Cold shock response of Bacillus subtilis: isoleucine-dependent switch in the fatty acid branching pattern for membrane adaptation to low temperatures.

Bacillus subtilis has developed sophisticated mechanisms to withstand fluctuations in temperature. Membrane fatty acids are the major determinants for a sufficiently fluid membrane state to ensure the membrane's function at all temperatures. The fatty acid profile of B. subtilis is characterized by a high content of branched fatty acids irrespective of the growth medium. Here, we report on the importance of isoleucine for B. subtilis to survive cold shock from 37 to 15 degrees C. Cold shock experiments with strain JH642 revealed a cold-protective function for all intermediates of anteiso-branched fatty acid biosynthesis. Metabolites related to iso-branched or straight-chain fatty acid biosynthesis were not protective. Fatty acid profiles of different B. subtilis wild-type strains proved the altered branching pattern by an increase in the anteiso-branched fatty acid content and a concomitant decrease of iso-branched species during cold shock. There were no significant changes in the fatty acid saturation or acyl chain length. The cold-sensitive phenotype of isoleucine-deficient strains in the absence of isoleucine correlated with their inability to synthesize more anteiso-branched fatty acids, as shown by the fatty acid profile. The switch to a fatty acid profile dominated by anteiso-C(15:0) and C(17:0) at low temperatures and the cold-sensitive phenotype of isoleucine-deficient strains in the absence of isoleucine focused our attention on the critical role of anteiso-branched fatty acids in the growth of B. subtilis in the cold.     

Regulation of the desaturation of fatty acids and its role in tolerance to cold and salt stress

The expression of cold-inducible genes is regulated by a two-component system in Synechocystis and Bacillus subtilis. The cold sensors are membrane-bound histidine kinases and it seems likely that they sense and transduce changes in the fluidity of membranes. Desaturation of fatty acids in membrane lipids has been implicated in tolerance to cold and salt stress.     

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 degrees C) and low (20 degrees 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 degrees 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 degrees C in the complex medium gave rise to the highest membrane fluidity with concomitant decrease of T(m) by 10.5 degrees C. In mineral media at 20 degrees C the corresponding changes of T(m) were almost negligible. After a temperature shift from 40 to 20 degrees 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.     

Ethanol-induced changes in the membrane lipid composition of Clostridium thermocellum

When ethanol is added to the growth medium of Clostridium thermocellum ATCC 27405 and C9, a different membrane composition is observed after the period of growth arrest. Changes in fatty acid composition and some unsaturated, branched hydrocarbons have been monitored by GLC-MS. There is a marked increase in normal and anteiso-branched fatty acids at the expense of isobranched fatty acids and an increase in short and unsaturated fatty acids. Thus, an adaptive response to growth in the presence of ethanol induces a membrane containing fatty acids with lower melting points and produces a more ‘fluid’ membrane. The suggestion is made that these membrane changes may be maladaptive to the performance of C. thermocellum.     

Nature and linkage type of fatty acids present in lipopolysaccharides of phase I and II Coxiella burnetii

The constituent fatty acids of lipopolysaccharides (LPS) of Coxiella burnetii (phase I and II) were qualitatively and quantitatively analysed by combined gas-liquid chromatography/mass spectrometry. The total fatty acid content (per mg LPS) was determined as 90.0 nmol (2.3 wt%) for LPS of phase I cells (LPS I) and 179.1 nmol (4.8 wt%) for LPS of phase II cells (LPS II). Of the 24 different acyl residues characterized (12 to 18 carbon atoms), nine were 3-hydroxy fatty acids (normal, iso- and anteiso-branched) which quantitatively predominated. All 3-hydroxylated fatty acids were found to possess the (R)-configuration, to be exclusively amide-linked and to be acylated at their 3-hydroxyl group. Ester-linked nonhydroxylated fatty acids (normal, iso- and anteiso-branched) were present but ester-bound 3-hydroxy- or 3-acyloxyacyl residues were lacking from C. burnetii LPS I and LPS II. As the major acyl group (R)-3-(12-methyl-tetradecanoyloxy)-12-methyl-tetradecanoic acid was identified. Our results show that the complex fatty acid spectrum of C. burnetii differs considerably from that of LPS of other Gram-negative bacteria. They further suggest an enormous heterogeneity of the lipid A component of C. burnetii LPS I and LPS II.     

