Antagonistic Interactions between Stress Factors during the Growth of Microorganisms under Conditions Simulating the Parameters of Their Natural Ecotopes

Two stress factors, hypoxia (microaerobic conditions) and a high salt concentration, if applied simultaneously to aerobic microorganisms, display an antagonistic mode of interaction. As a result, the NaCl level that is usually optimal for moderate halophiles (5–6 %) becomes optimal for the growth of weak halophiles (Rhodococcus erythropolis and Shewanella sp. CN32); the halotolerant yeast Yarrowia lypolytica acquires halophilic properties (with a growth optimum at a NaCl concentration of 10%), and the growth rate of the extremely halophilic Halobacterium salinarum increases at supraoptimal salt concentrations (25–34%). This phenomenon is apparently due to multiple changes in metabolic reactions. In particular, high salt concentrations suppress respiration and the formation of enzymes (superoxide dismutase and catalase) that protect the cell from toxic oxygen species. Therefore, establishment of microaerobic conditions compensates for the loss of these protective mechanisms and enables cell growth at higher salt concentrations than under aerobic conditions. Of some importance can also be the increase in the intracellular concentrations of osmoprotectants caused by the suppression of their intracellular oxidation. The implications of this phenomenon for the ecophysiology of microorganisms (including oil-oxidizing species) and for the classification of weak and moderate halophiles are discussed.     

Biology of Moderately Halophilic Aerobic Bacteria

The moderately halophilic heterotrophic aerobic bacteria form a diverse group of microorganisms. The property of halophilism is widespread within the bacterial domain. Bacterial halophiles are abundant in environments such as salt lakes, saline soils, and salted food products. Most species keep their intracellular ionic concentrations at low levels while synthesizing or accumulating organic solutes to provide osmotic equilibrium of the cytoplasm with the surrounding medium. Complex mechanisms of adjustment of the intracellular environments and the properties of the cytoplasmic membrane enable rapid adaptation to changes in the salt concentration of the environment. Approaches to the study of genetic processes have recently been developed for several moderate halophiles, opening the way toward an understanding of haloadaptation at the molecular level. The new information obtained is also expected to contribute to the development of novel biotechnological uses for these organisms.     

Effect of extreme salt concentrations on the physiology and biochemistry of Halobacteroides acetoethylicus.

Halobacteroides acetoethylicus grew in media with 6 to 20% NaCl and displayed optimal growth at 10% NaCl. When grown in medium with an [NaCl] of 1.7 M, the internal cytoplasmic [Na+] and [Cl-] were 0.92 and 1.2 M, respectively, while K+ and Mg2+ concentrations in cells were 0.24 and 0.02 M, respectively. Intracellular [Na+] was fourfold higher than intracellular [K+]. Since Na+ and Cl- ions were not excluded from the cell, the influence of high salt concentrations on key enzyme activities was investigated in crude cell extracts. Activities greater than 60% of the maximal activity of the following key catabolic enzymes occurred at the following [NaCl] ranges: glyceraldehyde-3-phosphate dehydrogenase, 1 to 2 M; alcohol dehydrogenase (NAD linked), 2 to 4 M; pyruvate dehydrogenase, 0.5 to 1 M; and hydrogenase (methyl viologen linked), 0.5 to 3 M. These studies support the hypothesis that obligately halophilic, anaerobic eubacteria adapt to extreme salt concentrations differently than do halophilic, aerobic eubacteria, because they do not produce osmoregulants or exclude Cl-. This study also demonstrated that these halophilic, anaerobic eubacteria have a physiological similarity to archaebacterial halophiles, since Na+ and Cl- are present in high concentrations and are required for enzymatic activity.     

Characteristics of the heterotrophic bacterial populations in hypersaline environments of different salt concentrations

Solar salterns, based on a multi-pond system, give a discontinuous gradient of salt concentrations. The heterotrophic bacterial populations of ponds containing from 10% salt to saturation have been studied. Saltern samples were spread on agar plates containing different media for halophilic bacteria and one medium made with water of the pond plus nutrients. Replica plating was done to determine the salt range for growth of the colonies. We studied 150 strains to determine the salt spectra of growth, the morphology, and nutrient requirements. The following conclusions were reached: (a) In salt concentrations above 10% (total salts), most bacteria are halophilic and few are halotolerant; (b) the two types of halophilic bacteria, moderate and extreme, show different distributions; in these ponds a narrow overlap exists between 25% and 32% salts with moderate halophiles predominating below this interval and extreme halophiles above it; (c) the populations of moderate halophiles are highly heterogeneous, and the salt concentration of their habitat affects their taxonomic composition, salt range for growth, and nutrient requirements. The population composition of extreme halophiles is less affected by the salt concentrations at which these bacteria are found.     

