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.     

Ion permeability of the cytoplasmic membrane limits the maximum growth temperature of bacteria and archaea

Protons and sodium ions are the most commonly used coupling ions in energy transduction in bacteria and archaea. At their growth temperature, the permeability of the cytoplasmic membrane of thermophilic bacteria to protons is high compared with that of sodium ions. In some thermophiles, sodium is the sole energy-coupling ion. To test whether sodium is the preferred coupling ion at high temperatures, the proton- and sodium permeability was determined in liposomes prepared from lipids isolated from various bacterial and archaeal species that differ in their optimal growth temperature. The proton permeability increased with the temperature and was comparable for most species at their respective growth temperatures. Liposomes of thermophilic bacteria are an exception in the sense that the proton permeability is already high at the growth temperature. In all liposomes, the sodium permeability was lower than the proton permeability and increased with the temperature. The results suggest that the proton permeability of the cytoplasmic membrane is an important parameter in determining the maximum growth temperature.     

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).     

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.     

Molecular mechanisms of water and solute transport across archaebacterial lipid membranes

Archaebacteria thrive in environments characterized by anaeobiosis, saturated salt, and both high and low extremes of temperature and pH. The bulk of their membrane lipids are polar, characterized by the archaeal structural features typified by ether linkage of the glycerol backbone to isoprenoid chains of constant length, often fully saturated, and with sn-2,3 stereochemistry opposite that of glycerolipids of Bacteria and Eukarya. Also unique to these bacteria are macrocyclic archaeol and membrane spanning caldarchaeol lipids that are found in some extreme thermophiles and methanogens. To define the barrier function of archaebacterial membranes and to examine the effects of these unique structural features on permeabilities, we investigated the water, solute (urea and glycerol), proton, and ammonia permeability of liposomes formed by these lipids. Both the macrocyclic archaeol and caldarchaeol lipids reduced the water, ammonia, urea, and glycerol permeability of liposomes significantly (6-120-fold) compared with diphytanylphosphatidylcholine liposomes. The presence of the ether bond and phytanyl chains did not significantly affect these permeabilities. However, the apparent proton permeability was reduced 3-fold by the presence of an ether bond. The presence of macrocyclic archaeol and caldarchaeol structures further reduced apparent proton permeabilities by 10-17-fold. These results indicate that the limiting mobility of the midplane hydrocarbon region of the membranes formed by macrocyclic archaeol and caldarchaeol lipids play a significant role in reducing the permeability properties of the lipid membrane. In addition, it appears that substituting ether for ester bonds presents an additional barrier to proton flux.     

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.     

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.     

Chloride dependence of endospore germination in Halobacillus halophilus

The ion requirement for germination and outgrowth of endospores from the moderately halophilic salt marsh bacterium Halobacillus halophilus was studied. Germination and outgrowth of endospores plated onto nutrient broth was dependent on the salt concentration in the artificial seawater used as the source of ions. Maximal germination and outgrowth were observed when double-concentrated artificial seawater was used. Replacement of chloride salts in the artificial seawater by other salts resulted in a complete loss of germination and outgrowth that was restored upon addition of chloride. To analyze the role of chloride more directly and quantitatively, a defined growth medium was used in which the artificial seawater was substituted by a solution of magnesium sulfate and sodium chloride. Spore germination and outgrowth were strictly dependent on the chloride concentration; maximal germination and outgrowth were observed at ≈ 1.3 M Cl^–. Chloride could be substituted by bromide, but not by sulfate or nitrate. Microscopic examinations of single spores clearly showed that germination is the chloride-dependent step. This first report on chloride dependence of spore germination in any endospore-forming bacterium adds another function to chloride in H. halophilus apart from its being essential for the physiology of the vegetative cell. Received: 21 May 1999 / Accepted: 26 July 1999     

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.     

Stability of pressure-extruded liposomes made from archaeobacterial ether lipids

Ether lipids were obtained from a wide range of archaeobacteria grown at extremes of pH, temperature, and salt concentration. With the exception ofSulfolobus acidocaldarius, unilamellar and/or multilamellar liposomes could be prepared from emulsions of total polar lipid extracts by pressure extrusion through filters of various pore sizes. Dynamic light scattering, and electron microscopy revealed homogeneous liposome populations with sizes varying from 40 to 230 nm, depending on both the lipid source and the pore size of the filters. Leakage rates of entrapped fluorescent or radioactive compounds established that those archaeobacterial liposomes that contained tetraether lipids were the most stable to high temperatures, alkaline pH, and serum proteins. Most ether liposomes were stable to phospholipase A2, phospholipase B and pancreatic lipase. These properties of archaeobacterial liposomes make them attractive for applications in biotechnology.     

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.     

