RESEARCH ARTICLE

Bacterial responses to fluctuations and extremes in temperature and brine salinity at the surface of Arctic winter sea ice Marcela Ewert & Jody W. Deming School of Oceanography, University of Washington, Seattle, WA, USA

Correspondence: Marcela Ewert, School of Oceanography, University of Washington, Campus Mailbox 357940, Seattle, WA 98195, USA. Tel.: +1 206 543 0845; fax: +1 206 543 0275; e-mail: [email protected] Received 27 December 2013; revised 30 May 2014; accepted 2 June 2014. Final version published online 30 June 2014. DOI: 10.1111/1574-6941.12363

MICROBIOLOGY ECOLOGY

Editor: Johanna Laybourn-Parry Keywords freeze tolerance; halotolerance; osmoprotection; compatible solutes; fragmentation.

Abstract Wintertime measurements near Barrow, Alaska, showed that bacteria near the surface of first-year sea ice and in overlying saline snow experience more extreme temperatures and salinities, and wider fluctuations in both parameters, than bacteria deeper in the ice. To examine impacts of such conditions on bacterial survival, two Arctic isolates with different environmental tolerances were subjected to winter-freezing conditions, with and without the presence of organic solutes involved in osmoprotection: proline, choline, or glycine betaine. Obligate psychrophile Colwellia psychrerythraea strain 34H suffered cell losses under all treatments, with maximal loss after 15-day exposure to temperatures fluctuating between
7 and
25 °C. Osmoprotectants significantly reduced the losses, implying that salinity rather than temperature extremes presents the greater stress for this organism. In contrast, psychrotolerant Psychrobacter sp. strain 7E underwent miniaturization and fragmentation under both fluctuating and stable-freezing conditions, with cell numbers increasing in most cases, implying a different survival strategy that may include enhanced dispersal. Thus, the composition and abundance of the bacterial community that survives in winter sea ice may depend on the extent to which overlying snow buffers against extreme temperature and salinity conditions and on the availability of solutes that mitigate osmotic shock, especially during melting.

Introduction The upper sea ice column is inhabited by a bacterial community, with a minor component of archaea, exposed to multiple physical, chemical, and biological stressors, including extremes of low temperature and high brine salinity (Collins et al., 2010). Bacteria inhabit the brine inclusions of the ice (Junge et al., 2004), which are interior microhabitats physically and chemically dynamic even in winter. Seasonal changes in atmospheric temperature expose sea-ice bacteria to a temperature decrease in winter and a temperature increase in spring (Collins et al., 2008; Ewert & Deming, 2013). According to the phase equations of sea ice (Cox & Weeks, 1983), these temperature changes result in seasonal changes in brine salinity. Even transient warming events, where the upper temperature still remains below freezing, can cause fluctuations in the brine salinity and volume fraction of the ice ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

(Petrich and Eicken, 2010) over timescales of a few hours to several days. The ice column is insulated from heat loss by the ice itself and by the overlying snow, but more exposed environments associated with the surface of the sea ice, such as the brine-skim layer, frost flowers, or saline snow layer, can exchange heat directly with the atmosphere. These more exposed environments are thus subjected to more extreme temperatures and salinities and wider fluctuations in both parameters (Ewert & Deming, 2013), with salinity expected to change with temperature in a manner similar to that in sea ice (e.g. Perovich & Richter-Menge, 1994; Barber et al., 2003). Temperature fluctuations of atmospherically exposed environments thus subject microorganisms in the brine phase to marked and frequent shifts in osmolarity (Ewert, 2013), yet the effects of such extreme conditions on bacteria present in these environments have not been examined. FEMS Microbiol Ecol 89 (2014) 476–489

Bacterial responses to winter surface sea ice conditions

Bacterial losses of 24–49% have been observed in the sea-ice column after 70–440 days under Arctic winter conditions (Krembs et al., 2002; Collins et al., 2008). The structure of the bacterial community, however, does not appear to change significantly during the winter period (Collins et al., 2010). Although possible changes in bacterial community structure over time are largely unknown for surface sea-ice environments, cell abundance can be expected to change (Ewert et al., 2013). Mortality in sea ice and associated environments may be caused by high osmolarity of the brine phase, reduced habitable space, viral lysis (Wells & Deming, 2006; Collins & Deming, 2011), cell puncturing by ice crystals, and the formation of intracellular ice crystals (Mazur, 1984). Cell loss has also been documented in freeze–thaw experiments using frozen soil communities (Morley et al., 1983) and sea ice bacterial isolates (Liu et al., 2013) where samples were fully melted at room temperature prior to refreezing. Furthermore, changes in the structure of bacterial communities in subarctic sea ice have been associated with shifts in salinity (Kaartokallio et al., 2005), implying selective mortality according to degree of halotolerance. To cope with an increase in osmolarity, whether under freezing or nonfreezing conditions, many bacteria accumulate compatible solutes, a category of organic compounds that can be concentrated in the cell without interfering with cellular functions (Roberts, 2005). Compatible solutes act as osmoprotectants by increasing the osmolarity of the cell, although their accumulation has also been associated with thermal stress in some microorganisms (e.g. Shockley et al., 2003; Hoffmann & Bremer, 2011). Examples of these solutes include glycine betaine (GB) and proline, which are of widespread use (Roberts, 2005; Empadinhas & da Costa, 2008; Wargo, 2013). Choline, although not a compatible solute per se, can also provide osmoprotection in organisms able to transform it into GB (Styrvold et al., 1986). Compatible solutes can be synthesized de novo, converted from precursors, or imported from the environment. Direct uptake from the environment is the most energetically favorable process (Oren, 1999) and can occur rapidly, on a scale of nM h
1 (as measured for choline in a temperate estuary; Kiene, 1998). Membrane transporters allowing the uptake of compatible solutes are widespread in Bacteria. In particular, GB, choline, and proline can be imported by primary transporters of the type ATP-binding cassette (ABC; ATP-driven). Secondary transporters of the betaine/choline/carnitine transporter family (BCCT), driven by proton or sodium motive force, can also transport betaine or choline and, in some instances, proline (Ziegler et al., 2010). BCCT transporters can respond to different stimuli, with the length of the N- and C-terminal FEMS Microbiol Ecol 89 (2014) 476–489

