Salt adaptation in chemolithotrophic alkaliphilic sulfur-oxidizing strains belonging to genera Thioalkalimicrobium and Thioalkalivibrio has been studied by determination of salt-dependent changes in fatty acid and compatible solute composition. In both alkaliphilic groups, represented by the low salt-tolerant Thioalkalimicrobium aerophilum strain AL 3T and the extremely salt-tolerant Thioalkalivibrio versutus strain ALJ 15, unsaturated fatty acids predominate over saturated fatty acids. In strain AL 3T, C18:1, C16:0 and C16:1 were the dominant fatty acids. In strain ALJ 15, the concentrations of C18:1 and C19cyclo were salt-regulated in an inverse proportional relationship, suggesting the stimulation of cyclopropyl-synthetase activity. Squalene has been found in substantial amounts only in strain ALJ 15. Ectoine and glycine betaine were found to be the main osmolytes in Thioalkalimicrobium aerophilum and Thioalkalivibrio versutus, respectively. The production of ectoine and glycine betaine was positively correlated with the salt concentration in the growth medium. A novel type of membrane-bound yellow pigments was uniformly detected in the extremely salt-tolerant strains of Thioalkalivibrio with a backbone consisting of C15-polyene, whose specific concentration correlated with increasing salinity of the growth medium. The results suggest that the mechanisms of haloalkaliphilic adaptation in Thioalkalimicrobium sp. and Thioalkalivibrio sp. involve the production of cyclopropane fatty acids, organic compatible solutes and, possibly specific pigments.
Soda lakes represent extreme environments characterized by moderate to high concentrations of salts (as Na2CO3/NaHCO3) and alkaline pH (9–10.5). The abundance of sodium carbonates leads to a highly buffered alkalinity . The organisms living in soda lakes are in general obligately haloalkaliphilic, requiring the presence of Na+ and at least pH 9 for growth. Their diversity and biology have been extensively reviewed [2–4]. Also, the physiology, biochemistry and molecular mechanisms that allow the adaptation of halophiles at high salt [5,6] and of non-halophilic alkaliphiles at high pH  are well documented. However, there are still questions concerning the adaptative features of double extremophilic salt-loving alkaliphiles.
Obligately chemolithoautotrophic sulfur-oxidizing bacteria (SOB) living in soda lakes have been recently discovered and classified into three genera in the gamma subdivision of the Proteobacteria. The genera Thioalkalimicrobium, Thioalkalivibrio and Thioalkalispira are all capable of autotrophic growth in highly alkaline saline media using inorganic sulfur compounds as electron donors and energy source [8–10]. The genetic and metabolic diversity, ecology and growth physiology of representatives of the haloalkaliphilic SOB have been studied and important differences have been found [8,11,12]. The low-salt tolerant Thioalkalimicrobium dominated in the hyposaline soda lakes in Central Asia. The Thioalkalivibrio group covered the complete salt spectrum, from moderate to saturated soda brines present in soda lakes of Central Asia, Africa and North America. The genetic and phenotypic diversity of this group is much greater than that of Thioalkalimicrobium. Yet, little is known about the mechanisms of salt adaptation in the new SOB.
The aim of this study was to reveal the structural differences in the two groups of haloalkaliphilic SOB from soda lakes represented by the extremely salt-tolerant Thioalkalivibrio and the low-salt tolerant Thioalkalimicrobium.
2Material and methods
2.1Strains and growth conditions
The low-salt tolerant Thioalkalimicrobium aerophilum strain AL 3T isolated from the hyposaline lake Hadyn in Tuva (Siberia)  and several extremely salt tolerant Thioalkalivibrio strains isolated from the hypersaline soda lakes in Kenya , Wadi Natrun in Egypt (D.Y. Sorokin, unpublished), Mongolia  and Mono Lake in California  have been used in this work.