Cold shock in Bacillus subtilis: different effects of benzyl alcohol and ethanol on the membrane organisation and cell adaptation

A temperature shift-down of Bacillus subtilis from 40 to 20 degrees C induces an 80 min growth lag. Benzyl alcohol reduced this period to 51 min, whereas ethanol prolonged it up to 102 min. The effect of the two alcohols on the membrane state was investigated by measuring the steady-state fluorescence anisotropy and analysing the lifetime distribution of diphenylhexatriene (DPH) and its polar derivative, TMA-DPH. As followed from the fluorescence anisotropy, the two alcohols exerted similar (fluidizing) effects on the cytoplasmic membranes of B. subtilis. However, benzyl alcohol significantly shortened the main DPH lifetime component and widened its distribution, while ethanol had no effect. The benzyl alcohol activity was interpreted in terms of an increased membrane hydration due to disordering of the membrane structure. Such an effect imitates the cold shock induced synthesis of unsaturated fatty acids in B. subtilis. The fatty acid analysis revealed that ethanol hindered this adaptive synthesis of fatty acids. At the same time, its effect on the membrane state (membrane order) was very low and could not substitute the physiological response as was the case with benzyl alcohol. It can thus be concluded that the adaptation of the membrane physical state contributes significantly to the cold shock response of B. subtilis.     

Complementation analysis of the cold-sensitive phenotype of the Escherichia coli csdA deletion strain

The cold shock response of Escherichia coli is elicited by downshift of temperature from 37 degrees C to 15 degrees C and is characterized by induction of several cold shock proteins, including CsdA, during the acclimation phase. CsdA, a DEAD-box protein, has been proposed to participate in a variety of processes, such as ribosome biogenesis, mRNA decay, translation initiation, and gene regulation. It is not clear which of the functions of CsdA play a role in its essential cold shock function or whether all do, and so far no protein has been shown to complement its function in vivo. Our screening of an E. coli genomic library for an in vivo counterpart of CsdA that can compensate for its absence at low temperature revealed only one protein, RhlE, another DEAD-box RNA helicase. We also observed that although not detected in our genetic screening, two cold shock-inducible proteins, namely, CspA, an RNA chaperone, and RNase R, an exonuclease, can also complement the cold shock function of CsdA. Interestingly, the absence of CsdA and RNase R leads to increased sensitivity of the cells to even moderate temperature downshifts. The correlation between the helicase activity of CsdA and the stability of mRNAs of cold-inducible genes was shown using cspA mRNA, which was significantly stabilized in the DeltacsdA cells, an effect counteracted by overexpression of wild-type CsdA or RNase R but not by that of the helicase-deficient mutant of CsdA. These results suggest that the primary role of CsdA in cold acclimation of cells is in mRNA decay and that its helicase activity is pivotal for promoting degradation of mRNAs stabilized at low temperature.     

Role of ferredoxin and flavodoxins in Bacillus subtilis fatty acid desaturation

The Bacillus subtilis acyl lipid desaturase (Δ5-Des) is an iron-dependent integral membrane protein able to selectively introduce double bonds into long-chain fatty acids. In the last decade since its discovery, the molecular mechanism of Δ5-Des expression has been studied extensively. However, the mechanism of desaturation, which must rely on unknown bacterial proteins for electron transfer, has not yet been explored. The B. subtilis genome encodes three proteins that can act as potential electron donors of Δ5-Des, ferredoxin (Fer) and two flavodoxins (Flds) (YkuN and YkuP), which are encoded by the ykuNOP operon. Here we report that the disruption of either the fer gene or the ykuNOP operon decreases the desaturation of palmitic acid by ～30%. Nevertheless, a fer ykuNOP mutant abolished the desaturation reaction almost completely. Our results establish Fer and the two Flds as redox partners for Δ5-Des and suggest that the Fer and Fld proteins could function physiologically in the biosynthesis of unsaturated fatty acids in B. subtilis. Although Flds have extensively been described as partners in a number of redox processes, this is the first report describing their role as electron donors in the fatty acid desaturation reaction.     

Responses to cold shock in cyanobacteria

Acclimation of cyanobacteria to low temperatures involves induction of the expression of several families of genes. Fatty acid desaturases are responsible for maintaining the appropriate fluidity of membranes under stress conditions. RNA-binding proteins, which presumably act analogously to members of the bacterial Csp family of RNA chaperones, are involved in the maintenance of the translation under cold stress. The RNA helicase, whose expression is induced specifically by cold, might be responsible for modifying inappropriate secondary structures of RNAs induced by cold. The cold-inducible family of CIp proteins appears to be involved in the proper folding and processing of proteins. Although genes for cold-inducible proteins in cyanobacteria are heterogeneous, some common features of their untranslated regulatory regions suggest the existence of a common factor(s) that might participate in regulation of the expression of these genes under cold-stress conditions. Studies of the patterns of expression of cold-inducible genes in cyanobacteria have revealed the presence of a cold-sensing mechanism that is associated with their membrane lipids. Available information about cold-shock responses in cyanobacteria and molecular mechanisms of cold acclimation are reviewed in this article.     

