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Applied and Environmental Microbiology, March 2006, p. 1784-1792, Vol. 72, No. 3
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.3.1784-1792.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Freeze-Thaw Tolerance and Clues to the Winter Survival of a Soil Community

Virginia K. Walker,1* Gerald R. Palmer,1 and Gerrit Voordouw2

Department of Biology, Queens University, Kingston, Ontario, Canada K7L 3N6,1 Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada T2N 1N42

Received 9 September 2005/ Accepted 14 December 2005


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ABSTRACT
 
Although efforts have been made to sample microorganisms from polar regions and to investigate a few of the properties that facilitate survival at freezing or subzero temperatures, soil communities that overwinter in areas exposed to alternate freezing and thawing caused by Foehn or Chinook winds have been largely overlooked. We designed and constructed a cryocycler to automatically subject soil cultures to alternating freeze-thaw cycles. After 48 freeze-thaw cycles, control Escherichia coli and Pseudomonas chlororaphis isolates were no longer viable. Mixed cultures derived from soil samples collected from a Chinook zone showed that the population complexity and viability were reduced after 48 cycles. However, when bacteria that were still viable after the freeze-thaw treatments were used to obtain selected cultures, these cultures proved to be >1,000-fold more freeze-thaw tolerant than the original consortium. Single-colony isolates obtained from survivors after an additional 48 freeze-thaw cycles were putatively identified by 16S RNA gene fragment sequencing. Five different genera were recognized, and one of the cultures, Chryseobacterium sp. strain C14, inhibited ice recrystallization, a property characteristic of antifreeze proteins that prevents the growth of large, potentially damaging ice crystals at temperatures close to the melting temperature. This strain was also notable since cell-free medium derived from cultures of it appeared to enhance the multiple freeze-thaw survival of another isolate, Enterococcus sp. strain C8. The results of this study and the development of a cryocycler should allow further investigations into the biochemical and soil community adaptations to the rigors of a Chinook environment.


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INTRODUCTION
 
Winter survival frequently requires organisms to exhibit at least a few of a range of adaptive behaviors and physiological adjustments. Although genes associated with migration, quiescence, or hibernation strategies have been studied for a variety of metazoans, microorganisms, particularly bacteria in temperate continental climate zones, have received considerably less attention. However, it is known that cryotolerance in some species is associated with fatty acid changes in membrane lipids (3, 8), as well as certain culture conditions (11, 21, 35). Studies of polar psychrophiles have revealed adaptations such as lipid modification to maintain membrane fluidity, accumulation of polyols, genome adaptations, and production of cold shock proteins and cold-active enzymes, including enzymes important for protein synthesis (4, 9, 19, 24, 26). Although it has been argued that polar bacteria do not require antifreeze proteins (AFPs) when they are living at the junction of ice crystals (9, 13, 20), the accumulation of molecules that inhibit ice recrystallization (IR) could be part of an adaptive response in Arctic and Antarctic microbes (12, 17, 18, 22, 27, 33). In contrast, certain Pseudomonas species have ice-nucleating protein complexes that promote the growth of ice (19) or cold acclimation proteins that are believed to contribute to freeze tolerance (23).

Particularly challenging temperate environments are found in the soil surface on the continental sides of large mountain chains located near the oceans. These regions frequently experience Foehn winds, also known as Chinook or Mistral winds, which develop when air descends from high elevations and heats due to compression. Chinook winds in North America can raise winter temperatures by 20°C in less than 1 h and frequently melt the snow cover. These conditions present additional challenges to microbial species that may endure summer temperatures as high as 35°C and winter temperatures that can dip to –35°C, as well as alternating freezing and thawing periods during the cold season. At these temperatures, microorganisms can be injured or killed as a result of cold shock, freezing, prolonged exposure to subzero temperatures, and subsequent warming (17), and injury or death is often due to damage to membranes or cell walls that results in permeability changes, as well as to damage to DNA (18). Indeed, the susceptibility of most enteric bacteria to freezing has allowed disposal of wastewater from polar research stations in a "sewage bulb" in the snowpack above glacial ice (28). In addition, commercial interests argue that the use of secondary wastewater for snowmaking leads to killing or irreversible damage of bacteria so that in the spring, runoff water is effectively "disinfected" (25). Given these challenges, the fact that soil consortia thrive in Chinook regions is a testimony either to environmental heterogeneity or to the remarkable adaptive abilities of microbial communities.

