Alteration in the contents of unsaturated fatty acids in dnaA mutants of Escherichia coli

DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli , has a high affinity for acidic phospholipids containing unsaturated fatty acids. We have examined here the fatty acid composition of phospholipids in dnaA mutants. A temperature-sensitive dnaA46 mutant showed a lower level of unsaturation of fatty acids (ratio of unsaturated to saturated fatty acids) at 42°C (non-permissive temperature) and at 37°C (semi-permissive temperature), but not at 28°C (permissive temperature), compared with the wild-type strain. Plasmid complementation analysis revealed that the dnaA46 mutation is responsible for the phenotype. Other temperature-sensitive dnaA mutants showed similar results. On the other hand, a cold-sensitive dnaAcos mutant, in which overinitiation of DNA replication occurs at low temperature (28°C), showed a higher level of unsaturation of fatty acids at 28°C. Based on these observations, we discuss the role of phospholipids in the regulation of the activity of DnaA protein.     

Effect of Unsaturated Fatty Acids on the Development of Respiration and on Protein Synthesis in an Unsaturated Fatty Acid Mutant of Saccharomyces cerevisiae

The extent of development of respiratory function induced by aeration of an anaerobically grown unsaturated fatty acid auxotroph of Saccharomyces cerevisiae is determined by the availability, endogenous or externally supplied, of unsaturated fatty acid. The synthesis of mitochondrial and cytoplasmic enzymes during aeration appears to have a similar basis of regulation by available unsaturated fatty acid. Levels of unsaturated fatty acid that permit the synthesis of mitochondrial enzymes also result in a substantial stimulation of cellular protein synthesis.     

Process of Infection with Bacteriophage φX174: XXXV. Cistron VIII

Twenty-two new amber and ochre mutants of phiX174 were isolated and classified into complementation groups. Three ochre mutants gave positive complementation tests with reference mutants in the seven previously defined groups and thus represent an eighth cistron. Studies of the physiology of infection in the nonpermissive condition for mutants in cistron VIII yielded the following information. (i) Replicative-form synthesis proceeds at a normal rate, and is turned off at the usual time. (ii) Synthesis of single-stranded deoxyribonucleic acid (DNA) is delayed until nearly 40 min after infection (in the absence of lysis), at which time a slow synthesis of infectious phage particles commences. The synthesis of infectious particles at late times is interpreted as a consequence of "leakage," and indicates that the cistron VIII product is required in very small quantities. (iii) During the normal period of single-strand synthesis, most of the replicative-form DNA is found in a form with properties similar to those of the transient intermediates of single-strand DNA synthesized during normal infection.     

Biogenesis of mitochondria: The effects of altered steady-state membrane lipid composition on mitochondrial-energy metabolism in Saccharomyces cerevisiae

A chemostat culture technique has been developed for the growth of an unsaturated fatty acid auxotroph of Saccharomyces cerevisiae. Any chosen steady-state cellular unsaturated fatty acid level between 75 and 15% of the total fatty acids could be established and maintained. In all cultures the steady-state glucose concentrations were maintained at levels below that which induces catabolite repression.The efficiency of oxidative phosphorylation as determined from the molar growth yield decreased as the cellular unsaturated fatty acid composition was lowered. The number of moles of ATP produced by oxidative phosphorylation per mole of glucose utilized was 7.2, 4.8, 0.7, and 0.4 for cells in which 75, 50, 44, and 34%, respectively, of the total fatty acids were unsaturated.The lesion in oxidative phosphorylation was a direct result of lowering the membrane unsaturated fatty acid composition as the respiratory activities and cytochrome content of cells and mitochondria were unaffected by a decrease in the cellular unsaturated fatty acid level from the wild-type value of about 75% down to about 34%.In cells which contained lipids with 22–28% unsaturated fatty acids, cyanide-sensitive respiration was absent, and the levels of all mitochondrial cytochromes were less than 10% of normal. The reduction in the levels of cytochromes aa₃ and b appeared to be a consequence of a loss of mitochondrial protein synthetic activity in such cells. The level of cytochrome c was also greatly decreased, indicating that the cellular unsaturated fatty acid composition was affecting either the synthesis in the cytoplasm of mitochondrial proteins or the assembly of these proteins in the mitochondria.     

