Download Desiccation Tolerance of Prokaryotes: Application of Principles to

INTEGR. COMP. BIOL., 45:800–809 (2005)
Desiccation Tolerance of Prokaryotes: Application of Principles to Human Cells1
MALCOLM POTTS,2 STEPHEN M. SLAUGHTER, FRANK-U. HUNNEKE, JAMES F. GARST,
AND
RICHARD F. HELM
Virginia Tech Center for Genomics, Department of Biochemistry, Virginia Tech, Blacksburg, Virginia 24061
SYNOPSIS.
The loss of water from cells is a stress that was likely imposed very early in evolution. An
understanding of the sensitivity or tolerance of cells to depletion of intracellular water is relevant to the
study of quiescence, longevity and aging, because one consequence of air-drying is full metabolic arrest,
sometimes for extended periods. When considering the adaptation of cells to physiological extremes of pH,
temperature or pressure, it is generally assumed that evolution is driven toward optimum function rather
than maximum stability. However, adaptation to desiccation has the singular and crucial distinction that
dried cells do not grow, and the time the cell is dried may represent the greater part of the life (the time
the cell remains viable) of that cell and its component macromolecules. Is a consideration of ‘‘function’’
relevant in the context of desiccated cells? The response of prokaryotic cells to desiccation, and the mechanisms they employ to tolerate this stress at the level of the cell, genome and proteome are considered.
Fundamental principles were then implemented in the design of strategies to achieve air-dry stabilization of
sensitive eukaryotic (human) cells. The responses of the transcriptomes and proteomes of prokaryotic cells
and eukaryotic cells (yeast and human) to drying in air are compared and contrasted to achieve an evolutionary context. The concept of the ‘‘desiccome’’ is developed to question whether there is common set of
structural, physiological and molecular mechanisms that constitute desiccation tolerance.
INTRODUCTION
Aside from the importance of understanding the role
of water (or lack of it) in the vitality of cells, and the
intellectual challenge of doing so, a knowledge of how
to dry cells (or their components), and to keep them
alive for long periods, is of intense societal and commercial interest. The worldwide market in stabilization
of cells and cell products is estimated to be some $500
billion (Brockbank, 2000). In contrast, recent world
events have now placed the inherent desiccation tolerance of many pathogenic bacteria, such as Staphylococcus aureus, in a new context in view of their
suitability as biowarfare agents. With what now seems
prophetic irony it was observed a decade ago that:
of prokaryotic cells is provided in recent studies (Potts,
1994, 1999, 2002, 2001; Billi and Potts, 2000, 2002;
van de Mortel and Halverson, 2004). It is clear that
much of the mechanistic basis for the concerted response of cells to water deficit remains cryptic. Some
unifying concepts and principles to do with desiccation
tolerance are developed in this brief account through
consideration of data from model prokaryotic and eukaryotic systems.
Mechanisms of desiccation damage
It seems that the energy changes involved in the loss
of cell viability upon drying are small and that even
desiccation-sensitive cells can cope with the removal
of a substantial fraction of their free water (Vf ; Potts,
1994). At the level of the bacterial community, gross
changes associated with air-drying include a change
(usually increase) in surface area, shrinkage, salt precipitation, change in texture, change in shape and
change in color through oxidation of pigments. At the
level of the cell, there is shrinkage of capsular layers,
increase in intracellular salt concentrations, crowding
of macromolecules, changes in volumes of cell compartments, changes in biophysical properties (e.g., surface tension), reduced fluidity (increased viscosity),
damage to external layers (e.g., pili, membranes), acquisition of static charge, and change in physiological
processes (e.g., growth arrest, 1994). DNA in metabolically active cells is thought to exist in the fully
hydrated B form and to have properties similar to
those measured for DNA in solution. The limited
chemical stability of DNA under these conditions is
cited as the major determinant of cell survival (Potts,
1999, 2001). Browning reactions presumably lead to
modification of nucleic acids and proteins during storage when dry. The Maillard (browning) reaction refers
to the formation of dark-brown products, originally re-
‘‘As the research community is aware, post offices
worldwide are unwittingly in the business of distributing samples of bacteria spotted on paper filters
and enclosed within envelopes after preparation. . . ’’ (Potts, 1994).
Many of the studies concerned with the mechanistic
basis for desiccation tolerance deal with prokaryotic
cells (other eukaryotic systems are described in accompanying papers of this issue). In fact, given the
historic and commercial importance of dried Saccharomyces cerevisiae (yeast), and the genetic and molecular biology resources available for this eukaryote
(http://www.yeastgenome.org/), the singular lack of
any global analysis of its response to desiccation (until
most recently; Singh et al., unpublished data) seems
anomalous. Background on the desiccation tolerance
1 From the Symposium Drying Without Dying: The Comparative
Mechanisms and Evolution of Desiccation Tolerance in Animals,
Microbes, and Plants presented at the Annual Meeting of the Society
for Integrative and Comparative Biology, 4–8 January 2005, at San
Diego, California.
2 E-mail: [email protected]
800
PROKARYOTE DESICCATION TOLERENCE: APPLICATION
TABLE 1.
1.
2.
3.
4.
TO
HUMAN CELLS
801
Strategies to achieve desiccation tolerance in otherwise sensitive cells.*
Selection—training through multiple exposures to stress (long term)
Accumulate intracellular protective agents—through uptake or via genetic engineering
Use extracellular protective agents—as additives or secretion via genetic engineering
Manipulate cell metabolism/physiology—prior to exposure to stress
* In practice more than one (or all) of these strategies is often needed, together with an understanding of the optimum conditions of
temperature (drying and rewetting), rates of drying (and rewetting) and conditions of storage. A further requisite is the ability to make a
comprehensive assessment of the recovery of the cells upon rewetting (viability, mutation rate, recovery of homeostasis etc.).
ferred to as melanoidins, in mixtures of sugars and
amino acids. The principal mechanism involved in the
formation of Maillard products is the condensation of
dicarbonyl compounds on reducing sugars with primary amines on proteins and nucleic acids. Metal-catalyzed Haber–Weiss and Fenton reactions, reactive oxygen species and free radicals, might also contribute
to DNA modification and ultimately killing of cells
(Potts, 1994). The mechanisms that lead to oxidative
damage of DNA, such as those mediated by Fenton
reactions, are complex and in many cases the details
remain lacking. Five-membered hydantoin rings in
DNA derive from the oxidative decay of six-membered pyrimidines and accumulate slowly as a function
of time and oxygen exposure. The occurrence of such
modified bases is problematic because neither Taq
DNA polymerase nor any other polymerase used in a
PCR assay can copy these damaged residues. Many of
the oxyradical-mediated processes that contribute to
nucleic acid damage also apply to proteins (Potts,
2001). However, it is hard to make generalizations
about protein stability in the context of desiccation.
