Download detection, recovery, isolation and characterization

DETECTION, RECOVERY, ISOLATION AND CHARACTERIZATION
OF BACTERIA IN GLACIAL ICE AND LAKE VOSTOK
ACCRETION ICE
DISSERTATION
Presented in Partial Fulfillment of the Requirement for
the Degree Doctor of Philosophy in the Graduate
School of The Ohio State University
By
Brent C. Christner, M.S.
*****
The Ohio State University
2002
Dissertation Committee:
Dr. John N. Reeve, Adviser
Dr. Ellen Mosley-Thompson
Dr. Lonnie G. Thompson
Dr. Olli H. Tuovinen
Dr. Charles J. Daniels
Approved by
__________________________
Adviser
Department of Microbiology
ABSTRACT
An extraction system has been constructed that melts
ice from the interior of ice cores and collects the
resulting melt water aseptically.
Using this system,
bacteria entrapped in modern and ancient glacial ice from
worldwide locations and in an ice core extending into
accreted Lake Vostok ice have been isolated using
enrichment culture and identified by amplification and
sequencing of DNA-encoding 16S rRNA genes.
In general, ice
cores from non-polar locations contained larger numbers and
species of cultivable bacteria than samples from polar
ices, presumably due to the closer proximity of terrestrial
biological ecosystems and exposed landscape.
When compared
with other polar locations, higher numbers of isolates were
obtained from ices adjacent to the Dry Valley complex of
Antarctica, consistent with the influx of airborne
biological particles from local environments serving as the
primary factor controlling the numbers of microorganisms
present.
The numbers of recoverable bacteria did not
correlate directly with the age of the ice, and isolates
ii
were recovered from the oldest samples examined (>500K
years old).
The 16S rDNA sequences from bacterial isolates
and amplicons obtained directly from samples position
within 6 different bacterial lines of descent (α−, β−, and
γ-proteobacteria, high and low G+C gram positive bacteria,
and the Cytophaga/Flavobacterium/Bacteroides).
Some of the
isolated bacteria have close phylogenetic relationships
with species originating from permanently cold
environments, and other ice core sites or different
portions (time periods) of the same core.
Macromolecular
synthesis was demonstrated in bacteria frozen under
conditions analogous to those in glacial ice, and the
possibility exists that metabolic activity and repair may
occur during extended periods of glacial entrapment.
Several of the species identified in Lake Vostok accretion
ice are also related to glacial isolates and species from
other cold environments.
These ice core studies have
provided a glimpse of the microorganisms likely to
inhabitant this potentially unique subsurface ecosystem.
Investigating microbial survival in ice and exploring
potential habitats for activity within the glacial and
subglacial environment has confirmed that these could have
served as refuge environments for life during periods of
iii
global glaciation (Snowball Earth), and has provided data
for extrapolations to the likelihood of microorganisms
surviving frozen in extraterrestrial habitats or during
interplanetary transport.
iv
ACKNOWLEDGMENTS
This study was made possible by financial support from
the National Science Foundation and the scientific
guidance, time and resources supplied by my advisers, John
Reeve, Ellen Mosley-Thompson and Lonnie Thompson.
I feel
very fortunate to have worked amongst this fine group.
Their scientific dedication and encouragement have served
as a constant source of motivation.
I am also grateful to
my thesis committee members Olli Tuovinen and Charles
Daniels, who provided an interesting subject for my general
exam, and have offered useful criticisms and comments on
project design.
Many thanks are also due to Victor
Zagorodnov, who designed and constructed the automated ice
core sampler.
I am indebted to the knowledge and friendship of past
and present members of the Reeve laboratory, including
Kathryn Bailey, Trevor Darcy, Brian Hanzelka, Wen-tyng Li,
Frederic Marc, Rod Morgan, Suzette Pereira, Kathleen
v
Sandman, Rachel Samson, Divya Soares, Mark Xie, and Li Yu,
a former technician on this project.
I would also like to thank Andrea Wolfe, Laurie
Achenbach, Tom Schmidt, and Joel Klappenbach for assistance
with phylogenetic analysis, Ahmed Yousef for help with the
disinfection procedures, Scott Rogers for recommendations
on PCR amplification from low biomass material, Wade
Jeffrey for advice on macromolecular synthesis, and Dorota
Porazinska and Allison Murray for obtaining ice core
samples from the Canada Glacier.
I am also appreciative of
funding supplied by NSF to participate in the Antarctic
Biology Course (2001, McMurdo Station, Antarctica) and by
OSU to participate in the 1999 Woods Hole Microbial
Diversity course.
vi
VITA
July 31, 1970......................Born – Mt.Pleasant,
Pennsylvania
1988-1992..........................B.S. Molecular Biology,
Westminster College, New
Wilmington, Pennsylvania
1992-1993..........................Research Technician, Eye
and Ear Institute
(UPMC), Pittsburgh,
Pennsylvania
1993-1996..........................M.S. Microbiology,
University of Dayton,
Ohio
1997-present.......................Graduate Student,
Department of
Microbiology, The Ohio
State University
PUBLICATIONS
Research Publications
1.
Christner, B.C., E. Mosley-Thompson, L.G. Thompson. V.
Zagorodnov, K. Sandman, and J.N. Reeve. 2000. Recovery and
identification of viable bacteria immured in glacial ice.
Icarus 144:479-485.
2.
Christner, B.C., E. Mosley-Thompson, L.G. Thompson,
and J.N. Reeve. 2001. Isolation of bacteria and 16S rDNAs
from Lake Vostok accretion ice. Environ. Microbiol. 3:570577.
FIELDS OF STUDY
Major Field:
Microbiology
vii
TABLE OF CONTENTS
Page
Abstract.............................................ii
Acknowledgments.......................................v
Vita................................................vii
List of Tables........................................x
List of Figures......................................xi
Chapters:
1. General introduction...............................1
Glacial ice, paleoclimatology, and
the cold biosphere...............................1
Microorganisms immured in glacial ice...........10
Astrobiology implications of
microbiological investigations of
terrestrial glacial ice.........................24
Objectives of this study........................35
2. Procedures used to prevent
contamination during
sampling and analysis.............................37
Introduction....................................37
Materials and methods...........................38
Results.........................................52
Discussion......................................55
viii
3. Isolation and characterization
of bacteria and 16S rDNA
sequences from glacial ice........................62
Introduction....................................62
Materials and methods...........................63
Results.........................................73
Discussion.....................................122
4.
Macromolecular synthesis under
frozen conditions...............................129
Introduction...................................129
Materials and methods..........................130
Results........................................132
Discussion.....................................141
5.
Isolation of bacteria and
16S rDNA sequences from Lake
Vostok accretion ice............................148
Introduction...................................148
Materials and methods..........................152
Results........................................153
Discussion.....................................168
6.
General discussion..............................171
List of References..................................179
ix
LIST OF TABLES
Page
Table
3.1
Inventory of glacial ice cores sampled..........77
3.2
Bacterial isolates
from glacial ice cores..........................98
3.3
Optimum growth temperature range
and antibiotic resistance in
isolated bacteria..............................110
3.4
16S rDNA molecules amplified from
>500,000 year old ice from Guliya, China.......118
5.1
Media inoculated with melt water
from ice core section 3593.....................154
5.2
Bacteria isolated from deep Vostok
ice core section 3593..........................156
5.3
16S rDNA molecules amplified from
core 3593 melt water...........................163
x
LIST OF FIGURES
Page
Figure
2.1
Construction of the sampling
head and prototype ice core sampler.............42
2.2
Final design of the automated sampler...........44
2.3
Location of primers used
to amplify and sequence 16S rDNA................49
3.1
Global locations of sampling sites
and ice cores available for study
at the Byrd Polar Research Center...............64
3.2
Spread plates of sample from the
Guliya ice cap on agar-solidifed media..........75
3.3
Microorganisms and particulates
filtered from glacial ice cores
visualized by SEM...............................80
3.4
Distribution of glacial isolates
based on phylogenetic assignment
to major bacterial divisions....................83
3.5
Phylogenetic analysis of αproteobacterial isolates recovered
from glacial ice................................84
3.6
Phylogenetic analysis of β- and γproteobacterial glacial isolates,
and a member of the C/F/B line of descent.......87
3.7
Phylogenetic analysis of low G+C
Gram positive glacial isolates..................90
3.8
Phylogenetic analysis of high G+C
Gram positive glacial isolates..................93
xi
3.9
Phylogenetic analysis of γproteobacterial sequences amplified
from >500,000 year-old ice from
Guliya, China..................................115
3.10 Freeze- tolerance in glacial
isolates, related species, and E.coli..........121
4.1
Incorporation of [3H]-thymidine and
[3H]–leucine into TCA-precipitated
material by Trans1 and G200-C1
during the freezing process....................133
4.2
Incorporation of [3H]-thymidine and
[3H]–leucine by strains Trans1 and
G200-C1 during a 206 day incubation at –15oC ...135
4.3
Incorporation of [3H]-thymidine and
[3H]–leucine by E. coli during a 102 day
incubation at –15oC ............................136
4.4
Incorporation of [3H]-thymidine and
the number of cfu ml-1 for Trans1 and
G200-C1 during a 100 day incubation
at –15oC .......................................138
4.5
Incorporation of [3H]-thymidine and
[3H]–leucine at –15oC by strains Trans1 and
G200-C1 over 23 days in the presence of
DNA and protein synthesis inhibitors...........139
4.6
Incorporation of [3H]-thymidine and
[3H]–leucine at –15o and –70oC by strains
Trans1 and G200-C1 over 50 days................142
5.1
Origin of deep Vostok ice
core section 3593..............................149
5.2
Phylogenetic analysis and scanning
electron micrographs of bacterial
isolates from core section 3593................157
5.3
Filaments in anaerobic enrichments.............160
xii
5.4
Phylogenetic analysis of the 16S rDNAs
amplified from core section 3593...............164
5.5
Scanning electron micrographs of
cells retained on the surface of
a 0.2 µm isopore filter after
concentration of core
section 3593 melt water........................167
xiii
CHAPTER 1
GENERAL INTRODUCTION
Glacial ice, paleoclimatology, and the cold biosphere
Distribution and formation of glacial ice
Snowfall accumulates into continental ice sheets in
the polar regions and globally at high altitudes.
Depending on the topological nature of the accumulation
environment, high elevation ice fields are termed valley or
alpine glaciers, and ice caps when a flat bedrock surface
or volcanic crater is completely covered in ice.
The
expansive ice sheets of Greenland and Antarctica cover ~10%
of Earth’s terrestrial surface with ice, and contain ~70%
of the fresh water on the planet (Patterson 1994).
Earth’s
climate is currently in an interglacial stage of a 100,000
year cycle, caused largely by episodic changes in the
planets axial tilt and ellipticity of its orbit around the
Sun.
During the last glacial maximum 18,000 years ago, sea
levels were ≈120 m lower than today and the north polar ice
1
cap advanced to cover 5 million square kilometers,
blanketing what is now Canada and half of the United States
(Hughes 1998).
The transformation of firn (granularized and compacted
snowflakes enduring a season without melting) to glacial
ice is a complex process that occurs at rates and depths
dependent on the air temperature, amount of snowfall
accumulation, moisture content of the snow, and whether the
glacial surface experiences annual cycles of melting and
freezing (Patterson 1994).
As overlying snowfall applies
pressure, firn crystals glide and bond to other crystal
planes, effectively squeezing intervening air spaces
together into ice-entrapped bubbles.
Gases are not able to
diffuse through solid ice; however, air within firn can mix
freely with the atmosphere.
Therefore, an air bubble
within glacial ice does not originate from precisely the
same time point as the surrounding ice, and differences
range from hundreds to several thousand years (Patterson
1994).
The firn depth is often only a few meters in
mountain glaciers, but can be a hundred meters or more in
polar ice sheets.
2
When a glacier accumulates to greater than 20 meters
in height, the ice flows much like a viscous liquid.
Gravity exerts a vertical force on the surface, causing the
ice to be pushed out laterally (Hughes 1998).
The highest
elevation of a mountain glacier and the interior of an ice
sheet are referred to as the zone of accumulation,
representing the regions where snowfall is added and
compressed into ice. The zone of ablation is the area where
material is lost from the glacier, by melting or the
calving of icebergs from an ice shelf.
Based on the
climatie-dependent ratio of gain to loss, or mass balance,
a glacier will expand, contract, or maintain equilibrium
(Patterson 1994; Hughes 1998).
High elevation glaciers, especially those in the
tropics, are limited by regional topography with flow to
lower altitudes and the ablated mass often serving as a
primary water source for many important rivers.
Polar ice
sheets, on the other hand, flow outward from a massive
dome-like accumulation zone to a thinner outer margin, and
are typical of the extensive glaciers that covered the
Earth during the climax of the last Ice Age (Hughes 1998).
Ice shelves are fed by glacial flow and rapidly moving ice
streams capable of transferring large quantities of inland
3
ice to costal calving zones.
Such remote and seemingly
inconsequential frozen environments have enormous impacts
on the global climate, and are vital to discussions of
apparent global warming trends.
An increase in global
temperature sufficient to melt only the West Antarctic ice
sheet would raise global sea level by 5 m (Alley and
Bindschadler 2001).
Paleoclimatic inferences from glacial ice cores
Archived chronologically within the Earth’s ice caps
and ice sheets are samples of the atmospheric constituents
from different times in the past.
Thus, by examining the
physical and chemical properties of ice cores, such proxy
data can be used to elucidate past regional and global
climatic conditions.
The ratio of
18
O/16O and D/H (δ18O and
δD, respectively) in precipitation is dependent on
temperature, anion/cation concentrations reflect the
atmospheric chemistry, dust concentrations reveal the
turbidity of the atmosphere, entrapped sulfates record
volcanic eruptions, changes in nitrate concentrations can
indicate the expansion and contraction of local vegetation,
4
and trace element concentrations can be used to monitor
anthropogenic emissions before and after the industrial
revolution (Patterson 1994).
Natural records such as deep sea sediments, coral
reefs, and tree rings also supply valuable information
about past climate change.
However, proxy data from ice
cores contain more detail about more paleoclimatic
parameters, including the precise gaseous composition of
ancient atmospheres.
Ice cores retrieved from Greenland
(Dansgaard et al. 1993; Grootes et al. 1993), Antarctica
(Lorius et al. 1985), and the Tibetan Plateau (Thompson et
al. 1997) have been used to reconstruct the last glacialinterglacial cycle, and the recent completion of the Vostok
(Antarctica) core in 1998 now extends that history back
through the last 4 glacial cycles, covering approximately
420,000 years of climate history (Petit et al. 1999).
The
immensity of polar ice sheets has a drastic effect on local
(as well as global) weather patterns, whereas smaller
tropical and subtropical glaciers are incapable of creating
a significant climatic disruption, and are therefore more
sensitive in recording higher frequency climatic
perturbations (Thompson et al. 1997, 1998, 2000).
5
As such,
data collected worldwide from ice cores are beginning to
provide a more detailed understanding of the mechanisms
which regulate global climate change.
The Neoproterozoic and Paleoproterozoic snowball Earth
The Earth’s climate system has always been dynamic,
but perhaps no degree of environmental fluctuation
parallels in severity of the conditions supposedly
responsible for two global periods of glaciation, that
occurred 600-800 million and 2.3-2.5 billion years ago
(Evans et al. 1997; Hoffman et al. 1998; Kirschvink et al.
2000).
In fact, the only evidence for glacial deposition
in 4 billion years of pre-Cambrian history comes from
geological sedimentary materials originating from these
time periods.
Budyko (1969) developed a mathematical
climate model that predicted a runaway freeze on the
Earth’s surface when ice covers latitudes >30o north or
south of the equator, due to a positive ice-albedo
feedback.
Such a drop in global temperature would create a
thick layer of ice that could then seal marine habitats
from the atmosphere, blocking photosynthetic primary
production and subsequently resulting in the world’s
ocean’s becoming anoxic.
The presence of tropical
6
Neoproterozoic glacial deposits at sea level locations in
the geological record followed immediately by cap
carbonates and banded iron formations (BIFs) are
interpreted as consistent with a snowball Earth scenario
(Kirshvink 1992; Hoffman et al. 1998).
But what could trigger such an extreme perturbation in
the climate system to allow a runaway freeze, and even more
perplexing, how could the process be reversed?
The
clustering of continents in the tropics may have induced
the snowball by remaining ice-free as the Earth cooled and
the poles covered with ice.
Under these conditions,
tropical carbon burial coupled with an increased polar
albedo might initiate a global freeze (Hoffman and Schrag
2000).
Decreased levels of atmospheric CO2 might also
result from the breakup of the Rodinia supercontinent,
which would have increased costal margins and carbon burial
(Hoffman et al. 1998).
Kirshvink (1992) noted that the
absence of a silicate weathering system on a snowball Earth
would not provide a sink for CO2.
Therefore, CO2 out-
gassing from volcanic sources would accumulate to very high
levels, eventually reversing the climate abruptly to hothouse conditions, as melting ice decreases albedo and
increases water vapor in the atmosphere, further amplifying
7
the warming effect.
Hoffman et al. (1998) report rapid
deposition of cap carbonates (~40 cm/year) after escape
from the global freeze, with pre- and post-snowball δ13C
data consistent with a significant decrease in biological
activity preceding glaciation, possibly due to the cooling
of the Earth, followed by a gradual rebound to preglacial
values.
Since a snowball Earth would take 4-30 million years
to reverse (Hoffman et al. 1998), such a long period of
global freeze and modified geochemistry would have had
drastic consequences on biological ecosystems established
prior to these events.
The oceans would have been covered
with 1-2 km of ice, although there was probably no more
than 10 m of ice in the tropics (Hoffman 2001, OSU Geology
Seminar), which may have provided potential ecological
niches for photosynthetic sea ice communities.
Sealed
beneath the sea ice, however, the world ocean was
geochemically modified by the circulation of reduced
hydrothermal fluids at mid-oceanic spreading zones, arguing
for the survival of only anaerobic chemoautotrophic or
heterotrophic communities.
Hot springs on the sea floor
and terrestrial surface may have served as oases for life
8
during these lengthy crises, providing thermal and chemical
gradients that could support a variety of microbial
lifestyles.
Microorganisms might also have survived frozen
within the expansive ice cover, until post-snowball melting
reintroduced environments suitable for activity.
It is noteworthy that the Cambrian explosion, a period
where all 11 animal phyla rapidly evolved, occurred
immediately after the last Neoproterozoic snowball Earth
(Hoffman and Schrag 2000).
Rather than representing some
kind of supposed biological pinnacle, etched from several
billion years of evolutionary fine tuning, the emergence of
metazoan life took place swiftly after a global
catastrophe, and may have been triggered by biological
innovations forged to survive on a frozen planet.
Ironically, the deep-rooted phylogeny of thermophilic
species on the tree of life, generally interpreted to mean
that life originated in a thermophilic setting, may instead
represent the consequence of an evolutionary “bottle-neck”
imposed by the extremes of multiple snowball Earth events
(Kirschvink et al. 2000).
9
Microorganisms immured in glacial ice
Cryogenic preservation of airborne biological
material deposited in glacial ice
Glacial ice sheets entrap and preserve aerosolized
biological material (i.e. insects, plant fragments, seeds,
pollen grains, fungal spores and microorganisms) deposited
in snowfall.
Ice cores extending thousands of meters below
the glacial surface can represent hundreds of thousands of
years of snowfall accumulation, and the assemblages of
microorganisms immured chronologically within a core are
species that were distributed in the atmosphere at
different times in history.
Studies indicate that the
topography, local and global environmental conditions, and
proximity of ecosystems contributing biological particles
to a particular air mass influence the concentration and
diversity of airborne microorganisms (Lighthart et al.
1995; Giorgio et al. 1996; Fuzzi et al. 1997; Marshall and
Chalmers 1997).
Thus, depending on the geographical
location and climate history of a glacial ice sheet,
microorganisms preserved sequentially within these strata
are mainly derived from nearby ecosystems, but may also
originate from distant ecological sources.
10
Microorganisms aerosolized from water, exposed soils
and rock surfaces can travel large distances on atmospheric
currents, often in a viable, but dormant state.
Amazingly,
some air conditions actually provide a medium for growth,
and microbial metabolism has been detected in fog particles
(Fuzzi et al. 1997) and clouds (Sallter et al. 2001).
For
an airborne microorganism to retain viability, the stress
associated with atmospheric transport, namely desiccation
and exposure to ultraviolet (UV) radiation, must not result
in a lethal level of unrepairable cellular damage.
During
desiccation, a decrease in the cellular water content
results in structural damage to the cell membrane and
protein denaturation (Marthi and Lighthart 1990; Geiges
1996).
The composition and structure of Gram-negative
bacteria make them more vulnerable to dehydration than
Gram-positive bacteria, which possess a more rigid cell
wall (Marthi 1994).
Many Gram-positive genera also form
spores, which are generally resistant to environmental
abuse.
Desiccation is also known to increase survival to
radiation, because much of the substantial damage induced
by exposure to ionizing radiation results from free-radical
formation, which is dependent on the presence of water
(Mattimore and Battista 1996).
11
Microbial strategies for
high levels of resistance to radiation include possessing
UV-absorbing carotenoid pigments, genetic redundancy, and
efficient enzymatic mechanisms to excise and repair
dimerized thymine bases (Marthi 1994; Atlas and Bartha
1998; Fogg 1998).
In addition to the severe conditions associated with
atmospheric transport, microorganisms deposited in glacial
ice are exposed to the physical and osmotic stress
associated with freezing and thawing, and low temperatures.
Ice crystals initially form in the extracellular phase, and
solute exclusion draws water from the cell, damaging the
integrity of the cell membrane (Fogg 1998).
The stress of
freezing is therefore a problem of water management.
Many
plants, animals, and microbes adapted to freezing
conditions therefore produce compatible solutes, like
betaine and glycine, which reduce the shock of such osmotic
imbalance (Marthi and Lighthart 1990).
Thermal hysteresis
antifreeze proteins have also been identified in bacteria,
and these cold-induced proteins function to prevent damage
initiated by intra- and extra-cellular ice crystal
formation (Willimsky et al. 1992; Duman et al. 1993).
Another consequence of low temperature is a decrease in
membrane fluidity, so for the cell membrane to avoid the
12
“gel-crystalline” state, many organisms increase the
proportion of unsaturated fatty acyl chains in the lipid
bilayer (Finegold 1996).
These injurious effects, however, are largely
dependent on the internal water content of the cell, and
since most airborne particulates would be desiccated, the
freezing process should cause minimal cellular damage,
considering the anhydrous nature of such microbial
aerosols.
On the other hand, this would suggest that
airborne microflora are dormant, and unlikely able to
induce the protection responses discussed above.
Intriguingly, when an organism is stressed under a
particular condition, such as starvation or osmotic stress,
the induced synthesis of stress proteins cross-protects the
cell from various other traumas (Morita 2000).
Similar
strategies are required for survival in both the atmosphere
and under frozen conditions, and indeed, there is a
relationship between freeze- and desiccation-resistance in
many organisms (Fogg 1998).
13
Longevity of microorganisms entrapped in ancient materials
Cells and macromolecules remain preserved for millions
of years in permafrost, (Gilichinsky et al. 1993; Shi et
al. 1997;) amber, (DeSalle et al. 1992; Cano et al. 1995;
Greenblatt et al. 1999) and salt crystals (Vreeland et al.
2000).
Inevitably during long-term preservation,
macromolecular damage accumultes, resulting from continuous
exposure to natural radioactive sources.
Temperature and
the hydration of nucleic acids and protein strongly
influence the rate of depurination and L-amino acid
racemization, respectively (Lindahl 1993; Bada et al.
1994).
Ancient samples in permanently cold regions
preserve DNA for ~105 years, whereas DNA survival in warmer
climates is restricted to a few thousand years (Poinar et
al. 1996).
Racemization rates of amino acid L-enantiomers
were correlated with the preservation of DNA in ancient
amber samples, and Poinar et al. (1996) were only able to
detect DNA templates of amplifiable length in samples with
low D/L ratios.
Amino acids in amber have retarded
racemization rates, and the observed stereochemical
preservation is likely a result of the anhydrous nature of
14
amber, which prevents abstraction of the amino acid αhydrogen (bound adjacent to carbonyl group) by water (Bada
et al. 1994).
In addition to preserving biological macromolecules,
viable microorganisms have been reported to survive in
ancient geological materials for millions of years
(Gilichinsky et al. 1992; Shi et al. 1997; Greenblatt et
al. 1999; Vreeland et al. 2000).
Estimations of microbial
longevity based on extrapolations of data collected over
rather short time frames predict survival ranges from
thousands to millions or even billions of years (Kennedy et
al. 1994).
