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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). 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