Thioalkalimicrobium aerophilum AL 3T is a low-salt tolerant and alkaliphilic strain that grows optimally in the presence of 0.3–0.5 M Na+ and at pH 9.5–10. For lipid and compatible solute analysis, strain AL 3T was grown in batch culture on alkaline mineral medium and thiosulfate as energy source. The mineral medium (0.6 M Na+) contained: 20 g/l Na2CO3, 6 g/l NaCl, 1 g/l K2HPO4, 1 g/l KNO3 and 8 g/l NaHCO3. After sterilization, this was supplemented with 40 mM thiosulfate, 1 mM MgCl2 and 1 ml/l of trace elements solution . The final pH was 10. For the growth at 0.2 and 1.2 M Na+, the medium contained less or more Na2CO3 and NaHCO3. The extremely salt-tolerant and alkaliphilic Thioalkalivibrio versutus strains can grow over a wide range of sodium concentration and alkaline pH with an optimum of 1–2 M Na+ and pH 10, respectively. For the analysis of fatty acid composition and pigment content, strains were grown in batch culture on mineral sodium carbonate-based medium containing 0.6, 2 and 4 M Na+, pH 10, with thiosulfate as energy source. All isolates were cultivated aerobically, on a rotary shaker at 30 °C (strain AL3T) and at 35 °C (Thioalkalivibrio versutus).
For determination of salt-dependent production of compatible solutes in strain ALJ 15, a thiosulfate-limited continuous cultivation was used. Continuous cultivation was performed in 1.5 l laboratory fermentors with a 1 l working volume, fitted with pH and oxygen controls (Applikon, Schiedam, The Netherlands). The pH was controlled at the level of 10.05–10.1 by automatic titration with 2 M NaOH and HCl. The dissolved oxygen concentration was controlled at a minimum level of 50% air saturation by the stirring speed. The temperature was set at 35 °C. The mineral media had identical composition with those used in batch cultivation. High-density cultivation of strain ALJ 15 for pigment analysis was performed in 15 l batch fermentor (Applikon) at pH 10 and 2 M total Na+. The initial volume of the medium was 10 l and at the end of cultivation it increased up to 12 l due to consequent additions of the substrate. After complete consumption of thiosulfate, new 40 mM portions were added, up to 200 mM in total. The growing consumption of oxygen was compensated by increasing the air pressure up to 0.8 atm to control the dissolved oxygen above 10% air saturation in order to prevent sulfur formation.
The growth of haloalkaliphilic strains was followed by thiosulfate consumption in the media. Micromolar thiosulfate concentrations were determined by cyanolytic procedure . Millimolar-range thiosulfate consumption in batch cultures was followed by standard iodimetric titration after neutralization of the medium with 50% (v/v) acetic acid.
For lipid analysis as well as for compatible solutes determination, cells grown under different sodium concentration, were harvested at 10,000g for 20 min. Biomass was washed twice in neutral, iso-osmotic saline solutions (e.g., 0.6, 2 and 3 M NaCl) to avoid cell lysis. The pellet was frozen overnight at −80 °C and freeze-dried. Lipids were ultrasonically extracted from the freeze-dried cell material using methanol, methanol/dichloromethane (1:1, v/v) and dichloromethane (3×). An aliquot of the total lipid extract, named as free lipids, was methylated with diazomethane in diethyl ether and filtered over a small SiO2 column with ethyl acetate as the eluent. To obtain the free and bound lipid fraction another aliquot of the total lipid extract was subjected to base hydrolysis (1 M KOH in 60% methanol under reflux for 1 h) and was subsequently processed in the same way. Alcohols in the ethyl acetate eluate of both fractions were converted to their trimethylsilyl ether derivatives with bis(trimethylsilyl)trifluoracetamide (BSTFA) in pyridine. Individual fatty acid methyl esters and trimethylsilyl derivatives of alcohols were quantified by gas chromatography (GC) and identified by GC–mass spectrometry (MS) as described in detail elsewhere .
2.3The analysis of organic and inorganic compatible solutes
Determination of potassium and chloride ions was performed by INAA (Instrumental Neutron Activation Analysis) at the Department of Chemistry, TU Delft. The “Hoger Onderwijs Reactor” was used as source for neutrons. The gamma spectrometer used a germanium semiconductor as detector and a computer controlled sample changer.