Although the degree of thermal decay (i.e.,
increase in entropy) in a microorganism dormant under known
environmental conditions over a given time frame can be
theoretically determined, it requires an estimate of the
minimum macromolecular decomposition needed to be lethal.
Decay rates are known to be drastically reduced when
proteins and nucleic acids are stabilized within materials
with low water activity, such as amber (Bada et al. 1994),
and this could also pertain to ice.
It is also possible that entrapped microbes carry out
a slow rate of metabolism, which allows repair of
macromolecular damage, but not growth.
15
Microenvironments
within salt crystals (Vreeland et al. 2000), amber
(Greenblatt et al. 1999), and permafrost (Gilichinsky et
al. 1992; Shi et al. 1997) appear to possess the conditions
necessary for active metabolism (i.e., liquid water,
carbon, electron donors and oxidants).
Therefore, it is
not possible to determine if biological preservation in the
abovementioned environments results from anabiosis (Morita
2000) or a low level of metabolism that counteracts the
dupurination and racemization processes.
Frozen microbial ecosystems
The biology of permanently cold environments in the
Arctic and Antarctic have received relatively little
investigation.
Similar to their high temperature
counterparts, frozen ecosystems are dominated by
microorganisms with trophic assemblages (such as microbial
mats) found only in other extreme environments.
In recent
years, a number of investigators have documented microbial
communities in permanently ice-covered lakes (Fritsen and
Priscu 1998; Olson et al. 1998; Paerl and Priscu 1998;
Priscu et al. 1998; Takacs and Priscu 1998; Brambilla et
al. 2001), Antarctic sandstone (Friedmann 1982; McKay and
Friedmann 1985; Hirsch et al. 1998; Sun and Friedmann
16
1999), permafrost (Gilichinsky et al., 1993; Shi et al.,
1997; Vorobyova et al. 1997; Rivkina et al. 2000), and sea
ice (Bowman et al. 1997; Gosink et al. 1997; Junge et al.
1998; Staley and Gosink 1999).
Many of these environments
never experience temperatures above the freezing point of
water; life nevertheless persists under arguably the
harshest conditions in the biosphere.
Despite the dry, cold conditions of Antarctica’s Dry
Valleys, microbiological surveys of this polar desert have
documented an abundance of cryptoendolithic lichen
(Friedmann 1982; McKay and Friedmann 1985), associations of
fungi and bacteria that together inhabit pores in the
dominating sandstone.
The endolithic communities so evades
exposure to abrupt temperature changes and solar
irradiation in these harsh surroundings, and estimates
indicate generation times as long as 104 years (Sun and
Friedmann 1999).
The Dry Valley complex is subjected to
persistent katabatic winds and is a substantial portion of
the Antarctic landscape that has exposed rock surfaces and
soil.
The vast majority of the continent (~98%) is covered
in kilometers of glacial ice.
The Dry Valleys have 24
hours of daylight during the austral summer, and rock
grains of aeolian origin on the surfaces of the permanently
17
frozen lakes, are warmed by the sun, and melt into the ice.
Pockets of liquid water are created on the surface and
within the ice, and these contain sufficient nutrients to
support the growth of microbial communities (Olson et al.
1998; Paerl and Priscu 1998; Priscu et al. 1998), many of
which are then frozen and trapped completely within the ice
during the austral winter.
The annual freezing of the north and south polar
oceans covers >7% of Earth’s surface with thick pack ice
(Staley and Gosink 1999), which drastically influences the
surface and underlying water column ecology.
During sea
ice formation, liquid brine pockets created during ice
crystal consolidation become increasingly concentrated.
Solute exclusion during freezing can elevate brine channel
salinities to >150‰ that of seawater, allowing supercooled
hypersaline solutions to exist at temperatures as low at
–150C (Bowman et al. 1997).
Planktonic microorganisms
incorporated into sea ice during crystallization
subsequently develop into spatially defined communities in
and on the ice surface.
Biological activity is highest in
the lower 10-20 cm of the ice at the seawater interface,
where the enriched microbial assemblage is termed the sea
ice microbial community (SIMCO).
18
This consists of diatoms,
protozoa, bacteria, fungi and invertebrates, contains
higher nutrient levels than seawater, and possesses
psychrophilic species that appear to be specialized for
survival and activity in the sea ice environment (Bowman et
al. 1997; Junge et al. 1998; Staley and Gosink 1999).
Subterranean layers of permafrost in polar locations
contain rocks, sediment, biological detritus, and organisms
(both fossilized and viable) that originated from
previously unfrozen soil environments.
Such frozen
deposits underlie ~20% of Earth’s terrestrial surface.
They contain 107-109 microbial cells per gram, and can be
millions of years old, with the oldest samples studied to
date originating from Siberia (Shi et al. 1997; Vorobyova
et al. 1997).
Gilichinsky et al. (1993) hypothesize that
unfrozen films of water present on mineral surfaces in
permafrost may protect microorganisms from freezing.
It is
also suggested that this refuge may serve as a habitat for
activity, given that many permafrost isolates grow at
temperatures as low as –100C.
The significance of liquid
water, which dependent on the permafrost temperature exists
in a film ranging from 5 to 75 Å (Anderson 1967), relates
to the physiological need of the cell to exchange ions,
metabolites, and waste products with the external milieu.
19
Rivkina et al. (2000) investigated metabolic activity in
permafrost by measuring [14C]-acetate incorporation into
lipids of samples incubated for 550 days at temperatures
from 5 to –200C.
A sigmoidal curve was observed, and
stationary phase occurred at different levels of
radioisotope incorporation, and this depended on the
incubation temperature.
Very little incorporation was
observed in samples at –15 and -200C.
Habitats for microorganisms in glacial environments
Glaciers are generally perceived as inhospitable
environments, yet microbial habitats are known to exist in
sediment-containing melt water depressions on glacial
surfaces [i.e., cryoconite holes (Wharton et al. 1985)] and
in the water generated through friction and geothermal
heating at the ice-bedrock interface (Skidmore et al.
2000).
Direct microscopic counts of melted snow from the
South Pole ranged from 102-103 cell ml-1, and low levels of
macromolecular synthesis were detected when the snow
samples were incubated at temperatures below -120C
(Carpenter et al. 2000).
Relatively few studies have
focused on microorganisms entrapped deep within permanent
ice sheets, although, Abyzov et al. (1993, 1998) did
20
examine the microbial species in samples from an ice core
drilled at Vostok Station, Antarctica.
They cultured both
prokaryotes and eukaryotes (yeast and fungi) from samples
<10,000 years old, whereas at depths >1500 meters below the
surface, corresponding to ice >100,000 years old, fewer
microorganisms were recovered (less than 5% of cultures
contained growth), and these were predominantly sporeforming bacteria (Abyzov 1993).
Based on these results, it
was concluded that cells within deep glacier horizons are
dormant (anabiotic), but that species resuscitated from
these ancient samples were apparently able to commence
metabolism and grow after thousands of years of inactivity.
Much attention is currently focused on the exciting
possibility that the subglacial environments of Antarctica
may harbor microbial ecosystems under thousands of
kilometers of ice, isolated for perhaps as long as the
continent has been glaciated (20-25 million years, Naish et
al. 2001).
The discovery of more than 70 subglacial lakes
in central Antarctica during the early 1970’s (Siegert et
al. 1996) went relatively unnoticed by the biological
scientific community, however, curiosity about the nature
of these environments has recently intensified as the
result of international interest in the largest such
21
subglacial lake, Lake Vostok.
The freshwater in Lake
Vostok originates from the overlying ice sheet which melts
into the lake at the ice-water interface in the north.
Ice
accretion occurs at the base of the ice sheet in the
central and southern regions, removing water from the lake
(Kapitsa et al. 1996 ; Jouzel et al. 1999; Siegert et al.
2001).
The Vostok ice core was extended in 1998 to a
record depth of 3623 m, but due to concerns regarding
contamination, drilling stopped at ~120 m above the lakeice interface.
However, 150 m of accretion ice was
recovered (Petit et al. 1999), and the presence of
microorganisms within Lake Vostok accretion ice was
confirmed by Priscu et al. (1999) and Karl et al. (1999),
with intact cells visualized by microscopy, and seven
different 16S rDNA sequences obtained after PCR
amplification.
Karl et al. (1999) also found evidence for
respiration by examining
14
CO2 release from samples
incubated with [14C]-acetate or glucose, however, there was
very little, if any,
14
C incorporation into macromolecules.
While it seems inevitable that viable microorganisms from
the overlying glacial ice and in sediment scoured from
bedrock adjacent to the lake are regularly seeded into the
lake, the question remains whether these or pre-existing
22
microorganisms have established a flourishing microbial
community within Lake Vostok.
Price (2000) has proposed that aqueous veins exist
between crystal boundaries in glacial ice that could serve
as habitats for microbial activity, possessing all the
necessary conditions, including liquid water, reductants
and oxidants.
Salts are insoluble in glacial ice, so only
acids deposited as aerosols become increasingly
concentrated when recrystallization occurs in deep ice.
Liquid water can also be found in lenses on grain
boundaries, around air bubbles, and at salt inclusions
(Patterson 1994).
Electron donors such as formate,
methysulfonic acid (MSA) and acetate, and the electron
acceptors SO4-2 and NO3- can be concentrated by several
orders of magnitude in these veins, depending on
temperature and grain size (Price 2000).
In contrast to
the highly acidic vein environments in glacial ice, veins
formed in Lake Vostok accretion ice contain concentrated
salts, much like sea ice, because this ice originated as
lake water that contained dissolved ions.
Based on the
minimum required input to avoid microbial carbon loss,
23
Price (2000) calculated that ~10-100 cells cm-3 could
survive for 400,000 years in glacial ice metabolizing the
available MSA alone.
Astrobiology implications of microbiological
investigations of terrestrial glacial ice
The origin and margins of the biosphere
It would be difficult, if not impossible, to find a
square millimeter of the Earth’s surface that is not
inhabited by life.
While knowledge of deep subsurface
ecology is scanty, it is clear that the boundaries of the
biosphere extend kilometers deep into the crust (Stevens
and McKinley 1995; Chandler et al. 1998), with
extrapolations predicting that the terrestrial subsurface
contains similar amounts of microbial biomass as the
surface (Whitman et al. 1998).
Environments with
severities of temperature, pressure, salinity, or pH are
labeled with the anthropocentric distinction of being
“extreme”, yet microorganisms nonetheless occupy, even
thrive in these niches.
The resident extremophiles have
evolved for these environmental conditions, with related
species often found only in similar ecological settings
(VanDover et al. 2001).
Hence, from a microbial
perspective, the planet’s zone of habitation extends from
24
high into the atmosphere (Sallter et al. 2001) to the inner
depths of the Earth, where temperatures rise with
increasing depth to exceed those assumed possible for known
carbon-based life.
Life arose early in Earth’s history, at least 3.9
billion years ago, and it seems unlikely that life could
have evolved much before this date.
The molten surface
took several hundred million years to solidify, and the
early planet was heavily bombarded with asteroids and
comets, which episodically evaporated the atmosphere and
any liquid water on the surface (Lunine 1999).
Sleep et
al. (1989) have suggested that deep marine hydrothermal
ecosystems, sustained by chemoautotrophic primary
production, could have endured these impacts that would
have sterilized the surface, perhaps as early as 4.0-4.4
billion years ago.
Stable carbon isotopic ratios (δ13C) in
>3.8 billion year old kerogen are consistent with
photosynthetic carbonate mineralization (Schidlowski 1987),
and stromatolithic communities fossilized in 3.5 billion
year old chert are strikingly similar to species common in
extant microbial mats (Schopf 1993).
Thus, when planetary
conditions were permissive, life arose in a geological
25
instant, with a 100-400 million year window for the
emergence of complex photosynthetic energy-generating
strategies and considerable species diversity.
Theories for an Earth-based origin of life are rooted
on the idea that cellular metabolism, based on
heterotrophy, photoautotrophy, or chemoautotrophy, and a
genetic system evolved spontaneously from an organic soup,
generated by photolysis, prebiotic chemical reactions and
cometary impacts.
The evolution of a dissipative chemical
system into the last common ancestor may have involved
intermediary stages, with primitive life possibly
possessing an inorganic crystalline genetic system (CairnsSmith 1985), existing as a self-replicating catalytic RNA
(Lazcano and Miller 1996), or perhaps consisting of a
genetically promiscuous ancestral community (Woese 1998).
No current hypothesis, however, is able to provide an
adequate explanation for the unprecedented degree of
biological innovation that occurred between 3.5-3.8 billion
years ago.
During life’s initial period of existence DNA
replication, transcription, and translation must have
evolved, as evidenced by the conserved genetic nature of
proteins involved in these core processes within the three
domains of life (Fitz-Gibbon and House 1999; Makarova et
26
al. 1999).
Yet, it would appear that these major
evolutionary challenges were solved in a geological
instant, with substantially evolved microbial species
living in complex photosynthetic communities by 3.5 billion
years ago (Schopf 1993).
Panspermia, the concept that life originated from an
extraterrestrial source, was first conceived and termed by
the Greek scientist Anaxagoras, and revisited in the early
twentieth century by the Nobel laureate Svanté Arrhenius,
who calculated that bacterial spores ejected into space
could travel on the solar wind to Alpha Centauri in 7,000
years (Margulis and Sagan 1995).
In recent years, Hoyle
and Wickramasinghe (1981) have been the most radical
proponents of panspermia theory.
They argue that life is
ubiquitous throughout the universe, and that the Earth is
bombarded at regular intervals with cometary materials
containing extraterrestrial cells and genetic material, and
these shaped the evolution of life on Earth.
While
theories for an extraterrestrial genesis of life have also
not supplied a tidy mechanistic explanation for spontaneous
generation, they do provide a cosmological context for such
a discussion, advocating the concept that planetary bodies
in the solar system are not biologically isolated.
27
Although such notions were once viewed as radical and
generally dismissed by the scientific community, recent
awareness of the tenacity of life and extremophile
diversity now place them in the realm of possibility.
Spores surrounded by radiation absorbing material have been
argued to have the longevity and durability needed to
survive transfer through interstellar space (Weber and
Greenberg 1985), and ejecta from large impacts of the Earth
containing viable microorganisms would provide sufficient
shielding from heat and lethal cosmic radiation to
facilitate transfer to Mars, and vice versa (Melosh 1988;
Weiss et al. 2000).
Remarkably, Streptococcus mitis
isolates were revived from a camera on Surveyor 3 that was
recovered by Apollo 12 astronauts after it had spent 2.6
years on the moon (Noever 1998).
The measures taken by NASA to control the forward
contamination of Mars during the Viking missions seemed
appropriate at the time.
However, 25 years of deliberation
and knowledge have made it necessary to reevaluate
appropriate protocols for the protection of planets and
satellites from contamination by Earth microorganisms,
28
prompting NASA to create a Planetary Protection Advisory
Committee to assess procedures used in solar system
exploration and sample return missions (Rummel 2001).
Prospects for frozen lifestyles in the solar system
A resurgence of interest in the field of Exobiology,
now often referred to as Astrobiology, is centered on the
notion that geological and physical settings in the
universe similar to those on Earth may also harbor life.
Thus identifying, characterizing, and understanding
terrestrial habitats that could be similar to
extraterrestrial habitats provides an experimentally
tractable system to evaluate the likelihood of
microorganisms surviving in extraterrestrial environments.
These may well offer important clues to lifestyles that
might be encountered on Mars or several of the jovian
moons, and perhaps even within comets or asteroids
containing liquid brine fluid inclusions (Zolensky et al.
1999).
The poles of Mars are covered by ice caps with some
features similar to those of terrestrial ice sheets (Budd
et al. 1986).
Although temperatures exceed the frost point
and water ice is unstable at lower latitudes, ice may also
29
still be present in the non-polar regions kilometers below
the surface (Squyres and Carr 1986).
Dust particles from
the surface, elevated into the atmosphere by wind, serve as
condensation nuclei for carbon dioxide and water which is
precipitated, perhaps seasonally, in the martian polar
regions (Clifford et al. 2000). The presence of alternating
clean and dusty layers within these polar ices could
reflect changes in the levels of atmospheric dust, or may
result from the sublimation of frozen volatiles during
periods of high obliquity.
Although organic molecules are
likely to be destroyed by the high levels of UV irradiation
and peroxides photochemically-generated in the martian
soil, biochemical traces of life or even viable
microorganisms may well be protected from such destruction
if deposited within polar perennial ice or far below the
planet’s surface.
During high obliquity, increases in the
temperature and atmospheric pressure at the northern pole
of Mars (McKay and Stoker 1989) could result in the
discharge of liquid water that might create environments
with ecological niches similar to those inhabited by
microorganisms in terrestrial polar and glacial regions.
Periodic effluxes of hydrothermal heat to the surface might
also move microorganisms from the martian subterranean,
30
where conditions may be more favorable for extant life
(McKay 2001).
The annual partial melting of the ice caps
might then provide conditions compatible with active life
or at least provide water in which these microorganisms may
be preserved by subsequent freezing (McKay and Stoker 1989;
Clifford et al. 2000).
The microfossils and chemical
signatures of potential biological origin that were
recently discovered in Alan Hills meteorite ALH84001,
reinvigorated the debate over the possibility of life on
Mars (McKay et al. 1996).
However, such circumstantial
evidence needs confirmation by explorative missions to the
martian surface.
In January 2000, the Galileo spacecraft measured
changes in Europa’s magnetic field, and provided the most
convincing evidence to date for the presence of a
subsurface liquid ocean (Kivelson et al. 2000).
Geothermal
heating and the tidal forces generated by orbiting Jupiter
are thought to maintain a 50-100 km deep liquid ocean on
Europa with perhaps twice the volume of the Earth’s ocean
(Chyba and Phillips 2001), but beneath an ice shell at
least 3-4 km thick (Turtle and Pierazzo 2001).
Cold
temperatures (<128 K; Orton et al. 1996) combined with
intense levels of radiation would appear to preclude the
31
existence of life on the surface, and the zone of
habitability, where liquid water is stable, may be present
only kilometers below the surface, where sunlight is unable
to penetrate (Chyba and Hand 2001).
Europa’s surface
appears strikingly similar to terrestrial polar ice floes,
suggesting that the outer shell of ice is periodically
exchanged with the underlying ocean.
The ridges in the
crust and the apparent rafting of dislocated pieces implies
that subterranean liquid water flows up through stressinduced tidal cracks, which may then offer provisional
habitats at shallow depth for photosynthesis or other forms
of metabolism (Gaidos and Nimmo 2000; Greenberg et al.
2000).
However, if one exists, the europan ecology must
differ significantly from any presently known on Earth, all
of which depend on photosynthesis for primary production.
Even communities at marine hydrothermal vents, which have
been widely described as self sufficient ecosystems based
solely on the metabolism of reduced geothermal energy,
ultimately require oxidants such as SO42-, CO2, and O2 from
the surface to function.
Currently, the only possible
example of a photosynthetically-independent ecological
community are the proposed subsurface lithoautotrophic
32
microbial ecosystems [SLiMEs]), with the primary producers
obtaining energy from geothermally produced hydrogen
(Stevens and McKinley 1995; Chapelle et al. 2002).
As discussed, photosynthetic organisms are usually
required to fix CO2 with reducing power generated by lightdriven reactions, and thus subsequently provide assimilable
carbon for heterotrophic consumption.
The oxidant
byproduct, however, is equally crucial for maintaining the
environmental redox gradients exploited by diverse
metabolic lifestyles.
Gaidos et al. (1999) argue that
without a source of oxidants, Europa’s subsurface ocean
would be destined to reach chemical equilibrium, making
biologically-dependent redox reactions thermodynamically
impossible.
However, the surface is continually bombarded
with high-energy particles, producing molecular oxygen and
peroxides, as well as formaldehyde and other organic carbon
sources (Chyba 2000; Chyba and Hand 2001), and thus it is
conceivable that europan microbial life might subsist
without employing photosynthetic or chemoautotrophic
lifestyles.
In this scenario, mixing between the crust and
subsurface need be the only mechanism required to provide
organics and oxygen at levels sufficient to support life
33
(Chyba 2000).
Tidal heat generation and electrolysis could
also provide sources of energy that could be coupled to
bioenergetic redox reactions (Greenberg et al. 2000).
Development of technology relevant to life detection in
extraterrestrial materials
NASA missions are already scheduled to examine Mars
and Europa for liquid water and prospective life, and the
first sample return missions from Mars could launch as
early as 2011, returning to Earth in 2014.
A major concern
is the forward contamination of these solar system bodies
with microbes or molecules transported on spacecraft from
Earth, which could then be misinterpreted as evidence for
extraterrestrial life.
Contamination of the Earth with non-terrestrial life
is an equally important consideration, and NASA is now
faced with the challenge of defining appropriate
containment measures (Rummel 2001), and constructing a
facility now designated the Mars Sample Receiving Facility.
To facilitate geochemical and microbiological analysis on
unaltered samples, the curation team has recommended
maintaining samples at below -300C (Meyer 2001).
The
challenge of identifying appropriate sampling sites and
developing protocols that maintain containment,
34
cleanliness, and cold will benefit from the experience
gained by sampling and studying frozen terrestrial
environments, analogs for Mars and Europa.
Objectives of this study
The Byrd Polar Research Center (BPRC) at Ohio State
collects ice cores from the polar regions, and from
mountain glaciers at low latitude.
Stored within this
archive are glacial ice cores ranging in age from <50 to
>750,000 years old.
The primary objective of this study
was to sample, isolate, and identify microorganisms
preserved in ice cores of defined ages, and to determine
the longevity of species entrapped in ice for hundreds of
thousands of years.
The availability of physical and
chemical data on each core provides the opportunity to
correlate the effect of climatic conditions on
microbiological deposition and survival.
As a second
project, the possibility that glacial and subglacial
environments represent microbial habitats was explored.
While establishing the lengths of microbial survival in ice
was of primary biological interest, it seems also possible
that there might be metabolism within these frozen
environments.
The data obtained are also relevant to
discussions about the survival of life through periods when
35
the Earth was completely covered in ice for millions of
years, and also might well offer valuable information in
considering exobiological lifestyles.
36
CHAPTER 2
PROCEDURES USED TO PREVENT CONTAMINATION
DURING SAMPLING AND ANALYSIS
Introduction
The major concern in any study employing culture-based
analyses and sensitive molecular amplification approaches
to examine ice cores is obtaining samples without
contamination by extraneous microorganisms and/or nucleic
acids.
Care also must be exercised in the subsequent
preparation and handling of media, glassware, and reagents,
with appropriate controls to monitor for contamination at
each experimental step.
Although protocols to eliminate
microbial contamination can be developed or implemented,
proving that a positive result is not an artifact is very
difficult.
To address this issue, and the inherent concern
it raises for this investigation, techniques and
decontamination procedures developed and employed are
described here in detail, together with the mechanisms used
to verify the authenticity of results obtained.
37
Materials and Methods
Bacterial strains and control ice cores
Escherichia coli, Bacillus subtilis and Serrratia
marcescens (OSU reference numbers 455, 848, and 234,
respectively) were cultured on tryptic-soy (Difco) broth or
1.5% solidified agar and used as indicator strains and in
the construction of control ice cores.
Cells were
suspended in deionized water at concentrations from 102-109
cfu ml-1, and the suspensions were frozen at -30oC in a 100
mm diameter cylinder.
Sterile cores of deionized water
were constructed in the same manner.
Expansion of the ice
formed within the cylinder made extraction of the cores
difficult.
The exterior surface was therefore melted by
running tap water over the cylinder and the extracted core
was then refrozen.
Stress fractures caused by freezing
from the outside to the inside limited the length of core
recovered by this method, but workable sample cores of 1020 cm were obtained.
Sterilization and removal of nucleic acids from reagents
and experimental materials
Liquid reagents and solid materials were autoclaved
for 30 min. at 120oC (20 psi).
Glassware, pipets, test
tubes, and all other solid items were then dried for at
38
least 72 h. at 55oC, and then exposed to ethylene oxide in
an Anprolene AN-74 sterilizer (H.W. Andersen Products Inc.,
Haw River, NC) for 12 h.
To remove nucleic acids from
water and reagent solutions used in PCR amplifications,
they were filtered through Microcon YM-100 (500 µl
capacity), Biomax-100 Centricon Plus-20 (20 ml capacity),
or Biomax-100 Centricon Plus-80 (80 ml capacity)
centrifugation devices following the manufacturers
directions (Millipore Corp. Inc., Cat. no. 42412, UFC2 LGC
02, and UFC5, LGC 02, respectively).
Bleach-rinsed gloves were worn throughout all
procedures.
All cultural manipulations and the preparation
of PCR amplification material were carried out in a BioGard
laminar flow hood (Baker Company, Sanford, ME) with a
germicidal UV-C lamp.
