Desiccation Tolerance in Prokaryotes

Desiccation Tolerance in Prokaryotes

Water is very important for life. Indeed, the processes of life, both external and internal even, at the cellular and the molecular level, are governed by water. Without water, most living organisms suffer from what is known as water stress.

This water stress can be due to the loss of water or dehydration. Desiccation is a special case of dehydration where drying takes place in air. Alternatively, another form of water stress is due to the excessive accumulation of salts. This is relatedly called osmotic stress. Osmosis seeks to reduce this accumulation by moving fluids across a concentration gradient. While most living beings cannot survive without water, lesser species belonging to the eukarya group -- that includes both bacteria and a more primitive organism archaea show remarkable tolerance to water stress.

Responses to water stress takes place at a supracellular level as well as a cellular level. This work will be concerned with the latter. Thirst response or the secretion of anti-diuretic hormones in animals indicates that there is water stress. This stress is remedied by either increasing the intake of water or reducing the loss of water. Plants also have a supracellular response. Closing of stomata on leaves indicate the need for decrease in transpiration rates. Another defense mechanism has origins in evolution. Cactus leaves have evolved into thorns to preserve water-loss. The green fleshy stems store water and conduct photosynthesis. This work will be dedicated to exploring the current research being done in explicating the idea of desiccation tolerance in prokaryotes -- namely bacteria and archaea (though not a lot of research is being done in the latter, these organisms having been only recently identified).

Desiccation tolerance is also called anhydrobiosis -- or a form of suspended animation. The concept was identified a few centuries ago. (Schierbeek, 1959). This suspended animation lasts until water is available to resume proper metabolic function. At its basic, mechanistic level, this process engenders the use of sugars (sucrose, etc.) to replace the hydrogen bonds that sustain the viability of protein and other molecules in cellular metabolism. Or, as will be shown in this work, other methods are employed to preserve the structure function of proteins and the integrity of DNA.

Bacteria are critical to most molecular biology research. If a DNA sample is to be studied or created in large quantities for expression purposes for (for instance) protein synthesis, researchers make use of Escherichia coli. The DNA is inserted into the E. coli chromosomal plasmid. The bacteria are then spotted on a plate that contains nutrients and moisture that allows the bacteria to grow and reproduce. Each spot is then allowed to multiply (almost exponentially) in a medium rich for bacterial growth. This medium is aqueous. It is kept at a temperature of 37 "C -- the ideal temperature for bacterial growth. E. coli then multiplies by the typical bacterial reproductive process of geometric cell division, thus creating large quantities of the DNA to be studied. The point for using, what is called as recombinant DNA techniques, is not only to illustrate that bacteria drive research, but that bacteria thrive under moist conditions at the right (and mild) temperature. The operative terms being -- moist and mild. As such, E. coli, despite its abundance is not a good candidate for desiccation tolerance and research has shown that this is indeed true.

On the other hand however, bacteria such as the cyanobacterium Nostoc commune and Deinococcus radiodurans thrive under extremely dry conditions of very high or very low temperature where water as a metabolite is not easily available. In fact, both these bacteria, especially D. radiodurans can withstand radioactivity that would easily kill all other living things. This correlation between tolerance to radioactivity and desiccation is important. Researchers believe that the mechanisms of both are intrinsically linked. That since radioactivity is not a natural phenomenon on earth, the tolerance to radioactivity is a manifestation of the tolerance to conditions of extreme dryness. In turn, therefore, to understand one form of tolerance, mechanistically, is to understand the other.

Prokaryotes have developed a mechanism for survival under extreme and harsh conditions that has earned them the moniker of extremophiles. These include theromophiles (able to exist at high temperatures) and acidophiles (able to survive under conditions of low pH) and halophiles (in conditions of high salinity). Thermophiles were first discovered in the depths of Yellowstone National Park's hot springs, where the temperature of the water exceeds that of its boiling point. Halophilic archaea have been discovered in salt crystals from millions of years ago. (Rothschild & Mancinelli, 2001).