Intracellular compatible solutes were extracted and analyzed following a modification of the method described by Galinski and Herzog . HPLC separation used an isocratic system from Thermo Separation Products (CA), a 3 μm Grom-sil Amino-1PR column (Grom Analytik, Rottenburg-Hailfingen, Germany) and a Shodex refractive index detector (model RI17, Showa Denko K.K., Tokyo, Japan). The mobile phase consisted of 80% (v/v) acetonitrile at a flow rate of 1 ml min−1. Natural abundance 13C NMR spectra of compatible solutes were recorded in the pulsed Fourier transform mode on a Bruker spectrometer (model Avance 3000 DPX) operating at 75.48 MHz (13C) and at 300 MHz for the proton decoupling channel relative to sodium trimethylsilylpropionate (TMSP).
Preliminary evidence on the presence of a pigment and its content were obtained by UV–visible spectrophotometry (Diode-array HP 8453, Amsterdam, The Netherlands) of methanol extracts from the wet cell pellets. Structural analysis of the pigments produced by the Thioalkalivibrio versutus strain ALJ 15 was performed after methanol extraction from dry biomass as described by Takaichi et al. .
3.1Lipid and fatty acid composition
The results of fatty acid analyses in Thioalkalimicrobium aerophilum strain AL 3T grown at 0.6 M Na+ (pH 10) and in Thioalkalivibrio versutus strain ALJ 15 grown at different sodium concentration (pH 10) are presented in Tables 1 and 2. From these results several differences between the two strains (both grown at 0.6 M Na+ and pH 10) could be outlined. When looking at the free and bound lipid composition, in strain AL 3T the concentration of monounsaturated fatty acids, and in particular C18:1 (80.4%), is higher than in strain ALJ 15 (49.5%). An unusual monounsaturated, possibly branched fatty acid C19 named “C19:1” was detected in strain AL 3T, but not in strain ALJ 15.
Table 1. Fatty acid composition in Thioalkalimicrobium aerophilum strain AL 3T grown at optimal salt concentration (0.6 M Na+), 30 °C and pH 10
% Free + bound
Table 2. Fatty acid composition in Thioalkalivibrio versutus strain ALJ 15 grown at different salt concentration, 35 °C and pH 10
% Free + bound
In Thioalkalivibrio versutus ALJ 15, the dominant fatty acids are the saturated C16:0, the monounsaturated C18:1 and the cyclic cyclopropyl-C19 (C19cyclo). The salt dependence of fatty acid distribution in strain ALJ 15 was best observed in the change of C18:1 and C19cyclo. The proportion of the C18:1 fatty acid remained relatively unchanged in cells grown at near-optimal salt concentration (0.6–2 M Na+), but was 10% lower at 4 M. At the same time C19cyclo showed an increase of approximately 20% from 0.6 to 2 M and approximately 50% from 0.6 to 4 M Na+. These changes were even more pronounced with the relative proportion of free fatty acids (80% and 300% increase, respectively). Overall, in both types of soda lakes SOB the total concentration of unsaturated fatty acids is higher than that of the saturated fatty acids.
Squalene, a non-polar lipid, was found in high amounts in cells of strain ALJ 15 grown over the whole range of Na+ concentration, but could not be related to salt adaptation. The relative squalene concentrations (% of squalene related to sum of squalene and all quantified fatty acids) in Thioalkalivibrio versutus strain ALJ 15 were: 41% (in 0.6 M Na+-grown cells), 52% (at 2 M Na+) and 28% (at 4 M Na+). The relative proportion of squalene is highest at near-optimum salt concentrations (0.6–2 M Na+) than at marginal salt concentration (4 M Na+). Another finding was that the relative concentration of the diglycerides in strain ALJ 15 is highest in 4 M Na+-grown culture (data not shown).
3.2Compatible solute composition
The main compatible solute in Thioalkalimicrobium aerophilum strain AL 3T was ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid) which accounted for 0.9–8.7% of the total dry weight depending on the growth conditions. There was a direct dependence of ectoine production with the Na+ concentration at which the organism was grown (Fig. 1). This result may suggest that ectoine is involved in the osmoregulation of strain AL 3T. Another organic compatible solute that could be detected in strain AL 3T in minor amounts was glutamate. The glutamate production decreased with increasing salt concentration (data not shown).