Before use, all interior surfaces of
the hood were wiped with a solution of 0.24% (w/v) sodium
hypochlorite.
Reagents, pipets, and tubes were placed
within the cleaned hood and exposed to UV irradiation for
at least 25 min. before use.
Disposable pipet tips with
hydrophobic barriers were used to decrease the likelihood
of aerosol generation and cross-contamination.
39
Rinse method for the decontamination of ice cores
The outermost layer was removed from ice cores using a
dust-free bandsaw, in a -5oC cold room, to expose previously
unhandled ice.
The ice was then equilibrated to the
sampling temperature (-5oC) overnight, to reduce the
likelihood of fracturing during washing procedures and
melting.
Sections of these ice cores were then held with
sterile forceps or scissor tongs within the laminar flow
hood and washed with ~200 ml of -5oC 0.2 µm filtered 95%
ethanol.
This disinfected and dissolved away the outermost
ice layer.
After a 2 cm annulus has been removed, a fresh
pair of forceps was then used to grasp a cleaned section of
the ice core, and the sample was rinsed with ~100 ml of
chilled, double autoclaved deionized water.
To evaluate
the effectiveness of the washing procedures, the wash
materials were collected and assayed for contaminants or
for the removal of bacteria deliberately placed on the ice
exterior in control experiments.
The cleaned ice was
placed in a sterile vessel and allowed to melt slowly at
4oC.
This took at least 16 h for most samples.
40
The automated ice core sampler
Heated sampling heads (Fig 2.1A) were constructed with
different diameters (10, 20, and 30 mm) to sample core
sections of varying sizes.
The sample head was housed in a
circular holder that guides the ice core position.
The
prototype holder was constructed out Teflon (Fig 2.1B), but
the experimental version was made using iodized aluminum
(Fig 2.2A).
Prior to sterilization, the device is fitted
to accommodate the size and shape of the ice core to be
sampled.
Movable dividers (Fig 2.2B) were fixed to
position the core so that the sampling head remained inside
the core throughout the melting process (Fig 2.2C).
The
sample unit accommodates 1/2 to 1/8 core sections >500 cm
in length.
The components of the unit were autoclaved for
30 min. at 120oC (20 psi), dried for at least 72 h. at 55oC,
and then exposed to ethylene oxide treatment [Anprolene AN74 sterilizer (H.W. Andersen Products Inc., Haw River, NC)]
for 12 h.
The components were assembled inside a laminar
flow hood housed within a –5o C walk-in freezer (Fig 2.2A).
The ice cores are archived at -30oC, but were equilibrated
to -5oC before sampling.
Using a dust-free bandsaw, used
only for this purpose, a cross-sectional cut removed a few
millimeters of ice from the end of an ice core exposing
41
Fig. 2.1
Construction of the sampling head and
prototype ice core sampler.
A. Picture of the sampling head. An external jacket
allows the circulation of water that heats the head, which
then melts through an ice core under the influence of
gravity. The melt water generated is collected through a
hole in the center of the melting head and pumped into an
external sterile container.
B. The housing for the prototype sampler was initially
constructed of Teflon. Here it is shown assembled inside a
laminar flow hood housed in a -5°C walk-in freezer. In the
device is a control core containing E.coli, used to
evaluate the recovery of viable bacteria and the sterility
of the melting procedure.
42
A
heated water
circulates through
sampling head
melt water
collected
externally
B
Figure 2.1
43
Fig. 2.2
Final design of the automated sampler.
A. The complete unit, constructed out of iodized aluminum,
with an ice core positioned perpendicularly in the device.
All components of the system were sterilized and then
assembled inside a laminar flow hood housed in a -5°C walkin freezer.
B. Moveable separation flanges facilitated melting 1/2 to
1/8 of a core section, permitting duplicate samples to be
collected from parallel regions of the same core.
C. The sampling head after movement through a core,
illustrating how the melting head removes a cylindrical
section from the core’s interior.
44
A
Figure 2.2
45
B
C
Figure 2.2 (Con't)
46
previously unhandled ice and provide a uniform, flat
surface for disinfection.
This surface was soaked in 0.2 µm
filtered 95% ethanol for 2 min. to sterilize and dissolve
away a portion of the exterior.
In control experiments,
indicator strains were intentionally applied to this
surface to assay the effectiveness of removal of microbial
contaminants by the disinfection procedure.
Exposing the
ice core surface to 0.24% sodium hypochlorite (bleach) and
short wavelength ultraviolet (254 nm) light were also
evaluated as sterilization procedures.
Prior to sampling,
the sterilized surface of the ice core was swabbed, the
material collected placed in 1 ml of sterile deionized
water, and inoculated into culture media, and included as a
control in molecular amplification procedures.
The ice
core was placed in the sampling device such that the
disinfected portion of the core contacted the sampling
head.
The melt water generated was collected through a
hole in the center of the melting head and pumped directly
into an external sterile container (Fig 2.1A).
47
Characterization of contaminating microbes and 16S rDNAs
Nucleic acids amplified and extracted from
microorganisms were analyzed by determining DNA-encoding
16S rRNA gene sequences.
A portion of an isolated colony
was resuspended, and the cells lysed directly in PCR
amplification mixtures according to Zeng and Kreitman
(1996).
The primers used for these amplifications are
named by the position of the homologous sequences in the
Escherichia coli 16S rRNA gene (Fig 2.3).
Combinations of
the forward primers 21F (complementary to most archaeal 16S
rDNA), 27F (bacterial 16S rDNA), 348F (archaeal 16S rDNA)
or 515F (universal 16S rDNA primer) were used with the
reverse primers 1392R (universal 16S rDNA primer), or 1492R
and 1525R (bacterial and archaeal 16S rDNA) (Lane 1991;
Reysenbach and Pace 1995).
Taq polymerases from GibcoBRL (catalog # 18038-042),
Sigma (ReadyMix™ Taq cat. no. P4475) and Eppendorf
(MasterTaq™ cat. no. 0032 002.650) were used for
amplification of DNA from cultured cells, and the
chemically-modified, thermally-activated AmpliTaq™ Gold DNA
polymerase or LD (low DNA) AmpliTaq™ Gold DNA polymerase
(Perkin-Elmer [PE] Biosystems, Foster City, CA [cat. no.
N808-0240 and N808-0107, respectively]) were employed for
48
Fig. 2.3
Location of primers used to amplify and
sequence 16S rDNA, referenced to the sense strand of
E.coli’s 16S rDNA sequence.
The positions of 16S rDNA oligonucleotides used as PCR
primers are shown schematically, with reference numbers
indicting the position of the 3’ nucleotide within the 16S
rDNA sequence of E.coli. Primer 341F had a 40 bp repeating
sequence of GC (GC clamp), and was to amplify DNA that
would be subsequently investigated by denaturing gradient
(DGGE) or temporal temperature (TTGE) gel electrophoresis
(Muyzner et al. 1993). Primers are designated by fill
pattern their complementarity to bacterial, eukaryotic,
and/or archaeal 16S rDNA sequences.
49
E. coli 16S rRNA gene
5’
3’
1000 bp
500 bp
1500 bp
21F
1525R
534R
27F
907R
348F
68F
1392R
341F
515F
Most bacteria
Most bacteria and archaea
Most bacteria, eukaryotes, and archaea
Most archaea
Figure 2.3
1492R
50
amplification of DNA recovered directly from melt water
samples.
Depending on the Taq polymerase utilized,
reaction mixtures (10X buffer supplied by the manufacturer,
2-4 mM MgCl2, and 5 pmol of each primer) were subjected to
30 or 43 (AmpliTaq [PE Biosystems]) cycles of amplification
by denaturation for 1 min at 94oC, annealing for 1 min at
50oC, and extention for 1 min at 60oC (AmpliTaq™ [PE
Biosystems]) or 72oC.
Before use, AmpliTaq™ requires heat
activation which was achieved by incubation at 95oC for 9
min.
Samples of each PCR product was evaluated by agarose
gel (0.8-2.5%) electrophoresis followed by staining with
ethidium bromide.
Amplicons of the expected length from colony isolates
were sequenced by using primers that hybridized within the
amplified DNA fragment.
Specifically, primers 515F, 534R,
907R, 1392R and 1492R ([Fig 2.3]; Lane 1991; Medlin et al.
1988) were used to prime ABI Prism BigDye™ terminator cycle
sequencing reactions (catalog# 4303149).
The products were
then analyzed on an ABI Prism 3700 DNA analyzer.
Some
amplicons were cloned using the pGEM-Teasy (Promega Corp.,
Madison, WI) vector system and sequenced using primers that
annealed the flanking T7 and SP6 promoter sequences.
sequences obtained were managed using the GeneTool™
51
The
(catalog# GT10-9808-0333-3452, BioTools Inc., Edmonton, AB)
software package, and were compared and aligned with all
sequences available in the Ribosomal Database Project
(Maidak et al. 2001) and GenBank (Benson et al. 2000).
Results
The effectiveness of removing S. marcescens cells
intentionally contaminated onto ice surfaces in visible
quantities, by rinsing samples with chilled sterile
deionized water, was assessed in preliminary experiments by
collecting the resulting wash-off, and spread plating 200 µl
of the material on agar-solidifed media to assay for the
easily identified indicator organism.
A rinse using 300 ml
of water for an oblong ice sample of ~50g was sufficient to
remove the S. marcescens contamination from sterile test
cores, with no colonies of S. marcescens observed in the
final 100 ml used to rinse the ice.
Using this method, ~20
ml of clean sample was obtained from the original 50g
sample.
A disadvantage of this procedure was that the
temperature difference between chilled water and ice
frequently resulted in core fractures that often formed so
rapidly, that the entire sample was lost.
52
The ice core
disintegrated into many fragments that were impossible to
secure with forceps.
Such fractures also provide an entry
point for surface contaminants to contaminate deeper into
the core, and S. marcescens cells were recovered from
cleaned ice-core samples that cracked during the initial
wash.
To overcome this problem, 95% ethanol equilibrated
to the ice temperature (-5oC) was subsequently used to wash
and disinfect cores, followed by a 100 ml rinse with
double-autoclaved distilled water, to dilute the ethanol to
nontoxic concentrations.
Ethanol is a potent disinfectant,
but it would not be expected to destroy endospores, but
contamination by endosporulating cultures of B. subtilis
was nevertheless removed by this washing procedure when
such cells were intentionally swabbed onto ice core
surfaces in control experiments.
An alternate method and instrumentation was developed
to melt and collect water only from the interior of an ice
core.
Disinfection of the cut surface of an ice core by
soaking in 95% ethanol, sodium hypochlorite, and exposure
to germicidal ultraviolet (UV) light irradiation were
evaluated.
Melt water from E. coli control cores treated
with sodium hypochlorite contained 105 cfu ml-1, which was 20
fold less than untreated samples.
53
Although no viable
cultures were generated from material collected from the
ice core exterior, toxic concentrations of bleach
apparently persisted within collected samples.
UV
irradiation of the surface was considered, but given that
ice is transparent to irradiance at wavelengths >190 nm
(Hobbs 1974), the exposure required to sterilize the core
surface would likely compromise the recovery of sublethally
injured species immured within the ice, and also destroy
preserved DNA.
Using 95% ethanol washes had the advantage
that the ethanol remained liquid at -20oC, the ice cores did
not fracture even when soaked for several minutes to
dissolve away potentially contaminated ice, and the ethanol
was easily diluted subsequently to non-toxic
concentrations.
When the ethanol disinfection strategy was
assayed to examine the removal of S. marcescens cells
swabbed on the ice and inoculated onto the saw blade used
to cut the ice core, S. marcescens was not then isolated
from the melt water generated and collected by the
automated sampler.
To investigate if there was DNA present on the cut
surface after the ethanol washing, the washed ice surfaces
were swabbed, the material collected was resuspended in 1
ml of sterile water, and used as template DNA in PCR
54
amplifications using universal and bacterial 16S rDNA
primers.
Amplicons were not generated from material
collected from cleaned ice core surfaces.
This was also
the case when sensitive two-stage nested amplification
strategies were utilized (e.g., one round of amplification
with primers 27F and 1525R, followed by a second round of
amplification using primers 515F and 1392R or 1492R).
The
amount of DNA that remained on the cut, ethanol treated ice
core surface must therefore have been below the level of
detection using these primers.
Discussion
Once the fracturing problem had been solved by the use
of ethanol for washing, the rinse method first used in the
initial stages of this project was fully effective at
removing cultivable contaminants from the cut ice surface.
Holding ice samples >100g with forceps proved difficult,
but by using scissor tongs, common food handling utensils,
such ice fragments could be held firmly after a series of
sharp ridges were filed on the grasping surface.
These
details are important when one considers the precious
nature and one-time availability of many of the ice cores
examined in this study.
55
Since PCR-based analyses of 16S rDNA was planned, it
was not clear if the ethanol washing approach would be
adequate to remove free nucleic acids, nor was there an
easy way to reliably assay for such contamination.
Bleach
destroys DNA, however, rinsing with an aqueous sodium
hypochlorite solution posed the risk of ice fracturing, and
control experiments demonstrated that toxic levels of
bleach could persist within cleaned ice samples.
To develop a more reliable sampling strategy, the
automated sampler (Fig 2.1 and 2.2) was designed,
constructed and tested.
The goal was that a heated melting
device with a reservoir to collect the generated melt water
will melt longitudinally through an ice core interior
without contacting the potentially contaminated outer
surface (Fig 2.2C).
The central feature is the heated
funnel-shaped sampling head (Fig 2.1A) that collects the
melt water, from which it is pumped to an external
collection system.
The advantages of this technology were:
(i) movable dividers (Fig 2.2B) facilitated repeat sampling
through of 1/4 to 1/8 segments of the same core; (ii) by
collecting the melt water as sequential fractions, a high
resolution sampling of a particular deposition period was
possible; (iii) melting only the interior avoided
56
contaminants present on the ice core exterior; (iv) only
the bottom of a freshly cut ice core needed to be
disinfected, reducing the risk of sample loss by
fracturing; and (v) the cut surface was easily swabbed and
assayed for the presence of contaminants.
During the
course of this study, only once was a culture obtained from
an ethanol-cleaned ice core surface, and this isolate had a
16S rDNAs sequence consistent with classification as an
endospore-forming Bacillus subtilis species.
If the same
species had subsequently been isolated from the interior of
the core it obviously would have been suspected as a
potentially introduced contaminant.
But, in the single
case of an apparent breach in sterilization procedures,
this was not the case.
The sample in question was
collected from a 200 year old ice core that originated from
the Guliya ice cap.
Although there was always a
possibility that microbial and/or nucleic acid
contamination would elude the decontamination used, by
employing quality control measures, and drawing conclusions
from the results obtained from more than one sample from an
ice core, this concern was addressed.
The most difficult aspect of using PCR-based
procedures to amplify 16S rDNAs present in low biomass
57
samples is that the Taq polymerase, a recombinant enzyme
isolated from a genetically engineered E. coli, may be
contaminated with small, but significant quantities of
chromosomal DNA.
All other components of the PCR (buffer,
MgCl2, primers, and water) were passed through Centricon and
Microcon filters (Millipore), which removed double-stranded
DNA molecules larger than 125 base pairs from the filtrate
solution.
The Taq polymerase preparations purchased from Sigma,
Gibco BRL, and Eppendorf possessed no detectable
chromosomal DNA contamination when assayed using universal
and bacterial-specific 16S/18S primer combinations in 30
cycles of amplification in template-free reaction mixtures.
Control experiments revealed that 10-100 pg of
Methanobacterium thermoautotrophicum chromosomal DNA,
equivalent to the amount of DNA present in 104-105 cells,
was required to yield a PCR product under these
amplification conditions.
However, with the low cell
numbers anticipated in glacial samples, the use of even
more sensitive PCR strategies, based on two rounds of
nested amplification was necessary, and spurious amplicons
were often generated in controls, namely in template-free
reaction mixtures using these PCR procedures.
58
Several of
these 16S rDNA amplicons were cloned and sequenced, and the
sequences obtained had >99% identity to E. coli and a γproteobacterial Pseudomonas species reported as a
contaminant of PCR reagents by Cisar et al. (2000).
Although the source was not fully established, the results
obtained suggest that the primary sources of contaminating
nucleic acids was the E. coli used to produce Taq
polymerase, and an indigenous microorganism that seems to
colonize laboratory reagents and water supplies in many
laboratories (Cisar et al. 2000; Tanner et al. 1998), based
on efforts to catalog the most frequently encountered
contaminants in negative control libraries.
Attempts to
treat the Taq polymerase with deoxyribonuclease I (DNase;
Gibco BRL) either compromised Taq activity or residual
DNase activity remained after heat treatment, and
amplification products were not obtained.
Finally, a
genetically modified, stringently purified Taq polymerase
(LD [low DNA] AmpliTaq™ Gold DNA polymerase, Perkin-Elmer
Biosystems) was used that the manufacturer claimed
contained <10 copies of bacterial 16S rDNA per 2.5 unit of
enzyme.
Consistent results were obtained using this enzyme
in nested amplifications protocols that indicated that the
DNA contamination was below the level of detection.
59
It is over 70 years from the time of the first reports
that claimed the recovery of viable microorganisms from
ancient geological specimens (Lipman 1928; Lieske 1932).
Since then, microbiological investigations have resulted in
similar revivals from ancient rock, salts, soils and
sediments (Kennedy et al. 1994).
Some extraordinary claims
have received much attention, but have also been viewed
with skepticism (Priest and Beckenback 1995; Hazen and
Roedder 2001).
Contamination from modern sources could not
always be definitively ruled out, and the ages of presumed
ancient specimens has been a source of debate (Hazen and
Roedder 2001).
For example, despite the stringent
precautions and controls used by Cano and Boruki (1995) to
examine the intestinal contents of a bee trapped in
Dominican amber, their recovery of a unique Bacillus
sphaericus species has been questioned (Priest and
Beckenback 1995).
Although the major criticisms focused on
the subsequent sequence analyses, this aggressive critique
underlies the issue central to all studies of this nature.
It is difficult to prove the authenticity of a single
result, even if the methods used have a very low
probability of introducing contamination.
60
Therefore, the
development and assays of the aseptic sampling procedures
used, and the extensive controls used to detect
contamination were of the highest priority for this study.
61
CHAPTER 3
ISOLATION AND CHARACTERIZATION OF BACTERIA AND 16S
rDNA SEQUENCES AMPLIFED FROM GLACIAL ICE CORES
Introduction
Studies of ice cores have established past climate
change and geological events, but rarely have these results
been correlated with the insects, plant fragments, seeds,
pollen grains, fungal spores, and bacteria that are also
present, and few attempts have been made to determine the
diversity and longevity of viable species entombed in
glacial ice.
Several studies have focused on recovering
viable microorganisms (Abyzov 1993, et al. 1998; Dancer et
al. 1997) and detecting virus particles (Castello et al.
1999) and eukaryotic species (Willerslev et al. 1999)
entrapped in polar ice.
However, a thorough evaluation of
the microorganisms preserved in glacial ices from different
geographical locations has not been undertaken, nor have
such isolates been characterized phylogenetically, examined
62
for features increasing survival and persistence while
frozen, or related to the local geography and environmental
conditions.
Fortunately, ice cores collected from a global
distribution of polar and non-polar glaciers (Fig 3.1) are
archived in the Byrd Polar Research Center (BPRC) cold
facility at The Ohio State University.
These ice cores
have been subjected to extensive physical and chemical
analysis, and provide the opportunity to investigate
microorganisms preserved in glacial ices formed at defined
dates, under known climatic conditions, and at
geographically very different locations.
Here, the results
obtained from ice cores originating from China, Bolivia,
Greenland and Antarctica that range in age from 50 to
>500,000 years old are reported.
Materials and Methods
Sample handling
The melt water, which ranged from 50 to >300 ml,
maintained at ~5oC during sampling, was collected during a
period of 1-2 h (see Chapter 2).
Less than 2 h after
melting, samples were placed in a BioGard laminar flow hood
(Baker Company) maintained at room temperature (~22oC) for
63
64
Figure 3.1 Global locations of sampling sites and ice cores available for study at the
Byrd Polar Research Center (BPRC). For each sampling site, the nearest ecosystems that
would most likely contribute the majority of airborne particles are very different.
processing.
The particulates in a large portion of the
melt water were concentrated by filtering through 0.2µm
Isopore filters (Millipore Corp., cat. no. GTTP04700), then
resuspended by vortexing in 5 ml of phosphate-buffered
saline.
In samples analyzed further by PCR amplification
procedures, the 0.2 µm filtrate was stored in a final
concentration 1 mM EDTA and 10 mM Tris.
Media and culture conditions
Aliquots of the 0.2 µm filter-concentrated samples were
spread on the surface of agar-solidified growth media that
specifically included tryptose blood, Actinomycetes
isolation, full strength and 1% nutrient broth, full
strength and 1% tryptic soy broth (all supplied by Difco),
R2 (Reasoner and Geldreich 1985), and M9 glucose minimal
salts (Sambrook et al. 1989).
Bovine liver catalase (1 KU;
Sigma) was filtered (0.22 µm) and added to cultures, as a
known enhancer in the recovery of sublethally injured
bacteria (Marthi et al. 1991).
Duplicate spread plates
were incubated aerobically at 4o or 10o and 22oC.
For
liquid cultures, a 1 ml aliquot of the unfiltered melt
water was inoculated into full strength and 1% nutrient
broth , full strength and 1% tryptic soy broth (Difco), R2
65
(Reasoner and Geldreich 1985), and M9 glucose minimal salts
(Sambrook et al. 1989).
Liquid enrichments were incubated
without shaking) at 4o and 22oC.
In some instances, medium designed to grow acetogens,
methanogens, and sulfate-reducers were also inoculated
directly with a sample of the melt water.
These media are
based on a bicarbonate-buffered salt solution that
contained (per liter) 0.4 g KH2PO4, 0.53 g Na2HPO4, 0.3 g
NH4Cl, 0.3 g NaCl, 0.1 g MgCl2·6H2O, 0.11 g CaCl2, 1 ml
trace element solution, 1 ml vitamin solution, 0.5 mg
resazurin, and 4 g NaHCO3, 0.25 g Na2S·7H2O.
The vitamin
and trace element solutions were as previously described
(Stams et al. 1992).
In addition, 0.16 mg of 2-mercapto
ethane sulfonic acid (MESA) and 0.27 µg of NiCl2 or 2.8 g of
Na2SO4 and 60 µg of FeSO4 were included to facilitate the
growth of methanogens and sulphate-reducing bacteria,
respectively.
These cultures were done in serum bottles
sealed with rubber stoppers, with a 20 psi gas phase of
N2/CO2 or H2/CO2 (4:1).
Carbon sources (methanol, lactate,
acetate, trimethyl amine syringic acid, and fructose) were
added from 0.5 M stock solutions to final concentrations of
10 mM.
Ammonia and nitrate salts media was also used to
enrich for methano- and methylotrophic species, and
66
prepared according to Patt et al. (1974), with the cultures
incubated in sealed serum bottles with a 1:4 methane-air
gas phase.
Duplicate enrichment cultures were incubated at
4o or 10o and 22oC.
16S rDNA amplification from bacterial isolates
A portion of an isolated colony was suspended and
lysed directly in PCR amplification mixtures according to
Zeng and Kreitman (1996).
Primer 27F and 1525R (Lane 1991;
Fig 1.3) were used to amplify 16S rRNA gene fragments with
Taq polymerase from GibcoBRL (cat. no. 18038-042), Sigma
(ReadyMix™ Taq cat. no. P4475) or from Eppendorf
(MasterTaq™ cat. no. 0032 002.650).
Reaction mixtures
contained the buffer supplied by the manufacturer, 2 mM
MgCl2, 0.05% Nonidet P-40 (Sigma, cat. no. N-6507) and 5
pmol of each primer, and were subjected to 30 cycles of
amplification by denaturing for 1 min at 94oC, annealing for
1 min at 50oC, and extending for 1 min at 72oC.
Samples
from each PCR reaction were evaluated by electrophoresis
through 0.8% agarose gels and subsequently stained with
ethidium bromide.
67
Direct amplification of 16S rDNA from melt water
Melt water volume (160 m)l was filtered through a 0.2µm
Isopore filter (Millipore Corp., cat. no. GTTP04700), and
DNA extracted from the collected particulates by using the
modified protocol of More et al. (1994), with hot detergent
treatment as described by Kuske et al. (1998).
Filters
were placed in a screw cap tubes containing 0.1 mm
zirconium beads (Biospec Products, cat. no. 11079101Z) and
a buffered lysis solution consisting of 4% SDS, 40 mM NaCl,
200 mM Tris (pH 8.0), and 20 mM EDTA.