Physicochemical study of the protein–liposome interactions: influence of liposome composition and concentration on protein binding

Abstract

The aim of the present study is to investigate the interactions between liposomes and proteins and to evaluate the role of liposomal lipid composition and concentration in the formation of protein corona. Liposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or hydrogenated soybean phosphatidylcholine (HSPC) with 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt) (DPPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-3000] (DPPE-PEG 3000), cholesterol (CH) or mixtures of these lipids, were prepared at different concentrations by the thin-film hydration method. After liposomes were dispersed in HPLC-grade water and foetal bovine serum (FBS), their physicochemical characteristics, such as size, size distribution, and ζ-potential, were determined using dynamic and electrophoretic light scattering. Aggregation of DPPC, HSPC, DPPC:CH (9:1 molar ratio), and HSPC:CH (9:1 molar ratio) in FBS was observed. On the contrary, liposomes incorporating DPPG lipids and CH both in a molar ratio of 11% were found to be stable over time, while their size did not alter dramatically in biological medium. Liposomes containing CH and PEGylated lipids retain their size in the presence of serum as well as their physical stability. In addition, our results indicate that the protein binding depends on the presence of polyethylene glycol (PEG), CH, concentration and surface charge. In this paper, we introduce a new parameter, fraction of stealthiness (F_s), for investigating the extent of protein binding to liposomes. This parameter depends on the changes in size of liposomes after serum incubation, while liposomes have stealth properties when F_s is close to 1. Thus, we conclude that lipid composition and concentration affect the adsorption of proteins and the liposomal stabilization.     

Lipid‐dependent pore formation by antimicrobial peptides arenicin‐2 and melittin demonstrated by their proton transfer activity

This work presents a comparative study of proton transfer activity (PTA) of two cationic (+6) antimicrobial peptides, β‐structural arenicin‐2 and α‐helical melittin. A new approach was proposed for the detection of passive proton transfer by using proteoliposomes containing bacteriorhodopsin, which creates a small light‐induced electrochemical proton gradient ?ΔpH. Addition of several nanomoles of the peptides lowers ?ΔpH that is proximately indicative of the pore formation. The quantitative analysis of sigmoidal dependences of ?pH on the peptides concentration was carried out using liposomes prepared from PC, PC/PE, PC/PE/PI and PC/PG. Substitution of PC‐containing liposomes with PE‐containing ones, having negative spontaneous curvature, reduced the PTA of α‐helical melittin and increased that of β‐structural arenicin‐2. This result indicates an essential difference in the pore formation by these peptides. Further increase of PTA in response to arenicin‐2 (in contrast to melittin) was observed in the liposomes prepared from PC/PE/PI. The data analysis leads to the conclusion that PTA is influenced by (i) efficiency of the pore assemblage, which depends on the structure of pore‐forming peptides, and the spontaneous curvature of lipids and (ii) the presence of mobile protons in the polar head groups of phospholipids. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

嗜盐耐盐微生物抗盐胁迫相关离子转运蛋白研究进展

离子转运蛋白在维持细胞内pH稳态、离子动态平衡等方面发挥着重要作用。钠离子转运体和钾离子转运体在嗜盐耐盐微生物中广泛存在,其"保钾排钠"机制是微生物抗盐胁迫的两大策略之一。近年来,嗜盐耐盐微生物中许多新型钠、钾离子转运体被陆续发现,如RDD蛋白、UPF0118蛋白、DUF蛋白和KimA蛋白等;Fe³⁺、Mg²⁺等其他金属离子的转运蛋白也被证实可通过影响微生物胞内相容性溶质的合成起到渗透调节的作用。本文综述了嗜盐耐盐微生物中抗盐胁迫相关的各类离子转运蛋白,分析其分子结构和工作机理,并对这些蛋白在农业方面的应用进行了展望。继续发现新的离子转运蛋白,探究抗盐胁迫相关离子转运蛋白的结构和机理,解析各转运系统的协同作用及分子调控机制,将进一步加深对嗜盐耐盐微生物抗盐胁迫调控的认识,并为盐碱地农作物的改良等提供新的思路。     

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.     

The cell membrane plays a crucial role in survival of bacteria and archaea in extreme environments

The cytoplasmic membrane of bacteria and archaea determine to a large extent the composition of the cytoplasm. Since the ion and in particular the proton and/or the sodium ion electrochemical gradients across the membranes are crucial for the bioenergetic conditions of these microorganisms, strategies are needed to restrict the permeation of these ions across their cytoplasmic membrane. The proton and sodium permeabilities of all biological membranes increase with the temperature. Psychrophilic and mesophilic bacteria, and mesophilic, (hyper)thermophilic and halophilic archaea are capable of adjusting the lipid composition of their membranes in such a way that the proton permeability at the respective growth temperature remains low and constant (homeo-proton permeability). Thermophilic bacteria, however, have more difficulties to restrict the proton permeation across their membrane at high temperatures and these organisms have to rely on the less permeable sodium ions for maintaining a high sodium-motive force for driving their energy requiring membrane-bound processes. Transport of solutes across the bacterial and archaeal membrane is mainly catalyzed by primary ATP driven transport systems or by proton or sodium motive force driven secondary transport systems. Unlike most bacteria, hyperthermophilic bacteria and archaea prefer primary ATP-driven uptake systems for their carbon and energy sources. Several high-affinity ABC transporters for sugars from hyperthermophiles have been identified and characterized. The activities of these ABC transporters allow these organisms to thrive in their nutrient-poor environments. This revised version was published online in August 2006 with corrections to the Cover Date.     

