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FEMS Microbiology Ecology 53 (2005) 103–115
www.fems-microbiology.org
Characterization of potential stress responses in ancient
Siberian permafrost psychroactive bacteria
Monica A. Ponder a,b, Sarah J. Gilmour a,c,d, Peter W. Bergholz a,b, Carol A. Mindock e,
Rawle Hollingsworth e, Michael F. Thomashow a,b,c,d, James M. Tiedje a,b,c,d,*
a
Center for Genomic and Evolutionary Studies on Microbial Life at Low Temperatures, Michigan State University, East Lansing, MI 48823, USA
b
Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48823, USA
c
Department of Crops and Soil Sciences, Michigan State University, East Lansing, MI 48823, USA
d
MSU-DOE Plant Research Lab, Michigan State University, East Lansing, MI 48823, USA
e
Department of Chemistry, Michigan State University, East Lansing, MI 48823, USA
Received 30 June 2004; received in revised form 4 December 2004; accepted 6 December 2004
First published online 27 December 2004
Abstract
Past studies of cold-acclimated bacteria have focused primarily on organisms not capable of sub-zero growth. Siberian permafrost isolates Exiguobacterium sp. 255-15 and Psychrobacter sp. 273-4, which grow at subzero temperatures, were used to study coldacclimated physiology. Changes in membrane composition and exopolysaccharides were defined as a function of growth at 24, 4 and
2.5 C in the presence and absence of 5% NaCl. As expected, there was a decrease in fatty acid saturation and chain length at the
colder temperatures and a further decrease in the degree of saturation at higher osmolarity. A shift in carbon source utilization and
antibiotic resistance occurred at 4 versus 24 C growth, perhaps due to changes in the membrane transport. Some carbon substrates
were used uniquely at 4 C and, in general, increased antibiotic sensitivity was observed at 4 C. All the permafrost strains tested
were resistant to long-term freezing (1 year) and were not particularly unique in their UVC tolerance. Most of the tested isolates
had moderate ice nucleation activity, and particularly interesting was the fact that the Gram-positive Exiguobacterium showed some
soluble ice nucleation activity. In general the features measured suggest that the Siberian organisms have adapted to the conditions
of long-term freezing at least for the temperatures of the Kolyma region which are 10 to 12 C where intracellular water is likely
not frozen.
2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Permafrost; Cryotolerance; Low temperature; Cold response; Psychroactivity; Salt response; Psychrobacter; Exiguobacterium
1. Introduction
The majority of the EarthÕs surface is permanently
cold, with approximately 70% of the surface covered by
oceans with an average temperature of 4 C and over
20% of the terrestrial area occupied by permafrost including, 85% of Alaska, 55% of Russia and Canada, 20% of
*
Corresponding author. Tel.: +517 355 0271ext.287; fax: +517 353
2917.
E-mail address: [email protected] (J.M. Tiedje).
China, and the majority of Antarctica. Soils, sediments
and rock exposed to temperatures of 0 C or below for
a period of at least 2 years are defined as permafrost [1].
A variety of microorganisms have been isolated from
buried permafrost of the Kolyma region of northeast
Siberia indicating that these organisms can survive subzero temperatures (10 to 12 C), low water activity
(aw = 0.9), low nutrient availability, and the cumulative
effect from background c radiation from soil minerals
that ranges from 1 to 6 kGy [2]. Depending on geologic
strata these microbes have been in a continuously frozen
0168-6496/$22.00 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsec.2004.12.003
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M.A. Ponder et al. / FEMS Microbiology Ecology 53 (2005) 103–115
environment for 20,000 to 3–5 million years [3]. In contrast to many oceanic isolates, permafrost isolates are
not psychrophiles, but psychrotolerant in that they can
grow at 4 C and above 20 C [4,5]. Most previous studies of permafrost microbes have been done with strains
that have been isolated and grown at room temperature
and in nutrient rich media. We previously isolated 238
bacterial strains from different age layers of Siberian permafrost without exposure to temperatures above 4 C
and using several isolation strategies, including low
nutrient media and cryo-protectants [3]. Previous characterization has indicated that many of these isolates
are psychroactive in that they grow at 2.5 C [6].
Physiological responses to low temperatures of some
mesophilic bacteria, psychrotrophic food-borne pathogens, and environmental isolates have been characterized [7]. The majority of studies have focused on
membrane composition changes and cold shock protein
production in response to lower temperatures. While
most studies have defined responses to temperatures below the organismÕs temperature optimum, only a few of
these mesophilic bacteria are capable of growth at temperatures below 4 C. Those that can are Listeria monocytogenes [8], several Pseudomonas [9] and Arthrobacter
strains [10], and some unidentified Arctic isolates [11]
from non-permafrost environments.
The permafrost environment in contrast to many surface environments, is very stable, with a constant set of
stresses that may have acted as selective factors on the
survivors, including adaptation to a very long-term frozen environment and the associated desiccation, and to
cumulative effects of c radiation which would be expected to damage cell DNA [2,12]. The Kolyma permafrost temperatures of 10 to 12 C would not be
expected to freeze the cytosol of bacterial cells [13] and
hence continued biochemical catalysis could be expected, albeit the fluid would be viscous and reaction
rates very slow. The objective of this study is to characterize features of a selected set of these isolates that may
play a role in or reflect their adaptation to permafrost
conditions. Particular emphasis is placed on a Gram
negative Psychrobacter strain isolated from a 20,000–
30,000 year old permafrost layer and a Gram positive
Exiguobacterium strain isolated from a 2–3 million year
old layer.
2. Materials and methods
2.1. Isolation and phylogenetic characterization
Permafrost samples were obtained from the polar
region of the Kolyma-Indigirka lowland (152–162E,
68–72N), located adjacent to the East Siberian Sea by
David Gilichinsky and team (Cryobiology Laboratory,
Russian Academy of Sciences, Pushchino). The isolation
conditions and characteristics of sampling sites and
cores chosen for bacterial isolation, were detailed previously by Vishnivetskaya et al. [3]. The first 500 bp of the
16S rRNA genes of the isolates were sequenced to determine phylogeny [6]. The isolates included members of
the order Actinomycetales (genus Arthrobacter and
Family Microbacteriacea) division Firmicutes (the genera Exiguobacterium and Planomicrobium), genus Flavobacterium, and division Proteobacteria (Psychrobacter
and Sphingomonas). Analysis of the complete 16S rRNA
gene of Psychrobacter sp. 273-4 and Exiguobacterium sp.