During a 20 min.
incubation at 70oC, samples were vortexed every 5 min for 10
s.
Tubes were then placed in a Biospec Mini-Bead Beater
for 5 min. at 5000 rpm, followed by centrifugation for 3
min. at 12,000xg to remove debris.
The supernatant was
recovered, and residual DNA still associated with the
pellet was extracted by adding 500 µl of TE (10 mM Tris and
1 mM EDTA), vortexing, and centrifuging the mixture as
before.
The SDS was precipitated by incubating samples at
4oC for 5 min in the presence of 2 M ammonium acetate and
removed after centrifugation at 12,000xg for 3 min.
Double-stranded DNA molecules >125 nucleotides present in
the extract were then concentrated and exchanged into TE
using Microcon YM-100 centrifugal filters (Millipore Corp.
68
Inc., cat. no. 42412), in accordance with the manufacturers
specifications.
Free nucleic acids in the cell-free 0.2µm
filtrate were also concentrated using Biomax-100 Centricon
Plus-80 (80 ml capacity) centrifugation devices (Millipore
Corp. Inc., cat. no. UFC5, LGC 02).
The thermally-activated LD (low DNA) AmpliTaq™ Gold
DNA polymerase (Perkin-Elmer [PE] Biosystems, cat. no.
N808-0107) was employed for PCR amplification of DNA
recovered directly from melt water samples.
Reaction
mixtures consisted of 2.5U of AmpliTaq™, the buffer
supplied by the manufacturer, 4 mM MgCl2, 5 pmol of each
primer (27F or 21F and 1392R, 1492R or 1525R), and 2 µl of
template.
AmpliTaq™ requires heat activation, achieved by
incubating the enzyme at 95oC for 9 min., which was followed
by 43 cycles of amplification by denaturing for 1 min at
94oC, annealing for 1 min at 50oC, and extending for 1 min
at 60oC.
An aliquot (2 µl) of the resulting product was
used as the template in a second PCR, with the forward
primer 515F (universal) or 348F (archaeal) combined with
either reverse primer 1392R (archaeal and bacterial) or the
universal reverse primer 1492R (Lane 1991; Reysenbach and
Pace 1995; Fig 2.3).
Samples from each PCR reaction were
evaluated by electrophoresis through 0.8% agarose gels and
69
staining with ethidium bromide.
Individual DNA molecules
were cloned from the resulting ~900 bp populations into
pGEM-Teasy (Promega Corp., Madison, WI), and the cloned
inserts, amplified using primers for the flanking T7 and
SP6 promoter sequences, were screened for restriction
fragment length polymorphisms (RFLP) using MspI and HinPI
(New England Biolabs).
Sequence and phylogenetic analysis
Nucleotide sequences were determined by using ABI
Prism BigDye™ terminator cycle sequencing (cat. no.
4303149) with an ABI Prism 3700 DNA analyzer.
For cultured
isolates, both DNA strands of the ~1500 bp amplicon (27F1525R) were sequenced by using the internally nested
primers 68F, 515F, 534R, 907R, 1392R and 1492R (Medlin et
al. 1988; Lane 1991; Fig 1.3).
Both DNA strands of inserts
cloned into pGEM-Teasy were sequenced using primers that
annealed to the flanking T7 and SP6 promoter sequences.
Multiple reads from sequences were edited and compiled
using the GeneTool™ software package (BioTools Inc., cat.
no. GT10-9808-0333-3452) and compared with those available
in the Ribosomal Database Project (Maidak et al. 2001) and
GenBank (Benson et al. 2000).
All sequences were imported
70
into the ARB software environment (Strunk et al. 1998),
aligned based on secondary structures using the ARB
sequence editor, and phylogenetic relationships were
evaluated using neighbor-joining, maximum likelihood (Olsen
et al. 1994), and maximum parsimony methods (Swofford
1999).
Electron microscopy
For electron microscopy, particulate from ~1 l of melt
water were concentrated onto an 0.2 µm filter, fixed for 16
h with 3% (v/v) glutaraldehyde in phosphate-buffered saline
and then for 1 h in 1% (w/v) osmium tetroxide.
The fixed
cells were dehydrated by sequential passage through
increasing concentrations of ethanol [50% to 100% (v/v)
ethanol in 10% increments], dried in a Pelco CPD-2 critical
point dryer (Ted Pella Inc., Redding, CA), coated with
gold-palladium for 60 s in a Pelco 3 sputter coater, and
visualized using a Philips XL30 scanning electron
microscope.
71
Nucleic acid quantitation
Cells collected by filtration of 750 to 1000 ml
aliquots of melt water were suspended in 3 ml of phosphatebuffered saline, and 500 µl of the suspension was lysed with
hot detergent treatment and bead-beating, as described
above.
The nucleic acids released, and similarly released
from 100-fold serial dilutions of an E. coli culture, were
denatured in 0.5 N NaOH and 6X SSPE (buffered hybridization
solution), transferred, and fixed by UV crosslinking onto
nylon membranes (Sambrook et al. 1989).
Following
hybridization to the universal 1492R 16S rDNA probe[γ-32P]ATP end-labeled, the blot was rinsed with a solution of 2X
SSPE and 0.1% SDS at 22oC, used to expose film, which was
developed after 4 days of exposure at -70oC.
Freeze-thaw experiments
Cells from colonies growing on agar-solidified media
were suspended in 1 ml of dH2O by vortexing, and the number
of cfu ml-1 determined by serial dilution plating.
Cell
suspensions were frozen at -15oC, thawed, and this cycle was
repeated 18X.
The number of cfu remaining was deterimined
after cycle 5, 10, and 18.
The known strains investigated
were Aureobacterium suaveolens (ATCC 958), Arthrobacter
72
globiformis (ATCC 8010), Brevibacterium linens (ATCC 9172),
Bacillus polymyxa (OSU ref. 443), Micrococcus luteus (OSU
ref. 122), and E. coli (OSU ref. 455).
Antibiotic-resistance assays
Antibiotic disks containing (µg/disk) ampicillin (10),
gentamicin (10), kanamycin (30), erythromycin (15),
tetracyclin (30), and clindamycin (2) [Dispens-O-Disk™
susceptibility system; Difco] were placed on plates spread
with cell suspensions of the test organisms and then were
incubated at the optimal growth temperature for that
isolate.
Zones of inhibition were measured to deterimine
susceptibility, as recommended by the disk manufacturer.
Results
Distribution, geographic, and chronological variation of
microorganisms recovered from glacial ice
A range of different enrichment media was used
during the course of this study, however, growth was
observed only under aerobic or microaerobic conditions
using culture media routinely used to enumerate
heterotrophic bacteria and fungi.
When these media were
solidified by inclusion of agar and inoculated with equal
73
amounts of a sample, the largest numbers of colonies were
routinely observed on media containing low levels of
nutrients, such as R2A, Actinomycetes isolation agar, and
nutrient and tryptic soy broth diluted 100-fold below the
manufacturers’ recommended concentrations (Fig 3.2). Often
colonies appeared only after 20 days of incubation at 22oC,
or after >70 days of incubation at 10oC.
However, most of
these isolates were subsequently able to form colonies
within 2 to 7 days when sub-cultured onto the same growth
medium at 22oC.
With melt water samples >500,000 years old
from the Guliya ice cap, colonies were never observed
growing after direct inoculation of agar-solidified media,
even after >100 d, but isolates were recovered from liquid
enrichments (full strength and 1:100 nutrient broth, 1:100
tryptic soy broth) after incubating at 4oC for 30-60 d.
Aliquots of the enrichment cultures were plated on agarsolidifed media and incubated at 4o and 22oC.
Based on the
numbers of cfu ml-1 retrieved from the primary liquid
enrichments, the bacteria did not multiply initially in
these cultures for 30 days, apparently requiring a
resuscitation period before any growth became possible.
74
TBAB
NA 1:100
75
Figure 3.2 Resulting growth after 200 ml of a 40-fold concentrated (0.2 mm filtered)
sample from 200-year old Guliya ice was spread onto rich (TBAB; tryptic blood agar base)
and low nutrient (NA 1:100; nutrient agar diluted 100-fold) media and incubated
aerobically at 22oC. There was a ~20-fold increase in recovery of cfu on low nutrient
media.
Ice cores of different ages from China, Bolivia,
Greenland, and Antarctica (Table 3.1) were sampled to
survey the abundance and range of different bacterial
species that could be revived.
No growth was observed in
samples from 150-year old ice from the Antarctic Peninsula,
nor from 1500-, 13K-, 14K-, 20K-, or 22K-year old ice from
the Sajama ice cap (Bolivia), whereas 180 cfu ml-1 were
recovered from 200-year old ice from the Guliya ice cap
(China).
Low but similar numbers of cfu (<20 cfu ml-1) were
recovered from both modern and 12,000 to 20,000-year old
ice from Sajama, indicating that age had little correlating
effect on the number of recoverable bacteria in ice from
this region.
Late Holocene (1800 years old) polar ice from
Taylor Dome (Antarctica) contained only 10 cfu ml-1, but
nevertheless, this was a higher number than from ice of the
same age from the Antarctic Peninsula or from Greenland
(Summit and Dye 2).
Similarly low numbers of isolates (1-5
cfu ml-1) were counted by Dancer et al. (1997) from surface
glacial ice from the Canadian high Arctic after enrichment
for coliform bacteria, and even lower numbers (<1 cfu ml-1)
were counted in earlier surveys of polar ice (Abyzov et al.
1993).
76
Ice Core
Sample
(sample
designation)
Guliya,
China (G)
Sajama,
Bolivia (SB)
Dye 2,
Greenland
Summit,
Greenland
Taylor Dome,
Antarctica
(M3C)
Table 3.1
Depth/Age
(mbsa/year)
# of
samples
?/50
?/200
295/>500K
296/>500K
1
1
1
1
40/100
2
45/150
5
76/1.5K
1
104/12K
3
110/13K
2
111/14K
112/14K
1
1
118/19K
2
121/20K
4
124/22K
3
?/200
1
203/1.2K
1
?/1.8K
191/3.8K
1
3
203/4.1K
3
219/4.7K
2
378/17.8K
2
452/52K
2
Vertical
distance/
melt
volume
(cm/ml)
?/180
34/200
78/170
72/170
5x10c/
24-35c
45-51/
140-190
36/75
6x10c,
3.5/ 2028c, 190
26, 17/
180, 114
28/187
31/191
36, 7/ 84,
180
14.5-43/
90-130
14-25/ 90155
Max. cfu ml-1
/unique
isolates
obtained
31/184
<1/0d
4x10 c/
18-35c
45/208
48/130-305
48/ 107308
310, 347/
48
126, 217/
20
307, 332/
48
Inventory of glacial ice cores sampled.
77
7/11
180/13
0/4b
0/14b
<1/7
<1/6
0
17/12
0
0
0
3/1
0
0
<1/0d
10/5
<1/0d
3/2
<1/1
<1/1
<1/0d
Table 3.1
Ice Core
Sample
(sample
designation)
Canada
Glacier,
Antarctica
(CanClean/
CanDirty)
Dyer,
Antarctica
Siple,
Antarctica
(SIA)
Ross Ice
Shelf,
Antarctica
(Trans)
Depth/Age
(mbs/year)
# of
samples
1/?
1
Vertical
distance/
melt
volume
(cm/ml)
50/600
1/?e
1
50/200
97/150
1
181/<1K
2
182/<1K
2
2/<10
1
5x10/
18-30c
41, 47/
257-272
25, 32/
157, 195
50/1000
a
mbs=meters below surface
b
Isolates obtained only from liquid enrichments.
Max. cfu ml-1
/unique
isolates
obtained
1.1 x 104/5e
8/2
0
2/3
<1/1
5/2
c
Melt water from 10 cm sections of each ice core was fractionated
in these experiments.
d
Only fungal growth observed.
e
Sample from a frozen cryoconite hole on the glacial surface.
?=information not available.
Table 3.1 (continued)
78
In an attempt to measure the total numbers of
microorganisms present in different glacial ices, DNA
released and concentrated from cells in large volumes (750
to 1000 ml) of melt water from 150 year old Dyer Plateau
ice, 200 year old Guliya and Dye 2 ice, 1500 and 12,000
year old Sajama ice were probed by slot blot hybridization.
Following hybridization using the [γ-32P]-ATP end-labeled
universal 16S rDNA probe (1510-1492), signals were observed
only from samples of the 200 year old Guliya ice and from
the E. coli controls (data not shown).
Based on comparison
of signal intensities with the E. coli controls, the Guliya
ice contained a number of rDNA copies equivalent to that
present in ~104 bacterial cells ml-1.
Consistent with this
result, the highest numbers of colonies recovered (~180 ml1
) also came from this particular section of the Guliya
core, although this number of cfu apparently represented
only ~1% of the cells present in the retentate, based on
the hybridization data.
Scanning electron microscopy (Fig. 3.3) of material
filtered from water from Taylor Dome ice revealed the
presence of microorganisms and some larger particles of
putative biological origin, but very little inorganic
debris.
In contrast, EM revealed much larger quantities of
79
Fig. 3.3
Microorganisms and particulates filtered from
glacial ice cores visualized by scanning electron
microscopy.
A. Particles trapped in (1) 1800 year-old ice from Taylor
Dome, Antarctica; (2) 12,000 year-old ice from Sajama,
Bolivia; (3) 200 year-old ice from Guliya, China.
B. (1) Coccal and (2) rod-shaped bacteria from 1800 yearold ice from Taylor Dome. (3) Diatom from 12,000 year-old
Sajama ice, and (4) pollen grain from 200 year-old Guliya
ice.
80
A1
A2
A3
Figure 3.3
B1
500nm
B2
1mm
B3
2mm
B4
5mm
20mm
20mm
20mm
81
apparently inorganic granules, in addition to pollen
grains, diatoms, and a variety of bacteria in material
filtered from similar volumes (~1 l) of water from Guliya
and Sajama ice.
Macroscopic particles were also visible in
these nonpolar glacial ices, typically forming dust layers
that apparently represented the annual deposition of
particles from nearby environments.
Relatedness of glacial isolates to known bacterial species
Isolates were designated by their geographical origin,
age of the ice in years, and strain number (e.g., G500K-78
is strain no. 78 isolated from Guliya glacial ice that was
500,000 years old), and compared to their closest
phylogenetic relatives by analysis of 16S rDNA sequences
corresponding to the region between nucleotides 27 and 1492
in the Escherichia coli 16S rDNA sequence (Fig. 3.4-3.8;
Table 3.2).
They were further categorized in terms of
growth temperature optima, ability to grow on different
media, and antibiotic resistance patterns (Table 3.3).
Based on having a 16S rDNA sequence >95% identical to that
of documented species residing in the α−, β−, and
γ−proteobacteria, and Cytophaga/ Flavobacterium/Bacteroides,
high, and low G+C Gram positive lines of descent, most
82
# of isolates
83
35
30
25
20
15
10
5
0
Nonpolar
Antarctic
a
b
g
CFB
Low G+C High G+C
GP
GP
Figure 3.4 Distribution of glacial isolates based on phylogenetic assignment to
major bacterial divisions. Recovered isolates from both polar and non-polar glacial
ice are members of the a-, b-, and g- proteobacteria, (CFB) Cytophaga/Flavobacterium/
Bacteroides line of descent, and low (Low G+C GP) and high (High G+C GP) G+C Gram
positive bacteria.
Fig. 3.5
Phylogentic analysis of a-proteobacterial
isolates recovered from glacial ice. The 16S rDNA
sequences obtained from single-colony isolates,
corresponding to nucleotides 27-1492 of the E. coli 16S
rDNA, were aligned based on secondary structure using the
ARB software package (Struck et al. 1998). Trees were
generated using a 1357 nucleotide mask of unambiguously
aligned positions. GenBank accession numbers are listed in
parentheses. Glacial isolates are in large font, and
designated by their geographical origin (G=Guliya, China;
SB=Sajama, Bolivia; M3C=Taylor Dome, Antarctica;
CanClear=Canada Glacier, Antarctica; SIA=Siple Dome,
Antarctica; and V=Lake Vostok accretion ice, Antarctica
[see Chapter 5]) age of the ice in years, and strain number
(e.g. G500K-78 is strain no. 78 isolated from Guliya
glacial ice that was 500,000 years old.
A. Maximum likelihood tree generated using fastDNAml
(Olsen et al. 1994). The scale bar indicates 0.1 fixed
substitutions per nucleotide position.
B. Maximum parsimony analysis performed with 100 bootstrap
replicates, with values shown at the nodes. The scale bar
represents 100 evolutionary steps. Of the 381 variable
characters in this alignment, 295 were parsimonyinformative.
84
Methylobacterium radiotolerans (D32227)
G500K-5 (AF395035)
(Af395035)
Methylobacterium fujisawaense (AJ250801)
Methylobacterium mesophilicum (D32225)
Methylobacterium sp. (Z23156)
unidentified bacterium (AJ223453)
V23 (AF324201)
Methylobacterium extorquens (D32224)
Methylobacterium rhodesianum (D32228)
M3C1.8K-TD4 (AF395030)
Methylobacterium sp. (U58018)
G500K-15 (AF395034)
Bosea thiooxidans (AJ250798)
Afipia sp. (U87779)
alpha proteobacterium (AF288309)
M3C1.8K-TD7 (AF479379)
Sphingomonas paucimobilis (X94100)
G500K-3 (AF395036)
Sphingomonas chlorophenolica (X87161)
Sphingomonas flava (X87164)
M3C1.8K-TD9 (AF479381)
Blastomonas natatorius (X73043)
Erythromonas ursincola (Y10677)
Blastomonas natatoria (AB024288)
Blastomonas ursinicola (AB02489)
SIA1K-1A1 (AF395032)
isolate "star-like" (AJ001344)
Blastobacter sp. (Z23157)
Sphingomonas pruni (Y09637)
Sphingomonas mali (Y09638)
Sphingomonas asaccharolytica (Y09639)
Sphingomonas sp. Ant. 20 (AF184221)
M3C4.1K-B5 (AF395031)
G500K-14 (AF395037)
V21 (AF324199)
Sphingomonas sp. (AB033945)
Sphingomonas parapaucimobilis (D13724)
Sphingomonas sp. MK355 (D84523)
M3C1.8K-TD1 (AF479378)
Sphingomonas sanguis (D13726)
0.10
Sphingomonas echinoides (AB033944)
CanClear1 (AF395038)
Sphingomonas paucimobilis (D16144)
Sphingomonas paucimobilis (D13725)
Figure 3.5A
85
Methylobacterium radiotolerans (D32227)
G500K-5 (AF395035)
Methylobacterium fujisawaense (AJ250801)
Methylobacterium mesophilicum (D32225)
83
Methylobacterium sp. (Z23156)
98
unidentified bacterium (AJ223453)
100
87
V23 (AF324201)
Methylobacterium extorquens (D32224)
80
Methylobacterium rhodesianum (D32228)
100
86
M3C1.8K-TD4 (AF395030)
Methylobacterium sp. (U58018)
96
G500K-15 (AF395034)
Bosea thiooxidans (AJ250798)
59
Afipia sp. (U87779)
100
alpha
proteobacterium (AF288309)
84
M3C1.8K-TD7 (AF479379)
Sphingomonas paucimobilis (X94100)
98
G500K-3 (AF395036)
100
100
Sphingomonas chlorophenolica (X87161)
96
Sphingomonas flava (X87164)
M3C1.8K-TD9 (AF479381)
100
Blastomonas natatorius (X73043)
100
Erythromonas ursincola (Y10677)
70
Blastomonas natatoria (AB024288)
74
Blastomonas ursinicola (AB02489)
70
SIA1K-1A1 (AF395032)
isolate "star-like" (AJ001344)
95
Blastobacter sp. (Z23157)
100
54
Figure 3.5B
86
Sphingomonas pruni (Y09637)
Sphingomonas mali (Y09638)
Sphingomonas asaccharolytica (Y09639)
Sphingomonas sp. Ant. 20 (AF184221)
100
M3C4.1K-B5 (AF395031)
86
G500K-14 (AF395037)
92
V21 (AF324199)
64
Sphingomonas sp. (AB033945)
Sphingomonas parapaucimobilis (D13724)
Sphingomonas sp. MK355 (D84523)
M3C1.8K-TD1 (AF479378)
Sphingomonas
sanguis (D13726)
100
Sphingomonas echinoides (AB033944)
CanClear1 (AF395038)
81
Sphingomonas paucimobilis (D16144)
72
Sphingomonas paucimobilis (D13725)
83 64
96
Fig. 3.6
Phylogenetic analysis of b- and gproteobacterial glacial isolates, and a member of the
Cytophaga/Flavobacterium/Bacteriodes line of descent
(CanDirty14). The 16S rDNA sequences obtained from singlecolony isolates, corresponding to nucleotides 27-1492 of
the E. coli 16S rDNA, were aligned based on secondary
structure using the ARB software package (Struck et al.
1998). Trees were generated using a 1395 nucleotide mask
of unambiguously aligned positions. GenBank accession
numbers are listed in parentheses. Glacial isolates are in
large font, and designated by their geographical origin
(G=Guliya, China; SB=Sajama, Bolivia; M3C=Taylor Dome,
Antarctica; CanClear/CanDirty=Canada Glacier, Antarctica;
and Trans=Ross Ice Shelf, Antarctica) age of the ice in
years, and strain number (e.g. G500K-78 is strain no. 78
isolated from Guliya glacial ice that was 500,000 years
old.
A. Maximum likelihood tree generated using fastDNAml
(Olsen et al. 1994). The scale bar indicates 0.1 fixed
substitutions per nucleotide position.
B. Maximum parsimony analysis performed with 100 bootstrap
replicates, with values shown at the nodes. The scale bar
represents 10 evolutionary steps. Of the 571 variable
characters in this alignment, 536 were parsimonyinformative.
87
Pseudomonas aeruginosa (U38445)
Pseudomonas oleovorans (D84018)
M3C4.7K-2 (AF479376)
Pseudomonas alcaligenes (Z76653)
Pseudomonas pseudoalcaligenes (Z76666))
Pseudomonas sp. from sea ice (U85869)
Pseudomonas sp. from sea ice (U85868)
M3C4.1K-B34 (AF479375)
Pseudomonas azotoformans (D84009)
Pseudomonas synxantha (Af267911)
Psychrobacter immobilis (U85880)
Psychrobacter glacincola (U85876)
Trans12 (AF479327)
Psychrobacter submarinus (AJ309940)
Psychrobacter marincola (AJ309941)
Acinetobacter junii (X81664)
Acinetobacter johnsonii (Z93439)
Acinetobacter haemolyticus (Z93436)
Acinetobacter calcoaceticus (AF159045)
M3C1.8K-TD8 (AF479380)
Acinetobacter radioresistens (X81666)
Acinetobacter sp. (Z93445)
G50-TB2 (AF479352)
CanClear23 (AF479323)
SB150-2A1 (AF479373)
CanDirty12 (AF479324)
Pseudomonas saccharophila (AB021407)
Matsuebacter chitosanotabidus (AB006851)
Leptothrix cholodnii (X97070)
Leptothrix discophora (L33974)
Janthinobacterium agaricidamno (Y08845)
Janthinobacterium lividum (Y08846)
Pseudomonas mephitica (AB021388)
CanDirty89 (AF479326)
Duganella ramigera (X74914)
Ralstonia sp. APF11 (AB045276)
G500K-6 (AF479337)
Ralstonia pickettii (AB004790)
Ralstonia solanacearum (U28233)
Haloanella gallinarum (AB035150)
CanDirty14 (AF479331)
0.10
Chryseobacterium balustinum (M58771)
Chryseobacterium gleum (M58772)
Riemerella columbina (AF181448)
Figure 3.6A
88
Pseudomonas aeruginosa (U38445)
Pseudomonas alcaligenes (Z76653)
Pseudomonas pseudoalcaligenes (Z76666))
Pseudomonas oleovorans (D84018)
100
M3C4.7K-2 (AF479376)
Pseudomonas sp. from sea ice (U85869)
59
Pseudomonas sp. from sea ice (U85868)
100
M3C4.1K-B34 (AF479375)
70
Pseudomonas azotoformans (D84009)
78
Pseudomonas synxantha (Af267911)
64
Psychrobacter immobilis (U85880)
Psychrobacter glacincola (U85876)
100
Trans12 (AF479327)
100
Psychrobacter submarinus (AJ309940)
80
Psychrobacter marincola (AJ309941)
78
67
Acinetobacter junii (X81664)
Acinetobacter johnsonii (Z93439)
74
65
Acinetobacter haemolyticus (Z93436)
100
Acinetobacter calcoaceticus (AF159045)
100
M3C1.8K-TD8 (AF479380)
Acinetobacter radioresistens (X81666)
Acinetobacter sp. (Z93445)
98
G50-TB2 (AF479352)
62
100
CanClear23 (AF479323)
61
SB150-2A1 (AF479373)
CanDirty12 (AF479324)
10
61
Pseudomonas saccharophila (AB021407)
61
Matsuebacter chitosanotabidus (AB006851)
100
Leptothrix cholodnii (X97070)
100
Leptothrix discophora (L33974)
Janthinobacterium agaricidamno (Y08845)
76
Janthinobacterium lividum (Y08846)
70
Pseudomonas mephitica (AB021388)
100
100
92
100
Figure 3.6B
CanDirty89
(AF479326)
Duganella ramigera (X74914)
Ralstonia sp. APF11 (AB045276)
89
G500K-6
(AF479337)
100
Ralstonia
pickettii
(AB004790)
100
Ralstonia solanacearum (U28233)
Haloanella gallinarum (AB035150)
93
CanDirty14
(AF479331)
89
Chryseobacterium balustinum (M58771)
93
Chryseobacterium gleum (M58772)
Riemerella columbina (AF181448)
56
89
Fig. 3.7
Phylogenetic analysis of Low G+C Gram positive
glacial isolates. The 16S rDNA sequences obtained from
single-colony isolates, corresponding to nucleotides 271492 of the E. coli 16S rDNA, were aligned based on
secondary structure using the ARB software package (Struck
et al. 1998). Trees were generated using a 1330 nucleotide
mask of unambiguously aligned positions. Glacial isolates
are in large font, and designated by their geographical
origin (G=Guliya, China; SB=Sajama, Bolivia; and V=Lake
Vostok accretion ice, Antarctica [see Chapter 5]) age of
the ice in years, and strain number (e.g. G500K-78 is
strain no. 78 isolated from Guliya glacial ice that was
500,000 years old.