477

extensions of the transporter playing a critical role in their response to either osmolarity or temperature (Ziegler et al., 2010). The bacterial response to a decrease in osmolarity is mediated by mechanosensitive channels, membrane channels that act as ‘emergency valves’. Mechanosensitive channels open when the intracellular pressure reaches a critical value, leading to a rapid and nonspecific release of solutes that does not require energy to be activated (Morbach & Kr€amer, 2002). Released solutes, including significant amounts of small organic molecules from the intracellular pool of the cell, can be recovered quickly from the surrounding media soon after the osmotic shock (Schleyer et al., 1993; Halverson et al., 2000). Depending on the membrane transporter, re-accumulation of compatible solutes may imply energy consumption for the cell. This paper presents a series of three experiments designed to assess the impacts of various freezing conditions on two cold-adapted Arctic isolates with different growth ranges for temperature and salinity. The first isolate examined, Colwellia psychrerythraea strain 34H (Cp34H), is an obligately psychrophilic gamma-proteobacterium that was isolated from cold (< 0 °C) marine sediments (Huston, 2003) and has relatives of the same genus and species found in sea ice (Bowman et al., 1997, 1998); its whole-genome sequence is available to explore for the potential to use compatible solutes (Methe et al., 2005; Collins & Deming, 2013). Cp34H grows at temperatures of
12 to 18 °C and salinities of 20–50 ppt (Huston, 2003; Wells & Deming, 2006). The second isolate is a psychrotolerant species of the genus Psychrobacter, not yet characterized at the species level but designated as strain 7E in our culture collection (P7E). P7E was isolated from upper sea-ice brine (salinity of 128 ppt by refractometer) collected in winter from a first-year ice floe in the southern Beaufort Sea. (Calculated brine salinities are reported in this paper in units of ppt, according to Cox & Weeks, 1983; as are laboratory-generated salinities from weighed amounts of salt.) Like other species in this genus (Romanenko et al., 2004), P7E can grow over a broad range of temperatures (<
8 to 25 °C) and salinities (17–125 ppt); higher temperatures and lower salinities have not yet been tested. Our working hypothesis was that bacteria with wider tolerances of salinity would better survive the more extreme and fluctuating conditions that characterize upper regions of the winter snow/sea ice system, compared with bacteria with narrower growth ranges. We also considered the related hypothesis that compatible solutes would offer protection against the extreme brine salinities and fluctuations inherent to the freezing conditions tested. ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

478

The objective of the first experiment was to identify first-order responses of Cp34H to freezing regimes that simulated the natural environment, followed by complete (direct) melting of the frozen samples. The second experiment included both organisms, used a wider range of test temperatures (
7 to
25 °C), and examined potential cell losses due to freezing conditions only, that is, by protecting against osmotic shock at the end of the experiment by melting into brine (with a direct-melt comparison). The third experiment reexamined the effects of the more extreme conditions on both organisms and considered whether compatible solutes offered either of them any osmoprotection. We tested proline and GB, as well as the GB precursor choline, based on genomic and physiological information available for Cp34H (Collins & Deming, 2013) and genomic information available for the permafrost isolate Psychrobacter arcticus, the closest relative of P7E for which a whole-genome sequence is available (Bakermans et al., 2006).

Materials and methods Environmental fluctuations in temperature and brine salinity

We analyzed in situ temperature records from the Mass Balance Observatory Site (MBS), established by the University of Alaska Fairbanks in coastal first-year sea ice near Barrow, Alaska (156.5°W, 71.4°N; Druckenmiller et al., 2009). Temperature (accurate to  0.2 °C) was recorded at 15-min intervals from January to June 2011. Data sets were downloaded from the MBS website (UAF, 2011). Brine salinity was calculated from temperature using phase equations for sea ice (Cox & Weeks, 1983). Analysis focused on three environments: the upper ice column (from 0 to 10 cm below ice surface), the ice– snow interface (0 cm), and the proximate snow– atmosphere interface (10 cm above the ice surface). Winter was defined as days 25–78 of the year, with day 25 being the first available measurement and day 79 the first inflexion point in the temperature data series determined by linear regression analysis, which also coincided with the spring equinox. Spring was defined as days 79–156, with day 156 corresponding to the second inflexion point in the temperature series, just a few days before the summer solstice on day 171. The intensity of the fluctuations in temperature and brine salinity was analyzed for each targeted environment by a power spectral analysis, using the Welch’s method (Emery & Thomson, 2001). Winter and spring time series were analyzed separately, dividing each time series into eight segments with 50% overlap and a Hamming window function. Each segment was treated as an independent time series. ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

M. Ewert & J.W. Deming

Preparation of isolates

Colwellia psychrerythraea strain 34H was subcultured from aliquots of the original sequenced isolate (Huston, 2003; Methe et al., 2005) stored at
80 °C since purification. Psychrobacter sp. strain 7E was newly isolated from winter sea ice brine that had accumulated overnight in bore holes drilled in a first-year ice floe, as described in Ewert (2013), and also stored at
80 °C following purification. Stock cultures of both organisms were grown and maintained in Marine Broth 2216 (Difco Laboratories) diluted to half organic strength but retaining full seawater salinity of 35 ppt (1/2 9 MB 2216), previously determined to provide optimal nutrient conditions. For the freezing experiments, we used the more defined medium of lactate and yeast extract (LYE) as the growth medium. LYE was prepared as in Marx et al. (2009) with 0.5% lactate and 0.0014% yeast extract in artificial seawater (ASW, 0.4 M NaCl, 9 mM KCl, 26 mM MgCl2, 28 mM MgSO4, and TAPSO buffer), with a final salt concentration of 35 ppt. For Experiment 1, the test organism was also grown in LYE supplemented with glycine betaine (LYEGB) by amending 500 mL of LYE with 0.25 mL of a 2 M solution of betaine hydrochloride (Sigma) in ASW, for a final concentration of 1 mM; NaOH was used to neutralize the LYE-GB medium to a pH of 7.3 before autoclaving. For the evaluation of genomic information associated with osmotolerance, we considered the whole-genome sequence for Cp34H (Methe et al., 2005) and, as a proxy for P7E, the available genome sequence of P. arcticus. Psychrobacter arcticus has 97% similarity with P7E (hypervariable V3–V5, 16S rRNA gene). Genes were accessed online at MicrobesOnline (Dehal et al., 2010; www.microbesonline.org) to check for homology, operons, and putative function. Prediction of N- and C-terminal length was performed by prediction of transmembrane helices in proteins (www.cbs.dtu.dk/services/TMHMM/). Bacterial abundance

