Regulation of the synthesis of unsaturated fatty acids by growth temperature in Bacillus subtilis

Bacillus subtilis synthesizes, almost exclusively, saturated fatty acids, when grown at 37° C. When cultures were transferred from 37° C to 20° C, a chloramphenicol- and rifampicin-sensitive synthesis of a C-16 unsaturated fatty acid was observed. Synthesis of this compound reached a plateau after 5 h at 20° C, reaching levels of 20% of the total fatty acid content. [¹⁴C]-labelled fatty acids attached as thioesters to acyl-carriers compounds, such as coenzyme A (CoA) or acyl-carrier protein (ACP) synthesized de novo by glycerol-requiring auxotrophs deprived of glycerol to arrest phospholipid synthesis, could not be desaturated at 20° C. Desaturation of these fatty acids was readily observed when glycerol was restored to the cultures allowing resumption of transfer of acyl-moieties from acyl-thioesters to phospholipid. It was also observed that depletion of the pools of CoA and ACP by starvation of pantothenate auxotrophs had no effect on the observed synthesis of unsaturated fatty acid at 20° C. The overall results indicate that synthesis of unsaturated fatty acids in B. subtilis is a cold-inducible process and that phospholipids are obligate intermediates in this fatty acid desaturation pathway.     

Temperature responses of growth, photosynthesis, fatty acid and nitrate reductase in Antarctic and temperate Stichococcus

Stichococcus, a genus of green algae, distributes in ice-free areas throughout Antarctica. To understand adaptive strategies of Stichococcus to permanently cold environments, the physiological responses to temperature of two psychrotolerants, S. bacillaris NJ-10 and S. minutus NJ-17, isolated from rock surfaces in Antarctica were compared with that of one temperate S. bacillaris FACHB753. Two Antarctic Stichococcus strains grew at temperature from 4 to 25°C, while the temperate strain could grow above 30°C but could not survive at 4°C. The photosynthetic activity of FACHB753 at lower than 10°C was less than that of Antarctic algae. Nitrate reductase in NJ-10 and NJ-17 had its optimal temperature at 20°C, in comparison, the maximal activity of nitrate reductase in FACHB753 was found at 25°C. When cultured at 4–15°C a large portion of unsaturated fatty acids in the two Antarctic species was detected and the regulation of the degree of unsaturation of fatty acids by temperature was observed only above 15°C, though the content of the major unsaturated fatty acid αC18:3 in FACHB753 decreased with the temperatures elevated from 10 to 25°C. Elevated nitrate reductase activity and photosynthetic rates at low temperatures together with the high proportion of unsaturated fatty acids contribute to the ability of the Antarctic Stichococcus to thrive.     

Substrate specificity of <Emphasis Type="Italic">Stenotrophomonas nitritireducens</Emphasis> in the hydroxylation of unsaturated fatty acid

An isolated bacterium that converted unsaturated fatty acids to hydroxy fatty acids was identified as Stenotrophomonas nitritireducens by API analysis, cellular fatty acids compositions, sequencing the full 16S ribosomal ribonucleic acid, and evaluating its nitrite reduction ability. S. nitritireducens has unique regio-specificity for C16 and C18 cis-9 unsaturated fatty acids. These fatty acids are converted to their 10-hydroxy fatty acids without detectable byproducts. Among the cis-9-unsaturated fatty acids, S. nitritireducens showed the highest specificity for linoleic acid. The cells converted 20 mM linoleic acid to 13.5 mM 10-hydroxy-12(Z)-octadecenoic acid at 30°C and pH 7.5 with a yield of 67.5% (mol/mol).     