Cognating the extremophilic behavior of prokaryotes has two-edged consequences. The first is that understanding the mechanism of desiccation tolerance allows researchers to understand the role of water in living things. On the other hand, pathogenic bacteria which sustain under harsh conditions can cause disease. In order to disinfect a laboratory bench of bacterial contaminants, an alcohol wipe is sufficient, since bacteria cannot grow in the presence of ethyl alcohol. Also, heating is supposed to kill bacteria and restrict its growth. Bacteria of the Acitenobacter genus: Acinetobacter baumannii (Jawad, Snelling et al., 1998). have shown remarkable desiccation tolerance. These bacteria have been found on the fingertips of medical staff and other dry surfaces in hospitals in the United Kingdom. These bacteria are responsible for cross-contamination outbreaks several times in these hospitals in the last twenty years.

From a mechanistic standpoint, the formation of trehalose has been implicated in the protection of the bacterium against desiccation. Trehalose is a reducing sugar. It is a disaccharide similar to sucrose and maltose. During the process of digestion, trehalose is broken down into two molecules of sucrose. Trehalose has approximately half the sweetness of sucrose when ten percent solutions were compared. From the standpoint of bacterial desiccation tolerance, two properties of trehalose stand out. It is not as hygroscopic as sucrose or maltose. Its hygroscopicity is less than one percent as opposed to seventy-five percent for sucrose. It has a relatively low melting point (97 "C, when compared to that of sucrose between 160 to 180 "C). Perhaps more relevant is its relative high glass transition temperature. Its Tg is 79 degrees as opposed to 52 degrees C. For sucrose. This high temperature allows trehalose to remain stable for wider temperature ranges. The high glass transition temperature also accounts for its significantly low hygroscopicity. Glass transition temperature is defined as temperature below which polymers achieve glass like status. This glass phase renders a compound inert to outside influences. It also slows down reaction rates. This inertness induces a "hibernation" of sorts in the bacterium thus allowing it to survive dry conditions. This means it also does not allow the exchange of material on either side of this "glass partition." Thus it preserves the remaining moisture on the inside of the bacterial cell.

Another theory suggests that the sugar domains of trehalose prevent internal desiccation by providing hydrogen bonding with the proteins inside the cell and with the phospholipid membrane bilayers in the cell membrane. Protein denaturation would have resulted if the water molecules that stabilize the tertiary structures of proteins (and hence the function) would have been removed due to desiccation. That however, does not happen; or, the process of drying is retarded. This theory however, has come under considerable criticism because it does not explain the advantage of trehalose over other sugars which would also replace water hydrogen bonding. In this case however, the chemical inertness of trehalose glass at a higher temperature prevents desiccation. By the same reasoning, the significantly lower Tg of sucrose becomes susceptible to the Maillard reaction between sugars and amino acids in proteins that result in the browning of food.

Researchers Welsh and Herbert showed the use of trehalose in protecting against desiccation by introducing trehalose externally into the medium surrounding E. coli and then subjecting it to short- and long-term drying. (Welsh & Herbert, 1999). E. coli showed resistance to drying in the presence of trehalose. Welsh and Herbert demonstrated that the desiccation-resistant efficacy of trehalose only occurs when it has reached a glasslike state. They measured the bacterial viability with respect to time and found that it decreased until a specific level of dryness. After that the trehalose began exerting its protecting mechanism.

Internal creation of trehalose within primitive systems is also not without precedence. Trehalose is naturally created in Saccharomyces cerevisiae when it is freeze-dried. While freeze-drying is not the same of air-drying or desiccation, it allowed researchers to test the notion that trehalose can also be manufactured by a species in response to water stress. In addition to external application of trehalose, Welsh and Herbert also sought to force E. coli to create trehalose in the presence of stress. When subjected to a medium of high salt content, E. coli -- to protect against this osmotic stress produced trehalose. The induced trehalose possessing bacteria then also resisted drying. The mere presence of a high glass transition temperature compound is not sufficient to create dessication tolerance. It is very possible that genomic and phenotypic factors (discussed later in this work) combine along with protectants such as trehalose. Different media concentrations induced E. coli to generate different compounds. Researchers induced the E. coli production of glycine betaine by varying the NaCl concentrations and the consequent osmotic stress. Glycine betaine has been implicated in desiccation protection for several higher plans such as grams and tobacco. Glycine betaine has also been suggested as a protectant against desiccation for certain lactobacilli. (Teunissen et al., 1992). from short-term viability experiments.