For each experimental time point, samples were fixed immediately after melting, using 0.2 lm filtered formaldehyde to a final concentration of 2%, and refrigerated in the dark until processing within 1–3 weeks. Bacterial abundance was determined by epifluorescence microscopy. An aliquot of fixed sample was brought to 5 mL volume by adding 0.2 lm filtered ASW, amended with 2–3 drops of surfactant (Triton-X) to disperse cells, and mixed by vortex for 10 s. Samples were filtered onto 0.2-lm pore size polycarbonate filters (Poretics) and stained for 10 min with the DNA-specific stain 40 -60 -diamidino2-phenylindole (DAPI), as in Marx et al. (2009). Filters, mounted onto slides, were examined with a Zeiss FEMS Microbiol Ecol 89 (2014) 476–489

479

Bacterial responses to winter surface sea ice conditions

Table 1. Summary of experiments. Samples were exposed to various freezing conditions for a short or long period of time (specified in parentheses). Samples were allowed to melt either directly or into a prechilled NaCl brine solution with final melt concentration of c. 100 ppt. Samples were amended with compatible solutes glycine betaine (GB) or proline (Pro) or the precursor choline (Cho). In Experiment 1, GB was added to the medium prior to bacterial growth; in Experiments 2 and 3, solutes were added just prior to freezing. Dashes indicate not performed

Exp.

Organism tested

Final melting protocol

Organic solute added

1 2 3

Cp34H Cp34H, P7E Cp34H, P7E

Direct only Direct and into brine Brine only

GB None Pro, Cho, GB


20 °C

Short

Long

Short

Long

Short

Long

U (3 days) U (40 min) –

U (15 days) U (15 days) –

U (3 days) U (40 min) –

U (15 days) U (15 days) U (15 days)

U (3 days) – –

U (15 days) U (15 days) U (15 days)

Universal epifluorescence microscope, with a minimum of 20 fields or 200 bacteria counted for each sample. One of the treatments from Experiment 3 consistently showed cell clumping despite the use of Triton-X; in this treatment, additional aliquots were processed with more Triton-X (five drops) and sonicated for 30 s, 1 min, or 5 min using a XL 2015 Sonicator (Misonix) at 20 kHz intensity. Cell loss or gain due to freezing treatment was evaluated as the difference between mean bacterial abundance measured at time 0 and at the end point of interest (see Statistical analysis). In the case of long-term freezing at
20 °C, cell size was estimated by image analysis of photographs taken from the DAPI-stained slides. Photographs were analyzed using the public domain Java image processing program IMAGEJ (http://imagej.nih.gov/ij/). The areas of a minimum of 200 bacteria were measured for each sample (n = 6). Experimental treatments

Table 1 presents a comparative summary of conditions used in our three experiments. In all cases, test organisms were grown at 8 °C (optimal for Cp34H) in LYE media to mid-exponential phase (5 days) before subjecting to freezing; for Experiment 1, Cp34H was also grown in LYE-GB medium. Cultures were placed in an ice bath (0 °C) for manipulations, and 1-mL aliquots were removed to measure initial bacterial abundance (n = 1 for Experiment 1; n = 6 for Experiments 2 and 3). Samples were then subjected to their freezing treatments. Although the use of cells in growth phase was to ensure viability at the start of an experiment, cell viability was also confirmed early in the study by documenting cell increases in control cultures held at 8 °C in parallel with freezing treatments. After the freezing treatments, samples were returned to the ice bath and allowed to melt directly (direct melt) or melted into brine. For brine melts, 0.35 mL of sterile, prechilled, 265 ppt NaCl brine was added to each microtube containing a 1.0-mL sample to achieve a final melt concentration of c. 100 ppt. This final melt salinity was chosen based on results from previous FEMS Microbiol Ecol 89 (2014) 476–489

Fluctuating between
7 and
25 °C

Above
20 °C

laboratory experiments, using bacterial isolates from frost flowers (collected from the same field site as P7E), showing no significant changes in cell numbers following a salinity shift from 220 to 100 ppt (data not shown; see Ewert et al., 2013). Immediately upon melting, samples were fixed for bacterial abundance measurements. Experiment 1 Triplicate aliquots of 5 mL of Cp34H culture, grown in LYE and LYE-GB medium, were transferred to sterile 15-mL tubes, frozen under different conditions (Table 1 and Supporting Information, Fig. S1) and directly melted. In the first short-term (3-day treatments) treatment, Cp34H was initially frozen at about
17 °C, after which the temperature was allowed to stabilize near
12 °C (treatment average of
13 °C), closely following values measured over time in the upper sea-ice column (Fig. S1a). In the second short-term treatment, samples were also initially frozen at about
17 °C, but the temperature was then allowed to fluctuate between
2 and
17 °C, closely following values measured in the snow near the atmosphere. Cells in the first treatment experienced one temperature cycle where the predominant temperature was relatively stable, whereas those in the fluctuating treatment experienced three cycles (Fig. S1a). For the two long-term 15-day treatments, Cp34H was held frozen at a stable temperature of
13 °C for one long cycle and under the same fluctuating temperature regime as before (between
17 and
2 °C) for six cycles (Fig. S1c). Brine salinities during the freezing periods (Fig. S1b and d) were calculated following Cox & Weeks (1983). Additional replicate aliquots (n = 2) were placed at stable
20 °C for comparison. Based on a power analysis of results of Experiment 1, the number of replicas for Experiments 2 and 3 was increased to six. Experiment 2 Aliquots of 1 mL of Cp34H and P7E cultures were transferred to sterile 1.5-mL microcentrifuge tubes and subjected to the following treatments. For short-term 40-min ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