Heat shock temperature acclimation of normal secretory protein synthesis in barley aleurone cells

Exposure of barley (Hordeum vulgare L. cv. Himalaya) aleurone layers to 40°C for a period of 3 h results in the selective suppression of the synthesis and secretion of hydrolytic enzymes; other normal cellular protein synthesis continues during heat shock. This suppression is correlated with secretory protein mRNA destabilization and the dissociation of stacked ER lamellae during heat shock (Belanger et al. 1986, Proceedings of the National Academy of Sciences USA 83, pp. 1354–1358). In this report we examined the effect of exposure to extended periods of heat shock. If exposure to 40°C was continued for a period of 18 h, the synthesis of α-amylase, the predominant secreted hydrolase, resumed. This was accompanied by increased α-amylase mRNA levels and the reformation of ER lamellae. Though initial exposure (3 h) to 40°C reduced protein secretion to ～10% of that observed in aleurone cells maintained at 25°C, exposure for prolonged periods (16–20 h) permitted the resumption of protein secretion to ～66% of non-heat-shocked control levels. The resumption of normal secretory protein synthesis during prolonged exposure to 40°C was correlated with an increase in the incorporation of [¹⁴C]glycerol into phosphatidylcholine and an increase in the ratio of saturated to unsaturated fatty acids in lipids isolated from ER membrane preparations. Increased fatty acid saturation has been demonstrated to enhance thermostability in biological membranes, and such changes in membrane composition may be important to the recovery of secretory protein synthesis at the ER.     

Complete auxotrophy for unsaturated fatty acids requires deletion of two sets of genes in Mycobacterium smegmatis

The synthesis of unsaturated fatty acids in Mycobacterium smegmatis is poorly characterized. Bioinformatic analysis revealed four putative fatty acid desaturases in its genome, one of which, MSMEG_1886, is highly homologous to desA3, the only palmitoyl/stearoyl desaturase present in the Mycobacterium tuberculosis genome. A MSMEG_1886 deletion mutant was partially auxotrophic for oleic acid and viable at 37°C and 25°C, although with a long lag phase in liquid medium. Fatty acid analysis suggested that MSMEG_1886 is a palmitoyl/stearoyl desaturase, as the synthesis of palmitoleic acid was abrogated, while oleic acid contents dropped by half in the mutant. Deletion of the operon MSMEG_1741‐1743 (highly homologous to a Pseudomonas aeruginosa acyl‐CoA desaturase) had little effect on growth of the parental strain; however the double mutant MSMEG_1886‐MSMEG_1741‐1743 strictly required oleic acid for growth. The ΔMSMEG_1886‐ΔMSMEG_1741 double mutant was able to grow (poorly but better than the ΔMSMEG_1886 single mutant) in solid and liquid media devoid of oleic acid, suggesting a repressor role for ΔMSMEG_1741. Fatty acid analysis of the described mutants suggested that MSMEG_1742‐43 desaturates C18:0 and C24:0 fatty acids. Thus, although the M. smegmatis desA3 homologue is the major player in unsaturated fatty acid synthesis, a second set of genes is also involved.     

Induction of respiratory incompetent mutants by unsaturated fatty acid depletion in Saccharomyces cerevisiae

Constant levels of cellular unsaturated fatty acids were obtained by growing a fatty acid desaturase mutant of $\text{Saccharomyces cerevisiae}$ in glucose limited chemostat cultures supplemented with various concentrations of Tween 80. An increase in the frequency of cytoplasmic respiratory incompetent mutants was observed in cultures growing at low cellular levels of unsaturated fatty acids. This effect has been shown to result from an increase in the rate of mutation as the cellular unsaturated fatty acid level is decreased. The majority of induced petite mutants are ?° (contain no mitochondrial DNA).     

Evidence for the involvement of unsaturated fatty acids in initiating chromosome replication in Escherichia coli