Biochemical aspects of the visual process XXXII. Movement of sodium ions through bilayers composed of retinal and rod outer segment lipids

The leakage of Na⁺ from sonicated liposomes, composed of rod outer segment lipids, retinal lipids and a 4 : 1 phosphatidylcholine/phosphatidylserine mixture, has been studied. Both retinal and rod outer segment lipid liposomes lose Na⁺ faster than Ca²⁺ which indicates that the observed leakage occurs from closed liposomal structures.Liposomes from rod outer segment lipids are extremely leaky, losing sodium about 10 times as fast as retinal lipid liposomes and twice as fast as the phosphatidylcholine/phosphatidylserine liposomes.This high permeability of rod outer segment lipid liposomes, as compared to retinal lipid liposomes, is probably due to both the higher degree of unsaturation of the fatty acid chains and their lower cholesterol content. In the rod outer segment lipid extract 48% of the fatty acid chains consists of docosahexaenoic acid (C_22:6) against only 24% in retinal lipid extract. Rod outer segment lipids contain 4.0% cholesterol against 12.3% in retinal lipids.The sodium leakage from rod outer segment lipid liposomes is little affected by the presence of 5 mM calcium in the external dialysis medium, but with the two other types of liposomes significant decreases in permeability of about 20% are observed.The results are discussed in connection with the role of cations in visual excitation.     

Could halophilic archaea improve the traditional salted anchovies (Engraulis encrasicholus L.) safety and quality?

Aims: The positive influence of two selected extremely halophilic archaea strains in the production of salted anchovies (Engraulis encrasicolus, L., 1758) was highlighted. Methods and Results: Anchovies produced with salt artificially contaminated with halophiles exhibited lower loads of staphylococci, Enterobacteriaceae and lactic acid bacteria, and a reduced content of histamine as well as an improved organoleptic acceptance. Conclusions: The findings of this survey are expected to enhance the safety of salted anchovies, with regard to the histamine formation during ripening, and to improve the sensory attributes of this product. Significance and Impact of the Study: This study represents the first report on the positive influence of halophilic archaea in traditional salted anchovies production, thus suggesting new perspectives about a conscious employment of properly selected haloarchaea strains in this traditional manufacture.     

Bioenergetics and cytoplasmic membrane stability of the extremely acidophilic, thermophilic archaeon Picrophilus oshimae

Picrophilus oshimae is an extremely acidophilic, thermophilic archaeon that grows optimally at 60°C and at pH 0.7. It is an obligatory acidophile that does not grow at pH values above 4.0. The proton motive force in respiring cells is composed of a large transmembrane pH gradient, inside less acid, and a reversed transmembrane electrical potential, inside positive. Cells maintain an intracellular pH at around 4.6 at extracellular pH values ranging from 0.8 to 4.0. Above pH 4.0 cells lyse rapidly and lose their viability. Liposomes prepared from lipids derived from P. oshimae have an extremely low proton permeability at acidic pH. However, at neutral pH, the lipids are unable to assemble into regular liposomal structures. These observations suggest that the loss of viability and cell integrity above pH 4.0 is due to an impairment of the barrier function of the cytoplasmic membrane. Received: July 18, 1997 / Accepted: November 25, 1997     

An ion switch regulates fusion of charged membranes

Here we identify the recruitment of solvent ions to lipid membranes as the dominant regulator of lipid phase behavior. Our data demonstrate that binding of counterions to charged lipids promotes the formation of lamellar membranes, whereas their absence can induce fusion. The mechanism applies to anionic and cationic liposomes, as well as the recently introduced amphoteric liposomes. In the latter, an additional pH-dependent lipid salt formation between anionic and cationic lipids must occur, as indicated by the depletion of membrane-bound ions in a zone around pH 5. Amphoteric liposomes fuse under these conditions but form lamellar structures at both lower and higher pH values. The integration of these observations into the classic lipid shape theory yielded a quantitative link between lipid and solvent composition and the physical state of the lipid assembly. The key parameter of the new model, κ(pH), describes the membrane phase behavior of charged membranes in response to their ion loading in a quantitative way.