480

treatments, replicas (n = 6) were placed at temperatures of
10,
15, and
20 °C, monitored every 30 min until frozen, and then left at stable-freezing temperature for 40 min. Estimated cooling rates were 0.2–0.7 °C min
1. Samples at
10 and
15 °C took longer to freeze (1 h) compared with samples at
20 °C (30 min); light shaking was required to promote nucleation. To end the short-term treatments, separate sets of replicas were melted directly and into brine for comparative purposes. Samples from the direct melts of P7E that had been frozen at stable
10 °C were lost due to procedural error. For long-term 15-day treatments, replicate aliquots were placed at stable temperatures of
10,
15, and
20 °C and also subjected to temperatures fluctuating between
7 and
25 °C every 12 h (24-h cycle). On one occasion, samples were left at
25 °C for 36 h (for a total of 14 temperature cycles). All long-term samples were melted into brine. Experiment 3 Aliquots of 0.9 mL of Cp34H and P7E cultures were transferred to sterile 1.5-mL microcentrifuge tubes and, just prior to freezing, amended with a 10 mM solution (in LYE) of choline (Cho), GB, or proline (Pro) to achieve a final concentration of 1 mM. The blank treatment received a 0.1-mL aliquot of sterile LYE. Samples were frozen under either stable (
20 °C) or fluctuating (
7 to
25 °C) temperatures for 15 days and then melted into brine. The warmer temperature was monitored and observed to oscillate between
5.5 and
9 °C (
6.8  1.1 °C, mean  SD, n = 15). The number of temperature cycles was as in Experiment 2. Statistical analyses

Means and 95% confidence intervals (CI) are shown in all figures. Normality and equal variance were tested using Shapiro and Bartlett tests. Experiment 1 was analyzed with a two-way analysis of variance (ANOVA) for unbalanced proportional design, with each medium analyzed separately. In Experiments 2 and 3, initial and final bacterial abundances were compared for each isolate using t-tests. Experiment 2 was analyzed with two-way ANOVAs to determine the effect of melting procedure on samples subjected to short-term freezing, as well as the effect of freezing time on samples frozen at stable temperature and melted into brine. Stable and fluctuating conditions were compared using a one-way ANOVA. Experiment 3 was analyzed with a two-way ANOVA comparing stable vs. fluctuating conditions, as well as type of amendment to the medium. When type of amendment had a significant effect, levels were analyzed with a post hoc pairwise comparison using ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

M. Ewert & J.W. Deming

Tukey’s honest significant differences test. The stable
20 °C treatment amended with GB was not included in this comparison due to cell aggregation; the fluctuating treatment with GB was compared directly with LYE medium using a t-test. Statistical analyses were performed using R v.2.13.1 (R Development Core Team, 2011).

Results Temperature and brine salinity regimes

The interquartile ranges for temperature and calculated brine salinity, representing the most frequent values in a given season, were wider at the snow–atmosphere interface than in the ice column and wider in spring than in winter (Fig. 1 and Table 2). Despite different absolute minima and maxima, the total range (maximum span) was similar in both seasons (Fig. 1, Table 2). Change in median values from winter to spring was also greatest at the snow–atmosphere interface (DT = 10.3 °C; DS = 71 ppt) than in the ice column (DT = 5.3 °C; DS = 40 ppt). Power spectrum analysis confirmed the snow–atmosphere interface as the environment with the most variable conditions, with significantly (95% CI) wider fluctuations of temperature and calculated brine salinity than in the ice column for periods between 1 h and 4 days. All three environments had milder fluctuations and increased noise level toward the shorter periods, as well as a significant diurnal spectral peak at 0.04 cycles per hour (1-day period) in the spring (Fig. S2). A small diurnal temperature fluctuation was noticeable in winter. Experiment 1

After direct melting, cell abundance was significantly lower for Cp34H samples subjected to long-term (15 days) freezing than for samples subjected to shortterm (3 days) freezing (P < 0.05; Fig. 2). This result was consistent across all freezing conditions. The greatest cell loss was observed in samples subjected to the most extreme treatment applied in this experiment: long-term freezing under fluctuating temperatures (between
2 and
17 °C), followed by direct melt. Short-term freezing showed the highest cell loss at the lowest temperature (
20 °C). There was no significant difference in end point data between the two growth media at either freezing period. Experiment 2

Unlike Experiment 1, where only end point comparisons of cell abundance were possible statistically, Experiment 2 allowed for robust comparison between the start and end FEMS Microbiol Ecol 89 (2014) 476–489

481

Bacterial responses to winter surface sea ice conditions

Winter

20

Winter

Spring

(a)

250

–10 –14

–20

–8.7

150

2.8

0

–10

–8.3

–16

–20

–17

–27 –40

250

150

(e)

233 191

182

134

143

50

0.1

0 250 (f) 244

(c)

3.7

0 –20

–6.7

–9.7 –25

–40

–37 0

50

100

150

231

211 150

140

–20

109

50 0

0.1 0

50

100

150

Calendar day

Maximum range

Interquartile range

Environment

Season

T (°C)

S (ppt)

T (°C)

S (ppt)

Snow–atmosphere interface

Winter Spring Winter Spring Winter Spring

30.1 28.5 16.5 20.0 12.4 15.9

136 232 90 191 83 179

6.7 13 4.6 9.6 3.6 7.8

42 136 28 105 23 87

of each treatment period. It also differentiated cell loss due to freezing conditions from that due to osmotic shock. Brine melting showed significant protection against cell loss when both organisms were considered together (i.e. two-way ANOVA; P < 0.05), although no significant effect was detected in P7E when data were evaluated separately (Fig. 3b). Direct melts caused significant cell losses of 22–30% for Cp34H (Fig. 3a). When comparing with initial conditions, no significant cell loss was FEMS Microbiol Ecol 89 (2014) 476–489