Biodiversity of poly-extremophilic Bacteria: Does combining the extremes of high salt, alkaline pH and elevated temperature approach a physico-chemical boundary for life?

Bacterial microorganisms that grow optimally at Na⁺ concentrations of 1.7 M, or the equivalent of 10% (w/v) NaCl, and greater are considered to be extreme halophiles. This review focuses on the correlation between the extent of alkaline pH and elevated temperature optima and the extent of salt tolerance of extremely halophilic eubacteria; the focus is on those with alkaline pH optima, above 8.5, and elevated temperature optima, above 50°C. If all three conditions are required for optimal growth, these microorganisms are termed "poly-extremophiles". However, only a very few extreme halophiles able to grow optimally under alkaline conditions as well as at elevated temperatures have been isolated so far. Therefore the question is: do the combined extreme growth conditions of the recently isolated poly-extremophiles, i.e., anaerobic halophilic alkalithermophiles, approach a physico-chemical boundary for life? These poly-extremophiles are of interest, as their adaptive mechanisms give insight into organisms' abilities to survive in environments which were previously considered prohibitive to life, as well as to possible properties of early evolutionary and extraterrestrial life forms.     

Growth Potential of Halophilic Bacteria Isolated from Solar Salt Environments: Carbon Sources and Salt Requirements

Eighteen strains of extremely halophilic bacteria and three strains of moderately halophilic bacteria were isolated from four different solar salt environments. Growth tests on carbohydrates, low-molecular-weight carboxylic acids, and complex medium demonstrated that the moderate halophiles and strains of the extreme halophiles Haloarcula and Halococcus grew on most of the substrates tested. Among the Halobacterium isolates were several metabolic groups: strains that grew on a broad range of substrates and strains that were essentially confined to either amino acid (peptone) or carbohydrate oxidation. One strain (WS-4) only grew well on pyruvate and acetate. Most strains of extreme halophiles grew by anaerobic fermentation and possibly by nitrate reduction. Tests of growth potential in natural saltern brines demonstrated that none of the halobacteria grew well in brines which harbor the densest populations of these bacteria in solar salterns. All grew best in brines which were unsaturated with NaCl. The high concentrations of Na⁺ and Mg²⁺ found in saltern crystallizer brines limited bacterial growth, but the concentrations of K⁺ found in these brines had little effect. MgSO₄ was relatively more inhibitory to the extreme halophiles than was MgCl₂, but the reverse was true for the moderate halophiles.     

Nutrition and growth of the moderately halophilic bacteria Micrococcus morrhuae K-17 and Micrococcus luteus K-15

Chemically defined media have been developed for the growth of two moderately halophilic bacteria, Micrococcus morrhuae K-17 and Micrococcus luteus K-15. M. morrhuae K-17 grows well in a synthetic medium (SM-1) which contains a number of salts, 0.21 M KCl, 2 M NaCl, D-mannose, five vitamins and ten amino acids. The synthetic medium (SM-2) for M. luteus K-15 contains a number of salts, 0.21 M KCl, 1 M NaCl, D-fructose, nine vitamins and nine amino acids. Nutritional studies show that M. morrhuae K-17 can utilize a large number of organic compounds as carbon and energy source while the ability of M. luteus K-15 in utilizing the organic compounds is rather limited. The minimum salt requirement is 0.5 M NaCl for both strains when growth at the optimum temperature of 30 degrees C. However, this requirement can be lowered to 0.2 M in M. luteus K-15 when grown at a lower temperature of 25 degrees C. It is concluded that the ability to grow in a wider range of salt concentrations in response to temperature is species specific in moderate halophiles. The salt range for growth to occur can be extended when cells of both strains are grown in complex medium which might provide the amino acids and growth factors that cannot be synthesized by these strains at high salt concentrations.     

Rhodospirillum salinarum sp. nov., a halophilic photosynthetic bacterium isolated from a Portuguese saltern

A new species of halophilic photosynthetic bacteria, Rhodospirillum salinarum, has been isolated and described. Its natural habitat are the terminal crystallization ponds of solar salt production plants. R. salinarum grows optimally at 42°C in the presence of 6–18% NaCl (w/v). Growth requirements are complex, yeast extract and peptone being required both for aerobic heterotrophic and for anaerobic phototrophic growth. Increasing concentrations of NaCl in the growth media did not give rise to any corresponding increase in intracellular concentrations of K⁺, Na⁺, polyalcohols or amino acids. Malate dehydrogenase from R. salinarum is not halophilic, being inhibited even at low concentrations of Na⁺ or K⁺. The GC mol % of DNA from R. salinarum is markedly higher than that for DNA from R. salexigens, the only previously described halophilic species of the genus Rhodospirillum.     

