Lengthening the side chain had the opposite impact on mutation of all three domains being investigated. Mutations increasing the negative charge of the domain, on the other hand, showed little impact on salt tolerance in any of the three protein domains studied. The researchers also investigated whether the tendency of halophiles' protein surfaces to have arginine rather than lysine had any effect on the proteins' ability to cope with salt.
They found that substituting lysines for arginines or other polar residues with small side chains increased the salt tolerance of ProtL. Introduction of lysine residues, with their long side chains onto the surface of Hv 1ALigN, decreased stability in salty solutions.
To further explore the connections between residue side chains and salt tolerance, the researchers used other types of mutagenic systems to alter a different set of residues on the surface of the proteins. The results indicated that it is the nature of a mutation, and not its location, that alters the proteins' ability to withstand a salt assault.
Interestingly, the researchers also discovered that salt tolerance is not the only characteristic conferred by the particular mix of residues found in halophilic proteins.
Their residue substitution experiments also showed that the abundance of aspartic acid and glutamic acid characteristics of halophilic proteins is good not only for salt tolerance but also for solubility—another valuable trait in conditions typical of high-salt environments. Concluding from their experiments that residue compactness and not charge is what matters most when it comes to surviving in salt, the researchers assessed salt tolerance and using high-resolution NMR the accessible solvent surface area in two multiply mutated versions of ProtL, in an attempt to quantify the relationship.
Halobacteria, however, have been detected in environments of fluctuating salinity such as coastal salterns and even around fresh water springs in the depths of the Dead Sea.
In order to identify the underlying mechanisms of low salt survival, we explored the reactivation capacity of Halobacterium Hbt salinarum sub-populations after incubation in low salt media and recovery in physiological salt. In vivo neutron scattering experiments showed that the recovery of Hbt salinarum sub-populations exposed to severe low salt conditions is related to a rapid retrieval of functional molecular dynamics in the proteome. In the hypothesis that the observations on Hbt salinarum have wider relevance, they could be of key ecological significance for the dispersion of extremophiles when environmental fluctuations become severe.
Extreme halophilic Archaea like Halobacterium salinarum are of special interest because they resist the external osmotic pressure mainly by the accumulation of correspondingly high intracellular KCl concentrations 3. A resolution to the puzzle was proposed from in vitro experiments on model enzymes, which revealed the requirement for high salt concentration to stabilize halophilic protein structures through solvation shells made up of hydrated salt ions 8.
The property is correlated with negatively charged protein surfaces, via an enrichment in acidic amino acids and marginal hydrophobic amino acids that favor repulsive interparticle forces to avoid aggregation in the high salt environment 9 , Site-directed mutagenesis of a halophilic protein from Haloferax volcanii and its mesophilic homologue and the subsequent NMR and thermodynamics characterizations indicated that surface aspartic and glutamic acids allow reducing the interaction surface between the protein and the solvent, which is beneficial in low water activity conditions to maintain an hydration shell Neutron scattering measurements of the molecular dynamic properties of the MalDH enzyme from Haloarcula.
It is likely, therefore, that most halophilic proteins display an obligatory requirement for high salt conditions in order to be active and stable. As a consequence, it is expected that intracellular ionic fluctuations in response to external concentration differences pose non-trivial problems for cellular biochemistry in extreme halophiles.
In coastal salterns extreme halophiles populations can be exposed to seasonal fluctuation in salt concentration, fresh water supply as well as transient rains and flooding episodes. Halophilic organisms exhibit a range of stress responses to counterbalance the deleterious effects induced by low salt.
Other responses include down-regulation of the translational apparatus and modulation of the expression of genes encoding for enzymes associated with the primary metabolism 19 , In its natural environment, however, Hbt salinarum is exposed to large salt fluctuations. In this context, we explored the reactivation capacity of Hs sub-populations after incubation in low salt media, 2.
The molecular dynamics of the proteome represents a good indicator of protein functionality. In vivo neutron scattering experiments showed that the recovery of Hbt salinarum sub-populations exposed to severe low salt conditions is related to a rapid retrieval of functional molecular dynamics.
The observations are of key ecological relevance in the context of climate change and the dispersion of extremophiles in fluctuating environments. Also, Dawson et al. Compatible solutes may also exist in extreme halophilic Archaea as suggested by comparative genomic analyses 24 but these organisms display a limited capacity to adapt to salt concentration below their optimum 4.