The analogue 3-decynoyl-N-acetylcysteamine inhibits the synthesis of unsaturated fatty acids in Escherichia coli, resulting in the accumulation of saturated fatty acids in the membrane (Kass, 1968).In the presence of this analogue, DNA, RNA and protein synthesis continue at a linear rate for approximately two doubling times, and then cease. On the other hand, the analogue will inhibit the formation of new replication forks (premature initiation), which normally arise as a result of thymine starvation.Unlike other temperature-sensitive DNA mutants, mutants that are defective in initiating DNA replication (dnaA or dnaC) are unable to replicate DNA at a permissive temperature if they terminate replication at 42 °C in the presence of 3-decynoyl-N-acetylcysteamine.When replication is terminated at 42 °C, cultures of dnaA or dnaC mutants normally will reinitiate replication upon lowering the temperature to 30 °C. For each mutant this reinitiation is characterized by a particular temperature sensitivity. Such mutants become more temperature sensitive if the temperature is lowered in the presence of 3-decynoyl-N-acetylcysteamine. All the effects of this analogue can be reversed by the addition of unsaturated fatty acids.These results are interpreted using a model in which replication is initiated at a particular lipid site on the membrane. In the absence of unsaturated fatty acids functional lipid sites are not made. Functional sites, however, can be used again provided they are not inactivated by interaction with an inactive dnaA or dnaC product.     

Competition between cis,trans and Cyclopropane Fatty Acid Formation and its Impact on Membrane Fluidity

The impact of cis, trans and cyclopropane fatty acids on membrane fluidity was investigated using batch‐grown Pseudomonas putida P8 and Comamonas testosteroni ATCC 17454. A major difference observed between the two investigated strains is the absence of the ability to synthesize trans‐unsaturated fatty acids in Comamonas. When grown exponentially at 30 °C, a shift to 35 °C increased the trans/cis ratios of the fatty acids of P. putida P8 from 0 to 0.81 and 0 to 1.07, in lipid extracts and cell hydrolyzates, respectively. After prolonged growth followed by nutrient deprivation for 48 h, both at 30 °C, trans fatty acids were absent, but the cyclo/cis ratios rose from 0.1 to 1.55 in lipid extracts, and from 0.1 to 1.54 in cell hydrolyzates. C. testosteroni ATCC 17454 contained no cyclo fatty acids when harvested in the exponential phase after 6 h, whereas after 72 h cultivation, the cyclo/cis ratios rose to 0.49 and 0.47, in lipid extracts and cell hydrolyzates, respectively. Trans fatty acids were never observed in this strain. Increased cyclo/cis and trans/cis ratios correlated with decreased fluidity measured by the fluorescence anisotropy of 1,6‐diphenyl‐1,3,5‐hexatriene (DPH) intercalated in the bilayers of liposomes and by Fourier Transform Infrared (FTIR) spectroscopy of lipids prepared from the cells. The specific effect of cyclopropane fatty acids on membrane fluidity was much smaller than that of trans fatty acids. FTIR‐measurements of intact cells of P. putida P8 confirmed the high potency of trans fatty acids to decrease the fluidity. In cells with induced cyclopropane fatty acid synthesis, the membranes remained more fluidized, indicating the lower importance of these fatty acids for homeoviscosis.     

EFFECTS OF VARIATION IN TEMPERATURE. II. ON THE FATTY ACID COMPOSITION OF EIGHT SPECIES OF MARINE PHYTOPLANKTON1

Eight species of marine phytoplankton showed significant variation in the relative amount of some fatty acids (FAs) in response to variation in temperature. Large changes in relative amounts of certain FAs occurred as a result of a 15° C change in growth temperature. For example, 14:0 increased from ?4% of total FAs at 10° C to > 20% at 25° C for Chaetoceros simplex and Isochrysis aff. galbana but decreased for Phaeodactylum tricornutum. The percentage of the polyunsaturated fatty acid (PUFA) 16:ω1 was consistently greater at 10° C than at 25° C, and the converse was usually true for 16: 4ω3. Calculated over all eight species, there was a modest but significant inverse relationship between the percentage of PUFAs and temperature. Only for Thalassiosira pseudonana was the percentage of either of the PUFAs and nutritionally essential fatty acids (EFAs) also an inverse function of temperature. For T. pseudonana, the percentage of the EFA 22:6ω3 decreased linearly with increasing temperature over the range from 10 to 25° C. For three species, the ratio of unsaturated/saturated FAs was correlated with growth rate when growth rate was controlled by variation in irradiance and temperature. Only for Thalassiosira pseudonana was the ratio of unsaturated/saturated FAs also an inverse function of temperature alone.     