We designed and constructed an apparatus that allows exposure of soil cultures to regular freeze-thaw cycles that mimic the slow warming and chilling of the Chinook temperature changes. Aided by this new apparatus, we hope that insights into the overwintering survival of soil communities may be obtained from an understanding of selected freeze-tolerant isolates in consortia.


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MATERIALS AND METHODS
 
Location, soil samples, and bacterial culture.
Soil samples were obtained from the upper 1 to 2 cm of soil in the northwest region of Calgary, Alberta, Canada (51°16.84'N, 115°4.78'W; elevation, 1,643 m). In the general area, 20 of the 90 coldest days of the year during December, January, and February routinely receive Chinook winds that raise the temperature from freezing to more than 5°C (Government of Alberta Agriculture Department website http://www1.agric.gov.ab.ca/$department/deptdocs.nsf/all/sag6295?OpenDocument#Chinook). A 1-g soil sample from each of three adjacent collection sites, obtained prior to the first autumn frost, was cultured in 3 ml of diluted tryptic soy broth (TSB) [diluted 10% (vol/vol) so that the final concentrations were 3 g tryptic soy broth from Difco (Sparks, Md.), 0.1 g KNO3, 0.1 g (NH4)2SO4, and 0.1 g K2HPO4 in 1 liter of water] with shaking at 22°C for 48 h. A single colony of a laboratory strain of the enteric bacterium Escherichia coli TG2 (29) or the plant pathogen Pseudomonas chlororaphis ATCC 13985 was used to initiate three 3-ml cultures in TSB or 10% TSB. After this, cultures were routinely incubated at 4°C for 6 to 14 h prior to plating or further treatment. The three cultures were pooled, and viable cell counts (in CFU per ml) were determined at 22°C on 10% TSB plates for the soil consortia and on TSB or 10% TSB plates for the cultures originating from E. coli and P. chlororaphis colonies.

Freeze-thaw treatments using a cryocycler.
An instrument for automatically subjecting cultures to programmed freeze-thaw treatments was constructed from two circulating baths, one at –18°C and the other at 5°C and filled with 40% (vol/vol) ethylene glycol (Fisher Scientific Company, Toronto, Canada). A diagram of this device, termed a cryocycler, is shown in Fig. 1A. Circulation is controlled using a programmable digital timer (RCT100; Garrison Co., Toronto, Canada) and four valves (Asco Valve Canada, Brantford, Canada), including two three-way universal valves (8320G176) and two two-way valves, one of which is normally open (8262G262) and one of which is normally closed (8262G208). The circulating ethylene glycol-water solution was directed through a jacketed beaker (Allen Glass Scientific, Boulder, CO) mounted on a magnetic stirrer and was kept at the appropriate bath temperature with plumbing insulation covering all tubing lines.


Figure 1
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  		FIG. 1. Design of the cryocycler apparatus that was used for automatically subjecting bacterial cultures to freeze-thaw cycles. (A) Ethylene glycol circulation through the jacketed sample chamber was controlled by a programmable timer and a panel of four valves, two of which were normally open (N.O.) and two of which were normally closed (N.C.), as described in the text. (B) Temperature profile of the cryocycler sample chamber, programmed as described in Materials and Methods, during the warming cycle (boxes) and the cooling cycle (triangles).

Cultures (2 ml) in 5-ml glass vials were placed in the jacketed beaker, which was filled with 40% ethylene glycol and stirred with a magnetic stir bar. The cultures were subjected to freeze-thaw cycles, with each 2-h cycle consisting of 1 h at –18°C and 1 h at 5°C. Samples were removed from each of the triplicate vials after 3, 12, 18 or 24, and 48 cycles, and the numbers of survivors were determined (CFU/ml). Counts were obtained after 48 h of incubation of the plates at 22°C and again after 72 h of incubation at 22°C. Colony diameters were determined after 72 h by using a minimum of 25 colonies, and means and standard deviations were calculated and analyzed using Student's t tests. Colony morphologies were also noted.

To assess the relative contribution of freeze-thaw treatments to cell viability, cultures of E. coli, P. chlororaphis, or soil samples were prepared as described above and treated in triplicate in the cryocycler for 72 h at –18°C, for 24 h at 5°C, and for 24 h at 5°C followed by 72 h at –18°C. The number of CFU/ml was determined for each experiment as described above, and the loss of viability was calculated by comparison with the cell counts in the initial cultures. It should be noted that in preliminary experiments, no cell growth was observed in any of the postlogarithmic isolates stored at 4°C to 5°C for several hours (not shown).