255-15 isolates confirmed their identity and established
their similarity to other previously studied members of
their respective clades (H. Ayala del-Rio and D. Rodrigues, respectively, unpublished observation). BOX PCR
profiles revealed that Psychrobacter sp. 273-4 and sp.
215-51 were different strains, likely different species. Isolates were chosen from the larger set for further studies
based on ease of culturability at 4 C, growth at
2.5 C, age of permafrost strata and for being representative of different taxa found.
2.2. Growth rates
Growth rates, as functions of temperature were measured for Exiguobacterium strain 255-15 and Psychrobacter strains 273-4 and 215-51 by optical density in
shaken flasks (200 rpm) of tryptic soy broth (TSB) (Difco, Detroit, MI), at temperatures from 45 to 0.5 C
using three biological replicates per temperature. Arrhenius plots of Exiguobacterium sp. 255-15 and Psychrobacter sp. 273-4 and sp. 215-51 were prepared by plotting
the natural logarithm of the growth rate against the reciprocal of absolute temperature [14] using Statview
with Lowess fitted lines (SAS Institute, Cary, NC). A
Bělehràdek growth model was also constructed by plotting the square root of the growth rate against the absolute temperature and the minimum temperature
determined by the method of Ratkowsky et al. [15].
Growth rates at 2.5 C were obtained by viable plate
count in triplicate non-shaken flasks. This data was
not included in the Arrhenius and Bělehràdek models
because of the different growth condition. Growth rates
were calculated from the slope of four or more time
points.
Growth rates as a function of low water activity were
measured by optical density in 1/10 TSB supplemented
with NaCl. Colony formation was scored after one week
on 1/10 TSA media supplemented with 0.125, 0.250, 0.5,
1.0, 1.5, 2.0, or 2.50 M NaCl. These concentrations correspond to internal osmotic pressures of 0.217, 0.433,
0.873, 1.78, 2.72, 3.70 and 4.70 osm, respectively, as
extrapolated from Rand et al. [16]. Several isolates were
chosen based on growth at high concentrations of NaCl
to further examine growth using sucrose as an alternate
osmolyte.
M.A. Ponder et al. / FEMS Microbiology Ecology 53 (2005) 103–115
2.3. Cell morphology and size
Psychrobacter sp. 273-4 was grown to an OD600 of 0.2
in 1/2 TSB and 1/2 TSB + 5% NaCl (1.61 osm) at 4 and
22 C. Twenty microliters of the culture was spotted
onto 10 agar coated microscope slides and viewed by
light microscopy. Digital images were analyzed for cell
size and morphology using the Center for Microbial
Ecology Image Analysis software (CMEIAS) [17].
Approximately 250 cells were analyzed for each
treatment.
2.4. Effect of temperature and salinity on lipid and
polysaccharide composition
Psychrobacter sp. 273-4 and Exiguobacterium sp. 25515 were incubated at 24, 4 or 2.5 C in 1/2 TSB or 1/2
TSB + 5% NaCl with shaking until an OD600 of 0.5 was
obtained. All cells were harvested by centrifugation and
washed four times in sterile 1X PBS. Total lipids were
extracted by the method of Mindock et al. [18]. The
aqueous layers were kept for polysaccharide analysis.
The organic layers containing polar lipids were analyzed
by NMR spectroscopy (using d4-methanol as the solvent) and by thin layer chromatography (TLC) on silica
plates using 10:4:2:2:1 chloroform: acetone: methanol:
acetic acid: water. The spots were sprayed either with
orcinol or 10% phosphomolybdic acid in ethanol and
heated at 120 C to visualize the organic components.
NMR spectroscopy provided confirmation of identities
obtained by TLC mobility compared to standards.
NMR spectra were recorded on a Varian VXR-500
spectrometer (500 MHz). Chemical shifts for samples
in d4-methanol are quoted relative to the proton resonances at 4.78 and 3.30 ppm, and for samples in D2O
relative to the proton resonance at 4.65 ppm. For the
double quantum filtered-correlated spectroscopy
(DQF-COSY) experiments, a total of 256 data sets with
24 transients at 2048 points each were acquired. The total correlated spectroscopy (TOCSY) experiments were
also performed by using a total of 256 data sets with
24 transients at 2048 data points each and with a mixing
time of 90 ms.
Fatty acid methyl ester analysis was done using a portion of the fatty acid containing organic layer of the total lipid extract. The extract was incubated in acidified
methanol at 75 C for 36 h, dried and resuspended in
2:1:0.7 hexane: chloroform: H2O. After vigorous shaking, the mixture was centrifuged, the organic layer removed and concentrated to dryness. The resulting
fatty acid methyl esters were analyzed by gas chromatography (GC) and GC–MS using a 30-m DB1 column
(0.32 mm inner diameter, 0.25 lm film). The temperature program increased from 50 to 150 C at 5/min,
150–185 at 2/min, 185–250 at 5/min. After an initial
analysis 2 ng of 2-OH dodecanoate (Sigma–Aldrich,
105
St. Louis, MO), not present in any of the samples, was
added to whole cells as an internal standard.
Polysaccharides were isolated from the aqueous layers of the total lipid extracts by precipitation with ethanol. The precipitated polysaccharide was recovered with
a glass rod and dissolved in 10 ml of water, 10 mg
MgCl2, and 10 units each of RNase A and DNase (Sigma–Aldrich) and incubated overnight at 22 C. The
polysaccharides were then dialyzed against water for
4–5 h, changing the water twice during that time, lyophilized and the resulting solids were weighed. These polymers were analyzed by gas chromatography–mass
spectrometry (GC–MS) after converting them to alditol
acetate derivatives [19]. The remaining aqueous alcoholic solution was centrifuged to remove any solids.
The supernatants which contained free amino acids
and carbohydrates were removed and also analyzed by
GC–MS.
2.5. Freezing tolerance
To determine if temperature acclimation influences
freezing survival, the strains were grown at 24 and at
4 C to a cell density of 108 CFU/ml in 1/2 TSB and subsequently frozen slowly (0.2 C/min) to 20 C. Viable
plate counts were performed initially before freezing and
compared to those obtained after slow thawing
(0.3 C/min) of samples held at 20 C for 1 year.
Plates were incubated at the same temperature at which
the strains were originally grown. An unpaired t test was
used to determine statistical significance (Statview version 5.0, SAS institute).
