Anhydrobiosis quotient: a novel approach to evaluate stability in desiccated bacterial cells

Aim:  The major objective of this study was the development of a methodology to quantify the anhydrobiotic ability of bacteria and its application to evaluate the stability of desiccated bacterial cells using the biocontrol agent Tsukamurella paurometabola C-924 as a model of anhydrobiote.
Methods and Results:  Tsukamurella paurometabola C-924 was desiccated by spray-drying. Samples of desiccated cells were stored at several temperatures and viability and residual moisture were measured at different intervals of time. The term anhydrobiosis quotient (ε) was defined, and a scale of anhydrobiotic ability for classifying micro-organisms in terms of tolerance to desiccation was established (1 ≤  ε  ≤ 15). The anhydrobiosis quotient was used to evaluate the stability of the anhydrobiotic cells. As a main result, changes in the anhydrobiosis quotient at several temperatures were fitted using a reparameterized Weibull model, which was found to be robust for the prediction of the stability at 4°C.
Conclusions:  A novel methodology was developed to evaluate the desiccated state in bacteria. The anhydrobiosis quotient allows the quantitative estimation of the anhydrobiotic ability, and the mathematical model developed allows the prediction of the desiccated state of bacterial populations.
Significance and Impact of the Study:  The new methodology could be applied in studying the anhydrobiosis state of bacterial populations as a predictive tool for industrial and environmental microbiology.     

Desiccation tolerance of prokaryotes.

The removal of cell-bound water through air drying and the addition of water to air-dried cells are forces that have played a pivotal role in the evolution of the prokaryotes. In bacterial cells that have been subjected to air drying, the evaporation of free cytoplasmic water (Vf) can be instantaneous, and an equilibrium between cell-bound water (Vb) and the environmental water (vapor) potential (psi wv) may be achieved rapidly. In the air-dried state some bacteria survive only for seconds whereas others can tolerate desiccation for thousands, perhaps millions, of years. The desiccated (anhydrobiotic) cell is characterized by its singular lack of water--with contents as low as 0.02 g of H2O g (dry weight)-1. At these levels the monolayer coverage by water of macromolecules, including DNA and proteins, is disturbed. As a consequence the mechanisms that confer desiccation tolerance upon air-dried bacteria are markedly different from those, such as the mechanism of preferential exclusion of compatible solutes, that preserve the integrity of salt-, osmotically, and freeze-thaw-stressed cells. Desiccation tolerance reflects a complex array of interactions at the structural, physiological, and molecular levels. Many of the mechanisms remain cryptic, but it is clear that they involve interactions, such as those between proteins and co-solvents, that derive from the unique properties of the water molecule. A water replacement hypothesis accounts for how the nonreducing disaccharides trehalose and sucrose preserve the integrity of membranes and proteins. Nevertheless, we have virtually no insight into the state of the cytoplasm of an air-dried cell. There is no evidence for any obvious adaptations of proteins that can counter the effects of air drying or for the occurrence of any proteins that provide a direct and a tangible contribution to cell stability. Among the prokaryotes that can exist as anhydrobiotic cells, the cyanobacteria have a marked capacity to do so. One form, Nostoc commune, encompasses a number of the features that appear to be critical to the withstanding of a long-term water deficit, including the elaboration of a conspicuous extracellular glycan, synthesis of abundant UV-absorbing pigments, and maintenance of protein stability and structural integrity. There are indications of a growing technology for air-dried cells and enzymes. Paradoxically, desiccation tolerance of bacteria has virtually been ignored for the past quarter century. The present review considers what is known, and what is not known, about desiccation, a phenomenon that impinges upon every facet of the distributions and activities of prokaryotic cells.     

Anhydrobiosis in tardigrades and its effects on longevity traits

Living in harsh and variable environments that are prone to periodic desiccation, tardigrades exhibit remarkable tolerance against physical extremes through a state known as anhydrobiosis. To study the effect of this state on the longevity and hence the lifecycle in the taxon Tardigrada for the first time, we exposed a tardigrade species, Milnesium tardigradum , to alternating periods of drying and active feeding periods in a hydrated state. Compared with a hydrated control, the periodically dried animals showed a similar longevity, indicating that the time spent in anhydrobiosis was ignored by the internal clock. Thus, desiccation can produce a time shift in the age of tardigrades similar to the model described for rotifers that has been termed 'Sleeping Beauty'.     