Brine salinity (ppt)

Temperature (°C)

(b)

Table 2. Maximum range (widest observed span) and interquartile ranges (representing most frequent conditions) of temperature (T) and brine salinity (S) recorded at three locations in the snow-ice system at the MBS site in 2011. Brine salinity was calculated in units of ppt according to Cox & Weeks (1983)

Upper ice column

0.1

0

20

Ice–snow interface

130

144

50

–40

20

179

171

–15

–23

Spring

227

0.7

0

Fig. 1. Seasonal temperatures (a–c) recorded at the MBS site during winter and spring of 2011 and corresponding brine salinity (d–f) for the upper sea ice column (a, d), ice–snow interface (b, e), and snow–atmosphere interface (c, f). For each season and environment, boxplots illustrate most frequent conditions; black lines and numbers indicate the median; gray lines and numbers indicate maximum and minimum. The vertical dashed line marks the spring equinox. The shaded vertical line in (a), (c), (d), and (f) highlights the warming event replicated in Experiment 1. (This figure was modified from Figs 3 and 4 in Ewert & Deming, 2013.)

(d)

observed for either test organism after short-term (40 min) freezing if the samples were melted into brine (Fig. 3). Among treatments melted into brine, freezing time and freezing temperature were the significant factors contributing to cell loss for Cp34H (P < 0.05; Fig. 3c). Cell losses were higher after 15 days, ranging from 21% to 53%, with the greatest losses observed when the freezing temperature fluctuated between
7 and
25 °C and when samples were held at
10 °C (53% and 45%, respectively; Fig. 3c). P7E showed significant difference in cell abundance due to the interaction of freezing time and freezing temperature (P ≤ 0.01), but no difference could be explained by a single factor only. In contrast to Cp34H, cell numbers for P7E samples appeared to increase (P = 0.06) when subjected to long-term (15-days) freezing at
20 °C and increased significantly (P < 0.01), by 66%, when temperature was allowed to fluctuate between
7 and
25 °C. The P7E replicas subjected to long-term freezing at
10 and
15 °C were not frozen at the end of the experiment (empty bars in Fig. 3). Cell abundance in these unfrozen treatments did not change significantly from initial conditions, although they did appear to be ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

482

M. Ewert & J.W. Deming

Brine salinity (ppt) Ini

166

209

38/191

(a)

3 days 15 days

11% 2

–3%

Cell abundance (107 cells mL–1)

–34%

–31% –47%

1

–52%

0 (b)

24%

2

10%

–41%

1

–29% –47%

0 Ini

C.–13

–20

–35%

–2/–17

(unamended or amended with Pro, Cho, or GB), temperature regime (stable vs. fluctuating), and the interaction of these two factors (P < 0.001). Freezing at stable
20 °C did not result in significant changes from the initial cell numbers (Fig. 4a), regardless of type of amendment. When exposed to fluctuating temperatures, however, significant cell losses (11–51%) occurred in the unamended samples and samples amended with GB and Pro, with the greatest loss detected in the absence of added compatible solute (Fig. 4c). Post hoc tests (including a separate analysis for GB-amended samples) showed higher numbers in samples amended with compatible solute compared with unamended samples (P < 0.001). No significant cell loss was detected in Cho-amended samples. For P7E, significant differences in bacterial abundance were observed due to temperature regime only (stable vs. fluctuating; P < 0.05), with all types of amendments having similar cell numbers except for GB-amended samples (P < 0.001, Fig. 4d). Cell numbers increased significantly in all types of amendments when the organism was exposed to long-term (15-days) freezing at stable
20 °C (P < 0.01, Fig. 4b) and in unamended and GB-amended samples when frozen under fluctuating temperatures (P < 0.05, Fig. 4d). Image analysis showed a significant 40% reduction in cell size when comparing cells in the long-term freezing at
20 °C, with cells in the initial samples (P < 0.05, t-test).

Temperature (°C) Fig. 2. Results from Experiment 1: initial (Ini) and end point cell abundances for Cp34H grown in LYE (a) or LYE-GB medium (b) and exposed to freezing/melting treatments. Samples were frozen for 3 days (light bars) or 15 days (dark gray bars) under relatively stable (c.
13 and
20 °C) or fluctuating (between
2 and
17 °C) temperatures (see Fig. S1 for the precise temperature trajectories). All treatments were followed by direct melting. Secondary (upper) labels indicate brine salinities corresponding to each freezing temperature. Error bars represent mean with confidence interval (n = 3, except for
20 °C, where n = 2). Numbers above bars indicate percent change relative to initial cell abundance.

slightly lower than in their frozen counterparts at
10 and
15 °C in the short-term treatment (Fig. 3b). Experiment 3

Samples amended with GB showed cell clumping for both test organisms when frozen at stable
20 °C, thus preventing accurate cell counts for this treatment. Additional efforts to disperse cells were unsuccessful, so this treatment was not included in the following statistical analyses. For Cp34H, significant differences in bacterial abundance were detected according to type of sample amendment ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

Discussion Effects of short-term freezing and melting

Short-term decrease in temperature, simulating upper sea ice and associated environmental conditions, resulted in minimal (statistically insignificant) cell loss for both organisms tested. In contrast, the increase in temperature associated with direct melts, also exposing organisms to steep salinity shifts (DS > 108 ppt), doubled the percentage of cell loss for Cp34H. Accordingly, Cp34H cell loss in 3-day treatments was highest when cells were subjected to the steepest changes in temperature and salinity as the samples were frozen and then subjected to a strong salinity shift (DS = 175 ppt). Transient freezing events in sea ice (decreasing temperature and increasing brine salinity) thus would not be as strong contributors to cell loss as the drastic decrease in salinity caused by complete melting of the ice. Of the two organisms tested, only Cp34H showed significant cell loss due to complete ice melting, presumably reflecting the more osmotolerant character of new isolate Psychrobacter sp. strain 7E. Higher tolerance of salinity decreases in P7E could be associated with the presence of FEMS Microbiol Ecol 89 (2014) 476–489