2.6. Temperature dependent nutrient utilization
Biolog plates (Biolog, Inc, Hayward, CA) were used
to assess whether temperature affected the utilization
of 95 different carbon sources. Psychrobacter sp. 273-4
and Exiguobacterium sp. 255-15 were grown at 24 or
4 C in 150 ml flasks, with 40 ml of 1/10 TSB with shaking until an OD600 of 0.5 was obtained. All cells were
harvested by centrifugation and washed four times in
sterile 1X PBS. Exiguobacterium sp. 255-15 was aliquoted into GP inoculating broth (Biolog Inc.) to match
the percent transmittance (±3%) of the GP standard prepared by Biolog. The same procedure was followed with
Psychrobacter sp. 273-4 with the exception that GN
(non-enteric) broth was used for the inoculation standard. After inoculating the GP (Exiguobacterium) and
GN (Psychrobacter) Biolog plates, they were then incubated at the temperature of inoculum growth. Six biological replicates (with two technical replicates each)
were used per strain and temperature.
The OD595 of the wells was determined with a microplate reader every 8 h until the absorbance did not increase (4 days at 24 C and 3 weeks at 4 C). Average
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M.A. Ponder et al. / FEMS Microbiology Ecology 53 (2005) 103–115
OD595 values for each carbon source were determined
for each set of replicates per time point. Replicates
which showed a standard deviation >15% were excluded
and the averages recalculated. Carbon sources which
showed at least an average absorbance change of 0.2
or more from the blank plate were considered utilized.
2.7. Antibiotic susceptibility
The effect of temperature on antibiotic resistance
was assessed using the impregnated disk method at
both 24 and 4 C of triplicate experiments [20]. Antibiotic disks (BBL Microbiology) were placed on lawns
of bacteria on Mueller Hinton agar, incubated for 2
days at 24 C or 2 weeks at 4 C. After this time
the size of clearings around the disks was measured.
Resistant cells grew significantly closer to the antibiotic disk than susceptible cells while clearing size cutoffs for each antibiotic were used to determine
resistance categories described by Barry and Thornsberry [20].
2.8. UVC survival
The effect of temperature on UVC survival was assessed using cultures of Psychrobacter sp. 273-4 and
Exiguobacterium sp. 255-15 grown to late-log phase
(OD600 = 0.6–0.9) in 1/2 TSB at 25 and 4 C. UVC
was chosen because it causes similar damage to low
doses of c irradiation [21,22], and the ease of application
of small doses. Escherichia coli B606 was used as a comparison strain, because it is a mesophilic c-Proteobacterium related to Psychrobacter sp. 273-4. Bacillus subtilis
PY79 was selected as a related mesophilic Firmicute for
comparison to Exiguobacterium sp. 255-15. Both mesophilic strains were grown to late log phase (OD600 = 0.9)
in 1/2 TSB at 24 C.
A 15 ml culture at late log phase was collected by centrifugation at their cultured temperature, re-suspended
in 15 ml 0.85% NaCl and stored on ice until use. Cells
were then pipetted into a sterile glass Petri plate and exposed to a UVC fluorescent lamp (equivalent dose
1.5 J m2 s1) with constant mixing. Psychrobacter sp.
273-4 and E. coli B606 were cumulatively exposed to
0, 25, 50, and 100 J m2. Exiguobacterium sp. 255-15
and B. subtilis PY79 were cumulatively exposed to 0,
100, 250, and 500 J m2. At each exposure level, plate
counts were performed on 1/2 TSA plates and incubated
in the dark at the respective culturing temperatures. All
steps from UVC exposure through incubation were carried out in the dark and all solutions were kept at the
same temperature at which cells were cultured. After
48 h at 24 C or 2 weeks incubation at 4 C, CFU/ml
were estimated. Average percent survival of four replicates at each of the UVC dosage levels was used to compare Psychrobacter sp. 273-4 grown at 24 C to both of
the other two samples using a one-tailed two sample t
test assuming unequal variances.
2.9. Ice nucleation activity
Ice nucleation studies were undertaken to determine
the effect of temperature on ice nucleation activity in seven permafrost strains. The strains were grown at 24 C
on 1/10 TSA and 1/10 TSA supplemented with 5% glycerol, which has been shown to optimize ice nucleation
activity (INA) in many bacteria [23,24]. INA was measured by the freezing drop method [25]. Suspensions of
bacterial cells were prepared in sterile buffer and 60
10 ll drops of the suspension were placed on an aluminum boat in a circulating ethanol cold bath set to
10 C. The freezing of droplets at or above 10 C
indicated INA. After initial determination of INA in
cultures grown at 24 C, the suspensions were subjected
to 3.5 h at 4 C and 1 h at 10 C and then reassessed
for INA by the freezing drop method. Pseudomonas
syringae ATTC 35421 and E. coli ATTC 39524 were
used as positive and negative controls, respectively, for
INA. The ice nucleation activity per cell was determined
with the method of Pooley and Brown [26].
3. Results
3.1. Effect of temperature on the maximal growth rate and
cellular morphology
The Exiguobacterium and two Psychrobacter strains
tested both grew at 2.5 C with generations times of
5.5 and 3.5 days, respectively, but the Exiguobacterium
strain had a much higher growth maximum (42 C) than
Psychrobacter strains (26 C) (Fig. 1). Arrhenius plots of
the Exiguobacterium growth profile reveal a change in
slope occurring at 24 C, while the Psychrobacter
showed changes in slope at 22 and 6 C. Minimum
growth temperatures of 15, 12, and 7 C were estimated using the Bělehràdek growth model for Psychrobacter sp. 273-4 and sp. 215-51 and Exiguobacterium,
respectively, and agreed with extrapolations from
Arrhenius plots.
Psychrobacter sp. 273-4 exhibited a change in cell size
and shape when subjected to either 5% salt or low temperature (Table 1). In general, the cells were slightly larger when grown at 4 C or in the presence of salt. At the
optimum growth temperature of 22 C, the cells are rod
shaped and 1.8 lm in length. The introduction of 5%
salt resulted in a significant increase in width and decrease surface/volume ratio of the cells. The average biovolume of Psychrobacter increased at low temperature
but most significantly in the presence of salt (Table 1).
Cells were somewhat more pleomorphic when grown
in the presence of salt. No differences in cell size or
M.A. Ponder et al. / FEMS Microbiology Ecology 53 (2005) 103–115
107
and low temperature. For Exiguobacterium, the lag
phase increased from 0.67 h at 24 C to 6.7 h at 4 C
(1/2 TSB), while the lag phase in high salt (1.78 m) increased more dramatically at 24 C to 21 h when compared to 4 C where an increase to 82 h occurred.
Psychrobacter lag times also increased at 4 C from 2.5
h to 22 h in 1/2 TSB, and incubation in 1.78 m salt increased lag times to 21 and 144 h at 24 and 4 C,
respectively.