Samples (100 µl) from cultures that contained viable cells after 48 freeze-thaw cycles were used to inoculate 8 ml of 10% TSB. The organisms were cultured for 48 h, and viable cell counts were determined as described above. These cultures, which originated from survivors of the initial freeze-thaw regimen, were then subjected to 48 additional freeze-thaw cycles, as described above, and viable cell counts were again determined.

Individual isolates, which had been putatively identified by partial 16S rRNA gene sequence analysis, were inoculated into 8 ml of medium, cultured for 48 h as described above, and then subjected to the freeze-thaw regimen. Although it was not a problem with mixed cultures or cultures in TSB, occasionally single isolates in 10% TSB supercooled rather than froze when the temperature dropped below 0°C. To ensure that all of the samples froze at the same temperature, a few sterilized AgI crystals were added to the vials at the start of the experiment (control experiments showed that there was no loss of CFU/ml with AgI addition). Presumably, endogenous nucleators (e.g., soil or silica particles) ensure that supercooling is not common in soil, although the cell contents likely do not freeze until the temperature is close to –15°C (24).

For fractionation experiments, cells were harvested by centrifugation at 6,000 x g for 10 min, and the cell pellet was washed with 10% TSB. Spent medium was obtained by centrifugation as described above and by passage through a 0.45-µm syringe filter. Starting cell counts, which were similar for all the cultures, were normalized to a common starting count of 1 x 108 CFU/ml. Counts were plotted, and standard errors were determined.

DNA isolation, PCR, and 16S rRNA gene sequence analysis.
Individual colonies, classified on the basis of morphology, were picked from plates and streaked on fresh 10% TSB plates. Single colonies from the streaked plates were subsequently used to inoculate 3 ml of 10% TSB and cultured for 48 h. Cells were centrifuged and resuspended in 200 µl 10 mM Tris-HCl-1 mM EDTA (pH 8.0). PCRs were performed with DNA templates released from 10 µl of cell suspension subjected to a 3-min heat treatment at 94°C. Alternatively, cells were resuspended in 180 µl of a solution containing 10 mM Tris-HCl, 5 mM sodium EDTA, and 1 mg/ml lysozyme (Sigma Chemical Co., St. Louis, MO) (pH 7) and incubated at 37°C for 1.5 h. DNA was then extracted using a DNeasy kit according to the manufacturer's instructions (QIAGEN, Stanford, Calif.). In this case, 2 to 10 µl of the resulting filtrate was used as the DNA template for PCR.

Sequences representing most of the small-subunit rRNA gene were amplified using a bacterium-specific 16S primer (f8 [5'-AGAGTTTGATCCTGGCTCAG]) and a reverse primer (r1406 [5'-ACGGGCGGTGTGTAC]), derived from the study of Lane et al. (16), as previously described (31). Amplified fragments were purified using a QIAquick PCR purification kit (QIAGEN), and the purity and amount of product were estimated by analysis on agarose gels containing 0.5 µg/ml of ethidium bromide or after electrophoresis on 1.5% agarose gels and staining with ethidium bromide (29, 31). Forward and reverse sequences for each product were determined, assembled, and subjected to web-based Basic Alignment Search Tool (BLAST) analyses (1) to identify the nearest phylogenetic relatives of the isolate.

Assay for inhibition of ice recrystallization.
Ice crystals were visualized in the jacketed beaker of the cryocycler apparatus, which was modified so that it had additional design features. The assay for IR inhibition was essentially the assay described previously (15, 32). Microcapillaries (10 µl) containing bacterial isolates in 10% TSB, which were sealed with vacuum grease and mounted on cut pieces of duct tape, were flash frozen in 40% ethylene glycol at –25°C for 15 min. They were subsequently placed in 40% ethylene glycol in the jacketed beaker and warmed to –6°C.

Ice crystal size was monitored by taking digital images (Canon PowerShot S45; Canon Canada Inc., Mississauga, Ontario, Canada) for ~18 h with a dissecting microscope (magnification, x10 to x40) illuminated from below, using crossed polarizing filters as previously described (32). In addition to soil isolates, the samples included 10% TSB, the E. coli and P. chlororaphis cultures described above, various buffers, and serial dilutions of type I fish AFP (A/F Protein Canada, St. John's, Newfoundland, Canada) or bovine serum albumin (BSA) in 10 mM Tris-HCl-150 mM NaCl (pH 7.5). Proteins were hydrolyzed in the same medium by digestion with a 20-mg/ml proteinase K solution (10%, vol/vol; QIAGEN) incubated at 25°C or 37°C for 1 h before the microcapillaries were loaded.