Effect of growth temperature on membrane fatty acid composition and susceptibility to cold shock of Bacillus amyloliquefaciens.

We investigated the fatty acid composition of the membrane of Bacillus amyloliquefaciens grown at different temperatures. A decrease in growth temperature was accompanied by an increase in the ratio of branched- to straight-chain fatty acids and a marked increase in the level of unsaturation of branched-chain fatty acids. When cells of this organism grown at 30 degrees C were cold shocked, viability and ability to secrete extracellular protease were lost. Growth of this organism at lower temperatures or addition of Tween 80 to cells caused the critical temperature zone for cold shocking to be lowered significantly. These results suggest a direct correlation between membrane fluidity and the susceptibility to cold shock.     

Genetic evidence for the temperature-sensing ability of the membrane domain of the Bacillus subtilis histidine kinase DesK

A decrease in environmental temperature leads to the synthesis of Delta5-unsaturated fatty acids in Bacillus subtilis by the fatty acid desaturase Des. Des is regulated by the two-component system DesKR. To understand the mechanism of cold signal perception and transduction by the membrane domain and the cytosolic domain of DesK, we expressed the cytosolic domain of DesK in trans under the control of a xylose-inducible promoter without the membrane domain. We performed growth experiments and a Northern blot analysis. Our results show that the kinase function of the cytosolic domain of DesK is temperature-independent, leading to a constitutive expression of the des gene. These findings support the conclusion that the membrane domain of DesK is the temperature-sensing element of the two-component system.     

Gene-engineered rigidification of membrane lipids enhances the cold inducibility of gene expression in synechocystis

A sudden decrease in ambient temperature induces the expression of a number of genes in poikilothermic organisms. We report here that the cold inducibility of gene expression in Synechocystis sp. PCC 6803 was enhanced by the rigidification of membrane lipids that was engineered by disruption of genes for fatty acid desaturases. DNA microarray analysis revealed that cold-inducible genes could be divided into three groups according to the effects of the rigidification of membrane lipids. The first group included genes whose expression was not induced by cold in wild-type cells but became strongly cold-inducible upon rigidification of membrane lipids. This group included certain heat-shock genes, genes for subunits of the sulfate transport system, and the hik34 gene for a histidine kinase. The second group consisted of genes whose cold inducibility was moderately enhanced by the rigidification of membrane lipids. Most genes in this group encoded proteins of as yet unknown function. The third group consisted of genes whose cold inducibility was unaffected by the rigidification of membrane lipids. This group included genes for an RNA helicase and an RNA-binding protein. DNA microarray analysis also indicated that the rigidification of membrane lipids had no effect on the heat inducibility of gene expression. Hik33, a cold-sensing histidine kinase, regulated the expression of most genes in the second and third groups but of only a small number of genes in the first group, an observation that suggests that the cold-inducible expression of genes in the first group might be regulated by a cold sensor that remains to be identified.     

Involvement of autolysin in cellular lysis of Bacillus subtilis induced by short- and medium-chain fatty acids.

The addition of saturated C6, C8, C10, and C12 fatty acids appeared to lyse actively growing cells of Bacillus subtilis 168, as judged by a decrease in the optical density of the culture. Of these fatty acids, dodecanoic acid was the most effective, with 50% lysis occurring in about 30 min at a concentration of 0.5 mM. These conditions also decreased the amount of peptidoglycan estimated by the incorporated radioactivity of N-acetyl-D-[1-14C]glucosamine. At concentrations above 1 mM, however, bacterial lysis was not extensive. Dodecanoic acid did not affect autolysis of the cell wall. The lytic action of dodecanoic acid was greatly diminished in cells in which protein synthesis was inhibited and in an autolytic enzyme-deficient mutant. The results suggest that fatty acid-induced lysis of B. subtilis 168 is due to the induction of autolysis by an autolytic enzyme rather than massive solubilization of the cell membrane by the detergent-like action of the fatty acids.     

