. Biol.

Chem. 259, 12025-12029.39. Nugent, J. H. A. & Atkinson, Y. E. (1984) FEBS Lett. 170,

89-93.40. Fletcher, N. H. (1959) J. Chem. Phys. 29, 572-576.41. Fletcher, N. H. (1963) J. Chem. Phys. 38, 237-240.42. Rogers, J. S., Stall, R. E. & Burke, M. J. (1987) Cryobiology

24, 270-279.

Proc. Natl. Acad. Sci. USA 85 (1988)