Effects of dehydration rate on physiological responses and survival after rehydration in larvae of the anhydrobiotic chironomid

Strategies to combat desiccation are critical for organisms living in arid and semi-arid areas. Larvae of the Australian chironomid Paraborniella tonnoiri resist desiccation by reducing water loss. In contrast, larvae of the African species Polypedilum vanderplanki can withstand almost complete dehydration, referred to as anhydrobiosis. For successful anhydrobiosis, the dehydration rate of P. vanderplanki larvae has to be controlled. Here, we desiccated larvae by exposing them to different drying regimes, each progressing from high to low relative humidity, and examined survival after rehydration. In larvae of P. vanderplanki, reactions following desiccation can be categorized as follows: (I) no recovery at all (direct death), (II) dying by unrepairable damages after rehydration (delayed death), and (III) full recovery (successful anhydrobiosis). Initial conditions of desiccation severely affected survival following rehydration, i.e. P. vanderplanki preferred 100% relative humidity where body water content decreased slightly. In subsequent conditions, unfavorable dehydration rate, such as more than 0.7 mg water lost per day, resulted in markedly decreased survival rate of rehydrated larvae. Slow dehydration may be required for the synthesis and distribution of essential molecules for anhydrobiosis. Larvae desiccated at or above maximum tolerable rates sometimes showed temporary recovery but died soon after.     

Postgenomic analysis of desiccation tolerance

Desiccation tolerance is the capacity to survive complete drying. It is an ancient trait that can be found in prokaryotes, fungi, primitive animals (often at the larval stages), whole plants, pollens and seeds. In the dry state, metabolism is suspended and the duration that anhydrobiotes can survive ranges from years to centuries. Whereas genes induced by drought stress have been successfully enumerated in tissues that are sensitive to cellular desiccation, we have little knowledge as to the adaptive role of these genes in establishing desiccation tolerance at the cellular level. This paper reviews postgenomic approaches in a variety of desiccation tolerant organisms in which the genetic responses have been investigated when they acquire the capacity of tolerating extremes of dehydration or when they are dry. Accumulation of non-reducing sugars, LEA proteins and a coordinated repression of metabolism appear to be the essential and universal attributes that can confer desiccation tolerance. The protective mechanisms of these attributes are described. Furthermore, it is most likely that other mechanisms have evolved since the function of about 30% of the genes involved in desiccation tolerance remains to be elucidated. The question of the overlap between desiccation tolerance and drought tolerance is briefly addressed.     

Resurrecting Van Leeuwenhoek's rotifers: a reappraisal of the role of disaccharides in anhydrobiosis

In 1702, Van Leeuwenhoek was the first to describe the phenomenon of anhydrobiosis in a species of bdelloid rotifer, Philodina roseola. It is the purpose of this review to examine what has been learned since then about the extreme desiccation tolerance in rotifers and how this compares with our understanding of anhydrobiosis in other organisms. Remarkably, much of what is known today about the requirements for successful anhydrobiosis, and the degree of biostability conferred by the dry state, was already determined in principle by the time of Spallanzani in the late 18th century. Most modern research on anhydrobiosis has emphasized the importance of the non-reducing disaccharides trehalose and sucrose, one or other sugar being present at high concentrations during desiccation of anhydrobiotic nematodes, brine shrimp cysts, bakers' yeast, resurrection plants and plant seeds. These sugars are proposed to act as water replacement molecules, and as thermodynamic and kinetic stabilizers of biomolecules and membranes. In apparent contradiction of the prevailing models, recent experiments from our laboratory show that bdelloid rotifers undergo anhydrobiosis without producing trehalose or any analogous molecule. This has prompted us to critically re-examine the association of disaccharides with anhydrobiosis in the literature. Surprisingly, current hypotheses are based almost entirely on in vitro data: there is very limited information which is more than simply correlative in the literature on living systems. In many species, disaccharide accumulation occurs at approximately the same time as desiccation tolerance is acquired. However, several studies indicate that these sugars are not sufficient for anhydrobiosis; furthermore, there is no conclusive evidence, through mutagenesis or functional knockout experiments, for example, that sugars are necessary for anhydrobiosis. Indeed, some plant seeds and micro-organisms, like the rotifer, exhibit excellent desiccation tolerance in the absence of high intracellular sugar concentrations. Accordingly, it seems appropriate to call for a re-evaluation of our understanding of anhydrobiosis and to embark on new experimental programmes to determine the key molecular mechanisms involved.     