Types and properties of some bacteria isolated from hypersaline soils

Five rhizosphere soil samples from the dominant xerophytic plants, and nearby root-free soil samples were obtained from a series of hypersaline soils (5.0–10.7% NaCl) from sites near Alicante in Spain. Physico-chemical analyses were made, and the bacterial flora estimated using three different plating media. Counts from rhizosphere soil were always significantly higher than those from root-free soils. A total of 211 strains isolated were purified and identified to genus level; 12 could not be classified. The range of salt concentration allowing growth was determined for each isolate, but this did not correlate with the salt content of the soil habitat. Most isolates appeared to be typical moderate halophiles (with optimum growth between 5 and 15% salts), but about half of them grew on normal media with only 0.9% naCl, a notable difference from moderately halophilic aquatic bacteria. Extreme halophiles were rare but this may have been due to an insufficient incubation period.     

Temperature and pH optima of extremely halophilic archaea: a mini-review

Archaeal microorganisms that grow optimally at Na⁺ concentrations of 1.7 M, or the equivalent of 10% (w/v) NaCl, and greater are considered to be extreme halophiles. This review encompasses extremely halophilic archaea and their growth characteristics with respect to the correlation between the extent of alkaline pH and elevated temperature optima and the extent of salt tolerance. The focus is on poly-extremophiles, i.e., taxa growing optimally at a Na⁺ concentration at or above 1.7 M (approximately 10% w/v NaCl); alkaline pH, at or above 8.5; and elevated temperature optima, at or above 50°C. So far, only a very few extreme halophiles that are able to grow optimally under alkaline conditions as well as at elevated temperatures have been isolated. The distribution of extremely halophilic archaea growing optimally at 3.4 M Na⁺ (approximately 20% w/v NaCl) is bifurcated with respect to pH optima, either they are neutrophilic, with a pH_opt of approximately 7, or strongly alkaliphilic, with pH_opt at or above 8.5. Amongst these extreme halophiles which have elevated pH optima, only four taxa have an optimum temperature above 50°C: Haloarcula quadrata (52°C), Haloferax elongans (53°C), Haloferax mediterranei (51°C) and Natronolimnobius ‘aegyptiacus’ (55°C).     

The growth and phospholipid composition of a moderately halophilic bacterium during adaptation to changes in salinity

The effect of a sudden change in NaCl concentration of the medium on the time course of alterations in growth rate and phospholipid composition of the moderately halophilic bacteriumVibrio costicola has been investigated. This organism and other moderate halophiles are known to contain a larger proportion of negatively charged phospholipids in their membranes when grown at higher salt concentrations. We show for the first time that the change in proportion of phosphatidylglycerol, relative to phosphatidylethanolamine, which occurs after a shift from 1M to 3M NaCl, or vice versa, is essentially completed during that period immediately following the salt shift when growth is zero or very slow, and before the cells have adopted the growth rate appropriate to the new salt concentration. It appears, therefore, that the alteration in membrane phospholipid composition may be a necessary physiological response for adaptation to change in salinity.     

Adaptive modifications in membranes of halotolerant and halophilic microorganisms

Halotolerant and halophilic microorganisms can grow in (hyper)saline environments, but only halophiles specifically require salt. Genotypic and phenotypic adaptations are displayed by halophiles; the halotolerants adapt phenotypically, but it is not established whether they show genotypic adaptation. This paper reviews the various strategies of haloadaptation of membrane proteins and lipids by halotolerant and halophilic microorganisms. Moderate halophiles and halotolerants adapt their membrane lipid composition by increasing the proportion of anionic lipids, often phosphatidylglycerol and/or glycolipids, which in the moderately halophilic bacteriumVibrio costicola appears to be part of an osmoregulatory response to minimize membrane stress at high salinities. Extreme halophiles possess typical archaebacterial ether lipids, which are genotypically adapted by having additional substitutions with negatively-charged residues such as sulfate. In contrast to the lipids, it is less clear whether membrane proteins are haloadapted, although they may be more acidic; very few depend on salt for their activity.     