Some proteins, such as those associated with pigment
antenna complexes, are very sensitive to the ravages
of oxidative damage. Others, such as elastin, show remarkable resistance to autoclaving, and industrialstrength acid and base. In addition, there are enzymes,
such as superoxide dismutase (SOD), that not only retain their structure but also remain fully active after
decades of desiccation (Shirkey et al., 2000).
The eoanhydrobiotes
For cells that are sensitive (or tolerant!; see above)
to desiccation four different approaches are available
to attempt to improve the capacity for dry storage in
air, at ambient temperature (Table 1). In large part,
these approaches derive from an understanding of how
a small but diverse group of organisms, the anhydrobiotes (Crowe and Crowe, 2000), withstand the removal of virtually all of their cellular water. The water
content in these cells is on the order of ;0.02 g H2O
per g cell solids), and this level is a prerequisite if
viability is to be maintained during long-term storage.
Of the four approaches, it is appropriate to consider
the first one (Table 1) here since it represents natural
selection, with its attendant evolutionary consequences.
There are many uncertainties and speculations about
the conditions under which life arose on Earth. It is
generally agreed that the atmosphere contained little
to no oxygen initially (Kasting, 1993), and that hydrogen sulfide and alkylthiols provided free radical scavenging mechanisms (Timmins et al., 2001). What
seems certain, however, is that primitive prokaryotes
(eoanhydrobiotes) were subjected to desiccation and
rehydration events, which suggests that desiccation
tolerance arose very early in evolution (Potts, 1995).
The dehydration, and subsequent rehydration, of a bacterial cell generates a continuum of states in a system
that is far from equilibrium (even without invoking a
consideration of glass transition theory). The consequences of the superimposition of water deficit, thermal stress, UV irradiation, and nutrient limitation
would certainly fulfill the paleological criteria thought
to have shaped early life. One important qualification
to the notion of an ancient origin of desiccation tolerance in the prokaryotes is that the air-drying of eoanhydrobiotes involved no, or minimal, oxidative damage. Did the ‘‘early’’ mode of desiccation tolerance
change as the atmosphere became hypoxic, and then
aerobic, following the advent of oxygenic photosynthesis? An important consideration in this context is
that a rise in oxygen levels, with a growing ozone
layer, led to diminished exposure to UV-irradiation
(Towe, 1996). One further attenuation of desiccation
tolerance in bacterial cells may have occurred in pathogenic bacteria that evolved to resist the stresses of
passage through host immune systems e.g., the lactoperoxidase system.
Ancestors of the eoanhydrobiotes
Adaptation to environmental oxygen is thought to
have arisen in microbial mat communities (Hoehler et
al., 2001), and this must have attenuated greatly the
cellular response to desiccation. Modern-day counterparts of these mat communities are widespread, especially in intertidal and hypersaline lagoon environments (Stal, 2000). Why is one of the key groups of
organisms in the Precambrian, the Cyanobacteria, still
one of the most widespread and important groups of
microorganisms today? Almost certainly their success
stems, in large part, from a capacity to withstand multiple, superimposed, environmental stresses, namely
desiccation, UV irradiation, heavy metals, temperature
and nutrient availability (Fig. 1A; Whitton and Potts,
2000). Not surprisingly these responses are interlinked
in many bacterial populations making it difficult to
draw a clear demarcation between them (Ramos et al.,
2001; Archuleta et al., 2002). For example, while the
extracellular polysaccharide (glycan) of the terrestrial
802
MALCOLM POTTS
FIG. 1. A. The terrestrial cyanobacterium Gloeocapsa alpina grows
on exposed surfaces and is subjected to desiccation, high levels of
UV-irradiation, temperatures as high as 958C, and heavy metals. The
pigmented cells are enveloped in a thick extracellular polysaccharide
(arrows). B. Changes in the proteome of desiccated cyanobacterium
Nostoc commune CHEN/1986 occur following rehydration. Panels
are from separate 2D SDS-PAGE gels (silver stained) and processed
as described (Potts, 1986).
cyanobacterium Nostoc commune provides protection
to cells during desiccation, it also generates potentially
damaging free radicals when exposed to UV irradiation (Shirkey et al., 2000). As a consequence cells secrete an Fe-SOD, and the synthesis of the enzyme is
induced by desiccation as well as UV-irradiation (Shirkey et al., 2000).
In general the most desiccation tolerant non-spore
forming bacteria tend to be Gram positive. Many include forms that also accumulate Mn(II), have a high
Mn(II) to Fe ratio and are radiation resistant (Daly et
al., 2004). While cyanobacteria have a Gram negative
type cell wall, they also show phylogenetic affinity
with Gram positive bacteria and are markedly tolerant
of both desiccation and gamma radiation (Potts, 1999;
Billi et al., 2000). Numerous technologies were used
to improve the stability of Gram negative bacteria cells
using approaches two and three listed in Table 1 (Linders, 1996; Kets, 1997; Manzanera et al., 2004a, b).
The effectiveness of different additives is, however,
often marginal during very long times of storage (Potts
and Goldsmith, unpublished data).
In a recent study an analysis was made of water
ET AL.
deprivation–controlled (wdc) gene loci in Pseudomonas putida (van de Mortel and Halverson, 2004). Induction of the wdc loci may contribute to desiccation
tolerance by maintaining the structural integrity of the
cell envelope, macromolecular biosynthesis and capacity for energy generation. It was suggested that maintenance of the integrity of the cell envelope was essential to counter the detrimental effects of matric
stress on protein folding. The importance of maintaining a fluid membrane during water stress was suggested to be critical in view of observations of the
degradation of damaged phospholipids, increased expression of locus wdc200, and modification in fatty
acid composition as evidenced by increased cis-totrans isomerase activity (Halverson and Firestone,
2000). Induction of alginate biosynthesis (wdc167;
algA) on PEG 8000-amended medium (van de Mortel
and Halverson, 2004) provided further support to the
suggestion that extracellular polysaccharide production
is a global mechanism for survival in low-water-content habitats (Hart et al., 1999; Helm et al., 2000).