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RESULTS
 
Cryocycler characterization.
The apparatus (Fig. 1A) allowed the soil samples to be subjected to precisely timed freeze-thaw cycles and also facilitated the IR assays. The jacketed beaker could keep samples at temperatures ranging from –15°C to just below the ambient temperature. During the freeze-thaw cycles, the temperature of the beaker solution increased from –15°C to 5°C (when the bath temperatures were set at –18°C and 5°C, respectively) in 45 min, making this a "slow warming" at an average rate of 0.5°C/min (Fig. 1B). All cultures had melted 40 min after the switch to the circulation from the warmer bath. The temperature of samples decreased from 5°C to –15°C in 15 to 20 min, making this a "slow chilling" at a rate of approximately 1°C/min. When AgI crystals were added to the single isolates, they uniformly froze at –4°C, as did the consortium samples. Thus, cultures were frozen for ≥90 min (40 min at the beginning of the "warm" cycle and 50 min at the end of the "cold" cycle) in every 2-h freeze-thaw cycle.

Selection for freeze-tolerant consortia.
Three samples from each soil collection were used for freeze-thaw treatments, and cultures from each of the three samples were pooled and the numbers of CFU/ml were determined before the consortia were subjected to multiple freeze-thaw cycles in triplicate. Samples removed after 3, 12, 18 or 24, and 48 cycles showed that the cell counts declined dramatically for each sampling period (Fig. 2). In the first three freeze-thaw cycles, the average number of CFU/ml was reduced by almost 2 orders of magnitude. The decline slowed with subsequent cycles. In addition to the decrease in numbers, there was a dramatic reduction in the complexity of the community, as reflected both in the colony morphology (Fig. 3) and in the diversity as assessed by sequencing of 16S rRNA gene fragments from isolates derived from the original samples (not shown). Thus, the reduction in the number of viable cells appeared to reflect injury or death of the more freeze-thaw-sensitive bacteria in the consortia. With the control bacterial cultures, E. coli and P. chlororaphis, there was a sharper decline in viability as the experiment progressed. Although P. chlororaphis was more resistant than E. coli, there was a 2-order-of-magnitude decrease in the number of P. chlororaphis cells after three freeze-thaw cycles and a further 100-fold decline after 12 cycles, and no viable bacteria remained after 48 cycles (Fig. 2). For E. coli cultures there was a 3-order-of-magnitude decrease in viability after three freeze-thaw treatments, and there were further dramatic declines with subsequent cycles until no viable bacteria remained after 24 cycles (Fig. 3). There was no difference in the decrease in cell viability, as calculated by using a known starting population, when the control E. coli or P. chlororaphis cultures were initially cultured in TSB or 10% TSB (not shown).


Figure 2
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  		FIG. 2. Decreases in viability of bacterial cultures as a function of the number of freeze-thaw cycles in the cryocycler. Cultures derived from single colonies of P. chlororaphis or E. coli were compared to a mixed culture derived from soil samples collected from a Chinook zone (Calgary soil). Bacteria from the Calgary soil culture that survived 48 freeze-thaw cycles were regrown and again subjected to 48 cycles (Calgary selected). The error bars indicate standard deviations.


Figure 3
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  		FIG. 3. Colony phenotypes before and after freeze-thaw cycles. The upper plate contained a typical sample (diluted 106-fold) with diverse colony morphologies in the mixed culture prior to freeze-thaw treatment. The lower left plate shows that after 48 freeze-thaw cycles the number of viable bacteria had decreased (sample diluted 102-fold; Calgary soil [Fig. 2]) and the phenotypic complexity was reduced. The lower right plate shows that when these bacteria were used to initiate freeze-thaw-resistant cultures (Calgary selected [Fig. 2]) and were subjected to 48 additional freeze-thaw cycles, the colony phenotypic diversity was further reduced (sample diluted 105-fold).

In addition to a decrease in viability, the colonies that grew on plates after three freeze-thaw treatments had smaller initial diameters than control, untreated colonies. There were significant differences in the two control strains. The average initial diameters of E. coli and P. chlororaphis colonies which had not been previously frozen were 2.7 times larger (1.6 ± 0.8 versus 4.3 ± 0.9 mm) and 1.9 times larger (2.7 ± 0.4 versus 5.0 ± 0.4 mm), respectively, than the diameters of colonies obtained from cultures that had previously been subjected to freeze-thaw treatments in the cryocycler. The diameters of colonies obtained from the soil consortia were also affected, but less so than the diameters of the E. coli and P. chlororaphis colonies; the colonies of soil bacteria that were never frozen were 1.4 times larger (2.5 ± 0.3 versus 3.4 ± 0.9 mm) than the colonies of bacteria that had been subjected to three freeze-thaw cycles.