483

Bacterial responses to winter surface sea ice conditions

Brine salinity (ppt) Ini

143

179

(a) Fig. 3. Results from Experiment 2: initial (Ini) and end point cell abundance for Cp34H (a, c) and P7E (b, d) grown in LYE medium and exposed to freezing/melting treatments. Samples were frozen for 40 min (a, b) or 15 days (c, d) under stable (
10,
15 and
20 °C) or fluctuating (between
7 and
25 °C) temperatures, followed by direct melting (light bars) or melting into brine (dark bars). Secondary (upper) axis as in Fig. 2. Error bars represent mean with 95% confidence interval (n = 6). Asterisks indicate significant change from initial cell abundance (*P < 0.05, **P < 0.01, t-test). Numbers above bars indicate percent change relative to initial cell abundance. See text for additional description of statistical comparisons among treatments. Missing bar in (b) (P7E, direct melt,
10 °C) corresponds to samples lost during processing; empty bars in (d) for treatments at
10 and
15 °C did not freeze during the 15-days incubation (see text).

Cell abundance (107 cells mL–1)

3

–14% ** –30%

–10% ** –22%

143

179

209

(c) –12%

* –21%

** –24%

2

109/232 Direct Brine

** –22%

** –45%

** –53%

1

0

(b)

(d)

** 66%

1.2

32% 8%

0.8

–11%

8% –24%

–7%

–22%

–9%

−10

−15

0.4 0.0

Ini

different types of mechanosensitive channels, potentially allowing a response to osmotic shifts of different intensity (Table 3, Morbach & Kr€amer, 2002). The observed differences in tolerance to steep osmotic changes highlight the need to melt into brine when processing sea-ice samples, especially from very cold environments, as discussed in Deming (2010). These in vitro results also agree with field observations by Ewert et al. (2013) that winter surface sea-ice samples suffer significant bacterial loss when allowed to melt directly. Effects of long-term freezing on cell abundance

For Cp34H, cell loss was greater when the freezing period was longer. Samples frozen at
10,
15, and
20 °C and melted in brine (without protective amendment of compatible solutes) resulted in cell losses of up to 45%, twice the losses experienced after short-term freezing. Whether Cp34H cell loss was due to prolonged exposure to freezing temperatures, to the corresponding high salinities or to both, cannot be deduced from these types of experiments in which the two factors change simultaneously. If due to freezing, the possible mechanisms of cell loss include the formation of extracellular (puncturing) or intracellular ice crystals (Mazur, 1984). If due to increased salinity, mechanisms include dehydration and irreversible plasmolysis (Scheie, 1969) and possibly lytic viral activity triggered by the salinity increase (Shkilnyj & FEMS Microbiol Ecol 89 (2014) 476–489

209

−10

−15

−20

−20

−7/−25

Temperature (°C)

Koudelka, 2007). Long-term freezing at
20 °C followed by direct melting was also highly deleterious for Cp34H, but most of this cell loss appeared due to the steep osmotic shift associated with direct melting, for that effect was milder when similarly treated samples were melted into brine. P7E presented a different response to long-term freezing, increasing cell numbers after 15 days at
20 °C. The observed decrease in cell size and increase in cell numbers suggest that P7E underwent a process of fragmentation prompted by the extreme conditions experienced during this prolonged freezing treatment. A fragmentation process (under nonfreezing conditions) has been observed for species of marine Vibrio, where exposure to starvation led to a 100–800% increase in cell numbers in 2 weeks accompanied by a reduction in cell size (Novitsky & Morita, 1977). An increase in cell numbers has also been reported for soil bacteria frozen at
9 °C for 9 days (Morley et al., 1983), while a reduction in bacterial cell size was reported for permafrost isolates grown at
10 °C (Bakermans et al., 2003). By increasing the total number of cells, cell miniaturization and fragmentation may increase the chances that a proportion of the population survives the period of harsh conditions to reproduce once the environment returns to a more favorable state. Achieving higher cell numbers without growth in an extreme environment may also increase the potential for dispersal to other settings that will experience more ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

484

M. Ewert & J.W. Deming

(a)

(c)

–4%

6%

Cell abundance (107 cells mL–1)

2

Unamended Amended

–6%

* –12%

–2%

* –11%

** –51%

1

0 (b)

** 69%

0.8

** 62%

(d)

** 84%

** 57% * 15%

15%

None

Pro

1%

0.4

0

Ini

None

Pro

Cho

GB

Sample amendment

clement conditions in later seasons; for example, winter saline snow at the sea-ice surface is subject to windinduced transport (Ewert et al., 2013). An analysis of viability in bacterial populations of interest would be needed to test these hypotheses. The finding that cell abundance for P7E had not changed significantly in samples that were unfrozen at
10 and
15 °C after 15 days supports the interpretation that the observed increases in cell number when samples did freeze at those temperatures (after 3 days) were associated with the ice-formation process itself. The lack of freezing may indicate that P7E produces an antifreeze substance with the ability to inhibit the growth of ice crystals. Antifreeze activity has been determined for other isolates of the genera Psychrobacter obtained from an Antarctic lake (Gilbert et al., 2004), but we have not made direct tests for such activity in our isolate. Effects of temperature and brine salinity fluctuations

The two organisms examined in this study also responded differently to fluctuating conditions during freezing treatments. When exposed to fluctuating temperatures in the absence of compatible solutes, Cp34H suffered cell losses within the range of 40–60% reported for soil samples subjected to repeated freeze–thaw cycles (Morley et al., 1983). A more extreme fluctuating regime was previously considered a likely cause for the lower cell abundance ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

Cho

GB

Fig. 4. Results from Experiment 3: initial (Ini) and endpoint cell abundance following freezing/melting treatments for Cp34H (a, c) and P7E (b, d) grown in LYE medium and amended, or not, with compatible solutes. Samples were frozen for 15 days under stable (
20 °C; a, b) or fluctuating conditions (between
7 and
25 °C; c, d), then melted into brine. Prior to freezing, samples were either amended with Pro, Cho, or GB to a final concentration of 1 mM (dark bars) or not amended (light bars). Missing bars indicate samples where cell abundance could not be determined due to cell aggregation. Error bars, asterisks and numbers above bars are as in Fig. 3 (n = 6). In (c), all amended treatments differed significantly from the unamended LYE treatment; in (d), only LYE-GB differed. See text for statistical details.