Microbial anhydrobiosis

The loss of cellular water (desiccation) and the resulting low cytosolic water activity are major stress factors for life. Numerous prokaryotic and eukaryotic taxa have evolved molecular and physiological adaptions to periods of low water availability or water-limited environments that occur across the terrestrial Earth. The changes within cells during the processes of desiccation and rehydration, from the activation (and inactivation) of biosynthetic pathways to the accumulation of compatible solutes, have been studied in considerable detail. However, relatively little is known on the metabolic status of organisms in the desiccated state; that is, in the sometimes extended periods between the drying and rewetting phases. During these periods, which can extend beyond decades and which we term ‘anhydrobiosis’, organismal survival could be dependent on a continued supply of energy to maintain the basal metabolic processes necessary for critical functions such as macromolecular repair. Here, we review the state of knowledge relating to the function of microorganisms during the anhydrobiotic state, highlighting substantial gaps in our understanding of qualitative and quantitative aspects of molecular and biochemical processes in desiccated cells.     

Anhydrobiotic engineering of gram-negative bacteria

Anhydrobiotic engineering aims to improve desiccation tolerance in living organisms by adopting the strategies of anhydrobiosis. This was achieved for Escherichia coli and Pseudomonas putida by osmotic induction of intracellular trehalose synthesis and by drying from trehalose solutions, resulting in long-term viability in the dried state.     

A new anhydrobiotic midge from Malawi,Polypedilum pembai sp.n. (Diptera: Chironomidae), closely related to the desiccation tolerant midge,Polypedilum vanderplanki Hinton

The sleeping chironomid (Polypedilum vanderplanki Hinton) lives on temporary rock pools in the semi‐arid tropical regions of Africa. Its larvae are able to survive the dry season in a completely desiccated ametabolic state known as anhydrobiosis. So far, P. vanderplanki was the only species among all insects showing demonstrated anhydrobiotic ability. Here, we show that a new related species originating from Malawi, Polypedilum pembai sp.n. , is also anhydrobiotic and that its desiccation tolerance mechanism is probably similar to what is observed in P. vanderplanki. The new species, P. pembai sp.n. , is described with special attention to the common and different morphological features, compared with P. vanderplanki. Phylogenetic analysis showed that both species are closely related, suggesting that anhydrobiosis evolved only once in their common ancestor about 49 Ma somewhere in Africa, before the divergence of two species, one in the sub‐Saharan area and another in southeastern Africa.     

Anhydrobiotic Engineering of Gram-Negative Bacteria

Anhydrobiotic engineering aims to improve desiccation tolerance in living organisms by adopting the strategies of anhydrobiosis. This was achieved for Escherichia coli and Pseudomonas putida by osmotic induction of intracellular trehalose synthesis and by drying from trehalose solutions, resulting in long-term viability in the dried state.     

Anhydrobiotic engineering of bacterial and mammalian cells: is intracellular trehalose sufficient?

Anhydrobiotic engineering aims to confer a high degree of desiccation tolerance on otherwise sensitive living organisms and cells by adopting the strategies of anhydrobiosis. Nonreducing disaccharides such as trehalose and sucrose are thought to play a pivotal role in resistance to desiccation stress in many microorganisms, invertebrates, and plants, and in vitro trehalose is known to confer stability on dried biomolecules and biomembranes. We have therefore tested the hypothesis that intracellular trehalose (or a similar molecule) may be not only necessary for anhydrobiosis but also sufficient. High concentrations of trehalose were produced in bacteria by osmotic preconditioning, and in mammalian cells by genetic engineering, but in neither system was desiccation tolerance similar to that seen in anhydrobiotic organisms, suggesting that trehalose alone is not sufficient for anhydrobiosis. In Escherichia coli such desiccation tolerance was achievable, but only when bacteria were dried in the presence of both extracellular trehalose and intracellular trehalose. In mouse L cells, improved osmotolerance was observed with up to 100 mM intracellular trehalose, but desiccation was invariably lethal even with extracellular trehalose present. We conclude that anhydrobiotic engineering of at least some microorganisms is achievable with present technology, but that further advances are needed for similar desiccation tolerance of mammalian cells.     