Cells that were viable after 48 freeze-thaw cycles were subsequently used to initiate cultures, termed freeze-thaw "selected" cultures, which were themselves subjected to additional treatments in the cryocycler. Selected cultures retained a higher level of viability when they were exposed to freeze-thaw cycles. Even after 48 freeze-thaw treatments, the cultures lost only approximately 1 factor of 10 in viability (CFU/ml). There was a further reduction in sample complexity, and only a few phenotypically distinct bacterial colonies remained at the conclusion of the experiment (Fig. 3).

The loss of viability attributable to multiple freeze-thaw treatments was determined by subjecting cultures to the freezing and thawing temperatures for periods corresponding to the times of exposure to these temperatures in the initial cycling experiments (24 and 72 h, respectively), as well as to a sequential regimen. There was little reduction in viability in any of the cultures after treatment at 5°C, but E. coli appeared to be five- to sixfold more susceptible to freezing than P. chlororaphis and the soil cultures (Table 1). Whereas the freeze viability of P. chlororaphis and the soil microorganisms was improved by prior treatment at 5°C (as has also been observed with other bacteria [8, 23]), which resulted in the loss of 1 log CFU/ml overall, this was not true for E. coli, which exhibited a ~200-fold decline in viability when the sequential regimen was used. However, even for this species, the reduction in cell number due to freezing was substantially less than the reduction in viability after 48 freeze-thaw cycles (Table 1).


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  		TABLE 1. Viability after freeze, thaw, and freeze-thaw conditions administered as block treatments

Microbial diversity and characterization of bacteria selected for freeze tolerance.
The microbial diversity of soil enrichments exposed to 96 freeze-thaw cycles (48 freeze-thaw cycles followed by regrowth and a further 48 freeze-thaw cycles) was determined by PCR amplification of rRNA sequences from single isolates. In 35 separate PCR amplifications for cultures inoculated with single colonies, five distinct sequences were putatively identified (Table 2). For convenience, the bacteria identified, designated Chinook strains, are referred to below by the names of their closest relatives in the database. The most common bacteria, which were found on every plate, were the Buttiauxella-related (gram-negative) cells (e.g., Chinook 7), which produced small white colonies with irregular boundaries, and Chryseobacterium-related (gram-negative) cells (e.g., Chinook 14), which produced small bright yellow colonies with a distinct pungent odor and regular boundaries. Less frequent were the Acinetobacter-related (gram-negative) cells (e.g., Chinook 3) that formed white colonies and the Enterococcus-related (gram-positive) cells that produced small pale yellow colonies (e.g., Chinook 9). Still rarer were the small white Carnobacterium-related (gram-positive) colonies (Chinook 4). All bacterial cultures yielded DNA that could be amplified with universal primers, although DNA was more difficult to obtain from some cultures. In addition, no rRNA sequences remained unidentified (<98% identity) after comparison with sequences in the database (Table 2). Because of the high degree of sequence identity, the isolates are referred to below by putative genus names with the species name not specified.


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  		TABLE 2. Bacterial isolates obtained from survivors of two freeze-thaw treatments (48 cycles each)

In order to characterize the freeze-thaw resistance of single-cell isolates, representatives of each of the different genera were subjected to 3, 12, 24, and 48 cryocycler cycles. Viable cell counts showed that although all of the isolates were more resistant than P. chlororaphis (not shown) and E. coli cultures placed in the cryocycler at the same time (Fig. 4), there were differences in the decreases in viability in response to freeze-thaw cycling. Chryseobacterium sp. strain C14, Buttiauxella sp. strain C1, Acinetobacter sp. strain C3 (not shown), and Acinetobacter sp. strain C5 lost little viability over the course of 48 freeze-thaw cycles, and they produced profiles that appeared to be similar to the profile of the mixed, selected consortium (Fig. 2 and 4). Carnobacterium sp. strain C4 was more freeze-thaw susceptible and lost 2 to 3 orders of magnitude of viability, but Enterococcus sp. strain C8 appeared to be much more susceptible and lost about 6 orders of magnitude of viability over the course of the experiment, which was even greater than the loss exhibited by the unselected soil samples.