A. Maximum likelihood tree generated using fastDNAml
(Olsen et al. 1994). The scale bar indicates 0.1 fixed
substitutions per nucleotide position.
B. Maximum parsimony analysis performed with 100 bootstrap
replicates, with values shown at the nodes. The scale bar
represents 10 evolutionary steps. Of the 326 variable
characters in this alignment, 294 were parsimonyinformative.
90
Bacillus subtilis (D26185)
G50-TB5 (AF479353)
G200-SD1 (AF479349)
Bacillus sp. (AB050667)
Bacillus sp. (AF411118)
G500K-16 (AF479336)
Bacillus pumilus (AB048252)
SB100-9-5-1 (AF479372)
Bacillus sp. 82352 (AF227852)
Bacillus sp. MK03 (AB062678)
G500K-19 (AF479330)
G500K-9 (AF479338)
Bacillus anthracis (AF176321)
Bacillus sp. BSID723 (AF027659)
Bacillus cereus (AF290548)
G50-TS3 (AF479356)
G500K-2 (AF479333)
SB12K-9-4 (AF479367)
Mariana Trench isolate (AB002640)
unidentified bacterium (AB004761)
Bacillus thuringiensis (D16281))
SB100-8-1 (AF479369)
Bacillus cereus (AF290555)
Bacillus cohnii (X76437)
G200-T16 (AF479350)
Bacillus sp. KSM-KP43 (AB055093)
Bacillus megaterium (D16273)
G500K-18 (AF479335)
Bacillus simplex (D78478)
Bacillus psychrosaccharolyticus (AB21195)
SB100-8-1-1 (AF479370)
SB105-2A2 (AF479374)
G500K-17 (AF479334)
Bacillus macroides (AY030319)
Bacillus maroccanus (X60626)
Antarctic isolate from geothermal soil (AJ250318)
Bacillus firmus (X60616)
Bacillus circulans (AY043084)
G200-T19 (AF479351)
Bacillus badius (X77790)
G200-N5 (AF479347)
Bacillus aminovorans (X62178)
SB100-8-1-2 (AF479371)
Exiguobacterium acetylicum (X70313)
SB12K-2-2 (AF479360)
Exiguobacterium undae (AJ344151)
Exiguobacterium acetylicum (AJ297437)
Bacillus benzeovorans (D78311)
Planococcus okeanokoites (D55729)
Planococcus mcmeekinii (AF041791)
G50-TS4 (AF479357)
Planococcus psychrotoleratus (AF324659)
Antarctic psychrophile SOS Orange (AF242541)
G500K-78 (AF395033)
Paenibacillus sp. 7-5 (AB043868)
Paenibacillus illinoisensis (D85397)
G50-TB9 (AF395027)
Bacillus longisporus (AJ223991)
Paenibacillus amylolyticus (D85396)
V22 (AF324200)
G200-C15 (AF395028)
SB150-2B (AF395029)
Paenibacillus polymyxa (D16276)
Paenibacillus peoriae (AJ320494)
0.10
G50-L2 (AF479345)
Paenibacillus sp. P51-3 (AJ297715)
Figure 3.7A
91
Bacillus subtilis (D26185)
G50-TB5 (AF479353)
G200-SD1 (AF479349)
86
Bacillus sp. (AB050667)
Bacillus sp. (AF411118)
G500K-16 (AF479336)
99
Bacillus pumilus (AB048252)
Bacillus benzeovorans (D78311)
Antarctic isolate from geothermal soil (AJ250318)
Bacillus firmus (X60616)
Bacillus circulans (AY043084)
65
G200-T19 (AF479351)
Bacillus badius (X77790)
SB100-9-5-1 (AF479372)
Bacillus sp. 82352 (AF227852)
Bacillus sp. MK03 (AB062678)
100
82
G500K-19 (AF479330)
G500K-9 (AF479338)
80
Bacillus anthracis (AF176321)
Bacillus sp. BSID723 (AF027659)
Bacillus cereus (AF290548)
100
G50-TS3 (AF479356)
G500K-2 (AF479333)
SB12K-9-4 (AF479367)
100
50
Mariana Trench isolate (AB002640)
unidentified bacterium (AB004761)
Bacillus thuringiensis (D16281))
SB100-8-1 (AF479369)
Bacillus cereus (AF290555)
Bacillus cohnii (X76437)
73
G200-T16 (AF479350)
100
Bacillus sp. KSM-KP43 (AB055093)
Bacillus megaterium (D16273)
99
100
60
G500K-18 (AF479335)
Bacillus simplex (D78478)
Bacillus psychrosaccharolyticus (AB21195)
SB100-8-1-1 (AF479370)
SB105-2A2 (AF479374)
G500K-17
(AF479334)
99
99
93
Bacillus macroides (AY030319)
Bacillus maroccanus (X60626)
G200-N5 (AF479347)
99
Bacillus aminovorans (X62178)
98
Planococcus okeanokoites (D55729)
Planococcus mcmeekinii (AF041791)
100
10
SB100-8-1-2 (AF479371)
G50-TS4 (AF479357)
53
64
Planococcus psychrotoleratus (AF324659)
Antarctic psychrophile SOS Orange (AF242541)
Exiguobacterium acetylicum (X70313)
95
71
SB12K-2-2 (AF479360)
100
Exiguobacterium undae (AJ344151)
Exiguobacterium acetylicum (AJ297437)
96
G500K-78 (AF395033)
100
69
Paenibacillus sp. 7-5 (AB043868)
Paenibacillus illinoisensis (D85397)
G50-TB9 (AF395027)
93
100
98
100
55
89
64
V22 (AF324200)
G200-C15 (AF395028)
SB150-2B (AF395029)
Paenibacillus polymyxa (D16276)
Paenibacillus peoriae (AJ320494)
75
100
100
Figure 3.7B
92
Bacillus longisporus (AJ223991)
Paenibacillus amylolyticus (D85396)
G50-L2 (AF479345)
Paenibacillus sp. P51-3 (AJ297715)
Fig. 3.8
Phylogenetic analysis of High G+C Gram
positive glacial isolates. The 16S rDNA sequences obtained
from single-colony isolates, corresponding to nucleotides
27-1492 of the E. coli 16S rDNA, were aligned based on
secondary structure using the ARB software package (Struck
et al. 1998). Trees were generated using a 1366 nucleotide
mask of unambiguously aligned positions. Glacial isolates
are in large font, and designated by their geographical
origin (G=Guliya, China; SB=Sajama, Bolivia;
CanDirty=Canada Glacier, Antarctica; SIA=Siple Dome,
Antarctica; Trans=Ross Ice Shelf, Antarctica; and V=Lake
Vostok accretion ice, Antarctica [see Chapter 5]) age of
the ice in years, and strain number (e.g. G500K-78 is
strain no. 78 isolated from Guliya glacial ice that was
500,000 years old.
A. Maximum likelihood tree generated using fastDNAml
(Olsen et al. 1994). The scale bar indicates 0.1 fixed
substitutions per nucleotide position.
B. Maximum parsimony analysis performed with 100 bootstrap
replicates, with values shown at the nodes. The scale bar
represents 100 evolutionary steps. Of the 463 variable
characters in this alignment, 375 were parsimonyinformative.
93
Mycobacterium alvei (AF023664)
Mycobacterium austroafricanum (X93182)
Mycobacterium aurum (X55595)
SB12K-2-16 (AF479359)
Gordona namibiensis (AF380931)
Gordona rubropertinctus (X80632)
High G+C isolate (X7318)
SIA1K-2A1 (AF479377)
Gordona terrae (X79286)
Rhodococcus sp. (AB010902)
Rhodococcus equi (X80614)
Nocardia corynebacteroides (X80615)
SB12K-2-5 (AF479363)
G200-C39 (AF479344)
Micromonospora rhodorangea (X92612)
Micromonospora echinospora (X92607)
Micromonospora inyoensis (X92629)
Friedmanniella lacustris (AJ132943)
SB12K-2-7 (AF479364)
Friedmanniella spumicola (AF062535)
Friedmannella antarctica (Z78206)
Nocardioides simplex (AF005011)
Nocardioides sp. OS4 (U61298)
Nocardioides plantarum (AF005008)
SB12K-2-4 (AF479362)
Brachybacterium tyrofermentans (X91657)
Brachybacterium sp. from sea ice (AF041790)
Brachybacterium faecium (X91032)
Brachybacterium conglomeratum (X91030)
V15 (AF324202)
Kocuria kristinae (X80749)
Trans4678 (AF479328)
Kocuria varians (X87754)
Kocuria rhizophila (Y16264)
deep subsurface isolate (X86608)
SB19K-1 (AF479368)
unidentified sludge eubacterium (Y15857)
Micrococcus luteus (AJ409096)
Arthrobacter sp. CAB1 (AB39736)
G200-A1 (AF479340)
CanDirty7 (AF479339)
Arthrobacter polychromogenes (X80741)
Arthrobacter oxydans (X83408)
Arthrobacter sp. from sea ice (U85895)
Arthrobacter sp. from sea ice (AF041789)
CanDirty1 (AF479325)
Arthrobacter agilis (X80748)
G50-TB7 (AF479354)
G200-C1 (AF479341)
clone from spacecraft (AF408269)
Arthrobacter sp. from sea ice (U85896)
0.10
Figure 3.8A
94
G200-C18
(AF479343)
Sanguibacter keddieii (X79450)
Sanguibacter inulinus (X79451)
Sanguibacter suarezii (X79452)
Cellulomonas sp. (X82598)
SB12K-6-2 (AF479365)
Promicromonospora enterophila (X83807)
Cellulomonas turbata (X83806)
Oerskovia paurometabola (AJ314851)
Agromyces mediolanus (D45056)
Agromyces cerinus (X77448)
Agromyces ramosus (X77447)
G500K-1 (AF479332)
G50-PD1 (AF479348)
Clavibacter michiganensis (AJ310415)
Clavibacter michiganensis (U30254)
Frigoribacterium faeni (Y18807)
Frigoribacterium sp. 227 (AF157478)
Frigoribacterium sp. 301 (AF157479)
G200-C11 (AF479342)
SB12K-2-1 (AF479358)
gram positive isolate W5P (AF323267)
Leucobacter komagatae (AB007419)
gram-positive bacterium str. 1 (AB008511)
G50-TB8 (AF479355)
Mycetocola sp. OM-A1 (AB020204)
Detolaasinbacter tsukamotoae (AB012646)
Detolaasinbacter muratae (AB012648)
Detolaasinbacter shiratae (AB012647)
Microbacterium arborescens (X77443)
SB12K-6-3 (AF479366)
Microbacterium sp. VKM Ac-1 (AB042072)
Aureobacterium testaceum (X77445)
Aureobacterium sp. (Y14699)
Aureobacterium liquefaciens (X77444)
Aureobacterium keratanolyticum (Y14786)
G500K-10 (AF479329)
G50-L3 (AF479346)
Microbacterium sp. MAS133 (AJ251194)
Microbacterium flavescens (Y17232)
SB12K-2-3 (AF479361)
0.10
Figure 3.8A (con't)
95
Mycobacterium alvei (AF023664)
Mycobacterium austroafricanum (X93182)
100
Mycobacterium aurum (X55595)
90 SB12K-2-16 (AF479359)
Gordona namibiensis (AF380931)
98 Gordona rubropertinctus (X80632)
100
High G+C isolate (X7318)
100 SIA1K-2A1 (AF479377)
Gordona terrae (X79286)
Rhodococcus sp. (AB010902)
56
Rhodococcus equi (X80614)
70
Nocardia corynebacteroides (X80615)
100
100 SB12K-2-5
(AF479363)
G200-C39 (AF479344)
100
Micromonospora rhodorangea (X92612)
81
Micromonospora echinospora (X92607)
55 Micromonospora inyoensis (X92629)
Friedmanniella lacustris (AJ132943)
75
100
SB12K-2-7 (AF479364)
80
Friedmanniella spumicola (AF062535)
52
Friedmannella antarctica (Z78206)
97
Nocardioides simplex (AF005011)
sp. OS4 (U61298)
100 Nocardioides
Nocardioides plantarum (AF005008)
52
100
SB12K-2-4 (AF479362)
Brachybacterium tyrofermentans (X91657)
100 Brachybacterium
sp. from sea ice (AF041790)
100
Brachybacterium faecium (X91032)
94
Brachybacterium conglomeratum (X91030)
100 V15 (AF324202)
Kocuria kristinae (X80749)
100 Trans4678
53
(AF479328)
100
Kocuria varians (X87754)
100 Kocuria
rhizophila (Y16264)
93
deep subsurface isolate (X86608)
(AF479368)
75
100 SB19K-1
unidentified sludge eubacterium (Y15857)
56 Micrococcus luteus (AJ409096)
Arthrobacter sp. CAB1 (AB39736)
71
99 G200-A1 (AF479340)
CanDirty7 (AF479339)
100
Arthrobacter polychromogenes (X80741)
74 Arthrobacter oxydans (X83408)
77
Arthrobacter sp. from sea ice (U85895)
89
Arthrobacter sp. from sea ice (AF041789)
97 CanDirty1 (AF479325)
96
Arthrobacter agilis (X80748)
100
G50-TB7 (AF479354)
100
G200-C1
(AF479341)
53
clone from spacecraft (AF408269)
Arthrobacter sp. from sea ice (U85896)
Figure 3.8B
96
68
85
67
65
92
G200-C18
(AF479343)
Sanguibacter keddieii (X79450)
69
Sanguibacter inulinus (X79451)
56
Sanguibacter suarezii (X79452)
Cellulomonas sp. (X82598)
100 SB12K-6-2
(AF479365)
Promicromonospora enterophila (X83807)
98
Cellulomonas turbata (X83806)
57
Oerskovia paurometabola (AJ314851)
gram-positive bacterium str. 1 (AB008511)
G50-TB8 (AF479355)
G200-C11 (AF479342)
Agromyces mediolanus (D45056)
Agromyces cerinus (X77448)
100
72
Agromyces ramosus (X77447)
74
G500K-1 (AF479332)
G50-PD1 (AF479348)
100
Clavibacter michiganensis (AJ310415)
80
Clavibacter michiganense (U30254)
Frigoribacterium faeni (Y18807)
99
Frigoribacterium sp. 227 (AF157478)
97
Frigoribacterium sp. 301 (AF157479)
SB12K-2-1 (AF479358)
gram positive isolate W5P (AF323267)
100
72
Leucobacter komagatae (AB007419)
99
58
89
Detolaasinbacter tsukamotoae (AB012646)
Mycetocola sp. OM-A1 (AB020204)
Detolaasinbacter muratae (AB012648)
91
Detolaasinbacter shiratae (AB012647)
Microbacterium arborescens (X77443)
SB12K-6-3 (AF479366)
Microbacterium sp. VKM Ac-1 (AB042072)
99
Aureobacterium testaceum (X77445)
64
100
67
59
65
Figure 3.8B (con't)
Aureobacterium liquefaciens (X77444)
Aureobacterium keratanolyticum (Y14786)
55
G500K-10 (AF479329)
Aureobacterium sp. (Y14699)
G50-L3 (AF479346)
Microbacterium sp. MAS133 (AJ251194)
Microbacterium flavescens (Y17232)
SB12K-2-3 (AF479361)
97
Guliya, China
Ice Core
Site
Strain
designationa/
GenBank
accession
no./# of
sequenced ntb
Nearest phylogenetic
neighbor/GenBank
accession no.
α proteobacteria
Sphingomonas
paucimobilis/X94100
G500K-3/
AF395036/
Sphingomonas
1395
chlorophenolica/
X87161
Methylobacterium
G500K-5/
radiotolerans/D32227
AF395035/
Methylobacterium
1402
fujisawaense/AJ250801
V21e
G500K-14/
Sphingomonas sp./
AF395037/
AB033945
1416
M3C1.2K-B5d
M3C1.8K-TD4d
G500K-15/
Methylobacterium sp./
AF395034/
U58018
1418
Methylobacterium
extorquens/D32224
β proteobacteria
Ralstonia sp./
G500K-6/
AB045267
AF479337/
Ralstonia pickettii/
1447
AB004790
γ proteobacteria
Acinetobacter
radioresistens/X81666
G50-TB2/
AF479352/
SB150-2A1d
1438
Acinetobacter sp./
Z93445
Table 3.2
Bacterial isolates from glacial ice cores.
98
%
identityc
98.4
96.8
99.3
98.7
99.8
99.1
97.2
99.5
99.5
99.1
100
99.8
99.8
99.8
99.8
Table 3.2
(continued)
Guliya, China
Ice Core
Site
Strain
designationa/
Nearest phylogenetic
GenBank
%
neighbor/GenBank
accession
identityc
accession no.
no./# of
sequenced ntb
Low G+C gram positive bacteria
Bacillus subtilis/
G50-TB5/
99.5
D26185
AF479353/
1464
Bacillus sp./AB050667
99.2
Bacillus cereus/
99.9
G50-TS3/
AF290548
AF479356/
G500K-2d
99.9
1450
d
SB12K-9-4
99.9
Paenibacillus
98.2
amylolyticus/D85396
G50-TB9/
AF395027/
V22e
98.0
1479
Paenibacillus
97.1
illinoisensis/D85397
Paenibacillus sp./
99.3
G50-L2/
AJ297715
AF479345/
Paenibacillus
1470
98.1
peoriae/AJ320494
Planococcus
98.2
okeanokoites/D55729
G50-TS4/
AF479357/
Planococcus
1465
98.1
psychrotoleratus/
AF324659
Bacillus sp./AB050667
99.7
G200-SD1/
d
AF479349/
G50-TB5
99.1
1443
Bacillus sp./AF411118
99.1
Bacillus sp./AB055093
99.8
G200-T16/
AF479350/
Bacillus cohnii/
97.1
1465
X76437
Bacillus circulans/
G200-T19/
97.3
AY043084
AF479351/
1447
Bacillus firmus/
97.0
X60616
Table 3.2 (continued)
99
Table 3.2
Ice Core
Site
Strain
designationa/
GenBank
accession
no./# of
sequenced ntb
G200-N5/
AF479347/
1442
G200-C15/
AF395028/
1470
(continued)
Guliya, China
G500K-16/
AF479336/
1454
G500K-19/
AF479330/
1450
G500K-9/
AF479338/
1458
G500K-2/
AF479333/
1452
G500K-18/
AF479335/
1444
G500K-17/
AF479334/
1436
Nearest phylogenetic
neighbor/GenBank
accession no.
%
identityc
SB100-8-1-2d
97.6
Bacillus aminovorans/
96.3
X62178
Bacillus badius/
95.1
X77790
SB150-2Bd
96.8
Paenibacillus sp./
94.8
AJ297715
G50-L2d
94.8
Bacillus pumilus/
100
AB048252
Bacillus sp./AB050667
97.4
Bacillus sp./AB062678
99.6
d
G500K-9
99.4
Bacillus sp./AF227852
98.8
d
G500K-19
99.4
Bacillus sp./AB062678
98.6
Bacillus sp./AF227852
98.2
Bacillus cereus/
100
AF290548
Bacillus cereus/
100
AF290555
SB12K-9-4d
100
Bacillus megaterium/
99.5
D16273
Bacillus simplex/
98.9
D78478
Bacillus maroccanus/
99.9
X60626
SB150-2A2d
99.4
Bacillus macroides/
99.3
AY030319
Table 3.2 (continued)
100
Table 3.2
Ice Core
Site
Strain
designationa/
GenBank
accession
no./# of
sequenced ntb
G500K-78/
AF395033/
1446
Nearest phylogenetic
neighbor/GenBank
accession no.
Paenibacillus sp./
AB043868
97.1
Paenibacillus
illinoisensis/D85397
95.2
(continued)
Guliya, China
High G+C gram positive bacteria
Arthrobacter agilis/
X80748
G50-TB7/
AF479354/
G200-C1d
1437
clone from
spacecraft/AF408269
G50-PD1/
AF479348/
1440
G50-TB8/
AF479355/
1424
G50-L3/
AF479346/
1404
G200-C39/
AF479344/
1394
%
identityc
Clavibacter
michiganensis/U30254
Clavibacter
michiganensis/
AJ310415
Detolaasinbacter
tsukamotoae/AB012646
Detolaasinbacter
shiratae/AB012647
SB12K-2-3d
Microbacterium sp./
AJ251194
Microbacterium
flavescens/Y17232
99.2
98.9
98.9
99.8
99.7
96.5
96.4
97.5
97.4
97.3
Micromonospora
rhodorangea/X92612
99.4
Micromonospora
echinospora/X92607
99.2
Table 3.2 (continued)
101
Table 3.2
Ice Core
Site
Strain
designationa/
GenBank
accession
no./# of
sequenced ntb
G200-C1/
AF479341/
1403
(continued)
Guliya, China
G200-A1/
AF479340/
1399
G200-C18/
AF479343/
1432
G200-C11/
AF479342/
1427
G500K-1/
AF479332/
1411
Sajama,
Bolivia
G500K-10/
AF479329/
1413
Nearest phylogenetic
neighbor/GenBank
accession no.
Arthrobacter sp./
AB39736
Arthrobacter
polychromogenes/
X80741
Arthrobacter oxydans/
X83408
Arthrobacter agilis/
X80748
Arthrobacter sp. from
sea ice/U85896
Sanguibacter
keddieii/X79450
Sanguibacter
inulinus/X79451
Frigoribacterium sp./
AF157478
Frigoribacterium sp./
AF157479
Agromyces ramosus/
X77447
Agromyces cerinus/
X77448
Aureobacterium
keratanolyticum/
Y14786
Aureobacterium sp./
Y14699
%
identityc
99.6
99.0
99.0
99.2
99.1
97.9
97.5
97.3
97.3
98.6
97.9
98.2
98.1
γ proteobacteria
Acinetobacter
99.9
SB150-2A1/
radioresistens/X81666
AF479373/
G50-TB2d
99.8
1435
d
CanClear23
99.6
Table 3.2 (continued)
102
Table 3.2
(continued)
Sajama, Bolivia
Ice Core
Site
Strain
designationa/
Nearest phylogenetic
GenBank
%
neighbor/GenBank
accession
identityc
accession no.
no./# of
sequenced ntb
Low G+C gram positive bacteria
Bacillus sp./AF227852
96.5
SB100-9-5-1/
d
AF479372/
G500K-19
96.3
1408
Bacillus sp./AB062678
96.2
Bacillus cereus/
100
SB100-8-1/
AF290555
AF479369/
Bacillus
1460
99.9
thuringiensis/D16281
SB150-2A2d
98.8
SB100-8-1-1/
Bacillus maroccanus/
98.8
AF479370/
X60626
1462
Bacillus macroides/
98.6
AY030319
Bacillus aminovorans/
98.4
X62178
SB100-8-1-2/
AF479371/
G200-N5d
97.6
1399
Bacillus badius/
95.1
X77790
Bacillus maroccanus/
99.8
X60626
SB150-2A2/
AF479374/
Bacillus macroides/
99.8
1459
AY030319
G500K-17d
99.3
d
G200-C15
96.8
SB150-2B/
Paenibacillus
AF395029/
94.6
illinoisensis/D85397
1463
G500K-78d
93.9
Bacillus cereus/
100
AF290548
SB12K-9-4/
AF479367/
Bacillus cereus/
100
1455
AF290555
G500K-2d
100
Table 3.2 (continued)
103
Table 3.2
Ice Core
Site
Strain
designationa/
GenBank
accession
no./# of
sequenced ntb
Nearest phylogenetic
neighbor/GenBank
accession no.