3.3. Membrane composition under stress
Fig. 1. An Arrhenius plot of growth of Exiguobacterium sp. 255-15
and Psychrobacter sp. 273-4 and 215-51 at a range of temperatures
between 42 and 0.5 C. No growth occurred in Psychrobacter isolates
at temperatures above 28 C.
morphology were seen in initial observations of Exiguobacterium sp. 255-15 in response to 5% NaCl or 4 C so
further CMEIAS analysis was not performed.
3.2. Effect of increased osmolarity on growth of two
Siberian permafrost isolates
Given the low water activity of permafrost, salt tolerance was assessed in Psychrobacter sp. 273-4 and Exiguobacterium sp. 255-15 using NaCl as an osmolyte.
Addition of salt dramatically reduced the growth rates
of both strains at 24 C and somewhat for Exiguobacterium at 4 C (Fig. 2). In contrast, the growth rate was
relatively constant as a function of salt concentration
for Psychrobacter sp. 273-4 at 4 C. Growth rate and
lag time did not differ significantly between cells grown
in sucrose or NaCl as an osmolyte ensuring that ion toxicity did not influence growth capabilities (results not
shown). As expected, lag phase increased for both
strains with exposure to increasing salt concentration
Requirements for membrane fluidity at permafrost
temperature and water activity should be reflected in
membrane composition for organisms adapted to these
conditions. Fatty acid profiles for Psychrobacter sp.
273-4 included both straight chain and unsaturated fatty
acids but branch chained fatty acids were not detected.
The overall composition of unsaturated fatty acids increases at lower temperatures and when grown in presence of 5% salt (Table 2a). The dominant fatty acid
was a C18, saturated at 24 C and unsaturated in the
presence of 5% NaCl and 4 C (Table 2a). A C18:2 fatty
acid was present between 1 and 3% under all the growth
condition (not shown). An unsaturated C16 was dominant at subzero temperatures alone. An increase in the
amount of C17 correlated with increasing salinity, at
the expense of C18 at 4 C. The second most abundant
fatty acid species were the C16 methyl esters. Saturated
C18:0 was predominant at 24 C, while in the presence
of NaCl or 4 C growth unsaturation was favored. However, the combination of NaCl and 4 C resulted in an
increase in the amount of C17:1 at 4 C rather than
C16:1 as seen in the single stress conditions (Table 2).
Phosphatidylglycerol and phosphatidylethanolamine
were detected at 4 and 24 C. Diacylglycerol and additional spots not corresponding to standards were seen
at 4 C under both conditions (data not shown). In addition, spots were present at the origins that were not soluble in the TLC solution.
In Exiguobacterium sp. 255-15, the presence of 5%
NaCl or low temperature conditions alone shifted the
fatty acids from saturated to unsaturated as expected
Table 1
Effect of salt and temperature on the cellular morphology of Psychrobacter sp. 273-4
Medium
Median length
(lm)
Median width
(lm)
Median length/width
Median biovolume
(lm3)
Median biosurface
(lm2)
Median surface/volume
4 C
1/2 TSB
+5%NaCl
1.80 ± 0.12
1.74 ± 0.09
0.77 ± 0.03
0.84 ± 0.03a
2.35 ± 0.15
1.99 ± 0.09a
1.00 ± 0.12
1.15 ± 0.13
5.23 ± 0.48
5.59 ± 0.50
5.00 ± 0.31
4.77 ± 0.14a
22 C
1/2 TSB
+5%NaCl
1.62 ± 0.13
1.86 ± 0.09
0.73 ± 0.03
0.82 ± 0.03a
2.25 ± 0.15
2.20 ± 0.09a
0.85 ± 0.12a
1.17 ± 0.13
4.44 ± 0.48
5.74 ± 0.59
5.26 ± 0.30
4.94 ± 0.14a
All results reflect the average of 250 cells analyzed by CMEIAS.
a
p-Value below 0.05 for 5%NaCl.
M.A. Ponder et al. / FEMS Microbiology Ecology 53 (2005) 103–115
0.45
Growth rate at 24C µmax (hr-1)
0.1
Exiguobacterium 255-15 24C
0.09
Psychrobacter 273-4 24C
Exiguobacterium 255-15 4C
0.4
0.08
Psychrobacter 273-4 4C
0.35
0.07
0.3
0.06
0.25
0.05
0.2
0.04
0.15
0.03
0.1
0.02
0.05
0.01
0
-1
0.5
Growth rate at 4C µmax (hr)
108
0
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
Osmolarity
Fig. 2. Comparison of average growth rates of two permafrost isolates at different temperatures and osmolarities. The lmax at 24 C of
Exiguobacterium sp. 255-15 was 0.9.
Table 2
Fatty acid methyl ester composition (% of average total peak area) of two permafrost isolates at different temperatures and osmolarities
FAME
TSB
24 C
TSB + 5% NaCl
4 C
2.5 C
(a) Average percent fatty acid methyl ester profiles of Psychrobacter sp. 273-4
1.3a
40.2c
C16:0
28.3a
C18:0
44.2a
12.4b
20.0b
a
b
C16:1
1.0
20.0
13.2b
C17:1
1.0a
13.0b
1.0a
a
b
C18:1
1.7
42.0
7.0c
Straight chain fatty acids
% of total
Unsaturated fatty acids
% of total
24 C
4 C
2.5 C
1.25a
1.2a
32.4b
2.63b
52.2a
2.8a
1.1a
1.30a
17.3b
72.0b
2.9a
23.4b
29.9b
1.0a
43.4c
72.5
13.7
60.2
2.5
3.9
26.3
3.7
75.0
21.2
87.2
90.6
74.3
31.3c
36.1c
10.5a
1.6b
6.6a
9.7b
14.0b
1.8a
4.7b
18.2b
4.0b
29.0c
31.8c
27.9b
11.3b
0.9a
7.5b
*
23.0a
13.6b
6.8b
4.2a
22.0b
2.5a
13.0b
(b) Average Exiguobacterium sp. 255-15 fatty acid methyl ester profiles
C16:0
C18:0
C15:0 Iso
C17:0 Anteiso
C17:0 Iso
C16:1
C18:1
26.3a
6.0a
7.0a
4.0b
16.5a
1.0a
5.4a
15.0b
5.0a
4.5a
7.0c
26.0b
13.0b
19.0b
*
*
*
Straight chain saturated fatty acids
% of total
32.3
20.0
67.4
36.6
23.7
597
Branched chain fatty acids
% of total
37.5
18.7
33.0
24.7
197
32
*
15.5
33
*
Unsaturated fatty acids
% of total
27.5
6.4
Average of three biological replicates.
a
Standard deviation < 0.5.
b
0.5 > Standard deviation < 2.0.
c
2.0 > Standard deviation < 5.0.