In this sense, both trehalose and glycine betaine can be considered compatible solutes because their presence (in response to the osmotic stress that draws water away from the cell) is compatible with the processes that occur within the cell. This compatibility arises no matter how high the concentrations of these solutes. One must remember however, that there is an artificial construct to attempting to render a desiccation-protection mechanism from osmotic stress. This is because osmotic stress occurs rarely in nature and the water loss from air-drying (or desiccation) is higher and relentless). On the other hand, one might consider osmotic stressors are necessary to understand desiccation protection under laboratory condition

In Welsh and Robert's experiments, glycine betaine had no discernible effect on desiccation tolerance. The action of glycine betaine as yet another mechanism of desiccation tolerance has been shown through the process of solute exclusion. (Lows, 1985). This concept proposed that, under water stress, solutes such as glycine betaine are excluded from the protein sphere and surrounding water, around which, this solute creates a protective barrier. It is possible that this would explain the short-term (but not long-term) desiccation tolerance. In addition to replacement of water, hydrogen bonds to prevent the denaturation of protein. It is also possible that the "glassy" trehalose also acts as a membrane barrier to the outside drying elements. E. coli does not produce trehalose in response to air-drying. But externally applied trehalose either diffuses into the cell or enters during structural phase changes in the outer membrane. (Leslie et al., 1995). It is also possible therefore, that bacteria that can produce trehalose in response to drying also have developed a mechanism by which trehalose produced by the bacterium can make its way to the surface of the cells.

It has been mentioned at least twice here that E. coli does not have a natural water-stress response. This does not make it particularly viable in dry conditions. Gowrishankar and co-workers have identified a third mechanism of this water stress. And it is referred to (in addition to solute inclusion or solute exclusion) by a term (coined by the authors) anhydrotic stress. This stress is related directly to the loss of water in the cell, and the consequent inhibition of bacterial growth due to permeable liquids such as glycols. This stress is alleged to be directly correlated with the increase of L-ornithine in the bacterial cell. It has also been mentioned above that genomic factors probably have a lot to do with resistance to desiccation. Gowrishankar and his co-workers have shown that in addition to L-ornithine correlation, certain transposons in the E. coli plasmid may also be associated with anhydrotic stress. (UmaPrasad & Gowrishankar, 1998). This has been observed in Gram-positive as well as Gram-negative bacteria. The authors believe that this additional information would be important in identifying why E. coli is not naturally tolerant but several other extremophile bacteria maintain their viability on drying.

The identifiable qualitative difference between Gram-positive and Gram-negative bacteria is that the former responds to the Gram stain which is a violet colored stain that interacts with iodine to produce a violet color. The Gram staining process also involves rinsing the bacterial sample with alcohol and counterstaining with saffranine dye. While positive bacteria turn violet, the negative bacteria which do not for complexes with the Gram stain are stained red to pink with saffranine. The quantitative difference between Gram-positive and Gram-negative bacteria is the amount of peptidoglycan in the bacterial cell wall. Positive strains of bacteria have almost five times as much as Gram-negative bacteria. The peptidoglycan layer complex with the Gram stain giving positive bacteria the distinct violet colors. This might have consequences to desiccation protection.

The osmolyte tetrahydropyramidine hydroxyectoine can also be used as a desiccation protectant. It is shown to work irrespective of the Gram-nature of the bacteria. Researchers Manzanera et al. aver that it produces better protection and desiccation tolerance than the ubiquitous trehalose, especially in Gram-negative bacteria. (Manzanera, Vilchez, & Tunnacliffe, 2004). They base their findings by comparing the protection strengths of hydroxyectoine vs. trehalose in the Gram-negative Pseudomonas putida. Their results show that hydroxyectoine produced better protection than trehalose in P. putida. When these experiments were conducted for (also Gram-negative) E. coli, the final results were varied. In the case of external applications, both compatible solutes performed equally well in providing dessication tolerance. Bacteria do not metabolize of synthesize hydroxyectoine, unlike they do trehalose. But there is osmotic uptake of hydroxyectoine into the cell body. Such an uptake naturally reduces the induced production of trehalose and it reduces the ability of E. coli to protect itself.