A First Record of Obligate Halophilic Aspergilli from the Dead Sea

The isolation of obligate halophilic aspergilli from the Dead Sea and the range of salt tolerance of halophilic fungi isolated, are reported here for the first time. The mycobiota of the Dead Sea isolated in this study, was dominated by Aspergillus and Penicillium species; Cladosporium were found in lesser numbers. All three genera were obtained from the water sample; however, Aspergillus was the only genus obtained from the sediment. There was significant difference in growth of each isolate at different salt concentrations and intraspecies analysis revealed dissimilarity in response of strains to different salt concentrations in the growth medium The isolates were euryhaline, with halotolerance up to 20–25% solar salt, Aspergillus and Penicillium species showing a higher level of halotolerance, as compared to that of Cladosporium. Halophilic fungi were found in greater numbers in the sediment sample as compared to that in the water sample. Penicillium and Cladosporium species were exclusively facultative halophiles, while some species of Aspergillus were facultative halophiles. All the obligate halophiles isolated, belonged to the genus Aspergillus and were identified as A. penicillioides and A unguis, the latter being a first record of the species from the Dead Sea.     

Survival of extremely and moderately halophilic isolates of Tunisian solar salterns after UV-B or oxidative stress

Adaptation to a solar saltern environment requires mechanisms providing tolerance not only to salinity but also to UV radiation (UVR) and to reactive oxygen species (ROS). We cultivated prokaryote halophiles from two different salinity ponds: the concentrator M1 pond (240 g·L(-1) NaCl) and the crystallizer TS pond (380 g·L(-1) NaCl). We then estimated UV-B and hydrogen peroxide resistance according to the optimal salt concentration for growth of the isolates. We observed a higher biodiversity of bacterial isolates in M1 than in TS. All strains isolated from TS appeared to be extremely halophilic Archaea from the genus Halorubrum. Culturable strains isolated from M1 included extremely halophilic Archaea (genera Haloferax, Halobacterium, Haloterrigena, and Halorubrum) and moderately halophilic Bacteria (genera Halovibrio and Salicola). We also found that archaeal strains were more resistant than bacterial strains to exposure to ROS and UV-B. All organisms tested were more resistant to UV-B exposure at the optimum NaCl concentration for their growth, which is not always the case for H(2)O(2). Finally, if these results are extended to other prokaryotes present in a solar saltern, we could speculate that UVR has greater impact than ROS on the control of prokaryote biodiversity in a solar saltern.     

Microbial life at high salt concentrations: phylogenetic and metabolic diversity

Halophiles are found in all three domains of life. Within the Bacteria we know halophiles within the phyla Cyanobacteria, Proteobacteria, Firmicutes, Actinobacteria, Spirochaetes, and Bacteroidetes. Within the Archaea the most salt-requiring microorganisms are found in the class Halobacteria. Halobacterium and most of its relatives require over 100–150 g/l salt for growth and structural stability. Also within the order Methanococci we encounter halophilic species. Halophiles and non-halophilic relatives are often found together in the phylogenetic tree, and many genera, families and orders have representatives with greatly different salt requirement and tolerance. A few phylogenetically coherent groups consist of halophiles only: the order Halobacteriales, family Halobacteriaceae (Euryarchaeota) and the anaerobic fermentative bacteria of the order Halanaerobiales (Firmicutes). The family Halomonadaceae (Gammaproteobacteria) almost exclusively contains halophiles. Halophilic microorganisms use two strategies to balance their cytoplasm osmotically with their medium. The first involves accumulation of molar concentrations of KCl. This strategy requires adaptation of the intracellular enzymatic machinery, as proteins should maintain their proper conformation and activity at near-saturating salt concentrations. The proteome of such organisms is highly acidic, and most proteins denature when suspended in low salt. Such microorganisms generally cannot survive in low salt media. The second strategy is to exclude salt from the cytoplasm and to synthesize and/or accumulate organic 'compatible' solutes that do not interfere with enzymatic activity. Few adaptations of the cells' proteome are needed, and organisms using the 'organic-solutes-in strategy' often adapt to a surprisingly broad salt concentration range. Most halophilic Bacteria, but also the halophilic methanogenic Archaea use such organic solutes. A variety of such solutes are known, including glycine betaine, ectoine and other amino acid derivatives, sugars and sugar alcohols. The 'high-salt-in strategy' is not limited to the Halobacteriaceae. The Halanaerobiales (Firmicutes) also accumulate salt rather than organic solutes. A third, phylogenetically unrelated organism accumulates KCl: the red extremely halophilic Salinibacter (Bacteroidetes), recently isolated from saltern crystallizer brines. Analysis of its genome showed many points of resemblance with the Halobacteriaceae, probably resulting from extensive horizontal gene transfer. The case of Salinibacter shows that more unusual types of halophiles may be waiting to be discovered.     