Decrease in beech (<Emphasis Type="Italic">Fagus sylvatica</Emphasis>) seed viability caused by temperature and humidity conditions as related to membrane damage and lipid composition

The aim of this study was to determine if the loss of germinability and viability of beech (Fagus sylvatica L.) seeds stored at different variants of temperature (4, 20, and 30 °C) and relative humidity (RH: 45 and 75 %) is associated with a loss of membrane integrity and changes in lipid composition. Beech seeds stored for 9 weeks gradually lost viability at a rate dependent on temperature and humidity. The harmful effect of temperature increased with growing humidity. The loss of seed viability was strongly correlated with an increase in membrane permeability and with production of lipid hydroxyperoxides (LHPO), which was regarded as an indicator of peroxidation of unsaturated fatty acids. The condition of membranes was assessed on the basis of their permeability and the state of lipid components: phospholipids and fatty acids. During seed storage we observed a decline in concentration of individual phospholipids and fatty acids, proportional to the loss of seeds viability. We also detected a decrease in concentrations of α-tocopherol and sterols, which play an important role in protection of membranes against the harmful influence of the environment. Our results show that the germinability of beech seeds declines rapidly at temperature above 0 °C and growing humidity. This is due mainly to the loss of membrane integrity, caused by peroxidation of unsaturated fatty acids.     

Separation and Identification of Fatty Acids

For the characterization of fatty acids, 2, 4-dinitrophenylhydrazones of p-bromophenacyl esters of even-numbered saturated fatty acids from C₂ to C₂₀ and several unsaturated fatty acids from monoethenoid to triethenoid were prepared. The derivatives of linoleic and linolenic acids as well as those of the other unsaturated and saturated acids, were successfully obtained in crystalline forms which showed sharp and high melting points, 72° and 69°, respectively. It was found that the derivatives of unsaturated acids were valuable for characterizing the parent acids, while those of saturated acids were unsuitable for this purpose owing to the similarity of their melting points.     

Improvement of polyunsaturated fatty acids synthesis by the coexpression of CYB5 with desaturase genes in Saccharomyces cerevisiae

Since Saccharomyces cerevisiae contains Δ9 fatty acid desaturase (OLE1) as a sole fatty acid desaturase, it produces saturated and monounsaturated fatty acids of 16- and 18-carbon compounds. We showed earlier that Kluyveromyces lactis Δ12 (KlFAD2) and ω3 (KlFAD3) fatty acid desaturase genes enabled S. cerevisiae to make also polyunsaturated fatty acids (PUFAs), linoleic (18:2n-6), and α-linolenic (18:3n-3) acids. Unlike Δ9 fatty acid desaturase Ole1p, the two added fatty acid desaturases (KlFAD2and KlFAD3) do not contain a cytochrome b5 domain, and we now report on effects of the overexpression of K. lactis and S. cerevisiae cytochrome b5 (CYB5) genes as well as temperature effects on PUFA synthesis. Without extra cytochrome b5, while PUFA synthesis is significant at low temperature (20 °C), it was marginal at 30 °C. Overexpression of cytochrome b5 at 20 °C did not affect the fatty acid synthesis so much, but it significantly enhanced the synthesis of PUFA at 30 °C.     

Impact of membrane fatty acid composition on the uncoupling sensitivity of the energy conservation of Comamonas testosteroni ATCC 17454