* Indicates trace amounts below 5% of total percentage.
at 4 C, however unsaturated fatty acids were not detected from three biological replicates grown at
2.5 C (Table 2b). C16:0 was the predominant fatty
acid in Exiguobacterium sp. 255-15 at mesophilic
and subzero temperatures but at 4 C a shift occurred
to isoC17:0. The interaction between 4 C and salt re-
M.A. Ponder et al. / FEMS Microbiology Ecology 53 (2005) 103–115
109
sults in a further increase in unsaturation with the
predominant fatty acid becoming C18:1. Fatty acids
below 5% of the total were not included in Table 2
and included: isoC13:0, anteisoC15:0, isoC15:0, isoC16:0,
C12:0, C13:0,C14:0, C17:0 and C22:0. The phospholipid
profile contained phosphatidylglycerol, diacylglycerol
and phosphatidylethanolamine at 24 and 4 C, with
additional unidentified spots. Unidentified carbohydrate containing spots were present when cells were
grown at 24 C. Only one spot, identified as phosphatidylglycerol, was shared in 4 and 24 C grown
cells (data not shown).
3.5. Freezing tolerance
3.4. Polysaccharide composition
3.6. Temperature dependent carbon utilization
The soluble polysaccharide composition of Psychrobacter sp. 273-4 and Exiguobacterium sp. 255-15 differed
with temperature and presence of 5% NaCl (Table 3).
Psychrobacter sp. 273-4 polysaccharides consisted of
rhamnose, glucose, mannose, galactose, xylose, fucose,
ribose and arabinose when grown at 24 C. When the
cells were grown in 5% NaCl arabinose increased and
fucose decreased. Lower temperatures resulted in an increase in predominance of glucose, mannose and a decrease in rhamnose (Table 3).
The predominant sugar in Exiguobacterium sp. 25515 at all three temperatures was glucose. Rhamnose,
galactose, mannose, ribose, fucose, arabinose, xylose
and an unknown amine sugar were also present (in
decreasing percentages). Ribose decreased in dominance
with low temperature, while arabinose and mannose increased. The presence of 5% NaCl resulted in a shift
with mannose and arabinose (2.5 C) increasing in percent composition at the expense of galactose and arabinose (Table 3).
Psychrobacter sp. 273-4 and Exiguobacterium sp. 25515 were tested for their ability to use 95 different carbon
sources at either 4 or 24 C (Table 5). Psychrobacter sp.
273-4 utilized 32 different carbon sources at 24 and 4 C.
Six of these carbon sources were used only at 4 C, while
12 were used only at 24 C. Exiguobacterium sp. 255-15
was able to utilize 42 different carbon sources at 24 C,
while at 4 C only 36 were used. Seven of these carbon
sources were used only at 4 C and 13 carbon sources
were used exclusively at 24 C (Table 5).
Twelve different permafrost isolates (including Psychrobacter and Exiguobacterium) were examined for their
ability to survive freezing at 20 C after a period of 1
year (Table 4). Most striking is that all strains showed
excellent survival rates with 105–108 CFU/ml found
from 108 CFU/ml after 1 year. Prior cold acclimation
(growth at 4 C) increased freeze survival in 9 of 12
strains, although only 3 strains (Planococccus sp. 21568, 45-18 and Rathayibacter sp. 190-4) were statistically
significant. Both results suggest that these strains are already adapted to subzero environments.
3.7. Temperature effect on antibiotic susceptibility
Six different permafrost isolates were tested for naturally occurring resistance at 4 and 24 C to five antibiotics (Table 6). Psychrobacter sp. 273-4 isolates showed no
resistance to any antibiotics tested. Arthrobacter sp. 45-3
resistance was maintained at 4 C with the five antibiotics tested, while Arthrobacter sp. 255-12 showed decreased resistance to ampicillin, chloramphenicol and
Table 3
Soluble polysaccharide composition of two permafrost isolates grown at different temperatures and osmolarities
Sugar
Psychrobacter sp. 273-4
TSB
Arabinose
Fucose
Galactose
Glucose 23b
Ua-1
Ua-2
Mannose
Rhamnose
Ribose
Xylose
Exiguobacterium sp. 255-15
TSB + NaCl
TSB
TSB + NaCl
24 C
4 C
2.5 C
24 C
4 C
2.5 C
24 C
4 C
2.5 C
24 C
4 C
2.5 C
2.2a
5.0b
8.9b
27.9c
ND
ND
14.6a
38.3c
3.8a
7.4b
2.0a
3.8a
8.8b
36c
ND
ND
11.9a
31.3c
5.3b
8.6b
4.1a
0.7a
8.0a
28b
ND
ND
22b
18b
6b
0.7a
Ô1.1a
2.8a
11.4
27.9c
ND
ND
13.3a
24.1b
4.1a
3.9a
1.0a
3.1b
8.9c
39.2c
ND
ND
14.7b
25.7c
3.4b
1.7b
2.38a
0.5a
6.7a
27.5b
ND
ND
22.3b
19.7b
ND
0.9a
1.6a
5.1a
15.7b
59c
1.8a
1.0a
1.4b
26.9c
9.0
3.1a
1.3a
3.5a
13c
50.4c
2.9a
ND
2.5c
21.4c
ND
2.4a
5.1a
5.1b
12.2b
22.5c
1.6a
3.7a
11.9b
17.7c
7.8b
1.8a
0.3a
2.0a
7.4c
35.9b
1.0a
1.0a
11.9b
19.1b
3.7b
3.1a
2.4a
3.3a
12.0c
16.7b
1.4a
1.0a
11.9a
22.3c
8.4a
1.9a
2.9a
10.7c
0.8c
Ua, unknown amine.
a
Standard deviation < 2%.
b
2% > Standard deviation < 5%.
c
5% > Standard deviation < 10%.
d
ND, not detected.
2.9a
NDd
19.9a
13.4b
0.41
2.5a
110
M.A. Ponder et al. / FEMS Microbiology Ecology 53 (2005) 103–115
Table 4
Effect of growth temperature on survival of permafrost isolates after one year at 20 C
Strain
Average cell loss after 365 days
at 20 C (log CFU/ml)
Growth temperature (C)
Arthrobacter sp.
Arthrobacter sp.
Arthrobacter sp.