Establishment of gene transfer and gene silencing methods in a desiccation-tolerant cell line,Pv11

Larvae of the African midge Polypedilum vanderplanki show extreme desiccation tolerance, known as anhydrobiosis. Recently, the cultured cell line Pv11 was derived from this species; Pv11 cells can be preserved in the dry state for over 6 months and retain their proliferation potential. Here, we attempted to expand the use of Pv11 cells as a model to investigate the mechanisms underlying anhydrobiosis in P. vanderplanki. A newly developed vector comprising a constitutive promoter for the PvGapdh gene allowed the expression of exogenous proteins in Pv11 cells. Using this vector, a stable Pv11 cell line expressing green fluorescence protein (GFP) was established and retained desiccation tolerance. Gene silencing with GFP-specific siRNAs significantly suppressed GFP expression to approximately 7.5–34.6% of that in the non-siRNA-transfected GFP stable line. Establishment of these functional assays will enable Pv11 cells to be utilized as an effective tool to investigate the molecular mechanisms underlying anhydrobiosis.

     

Hydroxyectoine is superior to trehalose for anhydrobiotic engineering of Pseudomonas putida KT2440

Anhydrobiotic engineering aims to increase the level of desiccation tolerance in sensitive organisms to that observed in true anhydrobiotes. In addition to a suitable extracellular drying excipient, a key factor for anhydrobiotic engineering of gram-negative enterobacteria seems to be the generation of high intracellular concentrations of the nonreducing disaccharide trehalose, which can be achieved by osmotic induction. In the soil bacterium Pseudomonas putida KT2440, however, only limited amounts of trehalose are naturally accumulated in defined high-osmolarity medium, correlating with relatively poor survival of desiccated cultures. Based on the enterobacterial model, it was proposed that increasing intracellular trehalose concentration in P. putida KT2440 should improve survival. Using genetic engineering techniques, intracellular trehalose concentrations were obtained which were similar to or greater than those in enterobacteria, but this did not translate into improved desiccation tolerance. Therefore, at least for some populations of microorganisms, trehalose does not appear to provide full protection against desiccation damage, even when present at high concentrations both inside and outside the cell. For P. putida KT2440, it was shown that this was not due to a natural limit in desiccation tolerance since successful anhydrobiotic engineering was achieved by use of a different drying excipient, hydroxyectoine, with osmotically preconditioned bacteria for which 40 to 60% viability was maintained over extended periods (up to 42 days) in the dry state. Hydroxyectoine therefore has considerable potential for the improvement of desiccation tolerance in sensitive microorganisms, particularly for those recalcitrant to trehalose.     

Morphological response of a bdelloid rotifer to desiccation

We desiccated bdelloid rotifers (Macrotrachela quadricornifera), submitting the animals to four desiccation procedures (protocols A, B, C, D) that differed in the rate of water evaporation, in the time of desiccation, and in the substrates provided. We observed external morphological changes of the rotifer bodies during drying with scanning electron microscopy and, in parallel, assessed rates of recovery after a 7-day period of dormancy. Two protocols produced disorganized morphologies of the anhydrobiotic animals, with no (A) or very poor (B) recovery. Protocols C and D gave rather high rates of recovery and dry rotifers appeared unaltered and well organized. The different protocols affected rotifer morphology during the 7-day anhydrobiosis and rates of recovery after the 7-day anhydrobiosis; high recovery rates corresponded to well-organized morphologies of anhydrobiotic bdelloids, suggesting that a proper contraction of the body into a tun shape and probably a rigorous packing of internal structures are necessary for survival after anhydrobiosis. These features are affected by the time between water shortage and full desiccation, but also by the surrounding relative humidity and by the nature of the substrate. Possible adaptations of anhydrobiotic rotifers are discussed.     