In the case of Hbt. In the same study, molecular dynamics parameters of the proteome were measured in vivo by neutron scattering for cultures exposed to various lower salt concentrations. Already at 2. It is assumed that such modification would cause irreversible damage, thus preventing cellular reactivation when optimal environmental hypersalinity is restored.
Considering the stress effect of low salt condition on halophilic systems, it is surprising that several studies highlighted the presence of halophilic Archaea in low salt conditions in soils, human or plant microbiomes and in the stratosphere 25 , 26 , 27 , 28 , In this work, we explored the reactivation capacity of Hbt.
We studied different cell properties after salt shock 2. Because Hbt. In previous work, effects of the environment on protein dynamics and its direct relevance for biological activity 30 was illustrated by neutron scattering studies of proteome molecular dynamics, in vivo , on bacterial cells adapted to different physiological temperatures In the study, the mean structural resilience of the proteome, expressed as an effective force constant, was found to be adapted in order to maintain flexibility appropriate for activity at the physiological temperature.
The measure of flexibility converged to a constant value at physiological temperature for all the organisms examined. Similarly, the current study established that molecular dynamics in Hbt. Here, the neutron scattering study revealed that, in the cytosol of reactivable cells, the necessary condition of functional molecular dynamics was rapidly restored after being significantly affected by low salt stress.
With this approach, we obtained evidence that sub-populations of Hbt. These observations may have general ecological significance in the context of dispersion of extremophiles in fluctuating environments.
In extreme halophiles, low salt conditions elicits the induction of stress response systems suggesting that part of the halophilic population could tolerate significant diminutions of environmental salt conditions.
To test this hypothesis, Hbt. Cells exposed to 2. The growth rates, however, were up to 5 to 8 times lower than for control cells in 4.
Growth then slowed down progressively, suggesting that cells entered a phase similar to a stationary phase. The stressful effect of low salt concentration appeared to be particularly pronounced at concentrations lower than 1. Below 1. Interestingly, we observed that all stressed cells inoculated into hypersaline physiological medium switch back to a normal growing cell population after a given time period Fig.
Cells incubated in 2. The observations suggest that part of the low salt stressed cells can undergo a reactivation process when they encounter optimal saline conditions.
A Normalized cell density of Halobacterium salinarum as a function of time for different salt NaCl concentrations. Cells grown in 4. B Growth kinetics of Hbt. Cells were shocked in 2. Growth was monitored as optical density of the culture. Each experiment was carried out in triplicate.
Errors bars represent standard errors. The viability of stressed Hbt. Mainly used for eukaryote and bacterial analysis, the extreme physico-chemical conditions around Hbt. Aspects of different cell populations are shown in scanning and transmission electron micrographs in Fig.
After moderate salt stress 2. However, for cells incubated in NaCl 0. A possible explanation for these results is that most cells are lysed under these extreme conditions, and the signal is dominated by a small fraction of surviving cells. To monitor reactivation, low salt 0. The tendency is confirmed in CRh, in which only one population is observed with undamaged DNA and intact membranes. Taken together, these results indicated that a significant sub-population of Hbt.
The cells were incubated in 4. In an exploration of Hbt. Stressed cells 0. Interestingly, rod-shaped cell formation did not follow classical binary fission, used by most Archaea for cell division. Cell division rather initiated from flat sacculi that eventually acquired conventional rod morphology.
The rod-shape cells then elongated and split into two daughter cells. The cycle was repeated as long as cells were cultured under favorable conditions, consistent with growth rates presented in Fig. Time-lapse light microscopy. The effect of low salt exposure on respiration is shown in Fig. We observed that the respiratory sensitivity of Haloarchaea is NaCl concentration and time-dependent. As illustrated in Fig. The longer the shock, the higher the minimum value of salt concentration for which respiration rate reached zero.
The O 2 -uptake rate of stressed cells decreased notably for cells exposed to below 1. The progressive loss of respiration, under these conditions, parallels slow or stopped Hbt. Figure 5 also shows the respiration activity of low salt stressed Hbt. It shows that the respiratory activity increases rapidly after transfer to 4. In all cases, respiration increases instantaneously including for the longest shock 7 days at the lowest salt concentration 0.