Exiguobacterium sp.
Exiguobacterium sp.
Exiguobacterium sp.
Flavobacterium sp.
Planococcus sp.
Planococcus sp.
Planococcus sp.
Psychrobacter sp.
Rathayibacter sp.
255-12
LTER
33-1
392-28
190-11
255-15
309-37
109-1
215-68
45-18
215-51
190-4
4
24
2.6
2.3
1.0
1.2
0.9
1.2
1.3
3.3
0.6
0.3
0.7
0.5
1.1
1.0
1.8
1.7
2.1
2.4
1.7
1.9
2.5
2.1
1.4
2.0
Significance
of growth
temperature
(p-value)
0.08
0.04
0.35
0.04
0.15
0.42
0.58
0.09
0.03
0.003
0.35
0.006
Table 5
Carbon sources uniquely utilized at the indicated temperatures
Psychrobacter sp. 273-4
Exiguobacterium sp. 255-15
24 C
4 C
24 C
4 C
D -Arabitol
D ,L -lactic acid
Propionic acid
a-Ketoglutaric acid
a-Ketovaleric acid
b-Hydroxybutyric acid
c-Aminobutyric acid
L -Fucose
a-Methyl D -glucoside
Palatinose
b-Hydroxybutyric acid
D -Malic acid
L -Malic acid
Pyruvic acid
D -Alanine
L -Alanine
L -Asparganine
L -Glutamic acid
Cellobiose
Glycerol
Inulin
Tween 40
Tween 80
Amygladin
Raffinose
D -Xylose
Acetic acid
D -Galactose
Gentibiose
m-Inositiol
a-D -Lactose
D -Glucuronic acid
D -Saccharic acid
Glucoronamide
L -Aspartic acid
L -Arabinose
D -Gluconic acid
Cellobiose
tetracycaline at 4 C. Only Exiguobacterium sp. 7-3 and
Planococcus sp. 215-68 showed decreased resistance to
streptomycin at 4 C. No strains showed differential
resistance to erythromycin with temperature. Resistance
was maintained at 4 C only in those strains possessing a
high level of resistance at 24 C (Table 6).
Psychrobacter sp. 273-4, in cells grown at 24 and 4 C.
Exiguobacterium sp. 255-15 grown at 4 C was more sensitive to UVC than when grown at 24 C at both 100
(p < 0.005) and 250 J m2 (p < 0.02). Exiguobacterium
sp. 255-15 at 24 C exhibited significantly greater
survival than B. subtilis PY79 at both the 100
(p < 7.9 · 105) and at the 250 J m2 (p < 8 · 104).
3.8. UVC resistance
3.9. Ice nucleation activity
Because ionizing and UV radiation share some similarities in DNA damage, we tested the capacity of two
permafrost isolates and reference mesophiles to remain
culturable after exposure to UVC. The percent survival
of Psychrobacter sp. 273-4 grown at 24 C was significantly greater than those grown at 4 C at all dosages
(Table 7). Psychrobacter and E. coli B606 had similar
sensitivities to UVC at mesophilic temperatures, except
at the highest UV dose where E. coli was more resistant
to UVC (p < 0.05). Exiguobacterium sp. 255-15, as well
as B. subtilis PY79, exhibited more UVC tolerance than
Nine different permafrost isolates were tested for
their ice nucleation activity, which may provide protection from harmful intracellular ice accumulation. Five
isolates (Flavobacterium sp. 309-37 and 23-9, Psychrobacter sp. 215-51 and 273-4, Sphingomonas sp. 190-14
and 3361-2) possessed weak ice nucleation activity when
grown at 24 C. Exposure to 4 C further increased ice
nucleation activity in all strains except Psychrobacter
sp. 215-51 and Sphingomonas sp. 3361-2 (Table 8). This
cold shock treatment also induced INA in Exiguobacte-
RS
RS
RS
RS
RS
S
RS
S
S
RS
S
S
4. Discussion
RS
RS
RS
RS
S
S
RS
RS
RS
RS
S
S
R
RS
RS
RS
S
S
RS
RS
RS
RS
S
S
c
S
RS
S
S
S
S
R
S
S
S
S
S
b
All three replicates gave consistent categories.
a
S, susceptible.
b
R, resistant.
c
RS, intermediate resistance.
45-3
255-12
255-15
7-3
215-68
273-4
S
S
S
S
S
S
a
Arthrobacter sp.
Arthrobacter sp.
Exiguobacterium sp.
Exiguobacterium sp.
Planococcus sp.
Psychrobacter sp.
111
rium sp. 7-3, which exhibited no measurable ice nucleation ability at 24 C. Cold shock at 10 C resulted
in a further increase in ice nucleation of in all six of
the above strains (Table 8).
RS
RS
RS
R
RS
S
24 C
4 C
30 lg
10 lg
4 C
4 C
15 lg
4 C
4 C
24 C
30 lg
10 lg
24 C
Chloramphenicol
Ampicillin
Strain
Table 6
The susceptibilities of selected permafrost strains to five classes of antibiotics
Erythromycin
24 C
Streptomycin
24 C
Tetracycline
M.A. Ponder et al. / FEMS Microbiology Ecology 53 (2005) 103–115
Permafrost provides an opportunity to obtain microbes that have experienced long term exposure to cold
temperatures, decreased water activities, c radiation and
low carbon availability. The Psychrobacter and Exiguobacterium strains, as diverse representatives of the permafrost community, should carry traits that have
allowed them to adapt to these conditions. The genomes
of these two organisms are currently being sequenced
and the characterization of physiological traits potentially important to cryo-adaptation is important for
beginning understand these adaptations at the genome
and proteome levels.
Psychroactivity is common for bacteria isolated from
cold environments such as sea ice. One such isolate, Psychromonas ingrahamii, grows at a temperature of 12 C
with a generation time of 240 h, the lowest growth temperature of any organism authenticated by a growth
curve [27]. Recently, growth of another Psychrobacter
from Siberian permafrost was reported at 10 C
(0.016 day1 [28]), the temperature of the Kolyma permafrost. Tolerance to low water activity coupled with
sub-zero growth, including the minimum growth temperature prediction of 7 and 15 C suggests that
Exiguobacterium sp. 255-15 and Psychrobacter sp. 2734 could be active in their native habitat.
Our Psychrobacter and Exiguobacterium isolates grew
over a moderate to broad temperature range, (15 C
calculated Tmin) 5 to 26 C and (7 C calculated
Tmin) 2.5 to 42 C, respectively, the later being especially interesting because of its temperature span of at
least 45 C. Permafrost strains typically must live for
thousands of years in the surface (active) soil layer with
annual freeze and thaw cycles of +10 C to 20 C,
which may have been the selective force for the broad
growth range typical of permafrost isolates.