Exiguobacterium
acetylicum/X70313
Exiguobacterium
acetylicum/AJ297437
High G+C gram positive bacteria
Mycobacterium aurum/
X55595
SB12K-2-16/
AF479359/
Mycobacterium
1427
austroafricanum/
X93182
Nocardia
corynebacteroides/
SB12K-2-5/
X80615
AF479363/
1428
Rhodococcus equi/
X80614
Friedmanniella
SB12K-2-7/
spumicola/AF062535
AF479364/
Friedmanniella
1428
antarctica/Z78206
Nocardioides simplex/
SB12K-2-4/
AF005008
AF479362/
Nocardioides
1416
sp./U61298
Cellulomonas sp./
SB12K-6-2/
X82598
AF479365/
Cellulomonas turbata/
1426
X83806
gram positive
SB12K-2-1/
isolate/AF323267
AF479358/
Leucobacter
1448
komagatae/AB007419
(continued)
Sajama, Bolivia
SB12K-2-2/
AF479360/
1470
%
identityc
99.4
97.8
99.6
98.9
99.6
96.8
99.1
98.3
94.8
94.8
99.1
97.7
96.6
96.1
Table 3.2 (continued)
104
Table 3.2
(continued)
SB12K-6-3/
AF479366/
1423
Taylor Dome, Antarctica
Sajama, Bolivia
Ice Core
Site
Strain
designationa/
GenBank
accession
no./# of
sequenced ntb
SB12K-2-3/
AF479361/
1433
SB19K-1/
AF479368/
1385
Nearest phylogenetic
neighbor/GenBank
accession no.
Aureobacterium
testaceum/X77445
Microbacterium sp./
AB042072
Microbacterium sp./
AJ251194
Microbacterium
flavescens/Y17232
Micrococcus luteus/
AJ409096
deep subsurface
isolate/X86608
α proteobacteria
G500K-15d
Methylobacterium sp./
M3C1.8K-TD4/
U58018
AF395030/
1373
Methylobacterium
extorquens/D32224
M3C1.8K-TD7/
AF479379/
1376
M3C1.8K-TD9/
AF479381/
1386
M3C1.8K-TD1/
AF479378/
1405
α proteobacterium/
AF288309
Afipia sp./U87779
Blastomonas
natatoria/AB024288
Blastomonas
ursinicola/AB024289
Sphingomonas sanguis/
D13726
Sphingomonas sp./
D84523
%
identityc
99.9
99.9
98.5
98.4
99.6
99.6
99.5
99.2
98.7
99.9
99.5
99.4
99.4
99.4
99.4
Table 3.2 (continued)
105
Table 3.2
(continued)
M3C4.1K-B5/
AF395031/
1408
Canada Glacier,
Antarctica
Taylor Dome, Antarctica
Ice Core
Site
Strain
designationa/
GenBank
accession
no./# of
sequenced ntb
Nearest phylogenetic
neighbor/GenBank
accession no.
Sphingomonas sp. from
Antarctica/AF184221
V21e
Sphingomonas sp./
AB033945
γ proteobacteria
Acinetobacter
haemolyticus/Z93436
M3C1.8K-TD8/
AF479380/
Acinetobacter
1434
calcoaceticus/
AF159045
Pseudomonas sp. from
sea ice/U85869
M3C4.1K-B34/
AF479375/
Pseudomonas
1447
synxantha/AF267911
Pseudomonas
oleovorans/D84018
M3C4.7K-2/
Pseudomonas
AF479376/
pseudoalcaligenes/
1446
Z76666
α proteobacteria
Sphingomonas
CanClear1/
paucimobilis/D16144
AF395038/
Sphingomonas
1419
paucimobilis/D13725
β proteobacteria
Pseudomonas
saccharophilia/
CanDirty12f/
AB021407
AF479324/
Matsuebacter
1430
chitosanotabidus/
AB006851
%
identityc
99.8
99.7
99.1
99.9
99.8
99.9
99.8
99.2
96.4
97.8
97.2
97.1
96.5
Table 3.2 (continued)
106
Table 3.2
Ice Core
Site
Strain
designationa/
GenBank
accession
no./# of
sequenced ntb
Janthinobacterium
lividum/Y08846
Pseudomonas
mephitica/AB021388
γ proteobacteria
Acinetobacter
radioresistens/X81666
CanClear23/
AF479323/
SB150-2A1d
1444
G50-TB2d
(continued)
Canada Glacier, Antarctica
CanDirty89f/
AF479326/
1442
Nearest phylogenetic
neighbor/GenBank
accession no.
Cytophaga/Flavabacterium/Bacteroides
Haloanella
f
gallinarum/AB035150
CanDirty14 /
AF479331/
Chryseobacterium
1426
balustinum/M58771
Riemerella columbina/
AF181448
High G+C gram positive bacteria
CanDirty7f/
AF479339/
1420
CanDirty1f/
AF479325/
1440
%
identityc
97.4
97.2
99.6
99.6
99.4
95.4
94.3
94.3
G200-A1d
98.8
Arthrobacter sp./
AB39736
98.7
Arthrobacter
polychromogenes/
98.5
X80741
Arthrobacter sp. from
96.4
sea ice/AF041789
Arthrobacter agilis/
96.0
X80748
Table 3.2 (continued)
107
α proteobacteria
Sphingomonas mali/
Y09638
SIA1K-1A1/
Sphingomonas pruni/
AF395032/
Y09637
1390
Sphingomonas sp. from
Antarctica/AF184221
High G+C gram positive bacteria
Gordona terrae/
X79286
SIA1K-2A1/
AF479377/
High G+C isolate/
1415
X7318
Ross Ice Shelf,
Antarctica
Ice Core
Site
Strain
designationa/
GenBank
accession
no./# of
sequenced ntb
Siple Dome,
Antarctica
Table 3.2
Nearest phylogenetic
neighbor/GenBank
accession no.
γ proteobacteria
Psychrobacter
Trans12/
submarinus/AJ309940
AF479327/
Psychrobacter
1426
marincola/AJ309941
High G+C gram positive bacteria
Kocuria kristinae/
X80749
Trans4678/
AF479328/
Kocuria rhizophila/
1434
Y16264
%
identityc
95.1
95.0
95.0
99.9
99.4
99.6
99.4
99.7
96.0
a
Isolates are designated by their geographic origin, age of
the ice in years or thousands (K) of years, and strain
number. (e.g. G500K-78 is strain no. 78 isolated from
Guliya glacial ice that was 500,000 years old.)
b
The number of 16S rDNA nucleotides sequenced for each
isolate.
c
The percentage identity with the 16S rDNA sequence of the
nearest phylogenetic neighbors.
Table 3.2 (continued)
108
Table 3.2
d
Isolate from this study.
e
Isolate from Lake Vostok accretion ice (see Chapter 5).
f
Isolate recovered from a frozen cryoconite hole on the
glacial surface.
Table 3.2 (continued)
109
Ice Core
Site
Strain
designationa
Optimum
growth
rangeb
(oC)
Growth
at 4oC
Sajama,
Bolivia
Guliya, China
γ proteobacteria
G50-TB2
30-37
NDd
Low G+C gram positive bacteria
G50-TB5
37-45
G50-TS3
45
G50-TB9
30
Antibiotic
resistancec
Tet, Amp, Cln
Amp
C
G50-L2
G50-TS4
37
30
-
-
G200-SD1
22-37
-
-
G200-T16
22-37
G200-T19
22-37
G200-N5
22-37
+
G200-C15
22-37
High G+C gram positive bacteria
G50-TB7
30
NDd
G50-PD1
30
NDd
G50-TB8
30
G50-L3
30
G200-C39
22-37
G200-A1
22
+
G200-C1
22
+
G200-C18
22
+
G200-C11
22
+
γ proteobacteria
SB150-2A1
22-37
+
Low G+C gram positive bacteria
SB100-9-5-1
22
+
SB100-8-1
37
SB100-8-1-1
22-37
+
SB100-8-1-2
22
+
ND
ND
Ery, Amp
Neo
Neo
Amp, Neo
Ery, Amp
Gnt, Amp, Neo
-
Table 3.3 Optimum growth temperature range and antibiotic
resistance in bacterial strains isolated from ice cores.
110
Table 3.3
(continued)
Taylor
Dome,
Antarctica
Sajama, Bolivia
Ice Core
Site
Strain
designationa
Optimum
growth
rangeb
Growth
at 4oC
(oC)
SB150-2A2
22-37
+
SB150-2B
22-37
+
SB12K-9-4
22-37
SB12K-2-2
22-37
+
High G+C gram positive bacteria
SB12K-2-16
22-37
+
SB12K-2-5
22-37
+
SB12K-2-7
22
+
SB12K-2-4
22-37
SB12K-6-2
37
+
SB12K-2-1
22-37
+
SB12K-6-3
22-37
+
SB12K-2-3
22-37
+
α proteobacteria
M3C4.1K-B5
22
+
Antibiotic
resistancec
Gnt
Ery, Tet, Amp
Amp
Gnt, Amp
Gnt, Amp
Amp
Gnt, Neo
Amp
γ proteobacteria
M3C4.1K-B34
22-37
+
+
M3C4.7K-2
22-37
High G+C gram positive bacteria
SIA1K-2A1
22-37
-
Ery, Amp
Ery, Amp, Neo
ND
a
Isolates are designated by their geographic origin, age of
the ice in years or thousands (K) of years, and strain
number. (e.g. G500K-78 is strain no. 78 isolated from
Guliya glacial ice that was 500,000 years old.)
b
Optimum growth temperature range determined by
qualitatively assessing the growth of isolates on agarsolidifed media at 4o, 15o, 22o, 30o, 37o, and 45oC.
Table 3.3 (continued)
111
Table 3.3
c
Resistance based on agar diffusion test. Antibiotic disks
contained (designation; µg/disk) ampicillin (Amp; 10),
gentamicin (Gnt; 10), kanamycin (Kan; 30), erythromycin
(Ery; 15), tetracyclin (Tet; 30), and clindamycin (Cln; 2).
d
Growth at 4oC not determined, but strains did grow at
15oC.
ND=Not determined.
Table 3.3 (continued)
112
isolates were assignable to established bacterial genera,
including Acinetobacter, Agromyces, Arthrobacter,
Aureobacterium, Bacillus, Blastomonas, Cellulomonas,
Clavibacter, Detolaasinbacter, Exiguobacterium,
Friedmanniella, Frigoribacterium, Gordona, Kocuria,
Methylobacterium, Microbacterium, Micrococcus,
Micromonospora, Mycobacterium, Nocardia, Nocardioides,
Ralstonia, Paenibacillus, Planococcus, Pseudomonas,
Psychrobacter, Sanguibacter, and Sphingomonas.
Five
additional isolates (SB12K-2-1, M3C1.8K-TD7, CanDirty12,
CanDirty14, and CanDirty89) have >95% identity to their
nearest neighbors, but the taxonomic classifications within
these phylogenetic clades are currently in disarray.
They
are composed of multiple genera, unidentified isolates, or
misclassified species (Fig. 3.5, 3.6, 3.8).
While fungi
were cultured routinely from ice samples from all
geographical locations, their identification was not
undertaken.
16S rDNA amplification and sequencing
Populations of small subunit rDNA molecules were
amplified directly from material concentrated (Biomax-100
Centricon Plus-80 centrifugation devices; Millipore) from
113
160 ml of Guliya core section 295 filtrate (material
passing through 0.22 µm filter), but not from DNA
extractions conducted on cells retained on the 0.22 µm
filter.
Amplicons were generated using a combination of
Bacteria-specific and universal primers, but not when
Archaea-specific primers and universal primers were used.
Individual DNA molecules were cloned from these populations
and the sequenced determined for the 16S rDNA region
corresponding to nucleotides 515 through 1392 of the E.
coli 16S rDNA sequence.
Eight different γ-proteobacterial
16S rDNA sequences were obtained (Fig. 3.9; Table 3.4).
Six of the clones (pG500K-80, pG500K-85, pG500K-86,
pG500K-96, pG500K-98, and pG500K-106; Fig. 3.9) cluster in
the genera Pseudomonas, and share between 99.2-100%
identity with sequences from environmental isolates and
clones, with Pseudomonas putida (D86002) being the closest
related type strain (98.9-99.4% identity).
Two of the 6
clones are most similar to each other, and the remainder
are most similar to either a bacterium from a uranium mine
waste pile (AJ295653) or a Pseudomonas isolate studied for
possessing a unique pyoverdin structure (AF321239) [Table
3.4].
114
Fig. 3.9
Phylogenetic analysis of g-proteobacterial
sequences amplified from >500,000 year old ice from Guliya,
China. The sequences obtained corresponded to nucleotides
515-1392 of the E. coli 16S rDNA, and were aligned based on
secondary structure using the ARB software package (Struck
et al. 1998). Trees were generated using a 426 nucleotide
mask of unambiguously aligned positions. Clones appear in
large font (pG500K-), and glacial isolates are marked with
asterisk (*), and designated by their geographical origin
(G=Guliya, China; SB=Sajama, Bolivia; M3C=Taylor Dome,
Antarctica; and CanClear=Canada Glacier, Antarctica) age of
the ice in years, and strain number (e.g. G500K-78 is
strain no. 78 isolated from Guliya glacial ice that was
500,000 years old.
A. Maximum likelihood tree generated using fastDNAml
(Olsen et al. 1994). The scale bar indicates 0.1 fixed
substitutions per nucleotide position.
B. Maximum parsimony analysis performed with 100 bootstrap
replicates, with values shown at the nodes. The scale bar
represents 10 evolutionary steps. Of the 72 variable
characters in this alignment, 58 were parsimonyinformative.
115
Pseudomonas aeruginosa (U38445)
Pseudomonas alcaligenes (Z76653)
Pseudomonas oleovorans (D84018)
M3C4.7K-2* (AF479376)
Pseudomonas pseudoalcaligenes (76666)
Pseudomonas mandelii (AF058286)
Pseudomonas sp. from sea ice (U85869)
Pseudomonas sp. from sea ice (U85868)
M3C4.1K-B34* (AF479375)
Pseudomonas synxantha (AF267911)
Pseudomonas azotoformans (D84009)
pG500K-96 (AF479387)
uncultured sheep mite bacterium
pG500K-106 (AF479389)
uranium mine bacterium KF/GS-JG36-1 (AJ295653)
pG500K-98 (AF479388)
Pseudomonas sp. PM-2001 (AF321239)
uranium mine bacterium KF/GS-Gitt2-41 (AJ295644)
Pseudomonas putida (D86002)
pG500K-86 (AF479386)
pG500K-80 (AF479384)
pG500K-85 (AF479385)
Acinetobacter radioresistens (X81666)
CanClear23* (AF479323)
Acinetobacter sp. (Z93445)
G50-TB2* (AF479352)
SB150-2A1* (AF479373)
Acinetobacter junii (X81664)
Acinetobacter johnsonii (Z93439)
M3C1.8K-TD8* (AF479380)
Acinetobacter haemolyticus (Z93436)
Acinetobacter calcoaceticus (AF159045)
Acinetobacter lwoffii (U10875)
Mariana Trench isolate (AB002655)
Acinetobacter johnsonii (X95303)
Mariana Trench isolate (AB002658)
Acinetobacter sp. from subsurface (X86572)
0.10
clone from uranium mine (AJ296557)
clone from uranium mine (AJ301569)
Lake Baikal isolate (AJ222834)
Acinetobacter lwoffi (X81665)
pG500K-1 (AF479382)
pG500K-41 (AF479383)
Figure 3.9A
116
Pseudomonas aeruginosa (U38445)
Pseudomonas alcaligenes (Z76653)
Pseudomonas oleovorans (D84018)
M3C4.7K-2* (AF479376)
Pseudomonas pseudoalcaligenes (76666)
Pseudomonas mandelii (AF058286)
Pseudomonas sp. from sea ice (U85869)
Pseudomonas sp. from sea ice (U85868)
M3C4.1K-B34* (AF479375)
Pseudomonas
synxantha (AF267911)
98
Pseudomonas azotoformans (D84009)
pG500K-96 (AF479387)
uncultured sheep mite bacterium
pG500K-106 (AF479389)
uranium
mine bacterium KF/GS-JG36-1 (AJ295653)
52
pG500K-98 (AF479388)
Pseudomonas sp. PM-2001 (AF321239)
uranium mine bacterium KF/GS-Gitt2-41 (AJ295644)
Pseudomonas putida (D86002)
pG500K-86 (AF479386)
pG500K-80 (AF479384)
64
pG500K-85 (AF479385)
Acinetobacter radioresistens (X81666)
CanClear23* (AF479323)
75
Acinetobacter sp. (Z93445)
G50-TB2* (AF479352)
SB150-2A1* (AF479373)
100
Acinetobacter junii (X81664)
Acinetobacter johnsonii (Z93439)
51
50
10
59
72
91
95
Figure 3.9B
117
M3C1.8K-TD8* (AF479380)
Acinetobacter haemolyticus (Z93436)
Acinetobacter calcoaceticus (AF159045)
Acinetobacter lwoffii (U10875)
Mariana Trench isolate (AB002655)
Acinetobacter johnsonii (X95303)
Mariana Trench isolate (AB002658)
Acinetobacter sp. from subsurface (X86572)
clone from uranium mine (AJ296557)
clone from uranium mine (AJ301569)
86
Lake Baikal isolate (AJ222834)
Acinetobacter lwoffi (X81665)
pG500K-1 (AF479382)
87
pG500K-41 (AF479383)
Sequence
designation
(no. of
clones)a
pG500K-1
(6)
pG500K-41
(2)
pG500K-80
(2)
pG500K-85
(10)
pG500K-86
(8)
pG500K-96
(1)
GenBank
accession
no./# of
sequenced
ntb
AF479382/
515
AF479383/
498
AF479384/
818
AF479385/
819
AF479386/
823
AF479387/
841
Nearest phylogenetic
neighbor/GenBank
accession no.
pG500K-41
clone from uranium
mine/AJ301569
Lake Baikal isolate/
AJ222834
clone from uranium
mine/AJ301569
Lake Baikal isolate/
AJ222834
pG500K-1
pG500K-85
pG500K-96
Pseudomonas sp./
AF321239
uranium mine
bacteria/AJ295653
Pseudomonas sp./
AF321239
pG500K-80
uranium mine
bacteria/AJ295653
Pseudomonas sp./
AF321239
pG500K-96
pG500K-98
uranium mine
bacteria/AJ295653
Pseudomonas sp./
AF321239
%
identityc
98.8
98.8
98.4
99.2
99.2
98.8
99.6
99.5
99.5
99.6
99.6
99.6
99.8
99.8
99.8
99.8
99.8
99.8
Table 3.4 16S rDNA molecules amplified from >500,000 year
old ice from Guliya, China (Core 2, tube 295). All clones
classify within the γ−proteobacteria.
118
Table 3.4
Sequence
designation
(no. of
clones)a
pG500K-98
(9)
pG500K-106
(1)
a
GenBank
accession
no./# of
sequenced
ntb
AF479388/
831
AF479389/
765
Nearest phylogenetic
neighbor/GenBank
accession no.
uranium mine
bacteria/AJ295653
Pseudomonas sp./
AF321239
pG500K-96
pG500K-96
uranium mine
bacteria/AJ295653
Pseudomonas sp./
AF321239
%
identityc
100
100
99.8
99.6
99.6
99.6
Number of individual clones with this sequence
b
The number of 16S rDNA nucleotides sequenced for each
isolate.
c
The percentage identity with the 16S rDNA sequence of the
nearest phylogenetic neighbors.
Table 3.4 (continued)
119
Clone pG500K-1 and pG500K-41 have 96.3 and 99.2%
identity, respectively, to Acinetobacter lwoffi (X81665).
While pG500K-1 is equally similar (98.8%) to pG500K-41 and
to a clone from a uranium mine waste pile (AJ301569),
pG500K-41 is 99.2% identical to the latter clone and an
isolate from Lake Baikal (AJ222834) [Table 3.4].
Freeze-thaw resistance
The freeze-thaw tolerance of several non-sporulating
ice core isolates and one endospore-forming Bacillus
isolate was investigated by subjecting cell suspensions to
18 cycles of freeze-thaw, and compared with survival
(ability to form colonies on agar-solidified medium) of
type strains of related bacteria and E. coli (Fig. 3.10).
The glacial isolates and related type strain species tested
were very tolerant to repeated cycles of freeze-thaw,
whereas E. coli was very sensitive, with no cfu remaining
after 18 cycles.
Psychrotrophy
Fifty-three ice core isolates from Antarctica,
Bolivia, and China were tested for growth at low
temperature (Table 3.3).
The majority were psychrotrophic,
120
1010
cfu ml
-1
108
106
104
102
1
0
4
8
12
16
freeze-thaw cycles
SB150-2A2
SB12K-2-2
SB12K-9-4
SB12K-2-1
SB12K-2-3
SB12K-2-16
SB12K-6-3
SB20K-1
G200-A1
Bacillus polymyxa
Aureobacterium suaveolens
Arthrobacter globiformis
Micrococcus luteus
Brevibacterium linens
Escherichia coli
Fig. 3.10
Freeze-tolerance was examine in glacial
isolates, related species, and E. coli. Cell suspensions
were serially diluted and plated on agar-solidifed media
during 18 cycles of freezing and thawing.
121
with >55% capable of growth at 4oC, although most were
isolated, and all grew optimally, at mesophilic
temperatures (>20 oC).
Discussion
Ice samples from non-polar, low-latitude, highaltitude glaciers in the Andes and Himalayas generally
contained more colony forming units and a greater variety
of recoverable bacterial species than polar ices.
This is
consistent with their closer proximities to locations with
substantial vegetation and exposed soils.
Ice core
sections from Taylor Dome and from the Canada Glacier,
adjacent to the McMurdo Dry Valley complex of Antarctica,
similarly contained larger numbers (8-10 cfu ml-1) and
species of recoverable bacteria, than other polar ices
(Table 3.1).
Rock grains, eroded by the persistent winds
characteristic to this region, are warmed on ice surfaces
during the austral summer, and melt into the ice creating
pockets of liquid water with sufficient nutrients to
support the growth of microbial communities.
Such
ecosystems have been documented in lake ice (Olson et al.
1998; Paerl and Priscu 1998; Priscu et al. 1998; Takacs and
Priscu 1998) and on glacial surfaces (Wharton et al. 1985).
Notably two of the isolates from sediment originating from
122
a cryoconite hole on the Canada Glacier (Fig. 3.8) belong
to the same genera as many isolates obtained in this survey
of polar and nonpolar glacial ices (Arthrobacter).
For
example, the nearest neighbor of one species recovered from
this polar cryoconite environment (CanDirty7; 98.8%) is an
ice core isolate from Guliya, China.
There is no consistent, monotonic decrease in the
number of recoverable bacteria with increasing age within
an ice core.
Rather, the numbers of recoverable bacteria
isolated from ice samples from different positions in ice
cores appear to reflect the prevalent climate and
individual events that occurred at the time of deposition.
For example, there were more recoverable bacteria in Sajama
ice deposited ~12,000 years ago during cool, wet climate
conditions than in modern ice, deposited at the same
location during a warmer, dryer period (Thompson et al.
1998).
Interestingly, 6 of the 7 Sajama isolates from
modern ice belong to genera that form environmentallyresistant endospores, whereas only one of the 11 species
isolated from older ice deposited at this location during
the Last Glacial Maximum and deglaciation climate reversal
was a Bacillus relative.
Wet climate conditions increase
vegetation density and productivity, increasing the
123
concentration of large airborne biological particles, such
as pollen (Liu et al. 1998), and these, in turn, presumably
transport microorganisms.
Increased snowfall accumulation
during this period produced thicker annual layers, which
would have reduced exposure to damaging UV radiation at the
glacial surface.
The decreased desiccation rates predicted
under these climate conditions may have also contributed to
survival during travel through the atmosphere.
Cells revived from ice core samples have presumably
endured desiccation, solar irradiation, freezing, a period
of frozen dormancy and thawing.
Therefore, it is not
surprising that a large number of the isolates recovered
belong to bacterial groups that form spores or have thick
cell walls and polysaccharide capsules.
These structures
help overcome the stresses associated with water loss,
namely increased intracellular solute concentrations,
decreased cell size, a weakened cell membrane, and physical
cell rupture caused by freezing and thawing (Fogg 1998).