Mattimore and Battista first proposed that the extreme radioresistance of Deinococcus radiodurans was
a consequence of its capacity for desiccation tolerance
(Mattimore and Battista, 1996). Indeed subsequent
work demonstrated that of 72 genes that were upregulated during the first hour after a sub-lethal dose of
ionizing radiation, 33 of these loci were highly induced in cultures recovering from desiccation (Tanaka
et al., 2004). If there is a tangible link between gamma-radiation resistance and desiccation tolerance then
these studies provide comprehensive data on possible
mechanisms involved in the recovery from air-drying.
Mechanisms employed by D. radiodurans to recover
from radiation-induced DNA (and possibly desiccation) damage include the synthesis of DdrA, a protein
with an affinity for 39 ends of single stranded DNA
that protects those ends from nuclease digestion (Harris et al., 2004). Among the cyanobacteria, only the
genome of Gloeobacter violaceus contains a gene
(glr0712) whose predicted product shares significant
sequence similarity (40%) with DdrA. Interestingly, G.
violaceus was isolated from exposed limestone in the
Alps, near Kastanienbaum, Switzerland, in a habitat
exposed to intermittent wetting and drying and solar
radiation. In addition to DNA protection, and implementation of repair mechanisms following desiccation
(Tanaka et al., 2004), additional enzyme activities
must be required for the recovery since long-term storage of air-dried cells leads to covalent modification of
DNA, which must be removed before the DNA can be
replicated (Shirkey et al., 2003). High intracellular
Mn(II) concentrations are thought to facilitate the gamma-radiation and desiccation resistance of D. radiodurans through scavenging of superoxide ions (O22;
Daly et al., 2004). Mn(II) is not thought to participate
in Fenton reactions and must supplement, in a positive
fashion, the activities of superoxide dismutases that
figure so prominently (see below) in the desiccation
response (Shirkey et al., 2000).
PROKARYOTE DESICCATION TOLERENCE: APPLICATION
TABLE 2.
TO
HUMAN CELLS
803
Exogenous addition of Wsp confers stability on air-dried E. coli.
Conditions
Viable colonies per
plate/100-ml aliquot
Control; no desiccation, plated immediately
Control (1BSA); rehydration after 3 days of desiccation; controlled 2-step rehydration protocol
1Wsp; rehydration after 3 days of desiccation; no temperature control at rehydration
1Wsp; rehydration after 3 days of desiccation; controlled 2-step rehydration protocol
600 6 87
0
12 6 5
488 6 205
The partial acid proteome of cyanobacterium Nostoc
commune
A 36-kDa acidic water stress protein (WspA) is the
most abundant protein in cyanobacterium N. commune; it is secreted and accumulates in the glycan extracellular matrix (Scherer and Potts, 1989; Hill et al.,
1994). A description of the cloning of wspA, and a
study of its mode of expression in response to desiccation and UV-irradiation, will appear elsewhere
(Wright et al., unpublished data). Proteins in desiccated N. commune (;0.05 g H2O per g cell solids) remain
stable during decades of storage through as yet undefined mechanisms. However, upon rehydration, the
proteome undergoes complex and selective changes including a striking loss of proteins within the first minute (Fig. 1B). The staining character and resolution
of WspA changes with time as a consequence of its
interactions with the extracellular matrix; note the
presence of SodF remains more or less constitutive.
Use of Wsp for air-dry stabilization of Escherichia
coli
In view of the prevalence of Wsp in the extracellular
matrix of N. commune, we investigated if exogenous
addition of Wsp may stabilize sensitive cells during
desiccation. Escherichia coli strain DH10B cells were
transferred from a 2708C glycerol permanent to a
LuriaBertani (LB) agar plate and incubated at 378C,
overnight. A single colony was used to inoculate 3 ml
of LB medium at 378C and the OD600 was monitored
over 2–3 hr until an OD of ;0.45–0.50 was reached.
The cells were recovered by centrifugation then resuspended in M9 medium (at room temperature) lacking
glucose. The suspension was diluted 1023 and 1024 in
different aliquots of M9 medium lacking glucose. The
1024 dilution generates around 150 colonies when 10
ml of the dilution are plated immediately on a LB agar
plate and grown overnight at 378C. 10 ml of the dilution was placed in the center of a plastic Petri dish and
mixed with 50 ml of recombinant Wsp (100 mg ml21)
and 40 ml of M9 medium lacking glucose (Table 2)
all at room temperature (total 100 ml). Separate controls were made with the same concentration of bovine
serum albumin (BSA) and with suspensions that
lacked either Wsp or BSA. The lid of the Petri dish
was left partly open; the plate was transferred to the
desiccation cabinet.
A two-step rehydration protocol was developed.
First, the Petri dish (with lid partially open) was transferred to a sterile glass desiccator and placed above a
reservoir of water. The water was vacuum-filtered to
remove air and the desiccator was sparged with nitrogen prior to its sealing and pre-equilibration. The desiccator was covered to exclude light, and stored at
378C, overnight. Second, the desiccator was removed
to a clean-bench; plates were removed and cells were
resuspended in 100 ml of sterile distilled water at
378C, and plated on preheated LB agar plates (378C)
and grown overnight at 378C. Cells desiccated (;0.02
g H2O per g cell solids) for 3 days under the specified
conditions in the presence of BSA failed to survive
upon rehydration using the 2-step rehydration protocol
(Table 2). In contrast there was an approximately 80%
survival rate when cells were desiccated in the presence of Wsp followed by rehydration under the same
conditions. If the two-step rehydration protocol was
not followed and cells were rehydrated directly following storage, the survival rate dropped to approximately
12% irrespective of the presence of Wsp. These data
suggest that the uptake of water in a reviving organism
is somehow controlled, with rapid uptake being deleterious.
A modular approach to achieving desiccation
tolerance in sensitive human cells
Recent attempts to dry and revive nucleated mammalian cells employed the non-reducing disaccharide
trehalose, either as an additive or, through its endogenous de novo synthesis following cell transformation
with otsAB genes (de Castro and Tunnacliffe, 2000;
Guo et al., 2000; Matsuo, 2001; Puhlev et al., 2001;
Huang and Tunnacliffe, 2004). These different approaches, however, achieved limited success with variable cell viability for only very short periods of dry
storage. A common feature of the experimental design
for these studies was the use of cells grown in twodimensional (monolayer) culture.