These two isolates exhibit different growth patterns as
determined by the linear portion of the Arrhenius plot.
Switch points in the slope of growth rates, indicative
of a physiological shift in metabolism, occurred at
22 C in Psychrobacter sp. 273-4, 24 C in Exiguobacterium sp. 255-15 and also at 6 C in Psychrobacter. These
two isolates exhibit different growth characteristic as has
been reported in Pseudomonas fluorescens MFO where
the ‘‘thermometer temperature’’ of 17 C triggers a
change in physiological characteristics such as increased
membrane permeability to b-lactamine and an increase
in enzymatic activity of extracellular protease, lipase
and periplasmic phosphatases [29]. While cell sizes,
112
M.A. Ponder et al. / FEMS Microbiology Ecology 53 (2005) 103–115
Table 7
Average percent survival of permafrost isolates exposed to UVC
Strain and temperature
Psychrobacter sp. 273-4 4 C
Psychrobacter sp. 273-4 24 C
E. coli B606 24 C
Exiguobacterium sp. 255-15 4 C
Exiguobacterium sp. 255-15 24 C
B. subtilis PY79 24 C
UVC dose (J/m2)
0
25
50
100
250
100
100
100
100
100
100
26.9 ± 13.9a
74.7 ± 22.6a
68.6 ± 8.4
ND
ND
ND
13.3 ± 6.6a
37.1 ± 9.6a
34.2 ± 0.4
ND
ND
ND
0.29 ± 0.2b
1.46 ± 0.8b
5.30 ± 1.0b
2.20 ± .32c
91.1 ± 13.1c,d
11.7 ± 1.6d
ND
ND
ND
0.01 ± 0.01c
2.72 ± 1.97c,d
0.08 ± 0.01d
ND, not determined for this UVC dose.
a
p-Value (24 C > 4 C) < 0.1.
b
p-Value (E. coli > 273-4) < 0.05.
c
p-Value (24 C > 4 C) < 0.01.
d
p-Value (24 C > B. subtilis) < 0.02.
Table 8
Ice nucleation activity (·109) per cell of selected permafrost isolates after growth at 24 C followed by exposure to 4 and 10 C
Strain
Exiguobacterium sp.
Flavobacterium sp.
Flavobacterium sp.
Psychrobacter sp.
Psychrobacter sp.
Sphingomonas sp.
Sphingomonas sp.
Escherichia coli
Pseudomonas syringae
Exposure temperature
7-3
23-9
309-37
273-4
215-51
190-14
3361-2
Migula
Sijderius
24 C
4 C
10 C
0
3.4 ± 0.1
7.4 ± 0.4
252 ± 4.3
16.2 ± 3.4
10.4 ± 1.2
54.8 ± 5.6
0
411
10.5 ± 0.1
10.4 ± 0.05
17.5 ± 0.1
360 ± 18
0.8 ± 0.1
314 ± 20
19.4 ± 0.21
0
411
18.2 ± 0.2
61.5 ± 0.4
2.8 ± 0.04
170 ± 12
3.4 ± 0.05
17.0 ± 0.8
5.2 ± 0.1
0
411
Mean value of two replicates.
If no standard deviation given there was no difference in number of drops frozen between replicates.
antibiotic susceptibility and membrane composition
were not assessed at the switch points in our study,
marked differences in these traits were seen between cells
grown at 4 and 24 C.
Minor but significant cell morphology and size
changes in Psychrobacter sp. 273-4 occurred with exposure to low temperature and increased salt. The decrease
in surface to volume ratio observed in Psychrobacter sp.
273-4 with low temperature and salt is similar to that
seen in other bacteria isolated from low water activity
environments [30]. An increase in surface to volume ratio is seldom encountered in cells isolated from low
water activity environments since the presence of compatible solutes, which balance the external and internal
solute concentrations, tend to swell cells. In contrast, another permafrost isolate, Arthrobacter sp. 45-3, showed
a large decrease in cell size (14-fold) after incubation
at 4 C [18]. Members of the genus Arthrobacter are
known to change cellular morphologies with stress and
development stages, suggesting that this may be a common adaptation in this genus [31].
Low temperature and high osmolarity are known to
induce changes in membrane composition that maintain
membrane fluidity [32,13]. A decrease in temperature resulted in a decrease in the saturation of fatty acids and
acyl chain lengths in Psychrobacter sp. 273-4 and Exiguobacterium sp. 255-15, as occurs in other bacteria
shifted to lower temperatures. Together these changes
function to lower the gel-liquid crystalline transition
temperature, i.e., homeoviscous adaptation [33]. Unsaturated membrane fatty acids are less abundant in these
psychroactive Siberian permafrost isolates grown at
mesophilic temperatures compared to the psychrotolerant, Oleispira antarctica. This difference in unsaturation
may account for the Psychrobacters narrower range of
growth temperatures [34]. These Siberian permafrost
isolates show a more dramatic shift to unsaturated fatty
acids than previously described for a permafrost isolate,
Arthrobacter sp. 33-1 [18]. Exiguobacterium sp. 255-15
exhibits the same shift to shorter branched chain fatty
acids, with an increase in anteiso-C15:0 at the expense
of anteiso-c17:0, which was also seen in Arthrobacter
sp. 33-1 and L. monocytogenes [35].
Exposure to increased osmolarity resulted in increased fatty acid unsaturation and acyl chain length
in these Siberian permafrost isolates. Moderate halo-
M.A. Ponder et al. / FEMS Microbiology Ecology 53 (2005) 103–115
philes have been described as showing increased membrane fatty acid saturation [36] rather than the increased
unsaturation seen in this experiment. The present study
concurs with the trend shown in Vibrio costicola indicating that the combination of high salinity and low temperatures results in increased unsaturation of fatty
acids and an increased ratio of phosphotidylethanolamine: phosphotidylglycerol; both of these metabolic
changes are commonly seen only under low temperature
conditions [37]. The changes in sugar composition with
temperature and salinity reflect additional adaptations,
likely in the membrane-associated exopolysaccharide
that may improve survival. Recent reports of exopolysaccharide accumulation in sea ice supports the stabilizing role for the cold active Cowellia ColAP, thought to
aid environmental survival [38].