Figure 4
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  		FIG. 4. Decreases in viability of single-colony isolates obtained after 48 serial freeze-thaw cycles (Calgary selected [Fig. 2]) compared to the viability of E. coli cultures. The Chinook isolates were named after the closest relatives in the database (Table 2). The error bars indicate standard deviations.

Since Enterococcus sp. strain C8 was unexpectedly relatively freeze-thaw susceptible in isolated culture despite the fact that it had previously survived treatments in the cryocycler as a member of the consortium, experiments were performed to determine if the survival of this organism was influenced by the presence of another isolate. When pelleted cells of Enterococcus sp. strain C8 were resuspended in medium obtained from centrifuged and filtered Chryseobacterium sp. strain C14 cultures, the survival of Enterococcus sp. strain C8 improved, so that only 3 orders of magnitude of viability was lost after 48 freeze-thaw treatments (Fig. 5). In contrast, pelleted E. coli cells suspended in medium obtained from Chryseobacterium sp. strain C14 cultures did not survive more than 24 cycles, the same result that was obtained when unfractionated E. coli cultures were subjected to cryocycler treatments, although the viability of cells subjected to 12 freeze-thaw cycles improved (Fig. 5). Chryseobacterium sp. strain C14 that was resuspended either in its own filtered medium or in filtered medium derived from E. coli cultures showed the same high level of survival as nonfractionated cultures (results not shown). Actinobacter sp. strain C5 cells were similar in that they showed the same high level of viability after 48 freeze-thaw cycles in medium derived from Chryseobacterium sp. strain C14 cultures as the unfractionated cultures (data not shown).


Figure 5
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  		FIG. 5. Decreases in viability of single-colony isolates of selected Chinook strains after serial freeze-thaw cycles. Individual isolates that survived 48 freeze-thaw cycles (Calgary selected [Fig. 2]) were used to initiate cultures, and these cultures were subjected to further freeze-thaw treatments (Enterococcus sp. strain C8) or separated from the culture medium and resuspended in medium derived from Chryseobacterium sp. strain C14 cultures (Enterococcus sp. C8 +). Control cultures (E. coli) were also resuspended in medium derived from Chyseobacterium sp. strain C8 cultures (E. coli +). The viability of Chryseobacterium sp. strain C8 cultures (cells and medium) is shown, and the values were similar to the values for Chryseobacterium sp. strain C8 cells resuspended in E. coli medium (not shown). The error bars indicate standard deviations.

Ice recrystallization inhibition.
Type I fish AFPs showed good IR inhibition activity in the dilution series (0.002 to 2 mg/ml protein), and no large ice crystals were seen in the capillary tubes after the frozen solutions were left overnight at –6°C (Fig. 6A). In contrast, capillaries containing more dilute AFP solutions (0.0002 mg/ml) or BSA (0.0002 to 20 mg/ml) contained large ice crystals, indicating that there was no IR inhibition activity. There was no evidence of any IR inhibition activity in the P. chlororaphis (not shown) and E. coli cultures (Fig. 6B). In addition, there appeared to be no activity in any of the cultures of soil isolates with the exception of Chryseobacterium sp. strain C14 cultures, which showed IR inhibition that was as clear as that of positive controls containing 0.002 to 2 mg/ml fish AFP (Fig. 6A). There was no IR inhibition with proteinase K samples alone (data not shown). Treatment of Chryseobacterium sp. strain C14 cultures or fish AFP preparations with proteinase K at either 25°C or 37°C eliminated all IR inhibition activity (Fig. 6C).