Analysis of the genes flanking xabB: a methyltransferase gene is involved in albicidin biosynthesis in Xanthomonas albilineans

Transposon mutagenesis and complementation studies previously identified a gene (xabB) for a large (526kDa) polyketide-peptide synthase required for biosynthesis of albicidin antibiotics and phytotoxins in the sugarcane leaf scald pathogen Xanthomonas albilineans. A cistron immediately downstream from xabB encodes a polypeptide of 343aa containing three conserved motifs characteristic of a family of S-adenosyl-L-methionine (SAM)-dependent O-methyltransferases. Insertional mutagenesis and complementation indicate that the product of this cistron (designated xabC) is essential for albicidin production, and that there is no other required downstream cistron. The xabB promoter region is bidirectional, and insertional mutagenesis of the first open reading frame (ORF) in the divergent gene also blocks albicidin biosynthesis. This divergent ORF (designated thp) encodes a protein of 239aa displaying high similarity to several IS21-like transposition helper proteins. The thp cistron is not located in a recognizable transposon, and is probably a remnant from a past transposition event that may have contributed to the development of the albicidin biosynthetic gene cluster. Failure of 'in trans' complementation of thp indicates that a downstream cistron transcribed with thp is required for albicidin biosynthesis.     

Mechanism of the apparent regulation of Escherichia coli unsaturated fatty acid synthesis by exogenous oleic acid.

Starvation of strains of Escherichia coli which are glycerol auxotrophs and are also defective in beta oxidation results in the accumulation of large amounts of free fatty acid (Cronan, J. E., Jr., Weisberg, L. W., and Allen, R. G. (1975) J. Biol. Chem. 250, 5835-5840). We now report that addition of exogenous oleic acid to these cultures results in no decrease in the synthesis of the unsaturated acids of the free fatty acid fraction although a 40 to 60% decrease of [14C]acetate incorporation into phospholipid unsaturated acyl moieties occurs under these conditions. This result indicates that the decreased synthesis of phospholipid unsaturated acyl moieties observed by others during oleic acid supplementation can be attributed to competition between exogenous and endogenously synthesized unsaturated fatty acids rather than a curtailment of unsaturated fatty acid synthesis per se.     

Mechanism of ethanol-induced changes in lipid composition of Escherichia coli: inhibition of saturated fatty acid synthesis in vivo.

The in vivo effects of ethanol on lipid synthesis in Escherichia coli have been examined. Under conditions which uncoupled fatty acid synthesis from phospholipid synthesis, ethanol decreased the amount of saturated fatty acids synthesized but had little effect on the selectivity of their incorporation into phospholipids. In the absence of fatty acid degradation and unsaturated fatty acid synthesis, E. coli was still able to adapt its membrane lipids to ethanol, while the inhibition of total fatty acid synthesis eliminated this response. During growth in the presence of ethanol, strain K1060 (an unsaturated fatty acid auxotroph) incorporated an increased amount of exogenous heptadecanoic acid (17:0) to compensate for the reduction in palmitic acid (16:0) available from biosynthesis. Thus, our results indicate that the reduced levels of saturated fatty acids observed in the phospholipids of E. coli following growth in the presence of ethanol result primarily from a decrease in the amounts of saturated fatty acids available for phospholipid synthesis.     

Morphogenesis of Bacteriophage φ80: Identification of the Cistron 13 Product

An in vitro complementation reaction leading to the assembly of bacteriophage phi80 tails from component proteins is described. Tail assembly occurs when a lysate of any mutant in cistron 13 is mixed with a second lysate of a mutant in any of the other cistrons involved in tail formation. Lysates of mutants that are blocked in tail formation contain phage heads that can unite with free tails to form infective particles. The rate of the complementation reaction shows little dependence upon temperature, suggesting that the assembly depends largely upon the kinetic encounter of the interacting components. The tail component missing in cistron 13 mutant lysates was purified approximately 55-fold and shown to be, at least in part, a protein having a molecular weight of approximately 22,000. This protein was also released from highly purified infective phi80 particles after osmotic shock followed by heattreatment, suggesting that it most probably is an integral structural protein of the phage tail. Lysates of mutants of bacteriophage lambda that are defective in tail formation were shown to contain a tail component identical with or similar to the phi80 cistron 13 product.     

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).     