In Thioalkalivibrio versutus strain ALJ 15 grown in continuous culture under substrate limitation, glycine betaine was the main organic compatible solute. At the same dilution rate, glycine betaine showed a salt-dependent production: 1.5%, 7.5% and 9% of total dry weight at 0.6, 2 and 4 M Na+, respectively (data not shown). Sucrose was produced as a minor secondary organic compatible solute in this organism and its concentration was highest in cells grown at 2 M Na+. The sucrose counted for 0.3%, 2.5% and 1.7% of the total dry weight at 0.6, 2 and 4 M Na+, respectively. The quantitative analysis of the potassium and chloride ions, which are potentially involved in the osmoregulation process in Archaea and in bacteria belonging to Halanaerobiales and Salinibacter sp., showed that their contribution to salt adaptation in the two haloalkaliphilic SOB is minor (less than 2% of the total dry weight).
When the first isolates of the Thioalkalivibrio able to grow in saturated soda brines were obtained from the Kenyan hypersaline soda lakes, it was noticed that their biomass was different from the low-salt strains by having obvious yellow coloration. Extraction of the wet biomass with methanol, acetone or their mixture, yielded a bright yellow solution which gave an absorption spectrum with a maximum at 425–430 nm and small shoulders at 405 and 450 nm (Fig. 2). Ultracentrifugation of the cell extracts obtained by sonication, revealed that the pigment was bound to the membrane. Further work demonstrated that the same pigmentation was also present in all (i.e., more than 50) extremely salt-tolerant Thioalkalivibrio strains isolated from the hypersaline soda lakes in Wadi Natrun (Egypt), Mongolia and Altai (Russia). The only exception among the colorless low-salt tolerant soda lake SOB was the obligately microaerophilic Thioalkalispira microaerophila, which produced high concentration of the yellow pigment at excessive aeration . In the extremely salt-tolerant Thioalkalivibrio the specific pigment content positively correlated with increasing salt content up to 2 M Na+ (Fig. 3).
HPLC analysis of the pigment extracted from the cells of extremely salt-tolerant Thioalkalivibrio versutus strain ALJ 15, grown at pH 10 and 2 M Na+, revealed the presence of three different fractions. The structures of the two major fractions have been identified (Fig. 4). In contrast to carotenoids, the new pigments possess considerably less carbon atoms (i.e., total number of carbon atoms is 23) and have a straight polyene instead of an isoprenoid chain. This polyene chain is terminated with a hydrophilic methylated phenole and a carboxymethyl group. The compound (2) differs from the compound (1) by the presence of chloride in the phenol group, which makes it even more unusual. The names natronochrome and chloronatronochrome have been suggested for compounds (1) and (2), respectively .
The extremely halotolerant Thioalkalivibrio versutus strain ALJ 15 has a fatty acid composition comparable to extremely halophilic Ectothiorhodospira and Halomonas species [20,21]. The C16:0, C18:1 and C19cyclo fatty acids were also the dominant fatty acids in Ectothiorhodospira sp. and Halomonas sp. The relative abundances of the C16:0 and C18:1 fatty acids in strain ALJ 15 is in the same range (14–20% and 60–70%, respectively) as in Ectothiorhodospira, while the fatty acid C18:1 concentration is two times higher than that found in Halomonas salina. However, the concentration of C19cyclo in strain ALJ 15 was five times higher than that found in Ectothiorhodospira and Halomonas salina. Small amounts of saturated C17:0 and C20:0 fatty acids have been found in Thioalkalivibrio versutus strain ALJ 15 grown at high salt concentration that are absent in Ectothiorhodospira, but present in Halomonas salina, however, only when grown at 3 M Na+. The observation that increasing salt concentration induced the decrease of monounsaturated C18:1 and concomitant increase of C19cyclo was also made for Halomonas species (H. salina and H. halophila) [20,22] and in Lactobacillus strains . This is consistent with the knowledge that cis monounsaturated fatty acids like C18:1 and its cyclic derivative C19cyclo can be interconverted. This may suggest a salt-dependent activation of cyclopropane synthetase, an enzyme involved in the regulation of membrane lipid composition and fluidity. The increase of cyclopropane fatty acid content at the expense of unsaturated fatty acids would contribute to an increase of the membrane rigidity [5,24]. A stimulation of cyclopropane synthetase activity by addition of organic compatible solutes (e.g., glycine betaine) was observed in the moderately halophilic bacterium “Pseudomonas halosaccarolytica”.
Squalene was found in unusually high amounts in the extremely salt-tolerant and alkaliphilic strain ALJ 15. In the alkaliphilic strains of Bacillus it was reported that squalenes and its derivatives represent 9.9–11.25% (w/w) of total membrane lipids . Approximately same amounts of squalenes were reported in the extremely halophilic Halobacterium species [27,28]. Squalene may play an important role in the pH adaptation and/or osmoadaptation of the halotolerant strain ALJ 15. As an apolar lipid, squalene is likely to be located within the lipid bilayer, in the most hydrophobic part of the membranes. Here, this lipid can act as a barrier decreasing the membrane permeability for ions [26,29] or stabilizing some of the membrane proteins in the halophiles [27,28]. The constant presence of squalene in the cells grown at variable salt conditions indicate that squalene is constitutively produced in the obligate alkaliphilic and extremely salt-tolerant strain ALJ 15 but its role in salt or pH adaptation is still unclear.
We have not determined the phospholipid composition, but the presence of high amounts of diglycerides together with substantial amounts of C16:1 and C18:1 fatty acids may suggest a significant concentration of negatively charged cardiolipin (diphosphatidylglycerol) in the membranes of haloalkaliphilic strain ALJ 15. The alkaliphilic bacteria were shown to contain high concentrations of squalene and anionic phospholipids, especially cardiolipin .
Both Thioalkalimicrobium aerophilum strain AL 3T and Thioalkalivibrio versutus strain ALJ 15 produce organic compatible solutes that contribute to the osmotic balance. The results obtained for strain ALJ 15 are in close agreement with the taxonomic relatedness of Thioalkalivibrio and Ectothiorhodospira, a group in which glycine betaine is one of the main compatible solutes . Glycine betaine is synthesized de novo in strain ALJ 15 but it can also be accumulated from the surrounding environment .
The presence of a new type of pigment in bacteria usually referred to as “colorless” sulfur bacteria is clearly unusual. The presence of multiple conjugated C-C bonds suggests its possible function in a radical defense. The pigments are located in the membranes of those alkaliphilic sulfur-oxidizing isolates that can grow at extremely high salt concentration. Since it is the cell membrane which functions as a barrier to salts in haloalkaliphiles, it is tempting to speculate that the new compounds somehow participate in adaptation to extremely haloalkaline conditions. However, there was one exception – the low-salt tolerant Thioalkalispira, which also produced a yellow pigment. This bacterium is an obligate micro-aerophile and its specific pigment content increased in response to excessive oxygen supply. So the pigments might participate both in salt and oxygen radical defense. Moreover, since high salt conditions are characterized by low oxygen solubility, the bacteria living in salt brines might be more sensitive to excessive oxygen than the low-salt organisms. In this respect, the high-salt and the micro-oxic conditions are related and the bacteria adapted to these conditions might possess common defensive mechanisms. Another suggestion concerns a possible defensive function against excessive radiation, which is common in shallow hypersaline lakes located in hot climatic zones. Recently, another new group of extremely halophilic neutrophilic SOB have been discovered in hypersaline lakes of central Asia (D.Y. Sorokin, unpublished results). Several of these isolates have been found to produce a yellow pigment with an absorption spectrum similar to those found in the natronophilic sulfur bacteria from soda lakes. Therefore, such compounds are not restricted to the alkaline conditions, being common among the extremely salt-tolerant/halophilic SOB.
In conclusion, the salt and pH adaptations are reflected in the biochemical composition of alkaliphilic SOB from soda lakes. In addition to constitutively high concentrations of unsaturated fatty acids and of squalene, several osmoregulated processes take place. The synthesis of cyclopropane fatty acids and of diglycerides at the membrane level is accompanied by the increase of compatible solute concentration in the cytoplasm. It is therefore possible that the modification of the internal osmotic pressure is also reflected in the changes of the membrane lipid composition.
This work was supported by the Dutch Technology Foundation (STW) project WCB.5939, Russian Foundation for Basic Research (Grant 04-04-48647) and by the Program of the Russian Academy of Sciences “Molecular and Cell Biology”. We thank M. Stein for her technical assistance.