The fatty acid composition of pyruvate-grown Comamonas testosteroni ATCC 17454 was analyzed after growth at 30 and 20°C and after half-maximum growth inhibition caused by different membrane-active chemicals at 30°C. Palmitic acid (16:0), palmitoleic acid (16:1 ω7c) and vaccenic acid (18:1 ω7c) were the dominant fatty acids. At 20°C, the proportion of palmitic acid decreased and those of palmitoleic and vaccenic acid increased. Saturation degree was also lowered when half-maximum growth inhibition was caused by 4-chlorosalicylic acid, 2,4-dichlorophenoxyacetic acid and 2,4-dinitrophenol and, to a lesser extent, in the presence of 2,4-dichlorophenol, phenol and ethanol. It appeared that the dissociated forms of the former group of chemicals were preferentially incorporated near the head group region of the lipid bilayer, thereby somewhat extending the outer region of the membranes, and that the increased amount of bent, unsaturated fatty acids helped to maintain membrane integrity. Irrespective of how the decrease of the saturation degree was triggered, it caused electron transport phosphorylation (adenosine triphosphate synthesis driven by n-hexanol oxidation) to become more sensitive to uncoupling. Apparently, the viscosity and phase stability of the cytoplasmic membrane of C. testosteroni were maintained at the price of a reduced protection against energy toxicity.     

Flavonoids in the Flowers of Gnaphalium affine D. Don

Thermonsenstivie division mutants were derived from Bacillus subtilis Marburg 168 thy trp₂ by means of membrane filtration after nitrosoguanidine mutagenesis. Among them, ts42 requiring uracil for normal growth at 48°C was investigated.

In the absence of uracil, the mutant cells grew normally at 37°C and stopped dividing after temperature shift to 48°C resulting in filaments of two to four times length of normal rods. The total cell number after temperature shift from 37 to 48°C, increased two to three fold in 90 min and remained constant thereafter. The viable count after the temperature shift to 48°C, increased 1.5 to 2 fold in initial 60 min and then decreased exponentially. A rapid restoration of colony forming ability was shown when the mutant cells were shifted back to the permissive temperature after 120 to 180 min of incubation at 48°C or when uracil was introduced to the culture at 48°C. This recovery of viability was partly observed even in the presence of chloramphenicol. The synthesis of RNA of this mutant was shown to decline 20 min after the temperature shift to 48°C whereas the syntheses of DNA and protein proceeded for more than 80 min at that temperature.

No newly isolated uracil requiring mutants formed filaments in the medium lacking uracil or showed growth pattern like ts42.     

Maturation of bacteriophage 9NA DNA is influenced by the fatty acid composition of the host cell membrane

The fatty acid composition of the membrane of the conditional auxotroph fabB2 can be altered by allowing the cells to grow at non-permissive temperature (37°C) in the presence of a cis-unsaturated fatty acid. The phage 9NA, a virulent phage ofSalmonella typhimurium, can not multiply in fabB2. Synthesis and maturation of the phage DNA are differentially affected by variation in the fatty acid composition of the cell membrane. The replicating DNA associates with the membrane complex, the site of DNA synthesis. The association is comparatively weak in oleic, claidic, palmitoleic, palmitelaidic and linolelaidic acid enriched cells. When the cells are grown in the presence of palmitoleic acid, a large pool of concatemeric phage DNA accumulates in the cytoplasm within 10 min of infection. The conversion of concatemeric DNA to monomeric one i.e., mature phage length DNA, is inhibited in such cells. The presence of concatemeric DNA can be visualized by electron microscope. Such a situation is not observed when the cells are grown in media supplemented with other types of unsaturated fatty acids. The mechanism by which the host cell membrane lipid controls phage development is yet to be worked out.     

Phase transitions of phospholipid bilayers from an unsaturated fatty acid auxotroph of Escherichia coli

Total phospholipids were extracted from cells of temperature sensitive unsaturated fatty acid auxotrophs of Escherichia coli (K-12 UFA^ts) grown at 28°C (PL28), and at 42°C in the presence of 2% KCl as an osmotic stabilizer (PL42 (KCl)). From the analysis of fatty acids, it was shown that the content of unsaturated fatty acids of PL42 (KCl) is only 9% of the total fatty acids, while that of PL28 is 54%. The thermal phase transitions of the bilayers prepared from the phospholipid fractions were studied by proton magnetic resonance. The line widths of the methylene signals and the sums of the methylene and methyl signal intensities were plotted against reciprocal values of absolute temperature 1/T or temperature itself. From the plots phase transitions were detected at about 19°C for PL28 and at 43°C for PL42 (KCl). In spite of its complex composition of fatty acids a highly cooperative transition was observed in the case of PL42 (KCl). It was also suggested that the phospholipids bilayers in the biomembranes of this strain at the growth temperature (42°C) are in the state where the gel and liquid crystalline phases coexist.     