In an attempt to achieve stabilization of desiccation
sensitive human cells, approach three (Table 1) was
followed by adding the purified extracellular glycan of
cyanobacterium Nostoc commune to human embryonal
kidney 293 (HEK293) cells (Bloom et al., 2001). This
approach lead to structural preservation of the cells
during several weeks of desiccation (;0.02 g H2O per
g cell solids), although the cells were not viable. To
better understand the loss of viability of HEK293 we
performed a transcriptional analysis during the initial
stages of air-drying of the cells. Cells were grown as
2D-monolayer cultures under standard conditions, in
the presence of 6 mg ml21 glycan, 50 mM trehalose
804
MALCOLM POTTS
ET AL.
TABLE 3. Transcriptional analysis of human 2-dimensional HEK293 cells grown in the presence of sucrose, trehalose and cyanobacterial
glycan and following drying in air for 45 minutes.*
Probe set name
35303pat
32609pat
37018pat
153pfpat
36347pfpat
35576pfpat
36629pat
32125pat
37724pat
33702pfpat
33845pat
33703pfpat
38617pat
34270pat
41084pat
39934pat
40457pat
40767pat
32749pspat
1461pat
36040pat
38324pat
32676pat
36979pat
37210pat
32755pat
32588pspat
41476pat
33707pat
36987pat
37178pat
39180pat
948pspat
G1 vs. G2b fold change
Description
6.29
4.73
4.44
4.36
3.79
3.67
3.61
3.46
3.31
3.24
3.09
3.08
23
23.04
23.05
23.06
23.09
23.11
23.12
23.12
23.4
23.41
23.42
23.43
23.63
23.67
23.77
23.86
24.05
24.15
24.23
25.77
25.92
INSIG1-regulator of lipid synthesis
Histone H2A.1
Histone H1D
Histone H2B
Histone H2B.2
Histone H2B
unknown
unknown
c-myc oncogene
PCK1 phosphoenolpyruvate carboxykinase
unknown
PCK1 phosphoenolpyruvate carboxykinase
LIMK-2-serine/threonine kinase
U6 small nuclear RNA associated
unknown
small GTP-binding protein Rab36
unknown
lipoprotein-associated coagulation inhibitor (LACI)
unknown
MAD-3 encoding ikB-like activity
SH3-binding domain and glutamic acid-rich protein
unknown
methylmalonate semialdehyde dehydrogenase
glucose transporter-like protein III GLUT3
neurofilament-66
smooth muscle alpha-actin
ERF-2; regulating response to growth factors
unknown
cytosolic phospholipase A2-gamma
lamin B2
unknown
unknown
cyclophilin 40; peptidyl prolyl cis-trans isomerase
a
The Affymetrix HG-U95a chips were used each with 12,453 target probes.
RNAs for microarray studies were isolated from monolayers growing in liquid culture in the presence of glycan, sucrose and trehalose
(time point G1), and after removing liquid medium and exposure of monlayers to air for 45 min, at 378C, in a 5% v/v CO2 incubator (time
point G2). The latter had residual water contents of ;0.80 g per g cell solids. Fold changes are the changes in levels of expression at G2
versus G1; only fold changes . 13.0 or 23.0 were tabulated.
b
and 50 mM sucrose. After cells reached approximately
80% confluence the media was removed and the
monolayer was exposed to air, at 378C, for 45 minutes
(;0.6–0.7 g H2O per g cell solids; Table 3). Under
these conditions 594 probe sets were up regulated, 12
of which were up regulated .3 fold (3.08 to 6.29),
and 703 probe sets were down regulated, of which 21
were down regulated .3 fold (23 to 25.92). The most
conspicuous features of the transcriptional response
were a marked upregulation of Insig-1 and histone
genes. The Insig-1 protein regulates (prevents) lipogenesis (Janowski, 2002), while chromatin remodeling
through increased synthesis of histones is a feature of
many stress responses and one that that we have observed previously with more long-term storage of
HEK293 cells (Mead et al., unpublished data). Interestingly, the up-regulation of PCK1 was also observed
during the complete desiccation of Saccharomyces
cerevisiae (Singh et al., unpublished data), suggesting
that a potential greater flux of oxaloacetate into the
gluconeogenesis pathway may be a common feature of
the response of the two cell types cells to air-drying.
However, the marked down-regulation of the cyclo-
philin 40 gene suggests that the HEK293 cells were
undergoing a severe, potentially lethal stress response
under these conditions, since cyclophilin 40, a peptidyl
prolyl cis-trans isomerase, is a repressor of apoptosis
(Schubert and Grimm, 2004).
In subsequent studies, the conditions for storage
with the glycan were optimized (Fig. 2A) and approaches were developed to condition the cells physiologically prior to drying (Approaches 1, 3 and 4;
Table 1). HEK293 cells were grown on an agarose gel
overlaid with liquid DMEM media plates under the
following conditions: 1) control (no exogenous protectants added); 2) media supplemented with 0.3%
w/v glycerol, final concentration; 3) media supplemented with 6 mg ml21 glycan (final concentration)
from cyanobacterium N. commune commune; 4) media
supplemented with 0.3% w/v glycerol and 6 mg ml21
glycan. Previous work demonstrated that growth of the
cells on agarose prevented their adherence to the plastic surface of the culture vessels and led to the formation of 3D spheroids, which are more resistant than
2D monolayer cells to environmental stress (Fig. 2B;
Mead et al., unpublished data; Jack et al., unpublished
PROKARYOTE DESICCATION TOLERENCE: APPLICATION
TO
HUMAN CELLS
805
weeks of growth, the only flask exhibiting growth was
the aliquot from the 0.3% glycerol 1 6 mg ml21 glycan
treatment (Fig. 2C).
FIG. 2. A. Human 293HEK cells were grown as 2D monolayers in
the presence of 2, 6 or 10 mg ml21 of cyanobacterial glycan. Following removal of liquid medium and exposure to air for 18 hr the
cells were rehydrated and passaged (yellow indicates active growth);
18 hr is the maximum length of time the cells could withstand exposure to air. B. Live (green)/dead (red) stain of HEK293 spheroids
(prepared as a smear on a glass slide) grown on agarose under liquid
media. C. Live/dead stain of outgrowth of a HEK293 monolayer
following storage of spheroids for 5 weeks and after rehydration.
data). After two weeks the liquid suspension was removed from the agarose, cells were recovered and then
replaced on the surface of the agarose, and thus exposed to the air. Plates were transferred to anti-static
plastic bags, flushed with nitrogen gas for approximately five minutes, then vacuum-sealed in a second
bag for storage. Cells were stored in the dark, at room
temperature. After five weeks of storage (;0.6 g H2O
per g cell solids), the plates were rehydrated with
DMEM medium supplemented with 20% final concentration of fetal bovine serum, and passaged to tissue
culture flasks with normal DMEM medium (no agarose) under standard growth conditions. After two
A comparison of the response of yeast and human
cells to air-drying
Having developed conditions that permit survival of
human cells following storage in air (without complete
desiccation), we then compared the transcriptional response of human HEK293 cells under these conditions, to the response of Saccharomyces cerevisiae
upon desiccation. For procedural reasons, the conditions for growing and treating the two types of cells,
drying of the cells, as well as the final residual water
contents of the cells, differ. However, exposure to air
and loss of cellular water are features common to the
experimental protocols, which suggested that there
may be some similarities in the response of these eukaryotic cells to water deficit.