Figure 6
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  		FIG. 6. Inhibition of ice recrystallization in protein samples and bacterial cultures. Samples of protein or bacterial cultures were placed in microcapillaries (diameter, 740 µm), flash frozen, and incubated at –6°C overnight in the jacketed sample holder of the cryocycler (Fig. 1), and they were subsequently examined between crossed polarizing filters. The images are typical of the inhibition of ice recrystallization assay samples. (A) Assays with control proteins. The samples included water (microcapillaries 1 and 2), sample buffer (microcapillaries 3 and 4), 10-fold serial dilutions of fish AFP in buffer (microcapillary 5, 20 mg/ml; microcapillary 6, 2 mg/ml; microcapillary 7, 0.2 mg/ml; microcapillary 8, 0.02 mg/ml; microcapillary 9, 0.002 mg/ml; microcapillary 10, 0.0002 mg/ml), and 10-fold serial dilutions of BSA in buffer (microcapillary 11, 20 mg/ml; microcapillary 12, 2 mg/ml; microcapillary 13, 0.2 mg/ml; microcapillary 14, 0.02 mg/ml; microcapillary 15, 0.002 mg/ml; microcapillary 16, 0.0002 mg/ml). (B) Assays with various Chinook strain and control cultures. Microcapillaries 1 and 2 contained E. coli cultures (~2 x 10–8 CFU/ml). Prior to treatment in the cryocycler, the other samples were characterized. Microcapillaries 3 and 4 contained ~2 x 10–8 CFU/ml Chyseobacterium sp. strain C14, microcapillaries 5 and 6 contained samples from microcapillaries 3 and 4 diluted 50% with 10% TSB, microcapillaries 7 and 8 contained ~5 x 10–8 CFU/ml Acinetobacter sp. strain C3, microcapillaries 9 and 10 contained ~2 x 10–8 CFU/ml Buttiauxella sp. strain C, microcapillaries 11 and 12 contained ~5 x 10–7 CFU/ml Enterococcus sp. strain C8, and microcapillaries 13 and 14 contained ~5 x 10–7 CFU/ml Carnobacterium sp. strain C4. (C) Assays with bacterial cultures and protease. The samples included buffer or medium (capillaries 1 and 2), 0.02 mg/ml fish AFP in medium (capillaries 3 and 4), in proteinase K buffer (capillaries 5 and 6), or in proteinase K buffer and incubated at 37°C with buffer alone (capillaries 7 and 8) or with 2 mg/ml proteinase K (capillaries 9 and 10) before the assay, Chyseobacterium sp. strain C14 cultures (~5 x 10–7 CFU/ml) incubated at 25°C with buffer (microcapillaries 11 and 12) or with 2 mg/ml proteinase K (microcapillaries 13 and 14), and the same Chyseobacterium sp. strain C14 cultures incubated at 37°C with buffer (microcapillaries 15 and 16) or with 2 mg/ml proteinase K (microcapillaries 17 and 18).


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DISCUSSION
 
Successive freeze-thaw cycles, which are characteristic of soil in a Chinook path, offer challenges and produce selective environments for cold adaptation of microbial communities that likely rival those found on the tundra and glaciers in the polar regions. Fast warming and chilling regimens are less damaging to microorganisms and result in intracellular ice formation, perhaps even a "glassy state," with no ice crystals (24). However, a low cooling and warming rate, such as the "Chinook-like" paradigm (0.5 to 1°C/min) used here, is particularly damaging (18) since ice crystals form extracellularly, concentrating the solutes and resulting in cell dehydration and consequent membrane damage and protein denaturation (17). Only modest decreases in viability were seen when the soil consortium was exposed to temperatures used for the thawing conditions, followed by a freeze period equal to that used in the freeze-thaw cycling paradigm but with a single freeze-thaw cycle (Table 1). In contrast, 48 freeze-thaw cycles resulted in tens of thousand-fold decreases in cell counts. These results dramatically demonstrate that microbe viability was not substantially reduced by cold starvation and that it was the cycling freeze-thaw treatments that were largely responsible for declines in the microbial populations. E. coli and P. chlororaphis cultures did not survive 48 cycles, the size of the soil consortium population was reduced 30,000-fold, and the population diversity was also reduced.

Not all cells in the soil samples were killed or irreversibly damaged by the treatments, and there was selection for a relatively small number of freeze-thaw-resistant species. The observation that the average initial diameter of the soil-derived colonies was affected less than the average initial diameters of E. coli and P. chlororaphis colonies after three freeze-thaw cycles, possibly reflecting cell damage, appeared to foreshadow the greater survival of the consortia than of the control bacteria. Other workers showed that when bacterial mixtures were added to snow and subjected to –8°C to 14°C cycles that were erratic and varied diurnally, no viable coliforms could be detected, but some gram-positive and gram-negative bacteria survived (24, 25). Similarly, in our study cultures derived from survivors of 48 freeze-thaw cycles exhibited increased viability after a further 48 cycles, demonstrating that freeze-thaw-susceptible microbes had been removed from the consortia (Fig. 2 and 4). Survivors were identified as members of five different genera, representing both gram-positive and gram-negative cells. Some of these microbes have been studied previously in association with cold stress or polar studies, so their appearance as the most freeze-thaw-resistant bacteria under this selective regimen is perhaps not unexpected. Cold shock and cold acclimation proteins have been studied in the psychrotroph Acinetobacter sp., a bacterium that has also been found in glacial ice (2, 6). Cold-active enzymes have been reported in Carnobacterium piscicola (7), Buttiauxella has been classified as psychrotrophic, and Chryseobacterium (formerly Flavobacterium) has been grouped with psychrophiles that have been found in an Antarctic glacier (6).