(relative to brine content) observed in Arctic saline snow as compared to the upper sea-ice column (Ewert et al., 2013). Temperature fluctuations expose cells not only to the potentially adverse effects of repeated ice crystal formation but also to multiple shifts in osmolarity. Such fluctuations may also increase the energy requirements of the cell beyond the already high-energy demands associated with survival at low temperatures (Bakermans & Nealson, 2004) and high salinities (Oren, 1999). P7E, on the other hand, had significant increases in cell numbers (15–66%) when exposed to fluctuating temperatures. These increases were likely the result of cell fragmentation associated with the stressors involved, as discussed above. Our results suggest that bacterial responses to extremes in fluctuating temperature and salinity may be speciesspecific. When complemented with studies of cell viability or gene expression, such results can contribute to explaining previous findings of distinctive bacterial communities in sea-ice surface environments (Bowman et al., 2013). Protective role of compatible solutes

When exposed to fluctuating temperature and salinity, the presence of the compatible solute GB at the time of freezing clearly offered protection against cell loss in Cp34H and led to increased cell numbers in P7E. Proline also had a protective effect on Cp34H, though a neutral influence on P7E. When GB, though, was added to the growth medium for Cp34H days before the freezing FEMS Microbiol Ecol 89 (2014) 476–489

485

Bacterial responses to winter surface sea ice conditions

Table 3. Comparative candidate genes, with putative functions in responding to osmolarity changes, for Colwellia psychrerythraea strain 34H and for Psychrobacter arcticus. Numbers in parentheses for genes betP, betU, and betT indicate number of amino acids in the N- and C-terminal tails for BCCT family transporters (N-/C-terminus) as predicted by the TMHMM method following Chen & Beattie (2008); www.cbs.dtu.dk/ services/TMHMM/ Gene

Putative function

Cp34H

betP

Betaine choline carnitine transporter (BCCT)

CPS_2003 (42/9) CPS_4027 (42/22) CPS_3860 (19/16)

betU betT

BCCT transporter BCCT transporter

betI

Transcriptional regulator

betB

Betaine aldehyde dehydrogenase

betA

Choline dehydrogenase

ProX ProV ProW PutP

Amino acid ABC transporter ATP-binding protein Proline/glycine betaine ABC transporter Na+/proline symporter

MscS

Small-conductance mechanosensitive ion channel

MscL

Large-conductance mechanosensitive ion channel

event, its survival was not improved. This result can be explained by the ability of Cp34H to metabolize GB, leaving it unavailable at the time of freezing. Consumption of GB was confirmed by higher cell yields for Cp34H in LYE-GB medium after growing for 15 days at 8 °C (data not shown). The maximum effectiveness of compatible solutes in the sea-ice ecosystem may thus depend on the timing of their production and location in the ice column. The availability of choline, a GB precursor, also reduced cell loss in Cp34H under fluctuating temperature and salinity, confirming that Cp34H can transport choline and transform it into GB, as proposed by Collins & Deming (2013). The ability to metabolize GB (Collins & Deming, 2013) could also provide Cp34H with an additional energy source to compensate for the higher energy requirements of the fluctuating environment. FEMS Microbiol Ecol 89 (2014) 476–489

CPS_4009 (3/27) CPS_1335 (3/27) CPS_4012 CPS_1332 CPS_4011 CPS_1333 CPS_4010 CPS_1334 CPS_0670 CPS_3434 CPS_4933 CPS_4935 CPS_4934 CPS_0068 CPS_0939 CPS_3347 CPS_3419 CPS_3463 CPS_0104 CPS_1419 CPS_1746 CPS_4603 CPS_1934 CPS_0969 CPS_4829 CPS_4830 CPS_0961 CPS_0962

P. arcticus

Psyc_1301 (8/185) Psyc_0727 (18/177) Psyc_0826 (18/44) Psyc_0730 Psyc_0729 Psyc_0728

Psyc_1415

Psyc_0970 Psyc_0625

Psyc 0340

In the stable
20 °C treatments, GB promoted cellular aggregation in both test organisms. Exogenous GB has been reported to promote cellular aggregation in Vibrio cholerae when exposed to high-salinity conditions (Kapfhammer et al., 2005). Lack of cellular aggregation in the fluctuating treatment could be explained by the transient character of GB accumulation, as it would be likely released back to the medium with every decrease in osmolarity. Comparative genomics on osmoprotection

Differences in the response of C. psychrerythraea strain 34H and Psychrobacter sp. strain 7E to the various freezing regimes and associated salinity changes are consistent with the genomic differences between Cp34H and P. arcticus, a close relative of P7E, regarding putative genes ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