An important consequence of membrane changes at
cold temperatures is the effect on membrane transport
which could explain the differences in carbon source utilization and antibiotic sensitivity seen at low temperatures. Glucose utilization increased at 0 C in an
unidentified cold-adapted psychrotroph [39]. Uptake
rates of NHþ
4 in psychrophilic Vibrio increased when
the organism was grown at temperatures between 0
and 15 C compared to 24 C, resulting in an increased
Vmax for the NHþ
4 transport system [40]. Alternatively,
cold shock increased the activity of malate dehydrogenase and glucose-6-phosphate dehydrogenase in Lactococcus lactis and Rhizobium [41,42]. In addition to
alterations in enzyme efficiency and in Vmax of uptake,
a more universal change in membrane permeability may
explain the differential use of certain carbon sources in
the permafrost bacteria tested. Studies on glycerol uptake, which is transported both by facilitated and passive diffusion, indicate an increase in both types of
uptake in E. coli when membrane fluidity is increased
[43]. Molecules, such as cellobiose, may not enter the cell
at 4 C due to changes in transport-associated proteins
which are influenced by membrane composition. Additional substrates may also be bound when membrane
and protein flexibility increases as demonstrated recently
for the arabinose binding protein [44]. An increase in
flexibility commonly occurs in proteins at low temperatures raising the possibility that additional substrates
could be bound by other binding proteins. If a single
transporter handles a range of substrates then decreased
efficiency of such an enzyme would decrease uptake at
low temperatures. The presence of temperature dependent nutrient uptake efficiencies suggests that microbes
have developed adaptations to low temperatures which
will counteract the unfavorable effects of decreased diffusion, allowing adapted microbes to be more competitive for nutrients under unfavorable conditions.
Continual exposure to sub-zero temperatures within
the permafrost for organisms with long-term freezing
survival, a trait noted in all permafrost isolates tested.
113
Cryotolerance has been reported in another Arctic-isolated Pseudomonas when pre-conditioned at 4 C prior
to freezing at 20 C for 24 h [9]. All permafrost strains
possessed excellent survival rates with bacterial numbers
of 105–108 CFU/ml observed after 1 year at 20 C
from an original population of 108 CFU/ml. This is substantially higher than E. coli [45], although it is similar
to the survival reported for the food-associated, L. lactis
[46]. Pre-conditioning to cold temperatures is known to
increase freezing survival in many food-associated microbes, including E. coli O157:H7 [45] and L. lactis
[46] though after only 28 days a one log decrease in survival occurs. This effect may be due to expression of
cold-responsive genes and cryoprotectant molecules that
function to enhance the survival of the microbe through
the stress of freezing and thawing conditions [47–49].
However pre-conditioning to cold temperatures, does
not increase freeze survival in all bacteria, as seen in this
experiment and for lactic acid bacteria [47]. Permafrost
organisms could already be selected for life in this continuously frozen environment since they donÕt need to
respond to temperature cycles.
Ice nucleation activity, enhanced by exposure to lower
temperatures, may be another mechanism of survival in
permafrost. Ice nucleation activity results from outer
membrane proteins whose structure mimics an ice crystal, providing a lattice for crystallization of water [50] at
higher temperatures, and preventing harmful intracellular ice formation because the crystals are too large to
penetrate the membrane and initiate intracellular freezing [51]. The formation of these small, thermodynamically unstable molecules makes ice nucleation proteins
unlikely to offer long-term survival due to the crystalsÕ
tendency to reform into large, damaging structures
[52]. The discovery of a protein capable of both ice
nucleation and antifreeze activity in Pseudomonas putida
may present a solution to this dilemma. The ice nucleation domain is believed to cause the formation of extracellular ice, while the antifreeze domain maintains the
crystals at a non-damaging size [53]. The ability of some
plant and animal antifreeze proteins to inhibit microbial
ice nucleation activity suggests that antifreeze proteins
may lower the supercooling point of water within the
cells. However, no direct evidence supports this theory
[54,55]. The presence of small amounts of thermal hysteresis activity in Psychrobacter sp. 273-4 suggests it
may possess antifreeze proteins or a combination antifreeze/ice nucleation protein, as seen in c-Proteobacterium relative, P. putida (J. Duman, personal
communication). The detection of ice nucleation activity
in the Gram positive Exiguobacterium sp. 255-15 is surprising since the ice nucleation protein is located within
the outer membrane in all bacteria known to date. Ice
nucleation activity was maintained after filtering (results
not shown) suggesting a soluble protein may be responsible in Exiguobacterium sp. 255-15.
114
M.A. Ponder et al. / FEMS Microbiology Ecology 53 (2005) 103–115
Exposure to UVC under two growth temperatures
was used to evaluate the ability of Psychrobacter sp.
273-4 and Exiguobacterium sp. 255-15 to survive DNA
damage resulting from reactive oxygen species and occasional single stranded DNA breaks such as would occur
from ionizing radiation. A decrease in UVC survival at
4 C relative to 24 C was observed in both strains, and
UVC survival during growth at low temperature was not
greater than that of either mesophilic comparison strain.
This result appears to indicate that unusual capacity to
withstand DNA damage was not necessary for either
organism to survive and be resuscitated in the lab after
104–106 years in permafrost. One must conclude that
some level of repair must have been occurring in situ.
Recently, Price and Sowers compiled data indicating
that in situ survival metabolism, defined as a metabolic
state in which cells ‘‘can repair macromolecular damage
but are probably largely dormant’’, is higher than theoretical rates of DNA depurination over decreasing temperature [56] their calculated rates of survival
metabolism were found to be approximately 106 times
lower than that required for measured metabolic rates
required for growth at similar temperatures. This very
low metabolic requirement could be met in situ by
Psychrobacter sp. 273-4 or Exiguobacterium sp. 255-15
and hence allow them to repair DNA damage.
The ability of permafrost isolated bacteria to respond
to laboratory- simulated permafrost conditions suggests
that these organisms possess adaptations to low temperature, increased osmotica and have efficient repair mechanisms that allow for these and not other tundra
organisms to continue to live in permafrost.
Acknowledgement
This research was funded by the National Astrobiology Institute of NASA. We would also acknowledge the
assistance of Chia-Kai Chang, Gisel Rodriguez, Debora
Rodrigues and Alexa Turke. We also thank Marcia Lee
for assistance with the ice nucleation activity studies,
Frank Dazzo for CMEIAS expertise, George Sundin
for assistance with UVC exposure experiments and Rich
Lenski and Lee Kroos for gifts of strains E. coli 606 and
B. subtilis PY79 respectively. All GC/MS analysis was
performed by Beverly Chamberlin at the Center for
Mass Spectrometry of Michigan State University.
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