When the surviving cells were subjected to 48 freeze-thaw cycles as individual isolates, most had viability profiles that were similar to that of the selected, mixed population, in that there were decreases of 1 order of magnitude or less in the Acinetobacter sp. strain C3 or C5, Buttiauxella sp. strain C1, and Chryseobacterium sp. strain C14 cell numbers at the conclusion of the experiment. Carnobacterium sp. strain C4 was somewhat more susceptible, but it still survived better than the unselected soil consortium. In contrast, Enterococcus sp. strain C8 lost almost all viability after 48 cycles.

Since slow freeze-thaw treatments encourage the growth of large, damaging ice crystals, it is notable that one of the isolates having the largest proportion of viable cells after multiple freeze-thaw cycles, Chryseobacterium sp. strain C14, also showed IR inhibition. IR inhibition can be mediated by polysaccharides, such as xanthan gum, that are used commercially to prevent the formation of large ice crystals in ice cream or by AFPs that have been reported in a few bacteria, such as an Antarctic Moraxella (34), an Arctic Rhizobacterium (14, 22, 30, 33), cold-acclimated Micrococcus and Rhodococcus (10), and Antarctic lake bacteria (12). For the soil isolate examined here, it is likely that the effect on ice crystals was mediated by a protein, since the activity was destroyed when the cultures were treated with a protease.

It was hypothesized that IR inhibition activity could potentially contribute to the overall viability of the consortium under the "Chinook" conditions. Suspending Actinobacter sp. strain C5 cells in medium derived from Chryseobacterium sp. strain C14 cultures did not improve their already excellent viability (not shown), nor was the presence of viable Chryseobacterium sp. strain C14 (not shown) or medium derived from Chryseobacterium sp. strain C14 cultures able to "rescue" E. coli from lethal effects after 48 freeze-thaw cycles. Remarkably, however, when Enterococcus sp. strain C8 cultures were added to medium derived from Chryseobacterium sp. strain C14 cells, freeze-thaw survival increased dramatically (Fig. 5). There was some evidence of IR inhibition after 48 freeze-thaw treatments in these reconstituted cultures (not shown), but it was weak. Possibly, the death or damage of large numbers of the Enterococcus cells released proteases that might have effectively decreased the concentration of any antifreeze-like protein. Alternatively, other macromolecules derived from the freeze-thaw-resistant bacteria could have contributed to survival. Whatever the explanation, these two bacteria appear to be an example of commensalism, in which the less freeze-thaw-resistant organism benefits from an association with Chryseobacterium sp. strain C14. Enterococcus species are classic examples of commensal bacteria, and possibility this symbiotic facility extends to freeze-thaw tolerance. Certainly, there are examples of freeze survival by close associations, such as the report of Antarctic sea ice psychrotrophs that are commensals of diatoms (5). It would not be surprising if, in addition to Chryseobacterium sp. strain C14, other nonculturable cells contribute to the overall survival of susceptible microbes in Chinook region soils.

In conclusion, our cryocycler appears to allow systematic and reproducible investigation of microbial viability after freeze-thaw treatments. We hope that further detailed investigations of the adaptations of temperate isolates to the rigors of a Chinook environment will allow community associations and biochemical analyses of freeze-thaw survival in resistant bacteria.


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ACKNOWLEDGMENTS
 
This work was supported by a Queen's University Research Chair to V.K.W. and by NSERC (Canada) Discovery grants to G.V. and V.K.W. The University of Calgary is acknowledged for infrastructure support during V.K.W.'s sabbatical research period.

We thank Shelley Haveman, Garth Fletcher, Jack Gilbert, and Johanna Voordouw for materials, helpful advice, and encouragement, and we acknowledge comments from two anonymous referees. V.K.W. also thanks Brendan Palmer for first noticing the changes in colony size after freezing.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biology, Queen's University, Kingston, Ontario, Canada K7L 3N6. Phone: (613) 533-6123. Fax: (613) 533-6617. E-mail: walkervk{at}biology.queensu.ca. Back


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Applied and Environmental Microbiology, March 2006, p. 1784-1792, Vol. 72, No. 3
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.3.1784-1792.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Wu, Z., Qin, L., Walker, V. K. (2009). Characterization and recombinant expression of a divergent ice nucleation protein from 'Pseudomonas borealis'. Microbiology 155: 1164-1169 [Abstract] [Full Text]  

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