Approximately 3% of the genome of S. cerevisiae
(385 probe sets) underwent changes in the level of
transcription following drying and desiccation (0.02 g
H2O per g cell solids; Table 4). In contrast less than
0.5% of the human genome underwent changes in
transcription following exposure of spheroids to air,
equilibration to a residual water content of approximately 65%, and several weeks of storage. There were
12 genes in common to both cell types that underwent
up-regulation including two encoding mitochondrial
proteins, SOD2 (Mn superoxide dismutase), MDM1
(intermediate filament protein involved in mitochondrial organization), and CPR2 (cyclophilin; Table 5).
Note that the latter was the most down-regulated gene
when HEK293 2D monolayers were subjected to airdrying (see above). Some 33 genes in common to both
cell types underwent down-regulation under these conditions, including three encoding mitochondrial proteins (Table 5). We have also noted similarities in the
transcriptional responses of S. cerevisiae during entry
into stationary phase quiescence; [Martinez et al.,
2004] and following desiccation [Singh et al., unpublished data].
THE DESICCOME
Is there some commonality in the attributes of cells
that confer desiccation tolerance? If so, what are the
restrictions that limit this trait? More intriguing is the
observation that there is no example of a vertebrate
that can withstand desiccation; why? These, and other
relevant questions, were considered in different contexts on previous occasions (Potts, 1994, 2001) and
are considered further in other studies of this volume.
In retrospect one can emphasize that perhaps our inability to fathom why some cells resist desiccation and
others don’t, stems from a greater lack of understanding of the biophysical properties of cytoplasm. One
can make plausible suggestions why certain types of
prokaryotes show either a tolerance or a sensitivity to
water stress and, in doing so, can begin to speculate
on mechanisms that may contribute to desiccation tol-
806
MALCOLM POTTS
TABLE 4. A comparison of the transcriptional responses of Saccharomyces cerevisiae and human HEK 293 cells to air drying.*
Total probes in human array
Total probes in yeast array
Total probe set id matches, between both data sets
Total unique gene matches
Threshold log ratio cutoff
Up regulated in yeast ,and. human
Down regulated in yeast ,and. human
Up regulated in yeast ,not regulated. human
Down regulated in yeast ,not regulated. human
Up regulated in human ,not regulated. yeast
Down regulated in human ,not regulated. yeast
Up regulated in human down regulated in yeast
Down regulated in human up regulated in yeast
22,284
9,336
667
384
ABS 1.0
12
33
123
217
20
30
15
11
* Desiccation of S. cerevisiae was achieved in a custom-fabricated
desiccation chamber, at 22 to 258C, where matric water potential
(Cm) was controlled via a psychrometer. Compressed nitrogen gas
was circulated through a baffle system to achieve uniform drying
within the chamber, and the relative humidity (RH) set point in this
system was 30% 6 2. Aliquots were withdrawn from the cell suspensions 18 hr (T2) after the start of the drying process. Once dry
(42 hr, Tdry; 0.05 g residual H20/g of cell solids) the water potential
was adjusted to 20% RH for a period of 72 hr. Dry cells were
hydrated (22 to 258C) using 1:20 diluted YPD medium, with 15 ml
per plate. Cells were resuspended by gently rotating the plates, with
any cells attached to the dish surface being released by gentle repeated pipetting. Samples were collected for analyses at time points
of 15 min (T4), 45 min (T5), 90 min (T6) and 360 min (T7) after
the start of rehydration. Viability was assessed at T1, T2, Tdry, T6,
and T7 after serial dilution in sterile saline (0.85% sodium chloride,
pH 7.2), at 22 to 258C. Three experiments, with 7 time points in
each, and 3 replicates/time point, required 63 Affymetrix Yeast Genome S98 Array chips. Sample arraying and processing was performed at the University of Rochester Functional Genomics Center.
Probe set signals were processed by a Wilcoxon’s signed rank test
and consolidated using a two-step linear mixed model. Relative expression estimates were derived with respect to both T1 (6 comparisons) and Tdry (4 comparisons).
Human HEK293 cells were grown at 378C, in 5% CO2-humidified
incubators. Unless stated otherwise the media and solutions used
were DMEM (supplemented at 10% v/v with FBS, and 1% v/v with
non-essential amino acids), 20% v/v PBS, and trypsin/EDTA. Cells
were grown to ;40% confluence before the medium was removed;
for 185 cm2 tissue culture flasks (Nunc) the monolayer was rinsed
briefly with 8 ml 20% v/v PBS after which 3 ml trypsin/EDTA
solution was added. Cells were dislodged from the surface after 30–
45 seconds and 6 mL fresh medium was added. Spheroids were
grown in suspension over agarose prepared with fetal bovine serum
(FBS) supplemented DMEM containing 50 mM HEPES (pH 7.2)
and 13 Glutamax-1 (Invitrogen). In the case of monolayer cells, all
free liquid was removed from the flask with a pipette before drying
(see below). Spheroids were recovered from aliquots of cell suspensions either by allowing them to settle in solution or, by centrifugation at 200 3 g for 10 min. Spheroids were then transferred back
to the surface of the agarose and distributed as evenly as possible.
