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.     

Membrane fluidization by alcohols inhibits DesK-DesR signalling in Bacillus subtilis

After cold shock, the Bacillus subtilis desaturase Des introduces double bonds into the fatty acids of existing membrane phospholipids. The synthesis of Des is regulated exclusively by the two-component system DesK/DesR; DesK serves as a sensor of the state of the membrane and triggers Des synthesis after a decrease in membrane fluidity. The aim of our work is to investigate the biophysical changes in the membrane that are able to affect the DesK signalling state. Using linear alcohols (ethanol, propanol, butanol, hexanol, octanol) and benzyl alcohol, we were able to suppress Des synthesis after a temperature downshift. The changes in the biophysical properties of the membrane caused by alcohol addition were followed using membrane fluorescent probes and differential scanning calorimetry.We found that the membrane fluidization induced by alcohols was reflected in an increased hydration at the lipid-water interface. This is associated with a decrease in DesK activity. The addition of alcohol mimics a temperature increase, which can be measured isothermically by fluorescence anisotropy. The effect of alcohols on the membrane periphery is in line with the concept of the mechanism by which two hydrophilic motifs located at opposite ends of the transmembrane region of DesK, which work as a molecular caliper, sense temperature-dependent variations in membrane properties.     

Role of the Bacillus subtilis fatty acid desaturase in membrane adaptation during cold shock

In our attempt to understand the cold shock response of Bacillus subtilis, we report on the role of the B. subtilis fatty acid desaturase (FA-D) Des during membrane adaptation to low temperatures and demonstrate its importance during cold shock. A des null mutant was constructed and analysed in comparison with its parental strain. Growth studies and large-scale comparative fatty acid (FA) analysis revealed a severe cold-sensitive phenotype of the des deletion mutant during the absence of isoleucine and showed that four unsaturated fatty acid (UFA) species differing in length, branching pattern and position of the double bond are synthesized in B. subtilis JH642 but not in the des null mutant. Apart from the lack of UFA synthesis, the FA-D deletion strain showed a dramatically altered saturated fatty acid (SFA) profile at the onset of the stationary growth phase in the presence of exogenous isoleucine sources. Expression of des integrated in trans at the amyE locus of the des deletion strain not only cured the cold-sensitive phenotype observed for the des mutant but allowed much better growth than in strain JH642 after a shift from 37 degrees C to 15 degrees C. These results show that, during cold shock adaptation, des expression can completely replace the isoleucine-dependent, long-term, FA branching adaptation mechanism. We conclude that the crucial aspect in cold adaptation of the cytoplasmic membrane is not its specific molecular composition but rather its physical status in terms of its fluidity.     

Exogenous isoleucine and fatty acid shortening ensure the high content of anteiso-C15:0 fatty acid required for low-temperature growth of Listeria monocytogenes

Previous studies have demonstrated that the branched-chain fatty acid anteiso-C15:0 plays a critical role in the growth of Listeria monocytogenes at low temperatures by ensuring sufficient membrane fluidity. Studies utilizing a chemically defined minimal medium revealed that the anteiso fatty acid precursor isoleucine largely determined the fatty acid profile and fatty acid response of the organism to lowered growth temperature. When isoleucine was sufficient, the fatty acid profile was very uniform, with anteiso fatty acids comprising up to 95% of total fatty acid, and the major fatty acid adjustment to low temperature was fatty acid chain shortening, which resulted in an increase of anteiso-C15:0 solely at the expense of anteiso-C17:0. When isoleucine was not supplied, the fatty acid profile became more complex and was readily modified by leucine, which resulted in a significant increase of corresponding iso fatty acids and an inability to grow at 10 degrees C. Under this condition, the increase of anteiso-C15:0 at low temperature resulted from the combined effect of increasing the anteiso:iso ratio and chain shortening. A branched-chain alpha-keto acid dehydrogenase-defective strain largely lost the ability to increase the anteiso:iso ratio. Cerulenin, an inhibitor of beta-ketoacyl-acyl carrier protein synthase (FabF), induced a similar fatty acid chain shortening as low temperature did. We propose that the anteiso precursor preferences of enzymes in the branched-chain fatty acid biosynthesis pathway ensure a high production of anteiso fatty acids, and cold-regulated chain shortening results in a further increase of anteiso-C15:0 at the expense of anteiso-C17:0.     

Thermal regulation of the fatty acid composition of lipopolysaccharides and phospholipids of Proteus mirabilis.