The high frequency of pigment production (Fig 3.2) is also
consistent with the need to absorb toxic solar irradiation
and so prevent lethal DNA damage.
Even though the
surviving cells may have resistant structures and
protective pigments, during extended periods of inactivity,
124
they must still incur some radiation and chemical damage.
Long periods (20-70 days) of incubation were often
necessary before visible colonies appeared, and no colonies
were obtained directly from ancient samples (>500,000 years
old) from the Guilya ice cap.
Bacteria were resuscitated
from these samples by inoculation of low-nutrient liquid
enrichments and incubation at 4oC for 30 days.
This
observation is consistent with aged cells needing time,
before beginning reproductive growth, to repair cellular
damage accumulated during extended periods of dormancy.
The majority of the glacial isolates obtained have
close phylogenetic relationships to either endosporeforming Bacilli, spore-forming or non-sporulating
Actinomycetes, several of which are known to have life
cycles with radiation and desiccation resistant resting
stages (Morita 1997), or to species from the α− and γproteobacterial lines of descent.
Bacillus and
Paenibacillus relatives of strains prevalent in soils were
most commonly isolated from nonpolar glacial ices, and
species of Sphingomonas, Methylobacterium, Acinetobacter,
and Arthrobacter were also ubiquitous, and recovered from
both polar and nonpolar locations.
125
The nearest neighbors
of 14 these isolates were isolates from other ice core
locations, or from other portions of the same core, based
on 16S rDNA identity (Table 3.2, in bold).
While most of the isolates are similar to species
frequently found in environmental surveys from around the
world, some are most closely related to species of
Arthrobacter, Brachybacterium, Exiguobacterium,
Friedmanniella, Frigoribacterium, Janthinobacterium,
Planococcus, Pseudomonas, Psychrobacter, and Sphingomonas
recovered previously from Antarctic lake mats (Brambilla et
al. 2001), sea ice (Gosink and Staley 1995; Bowman et al.
1997; Junge et al. 1998), and other predominantly cold
environments (Shi et al. 1997; Benson et al. 2000).
It
seems also noteworthy that relatives of the radiationresistant type strains Methylobacterium radiotolerans and
Acinetobacter radioresistens were also commonly
encountered.
Having very efficient DNA repair mechanisms
is likely to be valuable in terms of extended survival in
all environments including glacial ice.
The isolation of
related microbes from many geographically diverse but
predominantly frozen environments argues that these species
probably have features that confer resistance to freezing
and survival under frozen conditions.
126
The enhanced freeze-
thaw tolerance (Fig 3.10) and psychrotrophy (Table 3.3)
observed for many glacial isolates is consistent with this
notion.
Interestingly, all the 16S rDNA sequences amplified
from a >500,000 year old ice core from Guliya, China are
most similar to clones retrieved from other subsurface
environments.
Comparisons of the numbers of organisms and
types isolated by conventional enrichment strategies with
the numbers and types detectable by non-culture based
molecular approaches consistently demonstrate major
discrepancies between how many and who is there, with who
grows under laboratory conditions (Hugenholtz et al. 1998).
However, 16S rDNA fragments amplified from the >500,000
year old ice core from Guliya did have DNA sequences
phylogenetically-related on the genus level to species of
Pseudomonas and Acinetobacter (highest identity being 99.0
and 97.6%, respectively) recovered from both polar and
nonpolar glacial ices (Fig. 3.9; Table 3.4).
Identifying microorganisms deposited in glacial ice
has provided an indication of the influence of climate and
geography on the composition of entrapped microbial
species.
Investigating the isolates has identified common
survival strategies, and provided data confirming microbial
127
longevity over thousands of years.
These isolates, which
originate from different times in the past, have been
deposited in the DOE sponsored Subsurface Microbial Culture
Collection (Balkwill et al. 1997).
study at request.
128
They are available for
CHAPTER 4
MACROMOLECULAR SYNTHESIS UNDER FROZEN CONDITIONS
Introduction
For a microorganism to remain viable during periods of
dormancy, the damage incurred to the cell must not exceed a
level where effective repair is no longer possible.
Amino
acid racemization rates are retarded in amber (Bada et al.
1994), and decreased rates of macromoleular decay could
also occur in ice.
It is also possible, however, that
microorganisms entrapped within ancient specimens are in
fact active, and able to carry out a low level of
metabolism to facilitate the repair of accumulated
macromolecular damage.
Thin veins of liquid water between ice crystals could
potentially provide a microbial habitat within apparently
solid ice (Price 2000), and studies of permafrost (Rivkina
et al. 2000) and surface snow (Carpenter et al. 2000) have
demonstrated low levels of metabolic activity at subzero
temperatures.
Therefore, to explore the concept that
129
glacially-entrapped microorganisms could repair incurred
damage in situ, experiments were undertaken to determine if
macromolecular synthesis could be demonstrated in ice.
Materials and Methods
Bacterial strains and culture conditions
Isolate Trans1 and E. coli (OSU ref. no. 422) were
grown in Luria-Bertani medium (Sambrook et al. 1989), and
G200-C1 was cultured in R2 medium (Reasoner and Geldreich
1985).
All cultures (25 ml) were incubated aerobically
(200 rpm) at 22oC in 125 ml Erlenmeyer flasks.
Cells from
late exponential growth phase were used to inoculate
cultures at an initial A600 of 0.2, which were grown to A600
0.6-0.8.
Cells were then harvested by centrifugation at
17,000xg for 5 min., washed twice with distilled water, and
resuspended at an A600 of 0.2 in distilled water
(representing 1.3 and 2 x 108 cfu ml-1 of Trans1 and G200-C1,
respectively).
Aliquots (500 µl)of these cell suspensions
were placed in 1.5 ml Eppendorf tubes and chilled to 4oC.
Procedure for macromolecular synthesis assay
Cell suspensions were maintained on ice and used less
than 1 h after harvesting.
When added to cell suspensions,
130
chloramphenicol (Sigma, cat. no. 100K9113), nalidixic acid
(Sigma, cat. no. N-3143), and ciprofloxacin (ICN
Biomedicals, cat. no. 199020 were used at a final
concentration of 15 µg/ml.
In each experiment, a control
was included in which the cell and reaction components were
incubated in the presence of 7% trichloroacetic acid (TCA).
Either [3H]-thymidine (ICN Biomedicals, catalog# 24060;
1 µCi; final concentration 23 nM) or [3H]-leucine (ICN
Biomedicals, catalog# 20036E; 1 µCi; final concentration 17
nM) was added to the 500 µl samples.
The mixture was then
frozen by incubation at -70oC or by immersing the tube in
liquid nitrogen.
The samples frozen in liquid nitrogen
were then placed at -70oC.
After 1 h, all tubes were
transferred to -15oC for the duration of most experiments.
In one experiment, the tubes were maintained at -70oC for
100 days.
At each experimental time point, 100 µl of 50% TCA was
added to inactivate the mixture, which was then allowed to
melt.
After 30 min at 4oC, the acid-insoluble
macromolecules were sedimented by centrifugation at
18,000xg for 15 min, the supernatant removed, and the
resulting pellet washed with 500 µl of 5% TCA.
Following
centrifugation for 10 min. at 18,000xg, the supernatant was
131
removed and the pellet washed with 500 µl of 70% ethanol.
Following centrifugation for 5 min. at 18,000xg, the
ethanol was decanted, and the pellet mixed and resuspended
in 1 ml of Ecoscint H scintillation fluid (Life Sciences
Inc., catalog# LS-275).
The Eppendorf tube was placed
directly into scintillation vial, and tritium incorporation
was quantitated by 10 min counting in a Beckman model LS7500 scintillation counter.
demonstrated that
3
Control experiments
H was measured at a 61% counting
efficiency.
Results
The possibility of incorporation of [3H]-thymidine and
[3H]–leucine during the freezing process was investigated by
comparing incorporation in samples frozen at -70oC with
samples frozen by submersion in liquid nitrogen (Fig 4.1).
The background level of [3H]-thymidine and [3H]–leucine
associated with TCA-prefixed cell suspensions was lower
than the incorporation obtained from samples frozen
instantly in liquid nitrogen after the addition of label,
and there was no clear difference between the incorporation
by cells frozen by the two methods, even though samples
placed at -70oC remained liquid for 5 to 10 min. before
132
2450
dpm
1950
1450
950
Trans1
TdR
Trans1
Leu
G200-C1
TdR
2
N
0Co
-7
L-
A
TC
2
N
0Co
-7
L-
A
TC
A
LN
-7 2
0Co
TC
2
N
0Co
-7
L-
TC
A
450
G200-C1
Leu
Figure 4.1
Incorporation of [3H]-thymidine (TdR) and
3
[ H]-leucine (Leu) into TCA-precipitable material by strains
Trans1 and G200-C1 during the freezing process.
The acid-insoluble macromolecular fraction was precipitated
1 h after freezing. The incorporation in 3 separate
reaction mixtures was determined for every data point,
including samples frozen in the presence of 7% TCA, frozen
by submersion in liquid nitrogen (L-N2), and frozen at
-70oC. The y axis error bars denote standard deviation from
the mean.
133
freezing solid. This suggests that the difference between
the samples and the TCA controls results from acid
conditions effecting chemical binding, rather than the
difference representing incorporation during freezing.
Sampling during the course of a ~200 day incubation at
-15oC (Fig 4.2) clearly indicated that both DNA and protein
synthesis occurred during the first 20 days of incubation
in both strains studied, which appeared to continue for the
next 130 days, although at reduced rates.
A similar
incorporation profile of these precursors into TCAprecipitated materials was also observed in frozen E.coli
cell suspensions incubated for 100 days under identical 15oC conditions (Fig 4.3).
Precursor incorporation began to level off after 150
days at -15oC (Fig 4.2), where the maximum amount of
incorporation was observed, ranging between 9-14 x 103 dpm.
After subtracting background counts (isotope count in
samples TCA fixed from the outset) from these values, the
estimated incorporation range for thymidine and leucine was
5850-9690 dpm.
Knowing the specific activity of the
precusor (60-90 Ci/mmol of [3H]-thymidine and 40-60 Ci/mmol
of [3H]-leucine) and given that all cells (107-108 cfu ml-1)
in each sample participate in the DNA and protein
134
Trans1
Trans1
Trans1
Trans1
Tdr-TCA
TdR
Leu-TCA
Leu
G200-C1
G200-C1
G200-C1
G200-C1
TdR-TCA
TdR
Leu-TCA
Leu
10
135
dpm x 10
-3
14
6
2
0
50
100
150
200
days
Fig. 4.2
Incorporation of [3H]-thymidine (TdR) and [3H]-leucine (Leu) by strains
o
Trans1 and G200-C1 during a 206 day incubation at -15 C. Each data point was done in
triplicate, and included identical samples prefixed in 7% TCA prior to the addition of
label. The y axis error bars denote standard deviation from the mean.
2 x 10
E. coli Leu
E. coli Leu-TCA
E. coli TdR
E. coli TdR-TCA
4
dpm
1.5 x 104
1 x 104
5000
0
20
40
60
80
100
days
Figure 4.3
Incorporation of [3H]-thymidine (TdR) and
3
[ H]-leucine (Leu) by E.coli during a 102 day incubation at
-15oC.
Two samples were taken at each data point. These included
samples prefixed in 7% TCA prior to the addition of
radioactively labeled precursor. The y axis error bars
denote standard deviation from the mean.
136
syntheses, then it can be calculated that between 25-500
molecules of thymidine and leucine were incorporated per
cell after 206 days of incubation (for 107-108 cells, 1000
dpm represent 5-50 molecules of thymidine or leucine/cell).
The change in the number of cfu ml-1 was determined
over 100 days concurrently with measurements of [3H]thymidine incorporation (Fig 4.4).
The results indicate
that the low level of isotope incorporation observed during
this period of incubation was not accompanied by an
increase in the viable number.
The number of cfu ml-1
actually decreased >4-fold during the first 20 d of G200-C1
incubation.
Unfortunately, due to a technical error, data
on the 2 day time point are the first available for Trans1.
For both isolates, the number of cfu ml-1 decreased to a
minimum between 10-25 days post-freezing, and then
increased, appearing to reach a steady-state by 50-100
days, as also observed for thymidine incorporation (Fig
4.4).
Addition of ciprofloxacin (CFX) and chloramphenicol
(CM) decreased levels of precursor incorporation (Fig 4.5).
The presence of CFX, an inhibitor of DNA gyrase, reduced
thymidine incorporation by 30-40% (Fig 4.5A), and CM, an
inhibitor of protein synthesis, reduced leucine
137
2 x 104
Trans1-TdR
5 x 107
G200-C1-TdR
1.5 x 104
Trans1 cfu ml
5 x 10
3 x 10
7
2 x 10
7
1 x 10
7
G200-C1 cfu ml-1
4
3
0
20
40
60
80
100
-1
138
1 x 10
-1
cfu ml
dpm
4 x 107
0
days
3
Fig. 4.4
Incorporation of [ H]-thymidine (TdR) and the number of cfu ml-1 for Trans1
o
and G200-C1 during a 100 day incubation at -15 C. Data at each point was obtained in
triplicate. For Trans1, the 2 day cfu ml-1 value represents the first data point in the
series, due to a technical error. The y axis error bars denote standard deviation from
the mean.
3
Figure 4.5
Incorporation of [ H]-thymidine (TdR) and
3
[ H]-leucine (Leu) at -15oC by strains Trans1 and G200-C1
over 23 days in the presence of (A) ciprofloxacin and (B)
chloramphenicol.
Data at each point were obtained in duplicate, and the y
axis error bars denote standard deviation from the mean.
(A) ciprofloxacin [CFX] and (B) chloramphenicol [CM] were
added at a concentration of 15 mg/ml, before the addition
of radioactively-labeled precursor. Cell suspensions with
(.....) and without (_____) antibiotics, and prefixed in
TCA (_ _ _ _) were frozen an incubated under identical
conditions.
139
A
1.2 x 104
G200-C1 TdR
G200-C1 TdR/CFX
G200-C1 TdR-TCA
Trans1 TdR
Trans1 TdR/CFX
Trans1 TdR-TCA
1 x 104
3
6 x 10
3
4 x 10
3
dpm
8 x 10
2 x 103
0
4
8
12
16
20
24
days
B
1.2 x 10
4
1 x 10
4
G200-C1 Leu
G200-C1 Leu/CFX
G200-C1 Leu-TCA
Trans1 Leu
Trans1 Leu/CFX
Trans1 Leu-TCA
dpm
8 x 103
6 x 103
4 x 103
2 x 103
0
4
8
12
days
Figure 4.5
140
16
20
24
incorporation 50-60% (Fig 4.5B).
Curiously for both Trans1
and G200-C1, incubations in the presence of a second DNA
synthesis inhibitor, nalidixic acid, resulted in a 2-fold
increase in thymidine incorporation (data not shown).
Based on growth, G200-C1 was resistant to this antibiotic
at concentrations of 15 µg/ml, but Trans1 was inhibited by
nalidixic acid.
In contrast to the incubations at -15oC, neither
thymidine nor leucine incorporation was detected in cells
incubated at -70oC for 50 days (Fig 4.6).
In a repetition
of this experiment, no incorporation was observed at -70oC
after 100 days (data not shown).
Discussion
Having demonstrated that microorganisms remain viable
during entrapment in glacial ice for hundreds of thousands
of years, the question remained whether metabolic activity
was possible under these frozen conditions.
Alternatively,
the species recovered may be particularly successful at
surviving although metabolically dormant over extended time
frames.
The presence of liquid water is generally
considered essential for active metabolism, and within ice,
films of water exist on salt inclusions, air bubbles, and
141
3
Figure 4.6
Incorporation of [ H]-thymidine (TdR) and
3
[ H]-leucine (Leu) at -15 and -70oC by strains Trans1 and
G200-C1 over 50 days.
Duplicate samples were taken for each data point, and the y
axis error bars denote standard deviation from the mean.
Aliquots of the same cell suspension were frozen and
o
o
incubated at either -15 or -70 C, and samples frozen in the
presence of 7% TCA (background) were incubated at -15oC.
The effects of temperature on both the incorporation of
thymidine (A) and leucine (B) are illustrated.
142
A
1.8 x 104
Trans1 TdR -15oC
o
Trans1 TdR -70 C
Trans1 TdR-TCA
1.6 x 104
o
G200-C1 TdR -15 C
o
G200-C1 TdR -70 C
G200-C1 TdR-TCA
1.4 x 104
dpm
1.2 x 104
10
4
8 x 103
6 x 10
3
4 x 103
2 x 10
3
0
10
20
B
1.2 x 10
days
Trans1 Leu -15oC
o
Trans1 Leu -70 C
Trans1 Leu-TCA
4
30
40
50
o
G200-C1 Leu -15 C
o
G200-C1 Leu -70 C
G200-C1 Leu-TCA
1044
dpm
8 x 103
6 x 103
4 x 103
2 x 103
0
Figure 4.6
10
20
143
days
30
40
50
between ice crystals (Patterson 1994; Price 2000).
Water
has been shown to exist in permafrost at temperatures above
-60oC (Ostroumov and Siegert 1996).
Terrestrial glacial ice
is generally at temperatures above this value, and
therefore the possibility exists that microorganisms could
be metabolically active within liquid water in veins
between ice crystals, as proposed by Price (2000).
The two glacial isolates used in this study [Trans1
(Fig 3.6) and G200-C1 (Fig 3.8)] were chosen because of
their apparent close phylogenetic relationships to species
native to brine channels in sea ice [>99% 16S rDNA identity
to isolates reported by Junge et al. (1998) and Bowman et
al. (1997)], demonstrated ability to grow at 4oC
(psychrotrophic), and significantly different cell wall
structure (Trans1 is a Gram negative Psychrobacter species,
whereas G200-C1 is a Gram positive Arthrobacter species).
Incorporation of radioactive precursors in TCA-precipitated
material was investigated for cell suspensions held under
frozen conditions (-15oC) for ~30 weeks (Fig 4.2).
In both
cases after an initial increase, the rate of incorporation
gradually slowed and appeared to reach a steady-state after
200 days (Fig 4.3).
The inhibition of precursor
incorporation by CFX and CM was consistent with
144
macromolecular synthesis occurring at -15oC (Fig 4.5).
By
definition, unfrozen water is predicted not to exist below
-60oC in water ice or within cells (Ostroumov and Siegert
1996).
Consistent with liquid water being present in
samples at -15o but not -70oC, no thymidine or leucine
incorporation into the macromolecular TCA-precipitated
material was observed in cell suspensions at -70oC.
The cells used in these frozen activity studies were
taken from populations in logarithmic growth.
Cell
division will have stopped, but some metabolic activity
within the cell will have continued.
The difference
between decreased anabolism and continuing catabolism
results in the production of free radicals that damage DNA
and protein (Aldsworth et al. 1999; Stead and Park 2000).
Aldsworth et al. (1999) have termed this the “suicide
response”, and have shown that in contrast to cells
suddenly removed from a growth situation, cells from nondividing cultures in stationary phase are resistant to this
stress.
Their already reduced metabolism and stationary
phase-induced stress proteins apparently protect such cells
from incurring lethal damage.
145
In addition to the physical and osmotic stress imposed
on cells by ice crystal formation, the storage of E. coli
at -20oC has been demonstrated to cause single- and doublestranded chromosomal breakage (Alur and Grecz 1975; Grecz
et al. 1980).
Although substantial damage occurs during
the freeze process, the extent of double-stranded DNA
breakage increased over 4 months, but then began to
decrease by 12 months post-freezing.
The authors concluded
that “random reassociation and aggregation of the initial
DNA fragments” was the most likely explanation for the
unusual results (Grecz et al. 1980).
The data obtained are consistent with macromolecular
synthesis being possible when bacteria are within ice at
temperatures in which liquid films exist.
Inability to
grow at subzero temperatures does not preclude metabolic
activity at such temperatures.
E. coli was unable to grow
below 10oC, but nevertheless, significant precursor
incorporation was observed by suspensions of E. coli held
at -15oC (Fig 4.3).
The total incorporation of thymidine
and leucine into macromolecules observed after >200 days at
-15oC (Fig 4.2) corresponded to one cell assimilating
between 25-500 molecules of each labeled precursor.
Based
on E.coli’s genome and average protein size, this amount of
146
activity represents <0.01% of the genome or a total of 10
protein molecules.
Interestingly, 50-75% of this
incorporation occurred during the first 15-20 days of
incubation at -15oC; coincidentally, a period of time the
viable count (cfu ml-1) began to increase after initially
decreasing after freezing (Fig 4.4).
The amounts of
precursor incorporation detected were insufficient for
growth, and more likely represent incorporation during
cellular repair of damage incurred during the stress of
metabolic arrest and/or freezing.
These results obtained
add to the growing body of evidence for metabolism in
environments below freezing (Carpenter et al. 2000; Rivkina
et al. 2000).
They support the argument that bacteria
could continue with metabolic activity while entrapped in
water veins within glacial ice.
147
CHAPTER 5
ISOLATION OF BACTERIA AND 16S rDNA SEQUENCES FROM
LAKE VOSTOK ACCRETION ICE
Introduction
More than 70 subglacial lakes have been discovered in
Antarctica (Siegert et al. 1996).
The largest, Lake
Vostok, is covered with a layer of ~4000 m of glacial ice,
and has been isolated from direct surface input for at
least 420 K years (Petit et al. 1999).
The lake water is
derived from the overlying glacier, with ice melting into
Lake Vostok at the northern ice-water interface, and water
from the lake freezing as accretion ice, below the glacial
ice, over the central and southern regions of the lake
[Fig. 5.1; (Kapitsa et al. 1996; Jouzel et al. 1999;
Siegert et al. 2000)].
Glacier movement presumably must
transfer sediment from the adjacent bed rock into the lake,
and both eukaryotic and prokaryotic microorganism have been
detected in samples of glacial ice collected from above
Lake Vostok (Abyzov et al. 1998). It therefore seems
inevitable that viable microorganisms are seeded into Lake
148
Fig. 5.1
Origin of deep Vostok ice core section 3593.
Schematic illustrating subglacial Lake Vostok, based on
Bell (1998) and Siegert (2000). Chemical and isotopic
profiles established that the glacial ice-accretion ice
interface occurs at <3540 mbs below the surface (Jouzel et
al. 1999), and the deep Vostok ice core section used in
this study, section 3593, originated from 3591.965 to
3592.445 mbs. As illustrated, glacial ice melts into Lake
Vostok at the ice-water interface in the north, and
accretion ice accumulates at the base of the glacial ice
over the central and southern regions. Radar
measurements have detected the presence of a layer of
sediment below the lake water (Kapitsa et al., 1996), with
gas hydrates also predicted to be present (Doran et al.
1998).
149
Vostok
Ice flow
3538.7 m of
Glacial Ice
Core 3591.9653592.445 m
220 m of
Accreted Ice
~500 m of
Lake Water
Sediments and
Gas Hydrates?
Figure 5.1
150
Vostok, but the nature of the environment and ecosystem
within Lake Vostok remain uncertain.
Concerns for
contamination have resulted in a moratorium on direct
sampling of Lake Vostok water and ice core drilling has
been terminated above the ice-water interface.
An ice core
has, nevertheless, been retrieved in which the bottom ~150
meters are accretion ice (Fig. 5.1) and this therefore
provides a sample of Lake Vostok water (Petit et al. 1999).
Microbial cells in melt water from sections of this
accretion ice core that originated 3590 and 3603 meters
below the surface (mbs) have been detected by
epifluorescence and scanning electron microscopy (Priscu et
al. 1999; Karl et al. 1999), and seven small-subunit
bacterial ribosomal RNA-encoding DNAs (16S rDNA) were
amplified from the 3590 melt water that originated from αand β-proteobacteria and from an Actinomyces (Priscu et al.
1999).
Evidence for respiration was also obtained by
measuring
14
C-CO2 release during incubations at 3oC and 23oC
after the addition of
14
C-acetate or
14
C-glucose to melt
water from the 3603 section (Karl et al. 1999). However,
there was very little, if any,
14
macromolecules.
151
C-incorporation into
This chapter documents the results of experiments
undertaken to determine if viable bacteria could be
recovered directly from Lake Vostok accretion ice.
Four
different isolates have been obtained, and additional 16S
rDNAs have been amplified from a section of the accretion
ice core that originated at 3593 mbs.
The results are
consistent with the concept that Lake Vostok is seeded
regularly with bacteria initially immured in the overlying
glacial ice, and is likely to contain bacteria similar to
species found in other cold environments.
Materials and Methods
Ice core origin and sampling
A section of the deep Vostok ice core extending from
3591.965 to 3592.445 mbs, here designated core section 3593
(Fig. 5.1), was obtained from the National Ice Core
Laboratory (Denver, CO).