Sodium chloride affects propidium monoazide action to distinguish viable cells

Propidium monoazide (PMA) is a DNA-intercalating agent used to selectively detect DNA from viable cells by polymerase chain reaction (PCR). Here, we report that high concentrations (>5%) of sodium chloride (NaCl) prevents PMA from inhibiting DNA amplification from dead cells. Moreover, Halobacterium salinarum was unable to maintain cell integrity in solutions containing less than 15% NaCl, indicating that extreme halophilic microorganisms may not resist the concentration range in which PMA fully acts. We conclude that NaCl, but not pH, directly affects the efficiency of PMA treatment, limiting its use for cell viability assessment of halophiles and in hypersaline samples.     

Intracellular ion and organic solute concentrations of the extremely halophilic bacterium <Emphasis Type="Italic">Salinibacter ruber</Emphasis>

Salinibacter ruber is a red obligatory aerobic chemoorganotrophic extremely halophilic Bacterium, related to the order Cytophagales. It was isolated from saltern crystallizer ponds, and requires at least 150 g l(-1) salt for growth. The cells have an extremely high potassium content, the ratio K(+)/protein being in the same range as in halophilic Archaea of the order Halobacteriales. X-ray microanalysis in the electron microscope of cells grown in medium of 250 g l(-1) salt confirmed the high intracellular K(+)concentrations, and showed intracellular chloride to be about as high as the cation concentrations within the cells. A search for intracellular organic osmotic solutes, using (13)C-NMR and HPLC techniques, showed glutamate, glycine betaine, and N-alpha-acetyllysine to be present in low concentrations only, contributing very little to the overall osmotic balance. The results presented suggest that the extremely halophilic Bacterium Salinibacteruses a similar mode of haloadaptation to that of the Archaea of the order Halobacteriales, and does not accumulate organic osmotic solutes such as are used by all other known halophilic and halotolerant aerobic Bacteria.     

Lipid membranes from halophilic and alkali-halophilic Archaea have a low H+ and Na+ permeability at high salt concentration

The influence of pH and the salt concentration on the proton and sodium ion permeability of liposomes formed from lipids of the halophile Halobacterium salinarum and the haloalkaliphile Halorubrum vacuolatum were studied. In contrast with liposomes formed from Escherichia coli lipids, liposomes formed from halophilic lipids remained stable up to 4 M of NaCl and KCl. The proton permeability of the liposomes from lipids of halophiles was independent of the salt concentration and was essentially constant between pH 7 and pH 9. The sodium ion permeability increased with the salt concentration but was 10- to 100 fold lower than the proton permeability. It is concluded that the membranes of halophiles are stable over a wide range of salt concentrations and at elevated pH values and are well adapted to the halophilic conditions. Received: February 25, 1999 / Accepted: June 11, 1999     

Ecology and molecular adaptations of the halophilic black yeast Hortaea werneckii

Molecular studies on halophilic adaptations have focused on prokaryotic microorganisms due to a lack of known appropriate eukaryotic halophilic microorganisms. However, the black yeast Hortaea werneckii has been identified as the dominant fungal species in hypersaline waters on three continents. It represents a new model organism for studying the mechanisms of salt tolerance in eukaryotes. Ultrastructural studies of the H. werneckii cell wall have shown that it synthesizes dihydroxynaphthalene (DHN) melanin under both saline and non-saline growth conditions. However, melanin granules in the cell walls are organized in a salt-dependent way, implying the potential osmoprotectant role of melanin. At the level of membrane structure, H. werneckii maintains a sterol-to-phospholipid ratio significantly lower than the salt-sensitive Saccharomyces cerevisiae. Accordingly, membranes of H. werneckii are more fluid over a wide range of NaCl concentrations, indicating high intrinsic salt stress tolerance. Even H. werneckii grown in high NaCl concentrations maintains very low intracellular amounts of potassium and sodium, demonstrating the sodium-excluder character of this organism. The salt-dependent expressions of two HwENA genes suggest roles for them in the adaptation to changing salt concentrations. The high similarity of these ENA ATPases to other fungal ENA ATPases involved in Na⁺/K⁺ transport indicates their potential importance in H. werneckii ion homeostasis. Glycerol is the main compatible solute which accumulates in the cytoplasm of H. werneckii at high salinity, although it seems that mycosporines may also act as supplementary compatible solutes. Salt dependent increase in glycerol synthesis is supported by the identification of two copies of a gene putatively coding for glycerol-3-phosphate-dehydrogenase. Expression of only one of these genes is salt dependent.