Any residual liquid was removed by wicking the surface of the agarose with sterile filter paper. For storage experiments, flasks were
placed in antistatic bags (Dri-shield 2000 moisture barrier bag from
Surmount Inc., USA; Cat. # 70068), sparged with nitrogen gas for
one minute and sealed (inflated with N2). Each bag was then placed
inside an additional storage bag (20.3 3 30.5 cm, Keystone Manufacturing, USA), which was sealed immediately under partial vacuum (Deniw Magic VacTM, Champion model; Keystone Manufacturing, USA. Total RNA was extracted using RNAeasy kits
following the manufactuer’s instructions (Qiagen Inc.). Hybridization and processing of the U133A human genechips (Affymetrix)
were performed at the University of Rochester Functional Genomics
Center. Three chips were hybridized per experimental time point. A
data set of genes with statistically significant deviation of expression
levels from controls was generated using a multi-class F-Test with
variance stabilization encompassing 7 sample classes (Iobion Infor-
ET AL.
erance. It should be cautioned, nevertheless, that it is
unclear whether such discussion can be extrapolated
and applied effectively in a eukaryotic context. For
example, the pathogenic spirochete Treponema pallidum cannot survive outside the mammalian host and
is inactivated within seconds of exposure to air. Analysis of the genome sequence confirms morphologic
studies indicating a lack of lipopolysaccharide and lipid biosynthesis mechanisms, as well as a paucity of
putative outer membrane protein genes (Norris et al.,
2001). The metabolic capabilities and adaptability of
T. pallidum are minimal and it seems the balance of
oxygen utilization and toxicity is key to the survival
and growth of this microorganism T. pallidum. These
data appear to support suggestions that the structure
and composition of the cell wall and extracellular matrix, especially the production of extracellular polysaccharide, contribute to the capacity for desiccation tolerance, at least in prokaryotes (Potts, 1994). Further,
one of our findings from the transcriptional analysis of
Saccharomyces cerevisiae was the down regulation of
GAS1, GAS2 and FKS1 involved in cell wall organization, and up-regulation cell-wall spore-specific genes
(YNL196C and SPS1000; Singh et al., unpublished
data). Comparative genomics of T. pallidum and desiccation tolerant bacteria emphasize the probable importance of cell wall structure in desiccation tolerance.
For example, T. pallidum has only 9 genes that are
predicted to be involved in peptidoglycan biosynthesis
while Staphylococcus aureus and Vibrio cholerae have
14, and B. subtilis and Nostoc punctiforme each have
28 (http://www.genome.jp/kegg/; http://genome.ornl.
gov/microbial/npun/22dec03/).
Until relatively recently it was assumed that bacterial cells had a simple subcellular organization, with
no compartmentalization and little similarity to complex eukaryotic cells. This assumption may even have
been invoked to argue why the ability to withstand
water deficit is so widespread in prokaryotes and more
limited in eukaryotes. In fact the identification of homologs of actin and tubulin in bacteria mandate a radical reevaluation of the notion of a bacterial cytoskeleton (Lowe et al., 2004). FtsZ (the tubulin homolog)
and MreB/ParM (the actin homologs) are indispensable for cellular tasks that require the cell to accurately
position molecules, similar to the function of the eukaryotic cytoskeleton. FtsZ is the organizing molecule
of bacterial cell division and forms a filamentous ring
around the middle of the cell. Many molecules, including MinCDE, SulA, ZipA, and FtsA, assist with
←
matics). The false discovery rate (FDR) was corrected for multiple
testing using the Benjamini-Hochberg approach to multiple testing.
The Q-Value cutoff for the experiment was 0.09; signals for probesets (i.e., genes) with Q-values greater than 0.09 were considered
insignificant expressors across the experiment. This resulted in a list
of 251 probes that was then reduced further using a 6-fold change
(6) filter. The final data set contained 30 statistically significant
probesets that were either up- or down-regulated more than six fold
(log2) from the experimental multi-class mean.
PROKARYOTE DESICCATION TOLERENCE: APPLICATION
TABLE 5.
TO
HUMAN CELLS
807
Genes that are regulated in both S. cerevisiae and human HEK293 cells in response to air-drying and storage.*
ID (index key)
Function
Up-regulated
CDC23
CPR2
SOD2
PSP1
PEX3
TAF2
RGS2
TOP1
RAP1
GAD1
TAF7
MDM1
promotes metaphase to anaphase transition
peptidylprolyl isomerase (cyclophilin); positive control of cell proliferation
mitochondrial Mn-superoxide dismutase
protein contains RNA recognition motifs
peroxisome biogenesis
TATA-box binding protein-associated factor
negatively regulates G protein-coupled receptor signaling
topoisomerase I
telomere binding protein; affects telomere length
glutamate decarboxylase
TATA-box binding protein-associated factor
intermediate filament protein; mitochondrial organization
Down regulated
NUP133
PGM1
MOG1
NIT2
POP5
COX5B
ENO2
ARD1
MIG2
CHC1
RER1
PGK1
RPL5
DIM1
RBL2
MRPL23
IDH2
MSH2
COG5
TPM1
CYB5
MRPL19
MRPL17
GAS1
nucleoporin
phosphoglucomutase; carbohydrate metabolism
unknown
nitrilase; unknown
RNAse P subunit
cytochrome c oxidase
enolase
N-acetyltransferase; chromosome organization
putative Zn finger protein; cell proliferation
guanyl-nucleotide exchange factor; chromosome organisation
Golgi protein involved in retention of ER
phosphoglycerate kinase
ribosomal protein L5
thioredoxin-like; may be essential for mitosis
beta-tubulin binding protein
mitochondrial ribosomal protein L23
mitochondrial isocitrate dehydrogenase
mismatch repair during mitosis and meiosis
component of the Goligi complex
tropomyosin; control of cell polarity
cytochrome b-5
mitochondrial ribosomal protein L19
mitochondrial ribosomal protein L17
growth arrest specific protein
* Only those genes that had corresponding name designations in the two data sets were recovered through this analysis e.g., yeast COX5B
and human COX5B; nonannotated genes were not considered.
this process directly. Recently, genes much more similar to tubulin than FtsZ were identified in Verrucomicrobia (van den Ent et al., 2001; Lowe et al., 2004).
Through further consideration of the complexity of
prokaryotes, studies suggest that the E. coli chromosome is organized to form a compacted ring structure
with the Ori and Ter domains and these domains participate in the cell cycle-dependent localization of the
chromosome (Niki et al., 2000). Polar CckA functions
to activate CtrA just after the initiation of DNA replication, thereby preventing premature reinitiations of
chromosome replication. Thus, dynamic changes in
cellular location of critical signal proteins provide a
novel mechanism for the control of the prokaryote cell
cycle (Jacobs et al., 1999) These facts may suggest
that, a priori, the rapid removal of water from a prokaryotic cell would seem to guarantee consequences
as severe as those suffered by eukaryotic cells when
they are dried. Or do they? Despite the emerging appreciation for great similarities in the structure and organization of eukaryotic and prokaryotic cells, there
remains the fact that the structure and organization of
DNA in the two cell types differs considerably. Sufficiently so, that it was suggested the term ‘‘chromosome’’ should not be applied to prokaryotic cells
(Margulis, 2004). This was based on the argument that
nothing resembling mitosis has been found in prokaryotic cells and the 25-Å unit fibre (‘‘chromoneme’’), which is composed almost entirely of DNA,
does not react with Fuelgen or other cytological stains.