Core 3593 was broken in transit,
and the longer resulting section (~33 cm) was subjected to
automated melt water sampling, and the smaller fragment
washed with 95% ethanol and water, as described in Chapter
2.
152
Measurements made using a Finnigan Mat mass
spectrometer, revealed that the mean stable isotope ratios
in the core were –56.23‰ for δ18O and –446.24‰ for δD
(Henderson et al. 1999), similar to the values reported for
samples from 3540 to 3750 mbs by Jouzel et al. (1999), and
consistent with a water-freezing-to-ice origin.
Microbiological and molecular procedures
Enrichment cultures, colony isolations, 16S rDNA
amplification and sequencing, and electron microscopy were
conducted as described in Chapter 3.
Results
Enrichment isolates
Melt water that was obtained from inside Vostok deep
core section 3593 using the automated ice core sampling
system was used to inoculate a wide range of different
growth media (Table 5.1).
After 7 days, growth was
observed in M9 glucose-minimal salts (Sambrook and Maniatis
1989) and in R2 medium, a low nutrient medium designed to
recover stressed bacteria (Reasoner and Geldreich 1985), in
cultures incubated aerobically at 25oC.
Single colony
isolates were obtained from these enrichment cultures by
153
Table 5.1
Media inoculated with melt water from ice core
section 3593. When used in petri plates, media were
solidified by the addition of 1.5% (w/v) agar.
Used under aerobic conditions:
Designed to
Medium
Source
isolate/enumerate
Tryptose blood
Fastidious
Difco, Inc.
agar base
microorganisms
Nutrient agar
Oligotrophic
Difco, Inc.
diluted 1:100
microorganisms
R2A
Heterotrophic
Reasoner and
microorganisms in
Geldreich,
potable water
1985
Actinomycetes
Actinomycetes from
Difco, Inc.
isolation agar
soil or water
Low tryptone
Cytophaga/
E.
yeast extract
Myxobacteria
Leadbettera
M9 minimal
Auxotrophic mutants of Sambrook et
E. coli
al., 1989
Ammonia and
Methylotrophs
Patt et al.
nitrate minimal
1974
salts medium
Used under anaerobic conditions:
Medium
Designed to
Source
isolate/enumerate
Nutrient agar
Heterotrophic
Difco, Inc.
diluted 1:100,
nitrate-reducing/
R2A, M9 minimal
denitrifying bacteria
with 50 mM KNO3Basal salt medium Methanogenic archaea
C.M. Pluggea
with H2/CO2,
methanol,
acetate, and
fructose
Basal salt medium Acetogens and
C.M. Pluggea
with H2/CO2,
sulfate-reducers
methanol,
acetate,
fructose, BESb,
and 20 mM Na2SO4c
a
1999 Microbial Diversity Course, Woods Hole, MA.
b
BES (bromoethanesulfonic acid) inhibits the growth of
methanogenic archaea at a concentration of 5 mM.
C
Na2SO4 was added to cultures to facilitate the growth of
sulfate-reducing bacteria.
154
plating on agar-solidified M9-glucose and R2 media.
Three
isolates from the M9-enrichment culture, designated V15,
V18 and V19, that formed colonies with reproducibly
different morphologies were further investigated.
They
were all resistant to β-lactam antibiotics, and sensitive to
gentamycin, tetracyclin and neomycin but V19 alone was also
resistant to erythromycin.
Surprisingly, all three had the
same 16S rDNA sequence, indicating a close phylogenetic
relationship to Brachybacterium conglomeratum (Table 5.2;
Fig. 5.2A), and all of the single colony isolates
investigated from the R2 enrichment culture similarly also
had this Brachybacterium-related 16S rDNA sequence.
Media inoculated with core section 3593 melt water and
incubated for at 40 C under conditions used to enrich for
acetogenic bacteria and methanogenic archaea, appeared to
contain growth after 9 months.
These cultures contained
very long filaments, observed initially by light microscopy
and then confirmed by SEM (Fig 5.3), ranging from 100 to
300 µm in length, that were not present in cultures
incubated at 250C or in negative control cultures.
analyses have not detected methane or organic acid
155
To date
Table 5.2 Bacteria isolated from deep Vostok ice core
section 3593.
Isolate
V15
GenBank
accession
number
AF324202
Sequence
alignment
# of
base
pairsa
1454
%
identityb
99.4
99.8
V21
AF324199
1409
99.0
V22
AF324200
1485
99.2
99.3
V23
AF324201
1406
98.2
a
Nearest
phylogenetic
neighbor (GenBank
accession no;
origin)
Brachbacterium
conglomeratum
(X91030; cheese)
Guliya 500K-14
(AF395037; Guliya
ice core)
Sphingomonas sp.
(AB033945; not
available)
Paenibacillus
amylolyticus
(D85396; soil)
Unidentified
bacterium
(AJ223453; not
available)
Methylobacterium
sp. (Z23156;
biofilm on cooling
fan)
The number of 16S rDNA nucleotides used for the
alignment.
b
The percentage identity with the 16S rDNA sequence of the
nearest phylogenetic neighbour.
156
Fig. 5.2
Phylogenetic analysis and scanning electron
micrographs of bacterial isolates from core section 3593.
A. The 16S rDNA sequences obtained from single-colony
isolates, corresponding to nucleotides 27±1492 of the E.
coli 16S rDNA, were aligned based on secondary structure
using the ARB software package (Strunk et al., 1998), and a
phylogenetic tree was created with maximum likelihood
using a 1321 nucleotide mask of unambiguously aligned
positions and using FASTDNAML (Olsen et al., 1994).
Bootstrap values generated from 100 replicates using the
maximum parsimony method are shown at the nodes.
Evolutionary distance is defined as the number of fixed
nucleotide changes per position. The scale bar indicates
0.1 fixed substitutions per nucleotide position. Isolates
V21 and V23 position within the a-subdivision (a) of the
proteobacteria, V15 in the high-G+C-containing Grampositive (GP) group, and V22 in the low-G+C-containing GP
group. GenBank accession numbers and the percentage
identity of the corresponding 16S sequence with that of the
most similar Lake Vostok isolate are listed in
parentheses. Ice core isolates from Guliya (China) and
Sajama (Bolivia), and from Taylor Dome (TD), Canada glacier
(CanClear) and Siple Dome (SIA) in Antarctica are listed.
These are designated by their geographic origin, age of the
ice in years or thousands (k) of years and strain number,
e.g. Guliya500k-78 is strain no. 78 isolated from Guliya
glacial ice that was <500 000 years old.
B. Scanning electron micrographs of cells from cultures of
V15, V21, V22 and V23.
157
A
Figure 5.2A
158
Bacillus longisporus (AJ223991, 97.6%)
Paenibacillus amylolyticus (D85396, 99.2%)
Guliya50-TB9 (AF395027, 98.1%)
100 96 V22 (AF324200)
Low G+C GP
Guliya500k-78 (AF395033, 93.8%)
73
Sajama100-2B (AF395029, 93.6%)
100
Guliya200-C15 (AF395028, 94.2%)
90 Paenibacillus illinoisensis (D85397, 96.5%)
100
100 Brachybacterium tyrofermentans (X91657, 97.1%)
Brachybacterium sp. in sea ice brine (AF041790, 97.0%)
69
High G+C GP
V15 (AF324202)
100
100 Brachybacterium conglomeratum (X91030, 99.4%)
Brachybacterium faecium (X91032, 98.2%)
100
TD1.8k-4
(AF395030,
94.5%
)
100
Guliya500k-15 (AF395034, 94.9%)
100
94 Methylobacterium sp. (Z23156, 98.2%)
V23 (AF324201)
100 unidentified bacterium (AJ223453, 99.3%)
87
100 Guliya500k-5 (AF395035, 95.6%)
Methylobacterium fujisawaense (AJ250801, 95.7%)
76
Guliya500k-3 (AF395036, 92.8%)
a
57
SIA1k-1A1 (AF395032, 94.7%)
68
CanClear1 (AF395038, 94.5%)
TD4.2kB-5 (AF395031, 97.4%)
100 Sphingomonas sp. from Antarctic soil (AF184221, 97.1%)
99
Guliya500k-14 (AF395037, 99.8%)
60
V21 (AF324199)
0.10
Sphingomonas sp. (AB033945, 99.0%)
100
B
V21
V15
2 mm
2 mm
V22
V23
5 mm
Figure 5.2B
2 mm
159
2 mm
50 mm
2 mm
100 mm
Figure 5.3 Filaments in anaerobic enrichments. After 9
months of incubation at 4oC, filaments ranging in length
from 100 to 300 mm were observed in several anaerobic
enrichment cultures, but were not present in identical
cultures incubated at 25oC, nor in negative control
cultures. No methane or organic acid production was
observed, and attempts to isolate these filaments failed.
160
production, indicative of methanogenic and acetogenic
activity, respectively, and attempts to isolate and
characterize these filaments were not successful.
Colony isolations
Particulates, collected by filtration from 100-150 ml
of melt water, were resuspended at ~30-fold the original
concentration, and aliquots (200 µl) of the resuspended
materials were spread on the surface of agar-solidified
media.
A total of three colonies were obtained, all on
agar-solidified R2 medium on plates incubated aerobically
at 25oC.
The growth of colonies was never observed on any
other agar-solidified medium (see Experimental procedures),
even though the plates were incubated for >3 months at both
4oC and 25oC.
On subculture, all three isolates, designated
V21, V22 and V23, grew most rapidly on R2 medium at 25oC,
although V22 and V23 also grew at temperatures as low as
4oC, and V21 grew as low as 10oC.
Based on their 16S rDNA
sequences, V21, V22 and V23 are most closely related
phylogenetically to Sphingomonas, Paenibacillus and
Methylobacterium species, respectively (Table 5.2; Fig.
5.2A).
161
16S rDNA amplification and sequencing
Populations of small subunit rDNA molecules were
amplified directly from core 3593 melt water by using
universal and Bacteria-specific primers, but not when
Archaea-specific primers were used. Individual DNA
molecules were cloned from these populations and sequenced,
revealing the presence of bacterial 16S rDNAs from five
different phylogenetic lines of descent (Table 5.3; Fig.
5.4).
Sequence pA419 originated from an α-proteobacterium
whose nearest cultured neighbor was isolated from Lake
Baikal in Russia, and pA419 is also ~86% similar to a 16S
rDNA sequence retrieved by Benson et al. (2000) from an
Antarctic lake.
Although perhaps not so striking, in terms
of such very cold freshwater environments, the other 16S
rDNAs amplified from the 3593 melt water also have
freshwater-isolate relatives.
Specifically, sequences
pA3178 and pA42B412 are from β-proteobacteria and are most
similar to the 16S rDNA sequences of an Aquabacterium and a
facultative hydrogen-autotroph, formerly designated
Pseudomonas saccharophila, respectively.
Sequence pA47 is
93.6% identical to that of the 16S rDNA of Sphingobacterium
heparinum, a member of the Cytophaga/Flavobacterium/
Bacteroides lineage, and sequences pD12 and pD4 are 93.5%
162
Table 5.3
water.
Clone
(no.
obtained)a
16S rDNA molecules amplified from core 3593 melt
GenBank
accession
number
Sequence
alignment
# of
base
pairsb
%
identityc
pA3178
(4)
AF324205
842
98.7
pA419
(3)
AF324207
896
96.4
pA42B412
(12)
AF324206
839
99.0
pA47 (1)
AF324208
827
93.6
pD4 (2)
AF324203
845
98.9
pD12 (1)
AF324204
838
93.5
Nearest
phylogenetic
neighbor (GenBank
accession no;
origin)
Aquabacterium sp.
(AF089858;
drinking water
biofilm)
Lake Baikal
isolate (AJ001426;
400 mbsd in Lake
Baikal, Russia)
Pseudomonas
saccharophilia
(AB021407; not
available)
Sphingobacterium
heparinum (M11657;
not available)
Rubrobacter
xylanophilus
(AJ243871; hot
spring)
Alkalibacterium
olivoapovlitic
(AF143513; olive
wash water)
a
The number of individual clones sequenced that had this
sequence.
b
The number of 16S rDNA nucleotides used for the alignment.
c
The percentage identity with the 16S rDNA sequence of the
nearest phylogenetic neighbour.
d
mbs=meters below surface
163
Fig. 5.4
Phylogenetic analysis of the 16S rDNAs
amplified from core section 3593.
Sequences, PCR-amplified from melt water, that correspond
to nucleotides 515-1392 of the E. coli 16S rRNA-encoding
gene were designated pA419, pA3178, pA42B412, pA47, pD4 and
pD12. The phylogenetic tree was created with maximum
likelihood using an 813 nucleotide mask of unambiguously
aligned positions (Olsen et al., 1994). Bootstrap values
generated from 100 replicates using the maximum parsimony
method are shown at the nodes. Evolutionary distance is
defined as the number of fixed nucleotide changes per
position. The scale bar indicates 0.1 fixed substitutions
per nucleotide position. GenBank accession numbers are
provided in parentheses.
164
Figure 5.4
Leptothrix discophora (L33974)
99 pA42B412 (AF324206)
Pseudomonas saccharophila (AB021407)
strain B4 (AF035050)
76
pA3178 (AF324205)
89
Aquabacterium sp. (AF089858)
b
97
165
100
72
100
98
100
100
Antarctic lake clone CLEAR-13 (AF146237)
pA419 (AF324207)
100
Lake Baikal isolate (AJ001426)
Holospora obtusa (X58198)
Carnobacterium funditum (S86170)
Carnobacterium sp. (AF076637)
Carnobacterium alterfunditum (L08623)
pD12 (AF324204)
55
Dolosigranulum pigrum (X70907)
75
Alloiococcus otitis (S83561)
Alkalibacterium olivoapovlitic (AF143513)
Rubrobacter radiotolerans (X87134)
91
environmental clone #0319-7H2 (AF234151)
100
0.10
pD4 (AF324203)
Rubrobacter xylanophilus (AJ243871)
Sphingobacterium multivorum (D14025)
97
Flavobacterium mizutaii (M58796)
100
pA47 (AF324208)
100
Sphingobacterium heparinum (M11657)
a
Low G+C GP
High G+C GP
C/F/B
and 98.9% identical to the 16S rDNA sequences of
Alkalibacterium olivoapovlitic and Rubrobacter
xylanophilus, respectively, positioning them within the
low- and high-G+C Gram positive groups.
Extensive precautions were taken, but the
possibilities of contamination and of DNA molecules being
generated artifactually during the PCR amplifications were
always serious concerns.
Infrequently, an amplicon was
generated in a negative control reaction, and when these
were cloned and sequenced they had sequences almost
identical to a 16S rDNA sequence that has been shown
previously to arise in PCR controls (Cisar et al., 2000).
This sequence (GenBank Accession number AF195876) is
related to 16S rDNA sequences from γ-proteobacterial
pseudomonads and is not closely related to any of the
experimental sequences used in constructing Figs 5.2 and
5.4.
SEM analysis of Lake Vostok Accretion ice
Scanning electron microscopic analysis of 0.2 µm
filtered accretion ice section 3593 revealed the presence
of several morphotypes (Fig 5.5), and little, if any
166
1 mm
2 mm
1 mm
1 mm
Figure 5.5
Scanning electron micrographs of apparently prokaryotic cells, retained on the surface of
a 0.2 µm isopore (Millipore) filter after concentration of core section 3593 melt water.
167
inorganic or organic detritus.
Too few cells were present
on the filter sections analyzed to obtain a reliable cell
count.
Discussion
Based on the estimates of Siegert et al. (2000), the
48 cm ice core (section 3593) sampled in this study
accreted over a period of 10-25 years.
It did not contain
any macroscopically-visible solid inclusions, and therefore
most likely formed over a relatively deep portion of the
lake (Jouzel et al. 1999).
Scanning electron microscopy of
materials filtered from core 3593 melt water revealed
little inorganic debris, and although sparsely distributed,
a number of particles were identified with sizes and
morphologies consistent with bacterial cells (Fig 5.5).
Epifluorescence microscopy of DNA-stained samples also
revealed the presence of low cell numbers (2.3 x 103 and 2-3
x 102 cells ml-1) in melt water from flanking Vostok ice
core sections 3590 and 3603, respectively (Priscu et al.
1999; Karl et al. 1999).
Presumably, therefore, only a
very small percentage of the cells present in core 3593
were recovered, and it is perhaps noteworthy that V21, V22
and V23 are related, although not identical, to species
168
recovered previously from both polar and non-polar glacial
ices (Chapter 3).
These and related isolates survive
repeated cycles of freezing and thawing, even though V22 is
the only member of a bacterial group (Paenibacillus) that
is known to differentiate into cells (endospore) that
specifically facilitate airborne transport, resist
desiccation and provide long-term survival under non-growth
conditions (Cano and Borucki 1995; Vreeland et al. 2000).
The results obtained predict that representatives of
at least five bacterial lineages are likely to be present
in Lake Vostok, some of which are related, in terms of 16S
rDNA sequences, to isolates from other cold, potentially
very similar environments.
For example, sequence pD12 is
~92% identical to 16S rDNA sequences from two
Carnobacterium species that were isolated from ice-covered
Antarctic lakes (Franzmann et al. 1991; Bratina et al.
1998), and sequence pA419 clusters both with the 16S rDNA
sequence of an isolate recovered from 400 mbs of Lake
Baikal in Siberia and the sequence of an amplicon from a
frozen Antarctic lake (Fig. 5.4).
Extrapolations from
rDNA sequence similarities to similarities in life style
and physiology are clearly very tenuous, but these results
169
do argue that Lake Vostok probably contains bacteria
similar to species found in other permanently cold
environments.
170
CHAPTER 6
GENERAL DISCUSSION
The microorganisms recovered in this study from
glacial ice have endured desiccation, solar irradiation,
freezing, an extended period of no growth, and subsequent
thawing.
It is therefore not surprising that many of these
isolates belong to bacterial groups that form spores (Fig
3.4, 3.7, and 3.8), structures known to confer resistance
to environmental abuses.
Many also have thick cell walls
or polysaccharide capsules, and resist repeated cycles of
freezing and thawing (Fig 3.10).
The frequent isolation of
related genera from geographically different ice core sites
and from other frozen or permanently cold environments (Fig
3.5-3.8) suggests these are species adapted to surviving
freezing, and that persist under cold and non-growth
conditions.
Members of the genera Sphingomonas,
Acinetobacter, and Arthrobacter were often isolated from
glacial samples, and these are also the most frequently
isolated genera in enrichment surveys of terrestrial
171
subsurface environments (Balkwill et al. 1997).
This is
consistent with these genera having many species that can
survive for extended times under low nutrient, non-growth
conditions, and that similar survival strategies are in
effect in ice and in deep subsurface situations.
Ice cores from non-polar, low-latitude, high-altitude
glaciers generally contained more colony forming units
(Table 3.1) and a greater variety of bacterial species (Fig
3.4) than polar ices.
Similarly, the highest recovery of
viable bacteria from polar ice cores was obtained from
Antarctic regions adjacent to exposed soils and rock
surfaces of the McMurdo Dry Valley complex.
These results
are consistent with increased microbial deposition in
glaciers contiguous to environments that supply airborne
rock grains, soils, and biological particles.
Not
surprisingly, the highest numbers of bacteria were isolated
from sections of ice cores that were “dusty”, visibly
contaminated with macroscopic debris that presumably would
have transported and protected attached bacteria.
Abyzov
et al. (1998) also reported a correlation between the
number of total cells and the concentration of dust in the
Vostok core.
172
Analyses of DNA isolated directly from glacial ices
revealed that only non-polar ices containing
macroscopically visible particles possessed sufficient
biomass for DNA detection by slot blot hybridization
assays, and therefore nucleic acid-based quantitation
techniques and direct cell counting were not feasible.
The
information gathered on the microbiological content of
glacial ices was determined by more sensitive means,
specifically enrichment culturing (which can detect 1
viable cell) and the PCR.
Even with the amplification
potential of the PCR, isolates were often obtained from ice
samples for which no amplicons were subsequently generated
when extracted DNA was amplified from cells concentrated on
0.2 µm filters.
The very low biomass and possible presence
of PCR-inhibiting substances (Wilson 1997; Wintzingerode et
al. 1997) presumably limited the sensitivity of PCR
amplification in many of the ice cores samples examined.
Concentrating DNA released from cells in the melt water
during thawing and filtering provided more routinely
successful templates for PCR amplification than extractions
from cells concentrated on 0.2 µm filters.
It seems
possible that inhibitors may have been sufficiently diluted
in these filtrates, or removed during concentration in
173
these fractions, or that the quantity of released DNA in
these filtrates simply exceeded the DNA present in unbroken
cells.
It is remarkable that microorganisms can maintain
viability over hundreds of thousands of years trapped in
glacial ice.
The isolates obtained certainly appear to
possess features that might enhance their survival while
dormant, but the thermodynamic reality is that in the
absence of metabolic activity, cells must incur a
significant amount of macromolecular damage over such long
periods of time.
This point has often been raised in
discussions of reported microbial revivals from ancient
salt, amber, and permafrost (Gilichinsky et al. 1993; Cano
et al. 1995; Shi et al. 1997; Greenblatt et al. 1999;
Vreeland et al. 2000)
In contrast, however, to the
isolates from these once active environments that then
became impermeable geological materials, the microorganisms
deposited in glacial ice are most likely dormant and
sublethally injured, and then presumably still endure
thousands of years of additional damage while remaining
viable.
174
It was from this perspective that the experiments were
undertaken to determine if metabolic activity was possible
under conditions comparable to those in glacial ice.
DNA
and protein synthesis at -15oC were investigated to
determine if such macromolecular syntheses or possibly
repair were possible during apparently frozen storage.
According to the calculations of Price (2000), the supply
of organic carbon in the liquid veins within ice is
sufficient to support a small population of cells (~10-102
cells/cm3) for hundreds of thousands of years.
The evidence
presented here is consistent with macromolecular synthesis
occurring at ice temperatures in which water exists between
ice crystals, and on the surfaces of entrapped cells and
air bubbles (Fig 4.2-4.6).
Indirect evidence for microbial
activity in glacial ice was also obtained when analysis of
the air bubbles in cores from Vostok and Sajama revealed
isotopic fractionation profiles consistent with in situ
microbiological production of nitrous oxide and methane,
respectively (Sowers 2001; T. Sowers, personal
communication).
Geochemical anomalies attributed to
microbial activity in Greenland ice have also been reported
(Souchez et al. 1995; Souchez et al. 1998), and this issue
175
must now be experimentally addressed.
Perhaps
14
C-dating of
the microbial fraction in an ice core sequence might serve
as a practical first step.
Regardless of metabolic status, cells entombed in
glacial ice remain viable for >500,000 years, and the
possibility exists that microbes transported through ice
sheets have established unique ecosystems in the subglacial
environment.
Examination of an ice core recovered from
within the accretion zone of Lake Vostok has provided a
glimpse of the microbial inhabitants in a environment
isolated from the surface for at least 0.5 million years,
and perhaps as long as the continent has been glaciated.
Several of the isolates recovered from the lake water are
close phylogenetic relatives of bacteria commonly recovered
in this study of glacial ice.
The water in Lake Vostok
originates from the overlying glacier (Jouzel et al. 1999),
and our results are consistent with models of circulation
within the lake predicting the accreted ice is formed from
low-salinity water at the ice ceiling, which is composed
chiefly of the most recent glacial melt to enter the lake
(Siegert et al. 2001).
176
If a flourishing microbial ecosystem were found to
exist within the water or sediment of this subsurface
environment, it would represent one of the most extreme in
the biosphere, and ice core studies at OSU and by others
(Karl et al. 1999; Priscu et al. 1999) have made a
preliminary microbiological analysis possible.
Our results
predict that Lake Vostok contains viable bacteria, and it
seems noteworthy that several are most closely related to
species identified in glacial ice (Fig 5.2A), and
additional 16S rDNA sequences obtained are most similar to
species common to freshwater ecosystems (Fig 5.4).
Identifying bacteria recovered from glacial ice cores
originating from worldwide locations has provided a way to
examine the influence of geography and climate change on
the composition of entrapped species, observe common
survival strategies, investigate longevity while frozen,
and explore potential habitats for activity in the glacial
and subglacial environment.
Studying microbial survival
and preservation under some of the most extreme conditions
on the planet is intrinsically interesting, but has also
yielded clues about potential refuge environments for life
during periods of global ice cover, such as those predicted
by the snowball Earth hypothesis (Hoffman et al. 1998;
177
Kirshvink 1992).
The results obtained are clearly relevant
to discussions of the likelihood of microbial survival in
frozen extraterrestrial environments and during
interplanetary transport.
178
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