Hydroxyectoine Is Superior to Trehalose for Anhydrobiotic Engineering of Pseudomonas putida KT2440

Anhydrobiotic engineering aims to increase the level of desiccation tolerance in sensitive organisms to that observed in true anhydrobiotes. In addition to a suitable extracellular drying excipient, a key factor for anhydrobiotic engineering of gram-negative enterobacteria seems to be the generation of high intracellular concentrations of the nonreducing disaccharide trehalose, which can be achieved by osmotic induction. In the soil bacterium Pseudomonas putida KT2440, however, only limited amounts of trehalose are naturally accumulated in defined high-osmolarity medium, correlating with relatively poor survival of desiccated cultures. Based on the enterobacterial model, it was proposed that increasing intracellular trehalose concentration in P. putida KT2440 should improve survival. Using genetic engineering techniques, intracellular trehalose concentrations were obtained which were similar to or greater than those in enterobacteria, but this did not translate into improved desiccation tolerance. Therefore, at least for some populations of microorganisms, trehalose does not appear to provide full protection against desiccation damage, even when present at high concentrations both inside and outside the cell. For P. putida KT2440, it was shown that this was not due to a natural limit in desiccation tolerance since successful anhydrobiotic engineering was achieved by use of a different drying excipient, hydroxyectoine, with osmotically preconditioned bacteria for which 40 to 60% viability was maintained over extended periods (up to 42 days) in the dry state. Hydroxyectoine therefore has considerable potential for the improvement of desiccation tolerance in sensitive microorganisms, particularly for those recalcitrant to trehalose.     

Desiccation tolerance in bryophytes: a reflection of the primitive strategy for plant survival in dehydrating habitats?

Bryophytes are a non-monophyletic group of three major lineages (liverworts, hornworts, and mosses) that descend from the earliest branching events in the phylogeny of land plants. We postulate that desiccation tolerance is a primitive trait, thus mechanisms by which the first land plants achieved tolerance may be reflected in how extant desiccation-tolerant bryophytes survive drying. Evidence is consistent with extant bryophytes employing a tolerance strategy of constitutive cellular protection coupled with induction of a recovery/repair mechanism upon rehydration. Cellular structures appear intact in the desiccated state but are disrupted by rapid uptake of water upon rehydration, but cellular integrity is rapidly regained. The photosynthetic machinery appears to be protected such that photosynthetic activity recovers quickly. Gene expression responds following rehydration and not during drying. Gene expression is translationally controlled and results in the synthesis of a number of proteins, collectively called rehydrins. Some prominent rehydrins are similar to Late Embryogenesis Abundant (LEA) proteins, classically ascribed a protection function during desiccation. The role of LEA proteins in a rehydrating system is unknown but data indicates a function in stabilization and reconstitution of membranes. Phylogenetic studies using a Tortula ruralis LEA-like rehydrin led to a re-examination of the evolution of desiccation tolerance. A new phylogenetic analysis suggests that: (i) the basic mechanisms of tolerance seen in modern day bryophytes have changed little from the earliest manifestations of desiccation tolerance in land plants, and (ii) vegetative desiccation tolerance in the early land plants may have evolved from a mechanism present first in spores.     

A modulating role for antioxidants in desiccation tolerance

Most organisms depend on the availability of water. However, some life-forms, among them plants and fungi, but very few animals, can survive in the desiccated state. Here we discuss biochemical mechanisms that confer tolerance to desiccation in photosynthetic and non-photosynthetic organisms. We first consider damage caused by water removal and point out that free radicals are a major cause of death in intolerant tissue. Free radicals impair metabolism and necessitate protection and repair during desiccation and rehydration, respectively. As a consequence, desiccation tolerance and prolonged longevity in the desiccated state depend on the ability to scavenge free radicals, using antioxidants such as glutathione, ascorbate, tocopherols and free radical-processing enzymes. Some 'classic' antioxidants may be absent in lower plants and fungi. On the other hand, lichens and seeds often contain secondary phenolic products with antioxidant properties. The major intracellular antioxidant consistently found in all life forms is glutathione, making it essential to survive desiccation. We finally discuss the role of glutathione to act as a signal that initiates programmed cell death. The failure of the antioxidant system during long-term desiccation appears to trigger programmed cell death, causing ageing and eventual death of the organism. In turn, this suggests that a potent antioxidant machinery is one of the underlying mechanisms of desiccation tolerance.     

The effect of different periods of dehydrationhehydration upon the ability of second stage larvae of Anguina tritici to survive desiccation at 0% relative humidity

The effect of various desiccation regimes on the ability of second stage larvae of Anguina tritici (Steinbuch, 1799) to survive drying at 0% relative humidity has been studied. It was found that both repeated dehydrationhehydration cycles and the length of the desiccation period itself decreased viability of the larvae. The results are discussed in relation to the mechanisms which may facilitate anhydrobiosis.