Desiccation Tolerance in Prokaryotes and Extreme Environments

Introduction to Prokaryotic Structure and Classification

The microbial world is made up of prokaryotes and eukaryotes. Prokaryotes lack internal unit membranes and are self-sufficient, independent cells or organisms. The best-known prokaryotic organisms are bacteria. The cell membrane in prokaryotes serves as the cell's primary osmotic barrier and consists of a phospholipid unit membrane. Ribosomes, which carry out translation and protein synthesis, are present in the cytoplasm. Normally, the nuclear region consists of circular, double-stranded deoxyribonucleic acid (DNA).

Plasmids are accessory, self-replicating genetic structures present in many prokaryotes. They carry genes not essential to basic cell functions, such as those encoding proteins that inactivate antibiotics. By contrast, eukaryotic cells possess a nuclear membrane, well-defined chromosomes, mitochondria, a Golgi apparatus, an endoplasmic reticulum, and a variety of cell types. Prokaryotes lack this structural complexity but comprise millions of genetically distinct unicellular organisms and compensate through remarkable physiological diversity. Particular groups of prokaryotes are often distinguished by a specific physiological characteristic.

The groups of prokaryotes are classified on the basis of easily observed traits — such as morphology, Gram stain, motility, and structural features — as well as distinguishing physiological features, as outlined in Bergey's Manual. However, the most reliable method of classifying prokaryotes is on a genetic basis, specifically by comparing the nucleotide sequences of the small subunit ribosomal RNA found in all cellular organisms. Groups of prokaryotes can be placed under broader headings based on common structural, biochemical, or ecological properties. Some prokaryotes appear in more than one group, and some groups include both Archaea and Bacteria.

Based on RNA analysis, the Archaea consist of three phylogenetically distinct groups: Crenarchaeota, Euryarchaeota, and Korarchaeota. Archaea can also be arranged into three physiological types: methanogens (prokaryotes that produce methane), extreme halophiles (prokaryotes that exist at very high salt concentrations), and extreme thermophiles (prokaryotes that thrive at very high temperatures).

Prokaryotes display unique structural or biochemical attributes that adapt them to their particular habitats, in addition to the unifying archaeal features that differentiate them from bacteria. Phylogenetic analysis of Bacteria has confirmed that eleven distinct groups exist, though most contain members that are phenotypically and physiologically diverse. The major groups of Bacteria include photosynthetic purple and green bacteria, purple and green sulfur bacteria, spirochetes, cyanobacteria, myxobacteria, lithotrophs, pseudomonads, enterics and vibrios, nitrogen-fixing organisms, pyogenic cocci, lactic acid bacteria, endospore-forming bacteria, actinomycetes and related bacteria, rickettsias and chlamydiae, mycoplasms, and plant pathogenic bacteria.

The conventional classification of prokaryotic envelopes as either Gram-positive or Gram-negative is still used in modern reviews of bacterial cell wall properties. When stained with crystal violet, both envelope types show a characteristic difference based primarily on differences in their peptidoglycan architecture. In Gram-positive bacteria, the multilayered peptidoglycan normally forms a physical barrier that retains the dye. In Gram-negative bacteria, the dye can be easily washed out because of their comparatively thin sacculus. The peptidoglycan architecture largely determines the different properties of the two cell wall types in terms of mechanical stability, permeability, and resistance to chemical substances, as well as other traits such as the presence of accessory cell wall polymers or an outer membrane.

Desiccation Tolerance and Anhydrobiosis in Prokaryotes

Prokaryotes can be subjected to desiccation through the evaporation of cell-bound water by air-drying, followed by the subsequent addition of water to the air-dried cells. This process of air-drying is called desiccation. In some bacterial cells subjected to air-drying, the removal of free cytoplasmic water can occur rapidly. In such cases, the preferred balance between cell-bound water and the environmental water potential is reached almost immediately. The ability of an organism to endure this air-dried state is known as desiccation tolerance. For most cells, even a small loss of intracellular water is lethal. Some cells, however, survive extreme desiccation for extended periods — while certain bacteria can withstand desiccation for thousands or even millions of years, others survive only for a few seconds in the dried state.

The term used to describe the remarkable capability of certain organisms to withstand almost total dehydration is anhydrobiosis — a condition representing life without water. An anhydrobiotic cell is characterized by its singular lack of water, with contents as low as 0.02 g of H₂O per gram dry weight. At these levels, the monolayer coverage of macromolecules — including DNA and proteins — by water is disrupted. As a result, the mechanisms that confer desiccation tolerance upon air-dried bacteria are clearly different from those that preserve cellular integrity under salt stress, osmotic stress, or freeze-thaw stress, such as the preferential exclusion of compatible solutes.