The authors believe that mechanistically, hydroxyectoine does not possess enough hydroxyl groups that will hydrogen bond with cellular proteins replacing the water molecules and preventing denaturation. Indeed, a molecule of hydroxyectoine only possesses one -OH group, unlike trehalose. Manzanera and co-workers experimented with trehalose induction under high saline conditions vs. saline mixed with hydroxyectoine and glycine betaine. In the latter cases, perhaps due to osmolyte (compatible solute) uptake, the synthesis of adequate amounts of trehalose to tolerate drying adequately was not produced. This was seen in the results. These studies provide added substantiation for the water replacement model for mechanism of desiccation tolerance. One of the upshots of this study was also the development of a methodology for protecting E. coli to be used in laboratory settings. This includes inducing trehalose growth and then placing E. coli under desiccant conditions in the presence of a veneer of trehalose (in its glassy state).

Much like the acinetobacter family of prokaryotes, the adaptation to drying conditions (or conditions of osmotic stress) can give rise to pathogens. This means that the ability to protect against drying will become a health hazard. Enterobacter sakazakii can be found in baby formula powder. (Breeuwer et al., 2003). Its mechanism for dessication tolerance can also be traced to the internal production of trehalose in response to water stressful conditions.

As has been mentioned previously (first page), that non-reducing disaccharides substantiate the water replacement model. It is fairly obvious however, that sucrose and trehalose are only one part of the overall mechanism. This mechanism however, also cannot be universally applied to every species prokaryotes or otherwise. There is a lack of comprehensive research especially when it comes to Gram-positive bacteria. A study of the eukaryotic minute animal bdelloid (leech-like) rotifers (Phylum rotifera) indicates that other mechanisms must be at play. The results of such studies (on multicellular organisms) can do no better than further the cause of identifying mechanisms in prokaryotes.

Bdelloid rotifers are small animals that are found on films of water, especially in birdbaths. Though they live under moist conditions, they are remarkably well adapted to dry conditions and are ubiquitous in dry or very cold environments. When researchers fed, dehydrated and rehydrated bdelloid rotifers, the viability was greater than seventy-five percent. (Lapinski & Tunnacliffe, 2003). Under dehydrated conditions, the researchers discovered that there was no evidence of sucrose, trehalose, or any other mono- or polysaccharide. Lapinski and Tunnacliffe indicate that pursuing alternative mechanistic models might be necessary. The researchers also found (much like in the trehalose studies) that for full tolerance to take effect it took time. This meant that the organism (eukaryote or otherwise) undergoes physiological changes to counter the effects of drying. This is different from Deinococcus radiodurans, which is restored to full function just a few hours after stress. Indeed, they found that the viability of the rotifers decreased to fifty percent or less as soon if the drying process was rapid. This meant that the changes necessary for tolerance were not in place.

Lapinski and Tunnacliffe used the results of their research to posit questions of other models: namely, the protein restructuring to counter desiccation. They point to the identification of late embryogenic abundant (LEA) proteins as a potential protecting agent. An LEA like protein has also been isolated in nematodes. Indeed, it was relatively late that anhydrobiosis was identified in extremophilic bacteria. Nematodes and certain desert-viable plants and seeds were the first species on which anhydrobiosis-identifying research was conducted (Bartels & Salamini, 2001).

The research by Bartels and Salamini took a different approach. Realizing that organic sugar glasses and consequent water-replacement theory did not explain anyhydrobiosis in all species that exhibited it, they began seeking genomic information, a "protein-set" or a "gene-set." LEA proteins that are distributed throughout different species are perhaps one of these.