However, a differential susceptibility of the nuclear
material to air-drying can hardly be invoked as the
cause for the relative susceptibility of a cell to desiccation. The desiccation tolerance of Saccharomyces
cerevisiae, which has all the attributes of a eukaryotic
cell, is of historical recognition, while the DNA of
highly desiccation tolerant prokaryotic cells is subject
to considerable damage and modification during storage (Shirkey et al., 2003).
Genes that are co-regulated in yeast and human in
response to desiccation include some involved in cell
cycle progression, cell proliferation, transcription, and
808
MALCOLM POTTS
subcellular structure (Table 5). However, while there
are some clear similarities in the transcriptional response of yeast and human to air-drying, 2D monolayers of human HEK cells ultimately cannot withstand desiccation. This suggests there are cryptic differences in structure and/or function between these two
forms of eukaryotic cells. Mechanisms that circumvent
oxidative damage, such as the production of superoxide dismutase, seem to figure in the response of all
cells to desiccation (Fig. 1B; Table 5). And, it is of
interest that of the small number of genes that are coregulated in yeast and human 293HEK cells in response to air-drying, a number encode mitochondrial
proteins. Mitochondria probably evolved from oxygen-respiring, heterotrophic a-proteobacteria that were
phagocytosed by anaerobic motile protests (like today’s mastigamoebae); (Margulis, 2004). It will be
most informative to determine whether the mitochondria of some eukaryotes retain any possible vestiges
of a prokaryotic-like tolerance of desiccation.
ACKNOWLEDGMENTS
Our research and this account were made possible
by grants from DARPA-NRL (N00173-98-1-G005;
N00173-02-1-G016), a MURI award administered
through ONR (N00014-01-1-0852), and the NSF.
REFERENCES
Archuleta, R. J., P. Mullens, and T. P. Primm. 2002. The relationship
of temperature to desiccation and starvation tolerance of the
Mycobacterium avium complex. Arch. Microbiol. 178:311–314.
Billi, D., E. I. Freidmann, K. G. Hofer, M. G. Caiola, and R. Ocampo-Friedmann. 2000. Ionizing-radiation resistance in the desiccation-tolerant cyanobacterium Chroococcidiopsis. Appl. Environ. Microbiol. 66:1489–1492.
Billi, D. and M. Potts. 2000. Life without water: Responses of prokaryotes to desiccation. In K. B. Storey and J.M. Storey (eds.),
Environmental stressors and gene responses, pp. 181–192. Elsevier Science BV, Amsterdam, The Netherlands.
Billi, D. and M. Potts. 2002. Life and death of dried prokaryotes.
Res. Microbiol. 153:7–12.
Bloom, F. R., P. Price, G. F. Lao, J. L. Xia, J. H. Crowe, J. R. Battista,
R. F. Helm, S. Slaughter, and M. Potts. 2001. Engineering mammalian cells for solid-state sensor applications. Biosens. Bioelectron. 16:603–608.
Brockbank, K. G. M. 2000. Scientific and commercial challenges in
storage of living biomaterials. 37th Annual Meeting of the Society for Cryobiology, Boston, Massachasetts, Cambridge
Healthcare Institute, Cambridge, MA.
Crowe, J. H. and L. M. Crowe. 2000. Anhydrobiosis: A unique
biological state. Amer. Zool. 40:986–986.
Daly, M. J., E. K. Gaidamakova, V. Y. Matrosova, A. Vasilenko, M.
Zhai, A. Venkateswaran, M. Hess, M. V. Omelchenko, H. M.
Kostandarithes, K. S. Makarova, L. P. Wackett, J. K. Fredrickson, and D. Ghosal. 2004. Accumulation of Mn(II) in Deinococcus radiodurans facilitates gamma-radiation resistance. Science 306:1025–1028.
de Castro, A. G. and A. Tunnacliffe. 2000. Intracellular trehalose
improves osmotolerance but not desiccation tolerance in mammalian cells. FEBS Lett. 487:199–202.
Guo, N., I. Puhlev, D. R. Brown, J. Mansbridge, and F. Levine. 2000.
Trehalose expression confers desiccation tolerance on human
cells. Nat. Biotechnol. 18:168–171.
Halverson, L. J. and M. K. Firestone. 2000. Differential effects of
permeating and nonpermeating solutes on the fatty acid composition of Pseudomonas putida. Appl. Environ. Microbiol. 66:
2414–2421.
ET AL.
Harris, D. R., M. Tanaka, S. V. Saveliev, E. Jolivet, A. M. Earl, M.
M. Cox, and J. R. Battista. 2004. Preserving genome integrity:
The DdrA protein of Deinococcus radiodurans R1. Plos Biology 2:1629–1638.
Hart, T. D., A. H. L. Chamberlain, J. M. Lynch, B. Newling, and P.
J. McDonald. 1999. A stray field magnetic resonance study of
water diffusion in bacterial exopolysaccharides. Enzyme Microbial Technol. 24:339–347.
Helm, R. F., Z. Huang, D. Edwards, H. Leeson, W. Peery, and M.
Potts. 2000. Structural characterization of the released polysaccharide of desiccation-tolerant Nostoc commune DRH-1. J. Bacteriol. 182:974–982.
Hill, D. R., S. L. Hladun, S. Scherer, and M. Potts. 1994. Water
stress proteins of Nostoc commune (Cyanobacteria) are secreted
with UV-A/B-absorbing pigments and associate with 1,4-BetaD-xylanxylanohydrolase activity. J. Biol. Chem. 269:7726–
7734.
Hoehler, T. M., B. M. Bebout, and D. J. Des Marais. 2001. The role
of microbial mats in the production of reduced gases on the
early Earth. Nature. 412:324–327.
Huang, Z. and A. Tunnacliffe. 2004. Response of human cells to
desiccation: Comparison with hyperosmotic stress response. J.
Physiol. 558:181–191.
Jacobs, C., I. J. Domian, J. R. Maddock, and L. Shapiro. 1999. Cell
cycle-dependent polar localization of an essential bacterial histidine kinase that controls DNA replication and cell division.
Cell 97:111–120.