The fatty acid composition of the lipid A moiety of the lipopolysaccharide and phospholipid fractions of Proteus mirabilis changed significantly on varying the growth temperature. A decrease in the growth temperature from 43 degrees C to 15 degrees C resulted in a decrease in the palmitic acid content of the lipopolysaccharide from 19.4% of total fatty acids at 43 degrees C to 1.4% at 15 degrees C, and by the appearance of an unsaturated fatty acid residue, hexadecenoic acid. Changes in the 3-hydroxy-myristic acid content of the lipid A were minimal. The decrease in the growth temperature also resulted in a decrease in the saturated fatty acid content of the phospholipid fraction, which was accompanied by an increase in their fluidity, as measured by the freedom of motion of spin-labeled fatty acids incorporated into dispersions made of the phospholipids. Nevertheless, the fluidity obtained with membrane phospholipids extracted from the cells grown at various temperatures were essentially the same when fluidity was determined at the growth temperature, supporting the hypothesis that variations in the fatty acid composition of membrane phospholipids serve to produce membranes having a constant fluidity at different temperatures of growth.     

Fatty-acid biosynthesis in a branched-chain alpha-keto acid dehydrogenase mutant of Streptomyces avermitilis

Fatty-acid biosynthesis by a branched-chain alpha-keto acid dehydrogenase (bkd) mutant of Streptomyces avermitilis was analyzed. This mutant is unable to produce the appropriate precursors of branched-chain fatty acid (BCFA) biosynthesis, but unlike the comparable Bacillus subtilis mutant, was shown not to have an obligate growth requirement for these precursors. The bkd mutant produced only straight-chain fatty acids (SCFAs) with membrane fluidity provided entirely by unsaturated fatty acids (UFAs), the levels of which increased dramatically compared to the wild-type strain. The levels of UFAs increased in both the wild-type and bkd mutant strains as the growth temperature was lowered from 37 degrees C to 24 degrees C, suggesting that a regulatory mechanism exists to alter the proportion of UFAs in response either to a loss of BCFA biosynthesis, or a decreased growth temperature. No evidence of a regulatory mechanism for BCFAs was observed, as the types of these fatty acids, which contribute significantly to membrane fluidity, did not alter when the wild-type S. avermitilis was grown at different temperatures. The principal UFA produced by S. avermitilis was shown to be delta 9-hexadecenoate, the same fatty acid produced by Escherichia coli. This observation, and the inability of S. avermitilis to convert exogenous labeled palmitate to the corresponding UFA, was shown to be consistent with an anaerobic pathway for UFA biosynthesis. Incorporation studies with the S. avermitilis bkd mutant demonstrated that the fatty acid synthase has a remarkably broad substrate specificity and is able to process a wide range of exogenous branched chain carboxylic acids into unusual BCFAs.     

Role of membrane lipid fatty acids in cold adaptation.

Psychrophilic and psychrotolerant bacteria have evolved various strategies to adapt to low temperature. One important strategy, which is crucial to the survival of the cell at low temperature, relates to the ability of the cell to modulate the fluidity of the membrane. Bacteria in general modulate membrane fluidity by altering their fatty acid composition. But, bacteria could also achieve the same by various other strategies such as by altering the lipid head group, the protein content of the membrane, the type of carotenoids synthesized, the fatty acid chain length and the proportion of cis to trans fatty acids. In addition bacteria have a two-component signal transduction pathway consisting of a membrane-bound sensor and a soluble cytoplasmic response regulator involved in the perception and transduction of low temperature signals. This review on cold adaptation highlights the various strategies by which bacteria modulate the fluidity of the membrane and the process by which it senses and transduces the low temperature signal.     

Temperature-independent and -dependent expression of desaturase genes in filamentous cyanobacterium Spirulina platensis strain C1 (Arthrospira sp. PCC 9438)

The alteration of the degree of unsaturated fatty acids in membrane lipids has been shown to be a key mechanism in the tolerance to temperature stress of living organisms. The step that most influences the physiology of membranes has been proposed to be the amount of di-unsaturated fatty acids in membrane lipids. In this study, we found that the desaturation of fatty acid to yield the di-unsaturated fatty acid 18:2(9,12), in Spirulina platensis strain C1, was not regulated by temperature. As shown by the fatty acid composition and gene expression patterns, the levels of 18:1(9) and 18:2(9,12) remained almost constant either when the cells were grown at 35 degrees C (normal growth temperature) or 22 and 40 degrees C. The expression of desC (Delta9) and desA (Delta12) genes, which are responsible for the introduction of first and second double bonds into fatty acids, respectively, was not affected by the temperature shift from 35 to 22 degrees C or to 40 degrees C. Only the expression and mRNA stability of the desD gene (Delta6) that is responsible for the introduction of a third double bond into fatty acids were enhanced by a temperature shift from 35 to 22 degrees C, but not the shift from 35 to 40 degrees C. The increase in the level of desD mRNA elevated the desaturation of fatty acid from 18:2(9,12) to 18:3(6,9,12) at 22 degrees C. However, the increased level of 18:3(6,9,12) was observed after 36 h of incubation at 22 degrees C, indicating a slow response to temperature of fatty acid desaturation in this cyanobacterium. These findings suggest that the desaturation of fatty acids might not be a key mechanism in the response to the temperature change of S. platensis strain C1.     