Reorganization of membrane lipids during fast and slow cold hardening in Drosophila melanogaster

Abstract Rapid cold hardening is a naturally occurring phenomenon in insects that is thought to be responsible for increased cold tolerance during diurnal variations in temperature. The underlying physiological mechanisms are still not fully resolved but, in Drosophila melanogaster (Meigen 1830), rapid cold hardening is accompanied by specific changes in the membrane lipid composition. To further understand the link between rapid cold hardening and adjustments in the membrane lipid composition, the present study investigates how different rates of cooling affect thermotolerance and the composition of phospholipid fatty acids. Female Drosophila are cooled gradually from 25 to 0 °C at 0.01, 0.05, 0.1 or 0.5 °C min^?1, respectively, and, subsequently, phospholipid fatty acid composition and survival after a 1‐h cold shock at ?5 °C is measured. The rapid cold hardening treatments all influence cold tolerance differently so that short and intermediate rapid cold hardening treatments (0.05, 0.1 or 0.5 °C min^?1 cooling rates) increase cold shock survival, whereas the slow cooling treatment (0.01 °C min^?1) decreases survival relative to an untreated control. The intermediate rapid cold hardening treatments (0.05 or 0.1 °C min^?1) induce a similar type of response characterized by an increase in the molar percentage of linoleic acid, 18:2(n‐6), at the expense of 16:0 and 18:1(n‐9), which leads to an increase in the degree of unsaturation. The slowest cooling treatment (0.01 °C min^?1) results in a large increase in cis‐16:1(n‐7) and significant reductions in the saturated phospholipid fatty acids 16:0, 18:0 and the unsaturated 16:1(n‐9) and 18:2(n‐6) fatty acids. These changes cause a slight decrease in the average length of the phospholipid fatty acids and an increase in the overall ratio of unsaturated vs. saturated fatty acids. These findings demonstrate that the rate of cooling is important for both the reorganization of membrane lipids, and for the degree of acquired cold tolerance during rapid cold hardening, and they suggest an important role for rapid cold hardening during diurnal rather than seasonal temperature changes.     

Hydroxy fatty acids in Bacteroides species: D-(--)-3-hydroxy-15-methylhexadecanoate and its homologs.

Acid hydrolysates of 140 strains, representing 11 species of the genus Bacteroides, were analyzed by capillary gas-liquid chromatography for total cellular fatty acid. All samples contained components which appeared to be hydroxy fatty acid. The relative amount and chain length distribution of the hydroxy fatty acids, as well as the nonhydroxy fatty acids, varied according to species. To characterize the presumed hydroxy acids, a composite of some 40 of these samples was analyzed by thin-layer and capillary gas-liquid chromatography, mass spectrometry, infrared spectrophotometry, and polarimetry. The hydroxy acids were shown to be of the D-(--)-3-hydroxy acid family. The predominant component was the iso-branched D-(--)-3-hydroxy-15-methylhexadecanoic acid. Lesser amounts of the iso-branched 15-carbon, straight-chain 16-carbon, and anteiso-branched 17-carbon acids were also found.     

Changes in membrane lipid composition following rapid cold hardening in Drosophila melanogaster

Naturally occurring diurnal variations in temperature are sufficient to induce a rapid cold hardening (RCH) response in insects. RCH can increase cold tolerance by 1-2 degrees C and extend the temperature interval at which insects can remain active. While the benefits of RCH are well established, the underlying physiological mechanisms remain unresolved. In this study we investigated the role of RCH on expression of heat shock proteins (Hsp70) after a cold shock, and the effect of RCH on the composition of phospholipid fatty acids (PLFAs) in membranes of Drosophila melanogaster. These experiments were performed on both "control" flies and flies selected for cold resistance in order to additionally examine a possible target for selection for cold tolerance. RCH improved survival following cold shock at -4, -6 and -8 degrees C. No induction of Hsp70 was found following cold shock irrespective of the pre-treatment. In contrast, a 5h RCH treatment was sufficient to induce small, but significant, changes in the composition of PLFAs. Here, the polyunsaturated linoleic acid, 18:2(n-6), increased while monounsaturated (18:1) and saturated (14:0) PLFAs decreased in abundance. These changes were observed in both selection groups and caused a significant increase in the overall degree of unsaturation. This response is consistent with the membrane response typically found during cold acclimation in ectothermic animals and it is likely adaptive to maintain membrane function during cold. Cold selection resulted in PLFA changes (decrease of 18:0 and 18:1 and increase of 14:0 and 16:1), which may improve the ability to harden during RCH.