Janowski, B. A. 2002. The hypocholesterolemic agent LY295427
up-regulates INSIG-1, identifying the INSIG-1 protein as a mediator of cholesterol homeostasis through SREBP. Proc. Natl.
Acad. Sci. U.S.A. 99:12675–12680.
Kasting, J. F. 1993. Earths early atmosphere. Science 259:920–926.
Kets, E. P. W. 1997. Compatible solutes in lactic acid bacteria subjected to water stress. Industrial Microbiology. Wageningen,
Landbouwuniversiteit: 64.
Linders, L. J. M. 1996. Drying of Lactobacillus plantarum. Food
processing. Wageningen, landbouwuniversiteit: 101.
Lowe, J., F. van den Ent, and L. A. Amos. 2004. Molecules of the
bacterial cytoskeleton. Annu. Rev. Biophys. Biomol. Struct. 33:
177–198.
Manzanera, M., S. Vilchez, and A. Tunnacliffe. 2004a. High survival and stability rates of Escherichia coli dried in hydroxyectoine. FEMS Microbiol. Lett. 233:347–352.
Manzanera, M., S. Vilchez, and A. Tunnacliffe. 2004b. Plastic encapsulation of stabilized Escherichia coli and Pseudomonas putida. Appl. Environ. Microbiol. 70:3143–3145.
Margulis, L. 2004. Serial endosymbiotic theory (SET) and composite individuality; transition from bacterial to eukaryotic genomes. Microbiol. Today 31:172–174.
Martinez, M. J., S. Roy, A. B. Archuletta, P. D. Wentzell, S. S. AnnaArriola, A. L., Rodriguez, A. D. Aragon, G. A. Quinones, C.
Allen, and M. Wener-Washburne. 2004. Genomic analysis of
stationary-phase and exit in Saccharomyces cerevisiae: Gene
expression and identification of novel essential genes. Molec.
Biol. Cell 15:5295–5305.
Matsuo, T. 2001. Trehalose protects corneal epithelial cells from
death by drying. J. Opthalmol. 85:610–612.
Mattimore, V. and J. R. Battista. 1996. Radioresistance of Deinococcus radiodurans: Functions necessary to survive ionizing
radiation are also necessary to survive prolonged desiccation.
J. Bacteriol. 178:633–637.
Niki, H., Y. Yamaichi, and S. Hiraga. 2000. Dynamic organization
of chromosomal DNA in Escherichia coli. Genes Devel. 14:
212–223.
Norris, S. J., D. L. Cox, and G. M. Weinstock. 2001. Biology of
Treponema pallidum: Correlation of functional activities with
genome sequence data. J. Molec. Microbiol. Biotechnol. 3:37–
62.
Potts, M. 1986. The protein index of Nostoc commune UTEX 584
(Cyanobacteria): Changes induced in immobilized cells by water stress. Arch. Microbiol. 146:87–95.
Potts, M. 1994. Desiccation tolerance of prokaryotes. Microbiol.
Rev. 58:755–805.
PROKARYOTE DESICCATION TOLERENCE: APPLICATION
Potts, M. 1995. Ancient prokaryotes—water, water, everywhere?
ASM News 61:218–219.
Potts, M. 1999. Mechanisms of desiccation tolerance in cyanobacteria. Eur. J. Phycol. 34:319–328.
Potts, M. 2000. Nostoc. Kluwer Academic Publishers, Dordrecht,
Netherlands.
Potts, M. 2001. Desiccation tolerance: A simple process? Trends
Microbiol. 9:553–559.
Puhlev, I., N. Guo, D. R. Brown, and F. Levine. 2001. Desiccation
tolerance in human cells. Cryobiology 42:207–217.
Ramos, J. L., M. T. Gallegos, S. Marques, M. I. Ramos-Gonzalez,
M. Espinosa-Urgel, and A. Segura. 2001. Responses of Gramnegative bacteria to certain environmental stressors. Curr. Op.
Microbiol. 4:166–171.
Scherer, S. and M. Potts. 1989. Novel water-stress protein from a
desiccation-tolerant cyanobacterium—purification and partial
characterization. J. Biol. Chem. 264:12546–12553.
Schubert, A. and S. Grimm. 2004. Cyclophilin D, a component of
the permeability transition-pore, is an apoptosis repressor. Cancer Res. 64:85–93.
Shirkey, B., D. P. Kovarcik, D. J. Wright, G. Wilmoth, T. F. Prickett,
R. F. Helm, E. M. Gregory, and M. Potts. 2000. Active Fecontaining superoxide dismutase and abundant sodF mRNA in
Nostoc commune (cyanobacteria) after years of desiccation. J.
Bacteriol. 182:189–197.
Shirkey, B., N. J. McMaster, S. C. Smith, D. J. Wright, H. Rodri-
TO
HUMAN CELLS
809
guez, P. Jaruga, M. Birincioglu, R. F. Helm, and M. Potts. 2003.
Genomic DNA of Nostoc commune (Cyanobacteria) becomes
covalently modified during long-term (decades) desiccation but
is protected from oxidative damage and degradation. Nucl. Acids Res. 31:2995–3005.
Stal, L. J. 2000. Cyanobacterial mats and stromatolites. In M. Potts
(ed.), The ecology of cyanobacteria, pp. 61–120. Kluwer, Dordrecht.
Tanaka, M., A. W. Earl, H. A. Howell, M.-J. Park, J. A. Eisen, S.
N. Peterson, and J. R. Battista. 2004. Analaysis of Deinococcus
radiodurans’s transcriptional response to ionizing radiation and
desiccation reveals novel proteins that contribute to extreme radioresistance. Genetics 168:21–33.
Timmins, G. S., S. K. Jackson, and H. M. Swartz. 2001. The evolution of bioluminescent oxygen consumption as an ancient oxygen detoxification mechanism. J. Mol. Evol. 52:321–332.
Towe, K. M. 1996. Environmental oxygen conditions during the
origin and early evolution of life. Adv. Space Res. 18:7–15.
van de Mortel, M. and L. J. Halverson. 2004. Cell envelope components contributing to biofilm growth and survival of Pseudomonas putida in low-water-content habitats. Mol. Microbiol.
52:735–750.
van den Ent, F., L. A. Amos, and J. Lowe. 2001. Prokaryotic origin
of the actin cytoskeleton. Nature 413:39–44.
Whitton, B. A. and M. Potts. 2000. Ecology of cyanobacteria: Their
diversity in time and space. Kluwer, Dordrecht, Netherlands.