This cannot be the result of undesired contaminations, as demonstrated by control measurements of the recovery medium alone data not shown.
The observations suggested that a significant part of the cell population is still able to produce energy even during a brutal salt shock or when exposed to prolonged periods of stress at moderately low salt concentrations.
Respiration rate of Hbt. Respiration activity RA, light grey of cells in low-salt shock 2. The experiment starts with basal respiration of unstressed cells HS 4. Harvested cells were then exposed to various low-salt concentrations A 2. Finally, oxygen consumption of recovered cells was measured immediately after incubation of stressed cells in 4. Results are mean values for three independent experiments. Time on the x-axis corresponds to the low-salt incubation time.
Note that different scales were used in figure D. As discussed previously, the molecular dynamic state of the cellular proteome represents a robust indicator of cellular fitness 14 , 15 , Macromolecular denaturation through unfolding leads to lower effective force constants indicating less rigid structures.
According to in vitro experiments on a model halophilic protein and to our previous work on Hbt. Here, we performed in vivo neutron scattering experiments to characterize the molecular dynamics parameters of the Hbt. Sample preparation for the neutron experiments is described in Materials and Methods. A centrifugation step prior to sample load in the measurement cell eliminated most of cell debris and lysed material.
We considered, therefore, that the measured signal was dominated by the fraction of intact cells in the low salt stress samples to provide relevant information on their functional state. The experiments displayed in Fig. The scattering vector modulus Q range of IN13 extends from 0.
The analysis was limited to the low Q end, where the time-length window is appropriate for MSD of macromolecular internal motions as well as the larger fluctuation amplitudes involved in unfolding processes Fig.
Unfolded proteins have been measured by neutron scattering to display lower resilience than folded states 34 so that the effective force constants in the low salt samples in Fig.
Even if the unfolded structures tended to aggregate, it is not unlikely on the time scale of internal motions that such aggregates would be less resilient than the compact cores of native states Halophilic extremophiles, or simply halophiles, are a group of microorganisms that can grow and often thrive in areas of high salt NaCl concentration.
Halophiles have been found belonging to each domain of life but primarily consist of archaea. Anthropogenic hypersaline environments are commonly created by the salt industry. Salterns are large ponds that are filled with saltwater from the ocean or another source that are then evaporated away. The salt then precipitates out and is harvested. Halophiles take advantage of this environment and often their presence becomes visible due to pigments they produce.
Often in hypersaline environments, salts other than NaCl are also present. A salt profile for the Dead Sea is shown below. However, because halophiles are defined in relation to NaCl concentration, other salt content is not considered for halophilic classification.
Often in hypersaline environments, the salinity is just one extreme microbes must overcome. Hypersalinity often co-occurs with extreme temperature conditions, both hot and cold. In the case of Deep Lake, Antarctica, extreme cold and high salinity meet.
Similar overlaps of cold and hypersaline environments have been found in other places as well. In the case of the black smokers off the coast of Papua New Guinea, a hypersaline environment was created by a hydrothermal vent. Samples taken from the internal walls of the smoker contained Halomona and Haloarcula species. Such overlapping conditions of hypersalinity and extreme heat are also present in some hot springs and other hydrothermal vents. Some hypersaline environments have been found that overlap with other extremes, such as low and high pH, and dry, desiccating conditions.
Organisms in such conditions would be considered haloacidophiles, haloalkaliphiles, and haloxerophiles, respectively. The majority of extreme halophiles are archaea A thorough study of salt-crystallizing ponds from several places around the world by Oren in showed consistent communities between saltern ponds.
At lower degrees of salinity, diversity of genera present increases. At the extreme end of hypersalinity, eukarya are absent. However, there are a few moderately or slightly halophilic eukaryotes that can contribute to the halophilic communities.
There are also types of halophilic fungi, such as Debaryomyces hansenii and Hortaea werneckii. One mechanism halophiles use to survive in high concentrations of salt is the synthesis of osmoprotectants, which are also known as compatible solutes.
These work by balancing the internal osmotic pressure with the external osmotic pressure, making the two solutions isotonic, or close to it.
Compatible solutes are small-molecular weight molecules. A second, less common mechanism of defense against salt is through controlling potassium levels.
0コメント