486

associated with osmoprotection (Table 3). Both the protective effect of GB and the promotion of cellular aggregation are consistent with the presence of multiple GB transporters in the genomes of Cp34H and P. arcticus. The neutral influence of proline on P7E is consistent with the fewer copies and fewer types of proline transporters in P. arcticus compared with Cp34H. Mechanosensitive channels also differed in Cp34H and P. arcticus, with the latter having a copy of a large-conductance mechanosensitive channel, in agreement with our experimental results showing a greater tolerance of steep salinity shifts by PE7. The N- and C-terminal lengths of the BCCT transporters differed greatly between organisms (Table 3), with long C-terminal tails in P. arcticus and short tails in Cp34H. Putative betP transporters in Cp34H also had very short N-terminus, predicted to extend into the periplasmic space, not intracellularly as in other organisms. Based on work with Escherichia coli, Corynebacterium glu€ tamicum, and other bacteria (Ozcan et al., 2005; Tøndervik & Strøm, 2007; Ziegler et al., 2010), different lengths in the C-terminus suggest that the environmental cue leading to the activation of BCCT transporters, and consequent uptake of GB and choline, might differ between Cp34H and P. arcticus. A long C-terminal tail, as in genes Psyc_1301 and Psyc_0727 from P. arcticus, would suggest regulation primarily by osmolarity (possibly the case for our Psychrobacter sp. isolate as well); a short C-terminus, as in the same genes from Cp34H as well as Psyc_0826 from P. arcticus, would suggest regulation primarily by temperature. Proteomic work with P. arcticus provides some support for this hypothesis, as gene Psyc_0727 was not differentially expressed with temperature, while Psyc_0826 was downregulated at
6 °C, although Psyc_1301 was upregulated at
6 °C (Bergholz et al., 2009). As a note of caution, the close phylogenetic relationship between P. arcticus and P7E does not mean similarity across the genome. Genotypic and phenotypic differences are expected, especially given differences in the environments from which these two species were isolated. Psychrobacter arcticus came from Arctic permafrost with an estimated temperature of
9 to
11 °C considered stable over many thousands of years (Bakermans et al., 2006). P7E, on the other hand, was isolated from upper sea ice which, as described earlier, experiences extremes in brine salinity and temperature on the seasonal scale and extreme fluctuations in these parameters on shorter timescales. Despite these differences, both species grow across a wide range of salinities, from 0.6 to 76 ppt (range of 75 ppt) for P. arcticus and from 17 to 125 ppt (range of 108 ppt) for P7E. By comparison, Cp34H grows across a more restricted range of salinities (30 ppt), from 20 to 50 ppt. ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

M. Ewert & J.W. Deming

Environmental parameters and the sea ice microbial community

Median winter temperatures in the studied system are permissive for bacterial activity, and possibly growth, during winter (Huston, 2003; Breezee et al., 2004; Bakermans & Skidmore, 2011; and references within; Mykytczuk et al., 2013). Temperature, however, is not the only factor determining bacterial activity and growth in frozen environments (as reviewed by Bakermans & Skidmore, 2011). Limiting parameters such as high brine salinity, entrapment in a confined space, and short-term fluctuations in temperature and brine salinity differ depending on the location in the sea ice–snow–atmosphere continuum. Long-term freezing thus has the potential to select for different bacterial populations, particularly in the snow– atmosphere interface which presented the broadest interquartile ranges for temperature and salinity and the most energetic fluctuations. In fact, bacterial selectivity has been observed in saline frost flowers on the surface of new ice (Bowman et al., 2013). Organisms able to tolerate wide changes in both temperature and salinity would be favored in surface sea-ice environments with stronger fluctuation regimes, with osmolarity changes being the main stressor of the population. Organisms with more restrictive tolerances would survive best deeper in the ice column. Supporting this hypothesis, the two species evaluated in this study responded differently to long-term freezing under stable and fluctuating temperatures, with different susceptibilities to a decrease in osmolarity and different effects of compatible solutes. In fact, the higher tolerance of Psychrobacter species to a decrease in osmolarity might help them better persist through winter and spring, making possible their detection in summer sea ice (e.g. Bowman et al., 1997). Different growth ranges of the two species studied could also have played a role in their differential survival. Despite cold adaptation, growth of Cp34H at even moderate subzero temperatures may be limited by the corresponding brine salinity. P7E, on the other hand, even though possibly less cold-adapted, has the advantage of tolerating a wider range of salinities and thus may be active during the warming periods of the fluctuation. This suggestion is consistent with the recognition that simultaneous adaptation to multiple stressors and a broad range of environmental conditions is characteristic of species inhabiting frozen environments such as permafrost (Mykytczuk et al., 2013).

Conclusions As verified by observations near Barrow, Alaska, environmental conditions at the snow–atmosphere interface during winter are more extreme than those in the upper sea FEMS Microbiol Ecol 89 (2014) 476–489

Bacterial responses to winter surface sea ice conditions

ice column, with median winter temperatures colder by as much as 6 °C, brine salinities higher by as much as 40 ppt, and significantly wider fluctuations in both of these parameters. The results of laboratory experiments exposing the Arctic bacterium Colwellia psychrerythraea strain 34H to the most extreme conditions tested (temperatures fluctuating between
7 and
25 °C over 15 days) indicated that the better location for this organism to survive the winter would be deeper in the sea ice, where conditions are comparatively milder and more stable. The cell losses experienced by Cp34H under simulated winter conditions appeared to be due to osmotic shock associated with brine salinity changes, for providing compatible solutes at the time of freezing reduced the observed losses. The importance of compatible solutes to bacteria in sea ice may not be limited to intracellular osmoprotection, for the presence of the compatible solute GB, also induced cell clumping in both test organisms under stable-freezing conditions (
20 °C). In the case of Psychrobacter sp. strain 7E, which grows over a much wider range of salinities than Cp34H, a different strategy for surviving extreme winter conditions was uncovered. This organism underwent a process of miniaturization and fragmentation leading to increased cell number, potentially improving its survival and dispersal beyond the immediate environment. The overall abundance and composition of the bacterial community in surface sea ice and associated environments may be thus shaped by the differential effects of extremes in temperature and salinity and their fluctuations on organisms with varying environmental tolerances, as well as by the availability of compatible solutes in the inhabitable brine phase.

Acknowledgements This work was supported by NSF OPP awards 0908724 and 1203267 to J.W. Deming. J.S. Bowman extracted and sequenced the DNA for P7E. S.D. Carpenter and J. Schermer tested additional growth ranges for P7E. S. Lubetkin provided useful advice on statistical analysis and C. Peralta-Ferriz on spectral analysis. We thank our laboratory group colleagues for insightful discussion and three anonymous reviewers for comments that improved the manuscript. J. Baross and R. Woodgate provided valuable input to improve an earlier version of this manuscript.

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Supporting Information Additional Supporting Information may be found in the online version of this article: Fig. S1. Temperatures (a, c) and calculated brine salinities (b, d) used in Experiment 1 for short-term (a, b) and long-term (c, d) treatments. Fig. S2. Power spectral density for temperature (°C) fluctuations for winter (a) and spring (b) of 2011, calculated from data in Fig. 1.

ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved