Membrane fluidity and fatty acid comparisons in psychrotrophic and mesophilic strains of Acidithiobacillus ferrooxidans under cold growth temperatures

Psychrotrophic strains of Acidithiobacillus ferrooxidans have an important role in metal leaching and acid mine drainage (AMD) production in colder mining environments. We investigated cytoplasmic membrane fluidity and fatty acid alterations in response to low temperatures (5 and 15°C). Significant differences in membrane fluidity, measured by polarization (P) of 1,6-diphenyl-1,3,5-hexatriene (DPH), were found where the psychrotrophic strains had a significantly more rigid membrane (P range = 0.41–0.45) and lower transition temperature midpoints (T _m = 2.0°C) and broader transition range than the mesophilic strains (P range = 0.38–0.39; T _m = 2.0–18°C) at cold temperatures. Membrane remodeling was evident in all strains with a common trend of increased unsaturated fatty acid component in response to lower growth temperatures. In psychrotrophic strains, decreases in 12:0 fatty acids distinguished the 5°C fatty acid profiles from those of the mesophilic strains that showed decreases in 16:0, 17:0, and cyclo-19:0 fatty acids. These changes were also correlated with the observed changes in membrane fluidity (R ² = 63–97%). Psychrotrophic strains employ distinctive modulation of cytoplasmic membrane fluidity with uncommon membrane phase changes as part of their adaptation to the extreme AMD environment in colder climates.     

Cytoplasmic membrane fluidity and fatty acid composition of Acidithiobacillus ferrooxidans in response to pH stress

Strain variation in the acidophile Acidithiobacillus ferrooxidans was examined as a product of membrane adaptation in response to pH stress. We tested the effects of sub and supra-optimal pH in two type strains and four strains isolated from acid mine drainage water around Sudbury, Ontario, Canada. Growth rate, membrane fluidity and phase, determined from the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene, and fatty acid profiles were compared. The effect of pH 1.5 was the most pronounced compared to the other pH values of 1.8, 3.1, and 3.5. Three different types of response to lower pH were observed, the first of which appeared to maintain cellular homeostasis more effectively. This adaptive mode included a decrease in membrane fluidity and concomitant depression of the phase transition in two distinct membrane lipid components. This was explained through the increase in saturated fatty acids (predominantly 16:0 and cyclopropane 19:0 w8c) with a concomitant decrease in 18:1 w7c fatty acid. The other strains also showed common adaptive mechanisms of specific fatty acid remodeling increasing the abundance of short-chain fatty acids. However, we suspect membrane permeability was compromised due to potential phase separation, which may interfere with energy transduction and viability at pH 1.5. We demonstrate that membrane physiology permits differentiating pH tolerance in strains of this extreme acidophile.     

Effect of acidic conditions on fatty acid composition and membrane fluidity of<Emphasis Type="Italic">Escherichia coli</Emphasis> strains isolated from Crescenza cheese

The effect of low pH on minimum pH, growth kinetics and adaptive response to acidity conditions were studied on nineEscherichia coli strains having food origin and two collection strains. Moreover, the effects of low pH on the modification of fatty acid (FA) composition and membrane fluidity of the strains were studied. Also the effects of a pre-adaptation to pH 5 on acidic resistance and FA composition were considered. The results showed the crucial role of unsaturated FA in acid adaptation ofE. coli. Cyclization and isomerization of membrane FA represented the most interesting response to acidic growth conditions in theE. coli strains considered. In particularcis/trans isomerization was shown to play an important role in modulating membrane composition and fluidity under acidic conditions. Only one strain was able to adapt to acidic stress increasing its ability to grow at low pH. It responded to pH decrease, conversely to each other strain, by increasing its membrane fluidity at the lowest pH values considered. The results obtained showed that the determination of membrane fluidity in intact whole cells by fluorescence polarisation can represent a useful tool to study instantaneous stress-induced membrane fluidity modifications.     

Membrane fatty acid composition and fluidity are involved in the resistance to freezing of Lactobacillus buchneri R1102 and Bifidobacterium longum R0175

Determinations of membrane fatty acid composition and fluidity were used together with acidification activity and viability measurements to characterize the physiological state after freezing of Lactobacillus buchneri R1102 and Bifidobacterium longum R0175 cells harvested in the exponential and stationary growth phases. For both strains, lower membrane fluidity was achieved in cells harvested in the stationary growth phase. This change was linked to a lower unsaturated-to-saturated fatty acid ratio for both strains and a higher cyclic-to-saturated fatty acid ratio for L. buchneri R1102 alone. These membrane properties were linked to survival and to maintenance of acidification activity of the cells after freezing, which differed according to the strain and the growth phase. Survival of B. longum R0175 was increased by 10% in cells with low membrane fluidity and high relative saturated fatty acid contents, without any change in acidification activity. Acidification activity was more degraded (70 min) in L. buchneri R1102 cells displaying low membrane fluidity and high saturated and cyclic fatty acid levels. Finally, this study showed that membrane modifications induced by the growth phase differed among bacterial strains in terms of composition. By lowering membrane fluidity, these modifications could be beneficial for survival of B. longum R0175 during the freezing process but detrimental for maintenance of acidification activity of L. buchneri R1102.     

Metabolic control of the membrane fluidity in Bacillus subtilis during cold adaptation

Membrane fluidity adaptation to the low growth temperature in Bacillus subtilis involves two distinct mechanisms: (1) long-term adaptation accomplished by increasing the ratio of anteiso- to iso-branched fatty acids and (2) rapid desaturation of fatty acid chains in existing phospholipids by induction of fatty acid desaturase after cold shock. In this work we studied the effect of medium composition on cold adaptation of membrane fluidity. Bacillus subtilis was cultivated at optimum (40 °C) and low (20 °C) temperatures in complex medium with glucose or in mineral medium with either glucose or glycerol. Cold adaptation was characterized by fatty acid analysis and by measuring the midpoint of phospholipid phase transition T_m (differential scanning calorimetry) and membrane fluidity (DPH fluorescence polarization). Cells cultured and measured at 40 °C displayed the same membrane fluidity in all three media despite a markedly different fatty acid composition. The T_m was surprisingly the highest in the case of a culture grown in complex medium. On the contrary, cultivation at 20 °C in the complex medium gave rise to the highest membrane fluidity with concomitant decrease of T_m by 10.5 °C. In mineral media at 20 °C the corresponding changes of T_m were almost negligible. After a temperature shift from 40 to 20 °C, the cultures from all three media displayed the same adaptive induction of fatty acid desaturase despite their different membrane fluidity values immediately after cold shock.     

Proteomic insights into cold adaptation of psychrotrophic and mesophilic <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis> strains

Cold tolerant strains of Acidithiobacillus ferrooxidans play a role in metal leaching and acid mine drainage (AMD) production in northern latitude/boreal mining environments. In this study we used a proteomics and bioinformatics approach to decipher the proteome changes related to sustained growth at low temperatures to increase our understanding of cold adaptation mechanisms in A. ferrooxidans strains. Changes in protein abundance in response to low temperatures (5 and 15°C) were monitored and protein analyses of a psychrotrophic strain (D6) versus a mesophilic strain (F1) showed that both strains increased levels of 11 stress-related and metabolic proteins including survival protein SurA, trigger factor Tig, and AhpC-Tsa antioxidant proteins. However, a unique set of changes in the proteome of psychrotrophic strain D6 were observed. In particular, the importance of protein fate, membrane transport and structure for psychrotrophic growth were evident with increases in numerous chaperone and transport proteins including GroEL, SecB, ABC transporters and a capsule polysaccharide export protein. We also observed that low temperature iron oxidation coincides with a relative increase in the key iron metabolism protein rusticyanin, which was more highly expressed in strain D6 than in strain F1 at colder growth temperatures. We demonstrate that the psychrotrophic strain uses a global stress response and cold-active metabolism which permit growth of A. ferrooxidans in the extreme AMD environment in colder climates.     

Fluidization of Membrane Lipids Enhances the Tolerance of Saccharomyces cerevisiae to Freezing and Salt Stress

Unsaturated fatty acids play an essential role in the biophysical characteristics of cell membranes and determine the proper function of membrane-attached proteins. Thus, the ability of cells to alter the degree of unsaturation in their membranes is an important factor in cellular acclimatization to environmental conditions. Many eukaryotic organisms can synthesize dienoic fatty acids, but Saccharomyces cerevisiae can introduce only a single double bond at the Δ⁹ position. We expressed two sunflower (Helianthus annuus) oleate Δ¹² desaturases encoded by FAD2-1 and FAD2-3 in yeast cells of the wild-type W303-1A strain (trp1) and analyzed their effects on growth and stress tolerance. Production of the heterologous desaturases increased the content of dienoic fatty acids, especially 18:2Δ^9,12, the unsaturation index, and the fluidity of the yeast membrane. The total fatty acid content remained constant, and the level of monounsaturated fatty acids decreased. Growth at 15°C was reduced in the FAD2 strains, probably due to tryptophan auxotrophy, since the trp1 (TRP1) transformants that produced the sunflower desaturases grew as well as the control strain did. Our results suggest that changes in the fluidity of the lipid bilayer affect tryptophan uptake and/or the correct targeting of tryptophan transporters. The expression of the sunflower desaturases, in either Trp⁺ or Trp⁻ strains, increased NaCl tolerance. Production of dienoic fatty acids increased the tolerance to freezing of wild-type cells preincubated at 30°C or 15°C. Thus, membrane fluidity is an essential determinant of stress resistance in S. cerevisiae, and engineering of membrane lipids has the potential to be a useful tool of increasing the tolerance to freezing in industrial strains.     

Gene identification and functional characterization of a Δ12 fatty acid desaturase in Tetrahymena thermophila and its influence in homeoviscous adaptation to low temperature

Homeoviscous adaptation in poikilotherms is based in the regulation of the level of desaturation of fatty acids, variation in phospholipids head groups and sterol content in the membrane lipids, in order to maintain the membrane fluidity in response to changes in environmental temperature. Increased proportion of unsaturated fatty acids is thought to be the main response to low-temperature acclimation, which is mostly achieved by fatty acid desaturases. Genome analysis of the ciliate Tetrahymena thermophila and a gene knockout approach has allowed us to identify one Δ12 FAD and to study its activity in the original host and in a yeast heterologous expression system. The “PUFA index” -relative content of polyunsaturated fatty acids compared to the sum of saturated and monounsaturated fatty acid content- was ~57% lower at 15 °C and 35 °C in the Δ12 FAD gene knockout strain (KOΔ12) compared to WT strain. We characterized the role of T. thermophila Δ12 FAD on homeoviscous adaptation and analyzed its involvement in cellular growth, cold stress response, and membrane fluidity, as well as its expression pattern during temperature shifts. Although these alterations allowed normal growth in the KOΔ12 strain at 30 °C or higher temperatures, growth was impaired at temperatures of 20 °C or lower, where homeoviscous adaptation is impaired. These results stress the importance of Δ12 FAD in the regulation of cold adaptation processes, as well as the suitability of T. thermophila as a valuable model to investigate the regulation of membrane lipids and evolutionary conservation and divergence of the underlying mechanisms.     

Protein Profile of <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis> Strains Exhibiting Different Levels of Tolerance to Metal Sulfates

Strains of Acidithiobacillus ferrooxidans exhibited differences in the inhibition of Fe²⁺ oxidation in the presence of 250 mM of cadmium, zinc, and manganese sulfates in respirometric assays. Strains LR and I35 were practically not inhibited, whereas strains SSP and V3 showed significant inhibition (30–70%). Analysis by SDS-PAGE of total proteins from cells grown in the absence of metal sulfates showed different profiles between the more tolerant strains (LR and I35) and the more susceptible ones (SSP and V3). Total proteins of strains LR and V3 were also resolved by two-dimensional polyacrylamide gel electrophoresis (2-DE). A set of major proteins (40, 32, 22, and 20 kDa) could be identified only in the more tolerant strain LR. Our results show that protein profiles analysis could differentiate A. ferrooxidans strains that considerably differ in the tolerance to metal sulfates and present low genomic similarity as revealed by Random Amplified Polymorphic DNA (RAPD) data obtained previously in our laboratory.     

Survey of Extreme Solvent Tolerance in Gram-Positive Cocci: Membrane Fatty Acid Changes in Staphylococcus haemolyticus Grown in Toluene

We exploited the unique ecological niche of oil fly larval guts to isolate a strain of Staphylococcus haemolyticus which may be the most solvent-tolerant gram-positive bacterium yet described. This organism is able to tolerate 100% toluene, benzene, and p-xylene on plate overlays and saturating levels of these solvents in monophasic liquid cultures. A comparison of membrane fatty acids by gas chromatography after growth in liquid media with and without toluene showed that in cells continuously exposed to solvent the proportion of anteiso fatty acids increased from 25.8 to 33.7% while the proportion of 20:0 straight-chain fatty acids decreased from 19.3 to 10.1%. No changes in the membrane phospholipid composition were noted. Thus, S. haemolyticus alters its membrane fluidity via fatty acid composition to become more fluid when it is exposed to solvent. This response is opposite that commonly found in gram-negative bacteria, which change their fatty acids so that the cytoplasmic membrane is less fluid. Extreme solvent tolerance in S. haemolyticus is not accompanied by abnormal resistance to anionic or cationic detergents. Finally, six strains of Staphylococcus aureus and five strains of Staphylococcus epidermidis, which were not obtained by solvent selection, also exhibited exceptional solvent tolerance.     

Analysis of composition and structure of <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> membranes from wild-type and ethanol-adapted strains

Clostridium thermocellum is a candidate organism for consolidated bioprocessing of lignocellulosic biomass into ethanol. However, commercial use is limited due to growth inhibition at modest ethanol concentrations. Recently, an ethanol-adapted strain of C. thermocellum was produced. Since ethanol adaptation in microorganisms has been linked to modification of membrane lipids, we tested the hypothesis that ethanol adaptation in C. thermocellum involves lipid modification by comparing the fatty acid composition and membrane anisotropy of wild-type and ethanol-adapted strains. Derivatization to fatty acid methyl esters provided quantitative lipid analysis. Compared to wild-type, the ethanol-adapted strain had a larger percentage of fatty acids with chain lengths >16:0 and showed a significant increase in the percentage of 16:0 plasmalogens. Structural identification of fatty acids was confirmed through mass spectral fragmentation patterns of picolinyl esters. Ethanol adaptation did not involve modification at sites of methyl branching or the unsaturation index. Comparison of steady-state fluorescence anisotropy experiments, in the absence and presence of ethanol, provided evidence for the effects of ethanol on membrane fluidity. In the presence of ethanol, both strains displayed increased fluidity by approximately 12%. These data support the model that ethanol adaptation was the result of fatty acid changes that increased membrane rigidity that counter-acted the fluidizing effect of ethanol.     

Long-chain fatty acids and ethanol affect the properties of membranes inDrosophila melanogaster larvae

The larval fatty acid composition of neutral lipids and membrane lipids was determined in three ethanol-tolerant strains ofDrosophila melanogaster. Dietary ethanol promoted a decrease in long-chain fatty acids in neutral lipids along with enhanced alcohol dehydrogenase (EC 1.1.1.1) activity in all of the strains. Dietary ethanol also increased the incorporation of¹⁴C-ethanol into fatty acid ethyl esters (FAEE) by two- to threefold and decreased the incorporation of¹⁴C-ethanol into free fatty acids (FFA). When cultured on sterile, defined media with stearic acid at 0 to 5 mM, stearic acid decreased ADH activity up to 33%. In strains not selected for superior tolerance to ethanol, dietary ethanol promoted a loss of long-chain fatty acids in membrane lipids. The loss of long-chain fatty acids in membranes was strongly correlated with increased fluidity in hydrophobic domains of mitochondrial membranes as determined by electron spin resonance and correlated with a loss of ethanol tolerance. In the ethanol-tolerant E2 strain, which had been exposed to ethanol for many generations, dietary ethanol failed to promote a loss of long-chain fatty acids in membrane lipids. We are grateful for the support of National Institutes of Health Grant AA06702 (B.W.G.) and National Science Foundation Grant CHE-891987 (R.G.K.).     

Intracellular metabolic changes of Clostridium acetobutylicum and promotion to butanol tolerance during biobutanol fermentation

During the fermentation process, Clostridium acetobutylicum cells are often inhibited by the accumulated butanol. However, the mechanism underlying response of C. acetobutylicum to butanol stress remains poorly understood. This study was performed to clarify such mechanism through investigating the butanol stress-associated intracellular biochemical changes at acidogenesis phase (i.e., middle exponential phase) and solventogenesis phase (i.e., early stationary phase) by a gas chromatography-mass spectrometry-based metabolomics strategy. With the aid of partial least-squares-discriminant analysis, a pairwise discrimination between control group and butanol-treated groups was revealed, and 27 metabolites with variable importance in the projection value greater than 1 were identified. Under butanol stress, the glycolysis might be inhibited while TCA cycle might be promoted. Moreover, changes of lipids and fatty acids compositions, amino acid metabolism and osmoregulator concentrations might be the key factors involved in C. acetobutylicum metabolic response to butanol stress. It was suggested that C. acetobutylicum cells might change the levels of long acyl chain saturated fatty acids and branched-chain amino acids to maintain the integrity of cell membrane through adjusting membrane fluidity under butanol stress. The increased level of glycerol was considered to be correlated with osmoregulation and regulating redox balance. In addition, increased levels of some amino acids (i.e., threonine, glycine, alanine, phenylalanine, tyrosine, tryptophan, aspartate and glutamate) might also confer butanol tolerance to C. acetobutylicum. These results highlighted our knowledge about the response or adaptation of C. acetobutylicum to butanol stress, and would contribute to the construction of feasible butanologenic strains with higher butanol tolerance.     

Stability of copper tolerance inThiobacillus ferrooxidans

A strain ofThiobacillus ferrooxidans MAL-4-1 was adapted to grow at higher concentrations of copper by repeated subculturing in the presence of increasing levels of added cupric ions in 9K medium. The strains adapted to copper were found to be more efficient in bioleaching of copper from concentrates. When copper tolerant strains were back cultured repeatedly in 9K medium without cupric ions, the initially developed metal tolerance was observed to be lost. This indicates that the copper tolerance developed is stress-dependent and not a permanent trait of the adapted strain.     

Effect of ethanol on membrane fluidity of protoplasts fromSaccharomyces cerevisiae andKloeckera apiculata grown with or without ethanol,measured by fluorescence anisotropy

Summary Direct measurements of membrane fluidity by fluorescence anisotropy of protoplasts fromKloeckera apiculata andSaccharomyces cerevisiae, a low and a high ethanol tolerant strain respectively, are presented. The comparison of the behaviour of the two strains grown with or without ethanol enabled us to demonstrate the existing relationship between ethanol tolerance and membrane fluidity.     

Cadmium tolerance plasticity in Rhizobium leguminosarum bv. viciae: glutathione as a detoxifying agent

Rhizobium leguminosarum bv. viciae strains expressing different degrees of tolerance to metal stress were used in this work to study the basic mechanisms underlying heavy metal tolerance. We used various parameters to evaluate this response. The strains' growth responses under different Cd2+ concentrations were determined and we reported variation in Cd2+ tolerance. Total soluble protein content decreased drastically, revealing the toxic effects that intracellular Cd2+ imposes on cellular metabolism, but this decrease in protein content was particularly evident in sensitive and moderately tolerant strains. Tolerant strains presented the highest intracellular and wall-bound Cd2+ concentrations. Cd2+ induced increases in the expression of some specific proteins, which were identical in all tolerant strains. Glutathione levels remained unaltered in the sensitive strain and increased significantly in tolerant and moderately tolerant strains, suggesting the importance of glutathione in coping with metal stress. This work suggests that efflux mechanisms may not be the only system responsible for dealing with heavy metal tolerance. A clear correlation between glutathione levels and Cd2+ tolerance is reported, thus adding a novel aspect in bacteria protection against heavy metal deleterious effects.