Desiccation tolerance involves a complex range of interactions at the structural, physiological, and molecular levels. Although most of the mechanisms remain poorly understood, it is clear that they involve interactions — such as those between proteins and co-solvents — that arise from the unique properties of the water molecule. The water replacement hypothesis explains how the non-reducing disaccharides trehalose and sucrose maintain the integrity of membranes and proteins. Among prokaryotes, the cyanobacteria possess a distinct ability to survive as anhydrobiotic cells.

The study of the mechanisms by which some organisms tolerate complete desiccation represents an important branch of cell biology. To survive severe water shortage, desiccation-tolerant cells employ structural, physiological, and molecular mechanisms. Certain sugars, particularly trehalose, prevent dehydration damage by delaying fusion between neighboring membrane vesicles during drying and by preserving membrane lipids in the fluid phase in the absence of water. Water molecules contribute to the stability of proteins, DNA, and lipids and are significant components of many reaction mechanisms. Water may also have played a determinative role in the origin and development of the genetic code.

Role of Lipids and Fatty Acids in Stress Tolerance

The sensitivity to drying is associated with the accumulation of dehydration damage in a variety of organisms, and intracellular accumulation of non-reducing disaccharides — as well as the addition of exogenous trehalose or sucrose — can increase survival. The capacity to survive desiccation may be conferred on sensitive cells by transfecting them with genes that enable the synthesis of trehalose or sucrose. Since the synthesis of either disaccharide requires only two steps, involves only two gene products (a synthase and a phosphatase), and uses substrates found in all cells, this approach appears feasible.

Recent evidence shows that the phase transition temperatures and vibration frequencies (P=O stretch) of phospholipids rise in frequency by approximately 30 cm⁻¹ when the protein is dried without trehalose, but this frequency is reduced to or below that of hydrated P=O when the protein is dried with trehalose. Molecular modeling indicates that trehalose can be inserted between the phosphate groups of neighboring phospholipids. At low trehalose-to-lipid ratios, trehalose is not available to bind water, indicating a direct interaction between the sugar and the lipid. Free radicals initiate de-esterification of fatty acids from phospholipids when membranes are isolated.

During aging, free fatty acids typically accumulate in desiccation-sensitive cells and are a contributing factor in reduced membrane integrity. The number of free radicals in the dry state correlates well with the respiratory rate prior to desiccation, suggesting that suppression of respiratory metabolism before dehydration may be necessary for the maintenance of membrane integrity and desiccation tolerance. Because dehydration may involve a reverse phase transition of membrane lipids from the liquid crystal to the gel phase — which occurs in the presence of water — imbibition of viable, dry cells may lead to extensive leakage and death, particularly when it occurs at low temperatures.

Alterations in the lipid content of an organism's membranes in response to environmental stresses are of primary importance. The maintenance of membrane integrity in anhydrobiotic organisms represents a central mechanism of desiccation tolerance. The role of membrane fluidity and lipid composition in the survival of bacteria under extreme temperatures, salinity, and drying conditions has been well documented. A minor increase in the amount of total lipids was observed upon rehydration of dry mats of Scytonema geitleri. Trehalose can stabilize membranes; upon subsequent rehydration, membranes dried without trehalose undergo vesicle fusion, morphological changes, and loss of calcium transport activity.

The water replacement hypothesis, developed by Oliver et al., explains how the non-reducing sugar trehalose protects cells, membranes, proteins, and nucleic acids when they are dried. After desiccation, trehalose appears to lower the phase transition temperature of dry lipids and maintains them in the liquid-crystal state. A number of desiccation-tolerant cyanobacteria possess highly pigmented sheaths. Scytonemin, a yellow-brown, lipid-soluble pigment, is the only pigment distinctive to and limited to a few cyanobacteria. It is located in the extracellular polysaccharide sheath, has a molecular mass of 544 Da, a structure based on indolic and phenolic subunits, and is an optically inactive dimeric pigment.

Scytonemin has been proposed to serve as an ultraviolet sunscreen and has an absorption maximum at 386 nm. Once synthesized, scytonemin remains highly stable and carries out its screening activity without further metabolic investment from the cells. Its long persistence in terrestrial cyanobacterial crusts and dried mats demonstrates that rapid photodegradation of scytonemin does not occur. This approach may be invaluable to scytonemin-containing cyanobacteria that must survive long periods of metabolic inactivity while inhabiting harsh environments and enduring regular cycles of desiccation and rewetting. The means by which lipids and fatty acids confer desiccation tolerance in cyanobacteria thus involves complex interactions that cannot easily be resolved through genetic analysis alone.