Involvement of Membrane Lipids in Cold Shock-induced Autolysis of Bacillus subtilis Cells

Exponentially growing Bacillus subtilis cells autolysed when exposed to cold shock treatment in minimal medium followed by incubation at 37°C. From characteristics of the lysis, it was suggested that the cold-shock-induced cell lysis resulted from the perturbation of membrane organization that is initiated by rapid changes in temperature, lipid phase transitions. For maximum lysis induction to occur, in addition to rapid cooling to 5°C or lower, retention at temperatures lower than 10°C for at least 20 min is required. The cell sensitivity to the autolysis induction by cold shock was different between cells grown at 25°C and cells grown at 37°C. Analyses of the fatty acid composition and the phase transition temperature of membrane lipids suggested that the membrane fluidity may affect the autolysis induction. Experiments to discover the effects of cerulenin treatment and lipid addition on autolysis induction and the autolysin activity level support the hypothesis that membrane lipids are involved in cold-shock-induced cell autolysis.     

Diversity of Yeasts from Puddles in the Vicinity of Midre Lovénbreen Glacier, Arctic and Bioprospecting for Enzymes and Fatty Acids

A total of 132 yeast strains were characterised from 4 sediment samples collected from small puddles in the vicinity of Midre Lovénbreen glacier, Arctic. Based on the D1/D2 domain sequence similarity, the isolates could be categorised into 6 groups. The nearest phylogenetic neighbour of groups I to VI were identified as Cryptococcus gastricus, Cryptococcus terricolus, Rhodotorula muscorum, Mrakia psychrophila, Mrakia gelida and Rhodotorula glacialis, respectively. Strains representative of the six groups were psychrophilic and salt tolerant but varied in their ability to produce cold-active extracellular enzymes such as lipase, protease, pectinase, cellulase and amylase. C_{18:1 (w9C)} and C_{18:2 (w9,12C)} were the only two fatty acids common to all the yeasts and branched and (or) unsaturated fatty acids increased in yeasts growing at 8°C compared to 22°C, probably as an adaptation to low temperature. The present study establishes that psychrophilic yeasts are predominant in Arctic and could be used as work horses to produce cold-active enzymes and poly unsaturated fatty acids which have been implicated in low temperature adaptation and also for their use in biotechnology.     

The relationships between growth temperature,fatty acid composition and the physical state and fluidity of membrane lipids in Yersinia enterocolitica

The relationship between membrane lipid composition and membrane lipid phase transitions was investigated in Yersinia enterocolitica cells grown at 5, 22 and 37°C. The total phospholipid concentrations were 9.4, 7.3 and 6.3% of the cell dry weight for cells grown at 5, 22 and 37°C, respectively. The relative concentrations of the three major phospholipids, phosphatidylethanolamine (73–76%), phosphatidylglycerol (9–11%) and cardiolipin (11–13%) were essentially the same at all three growth temperatures. The ratios of unsaturated to saturated fatty acids were 2.2, 1.1 and 0.4 for cells grown at 5, 22 and 37°C, respectively. This change in the fatty acid composition in response to temperature changes is similar to the patterns reported for other organisms. Reversible thermotropic phase transitions were detected by calorimetric analysis in both pure lipid preparations and membrane preparations. The mid-points of the thermotropic phase transitions were at ?13, ?9 and 1°C for membranes from cells grown at 5, 22 and 37°C, respectively. The phase transitions of the membranes from cells grown at the three different temperatures occurred below the lowest growth temperature (5°C). The alternations in the fatty acid composition in Y. enterocolitica did not, therefore, appear to be required to adjust membrane fluidity but might rather be required for some other membrane function.