Interestingly enough, the push for identification of LEA proteins as putatively anhydrobiotic is supported by the fact that these proteins have been found in prokaryotes such as haemophilus influenzae and deinococcus radiodurans. It has been mentioned previously in this work, that since the stress from radioactive doses is not a natural phenomenon, these organisms evolved a protective mechanism to prevent desiccation. D. radiodurans can withstand radioactivity that would kill virtually all other living species. This means that this bacterium has evolved a method of quickly repairing itself. Understanding D. radiodurans' reaction to radioactive stress therefore, might (also) hold the key to understanding the mechanisms of desiccation tolerance. One of the reasons denaturation or breakdown occurs in the presence of extreme situations such as exposure to radioactivity is the breakdown of DNA. There are many repair mechanisms in place that involve the notion that downstream DNA recruit (or create) proteins that serve as surrogates during the repair processes. (Feng, Crasto, & Matsumoto, 1998).

Research in the repair mechanism of DNA of D. radiodurans indicates that there is nothing particularly different from the repair processes of radiodurans from those of most other bacteria. This means that another mechanism has to be involved in the process of protection against radioactivity/desiccation.

Karlin and Mrazek identified a family of proteins by their expression levels and functions (otherwise known as "codon usage"). (Karlin & Mrazek, 2001). These proteins, according to the authors, bear the moniker Predicted Highly Expressed (PHX) proteins.

Typical PHX genes express chaperone, detoxification, protease and degradation proteins. In radiodurans, detoxification and protease genes abound at levels far higher than those for other bacteria.

Most bacterial genomes consist of a circle of chromosome on a single plasmid. D. radiodurans' genome is unusual; it consists of three plasmids and possibly two chromosomes. (White et al., 1999). The repair mechanism is affected in such a manner that most radioactivity-induced damage is often repaired in less than three hours. According to Karlin and Mrazek, "The PHX chaperone, degradation, protease, and detoxification protein ensemble allow DEIRA to maintain the integrity of its essential macromolecules." Some of the specific genes engaged in the protective mechanism are PHX-S or surface proteins that protect the bacterial cell wall. In addition, protein abundance genes that are responsible for biolipid synthesis are also present on the surface of the bacterial cell. No doubt, these proteins fortify the plasma membrane and afford protection from the bulk phase or the environment. Other overtly expressed genes are associated with the tricarboxylic acid (TCA) cycle, particularly, aconitate hydratase, which is sensitive to oxygen radicals and therefore might help in preventing oxidative degradation.

The authors also describe the ubiquitous protein cyclophilin protein as necessary for radiodurans self-preserving abilities. This protein has been implicated not only in fostering rapid transcription but also rapid protein folding. This is important because rapid DNA repair and maintenance of protein integrity are necessary to restore function after damaging radioactivity (or desiccation).

As mentioned earlier, replicated copies of chromosomes in radiodurans might also allow for rapid replacement of a damaged DNA or its products while the repair of the damage takes place. Mrazek independently reported that D. radiodurans can repair more than 100 DNA breaks. This is different from other bacteria with multiple chromosomes that can only repair a few DNA breaks. (Lipton et al., 2002). When replaced by RecA from another bacterium, the radioactive resistance was lost. Mrazek also avers that the RecA is manufactured only after radiation damage and might actually be detrimental to bacterial cell under conditions of normality.

Another study cements the relationship between the resistance to ionizing radiation and its mechanistic correlations with desiccation tolerance. A study of forty-one strains of D. radiodurans modified with varying degrees of sensitivity to radiation. Samples of these strains as well as the wild-type radiodurans were subjected to typical laboratory desiccation. The results cemented two conclusions. (Mattimore & Battista, 1996). The first was that desiccation and its consequent lethality was due to the damage and breakdown in DNA and their products. The second indicated that the viability and recoverability from desiccation was significantly reduced in the radiation sensitive strains. The rehydration in the desiccated wild-type radiodurans species was significantly higher and in some cases, the bacteria was restored to full viability. Unlike previous studies by Mrazek and Karlin, this study does not delve into possible mechanisms. But it establishes a vital link between resistance to radioactivity and resistance to desiccation.