The RssAB two-component signal transduction system in Serratia marcescens regulates swarming motility and cell envelope architecture in response to exogenous saturated fatty acids

Serratia marcescens swarms at 30 degrees C but not at 37 degrees C on a nutrient-rich (LB) agar surface. Mini-Tn5 mutagenesis of S. marcescens CH-1 yielded a mutant (WC100) that swarms not only vigorously at 37 degrees C but also earlier and faster than the parent strain swarms at 30 degrees C. Analysis of this mutant revealed that the transposon was inserted into a gene (rssA) predicted to encode a bacterial two-component signal transduction sensor kinase, upstream of which a potential response regulator gene (rssB) was located. rssA and rssB insertion-deletion mutants were constructed through homologous recombination, and the two mutants exhibited similar swarming phenotypes on LB swarming agar, in which swarming not only occurred at 37 degrees C but also initiated at a lower cell density, on a surface with a higher agar concentration, and more rapidly than the swarming of the parent strain at 30 degrees C. Both mutants also exhibited increased hemolysin activity and altered cell surface topologies compared with the parent CH-1 strain. Temperature and certain saturated fatty acids (SFAs) were found to negatively regulate S. marcescens swarming via the action of RssA-RssB. Analysis of the fatty acid profiles of the parent and the rssA and rssB mutants grown at 30 degrees C or 37 degrees C and under different nutrition conditions revealed a relationship between cellular fatty acid composition and swarming phenotypes. The cellular fatty acid profile was also observed to be affected by RssA and RssB. SFA-dependent inhibition of swarming was also observed in Proteus mirabilis, suggesting that either SFAs per se or the modulation of cellular fatty acid composition and hence homeostasis of membrane fluidity may be a conserved mechanism for regulating swarming motility in gram-negative bacteria.     

大肠杆菌生长温度、膜脂肪酸组成和压力抗性之间的关系

通过对大肠杆菌生长温度、膜脂肪酸组成和压力抗性之间关系研究发现,10℃培养,对数期细胞有最大的压力抗性,随着培养温度的升高直到4 5℃,压力抗性呈下降的趋势;相反,10℃培养,稳定期的细胞对压力最敏感,随着培养温度的升高,压力抗性呈增加趋势,30～37℃时达到最大,之后到4 5℃有下降。对数期和稳定期细胞膜脂中不饱和脂肪酸的组成随温度的上升而下降,这与从全细胞中抽提的磷脂的熔点密切相关。因此,对数期细胞压力抗性随着膜流动性的增大而升高;但稳定期细胞,膜流动性与压力抗性之间不存在简单的对应变化关系     

Escherichia coli membrane fluidity as detected by excimerization of dipyrenylpropane: sensitivity to the bacterial fatty acid profile.

A coordinated study of membrane fluidity and fatty acid composition has been carried out in Escherichia coli W3110. The lipid acyl chain profile of the bacteria, altered by growing cells in steady state at 30, 37, 42, or 45 degrees C, was determined by gas chromatography of the fatty acid methyl esters. In parallel experiments, total membranes obtained from cells of the above-mentioned cultures were labeled with dipyrenylpropane and their relative fluidity was measured on the basis of the excimer to monomer fluorescence intensity ratio of the fluorophore. It has been found that, at constant assay temperature, fluidity determined with dipyrenylpropane decreases gradually with the growth temperature increment, from 30 to 45 degrees C. Interestingly, when fatty acid composition is taken into account, fluidity increases linearly in the range under study, with the proportion of unsaturated fatty acyl chains, both variables being highly correlated (0.924 相似文献   

The genome of <Emphasis Type="Italic">Bacillus aryabhattai</Emphasis> T61 reveals its adaptation to Tibetan Plateau environment

Tibetan Plateau is called ‘the Roof of the World’. Organisms survive there have to adapt to the high altitude environment. By shotgun method, we sequenced the genome of Bacillus aryabhattai T61, which inhabits in the soil at the altitude of 4123 m in Shigatse, Tibetan. Further, we explored the genomic basis for its adaptations to the plateau environment. The results showed that B. aryabhattai T61 has evolved an array of ROS defense systems and sporulation system for adaptations to the stresses caused by the plateau strong ultraviolet radiation, extreme oxygen limitation and low temperature. Specifically, B. aryabhattai T61 encodes the ResE–ResD two-component to sense the oxygen limitation and regulates COX15 for aerobic and anaerobic respiration. The two-component system DesK–DesR, which regulates the gene Des initiating the biosynthesis of unsaturated fatty acids, along with 33 temperature-shock proteins contribute to low temperature adaptation. With the comparative analysis, we deduced the novel gene cbiY may be involved in cobalamin biosynthesis. We also found that B. aryabhattai T61 may have novel regulatory mechanisms for sporulation initiation. B. aryabhattai T61 is the first Tibetan strain with high quality genome sequenced. The genome provides a paradigm for understanding the adaptations to the plateau environment in Bacteria kingdom.