Functional complementation of a periila ω3 fatty acid desaturase under the seed-specific SeFAD2 promoter

Functional characterization of the fatty acid desaturase genes and seed-specific promoters is prerequisite for altering the unsaturated fatty acid content of oilseeds by genetic manipulation. The ω-6 fatty acid desaturase (FAD2) and ω-3 fatty acid desaturase (FAD3) catalyze extra-plastidial desaturation of oleic acid to linoleic acid and linoleic acid to linolenic acid, respectively. These are major constituents in seed storage oils. Here, we report the complementation of a perilla linoleic acid desaturase (PrFAD3) cDNA under the seed-specific sesame FAD2 (SeFAD2) promoter in the Arabidopsis fad3 mutant. PrFAD3 is functionally active and the SeFAD2 promoter is applicable for modifying fatty acid composition in developing seeds. Transient expression of the GUS gene under that promoter in the developing seeds and leaves of sesame, soybean, and corn via microprojectile bombardment indicated that the SeFAD2 promoter likely will be useful for altering the seed phenotypes of dicot and monocot crops.     

Peroxidized unsaturated fatty acids stimulate Toll-like receptor 4 signaling in endothelial cells

AimAlthough unsaturated fatty acids are assumed to be protective against inflammatory disorders that include a pathway involving Toll-like receptor 4 (TLR4) activation, they might actually be toxic because of their high susceptibility to lipid peroxidation. Here we studied the effects of peroxidized unsaturated fatty acids on the TLR4–nuclear factor (NF)-κB pathway in endothelial cells.Main methodsConfluent cultured endothelial cells from bovine aorta were incubated for 1 h with fatty acids integrated into phosphatidylcholine vesicles. Lipopolysaccharide (LPS) or phosphatidylcholine vesicles without fatty acids were also applied as a positive control or a control for fatty acid groups, respectively. Activation of TLR4 and downstream signaling was assessed by membrane fractionation and Western blotting or immunofluorescent staining.Key findingsIn the same way as LPS, application of sufficiently peroxidized unsaturated fatty acids like oleic acid or docosahexaenoic acid, acutely caused TLR4 translocation to caveolae/raft membranes, leading to activation of NF-κB signaling in endothelial cells. In contrast, saturated fatty acids did not show such effects. Applying well-peroxidized unsaturated fatty acids, but not saturated fatty acids, acutely activates the TLR4/NF-κB pathway.SignificancePeroxidation of unsaturated fatty acid is essential for the acute activation of TLR4 by the fatty acids that follow the same pathway as the activation by LPS. Unsaturated fatty acids have been assumed to be protective against inflammatory disorders, and drugs containing unsaturated fatty acids are now developed and provided. Our result suggests that, for inflammatory disorders involving TLR4 signaling, using unsaturated fatty acids as anti-inflammatory drugs may cause contrary effects.     

Synthesis and utilization of fatty acids by wild-type and fatty acid auxotrophs of Caulobacter crescentus.

The fatty acid composition of the dimorphic bacterium Caulobacter crescentus was found to consist primarily of 16- and 18-carbon fatty acids, both saturated and monounsaturated, in agreement with the findings of Chow and Schmidt (J. Gen. Microbiol. 83:359-373, 1974). In addition, two minor but as yet unidentified fatty acids were detected. Chromatographic mobilities suggested that these fatty acids may be a cyclopropane and a branched-chain fatty acid. In addition, we demonstrated that the fatty acid composition of wild-type C. crescentus can be altered by growing the cells in medium supplemented with any one of a variety of unsaturated fatty acids. Linoleic acid, a diunsaturated fatty acid which is not synthesized by C. crescentus, was incorporated into phospholipids without apparent modification. In addition, we found that C. crescentus, like Escherichia coli, synthesizes vaccenic acid (18:1 delta 11,cis) rather than oleic acid (18:1 delta 9,cis). This result allowed us to deduce that the mechanism of fatty acid desaturation in C. crescentus is anaerobic, as it is in E. coli. Finally, we examined the fatty acid biosynthesis and composition of two unsaturated fatty acid auxotrophs of C. crescentus. Neither of these mutants resembled the E. coli unsaturated fatty acid auxotrophs, which have defined enzymatic lesions in fatty acid biosynthesis. Rather, the mutants appeared to have defects relating to the complex coordination of membrane biogenesis and cell cycle events in C. crescentus.     

The biogenesis of mitochondrial membranes in the yeast Saccharomyces cerevisiae.

Membrane lipids of yeast mitochondria have been enriched by growing yeast cells in minimal medium supplemented with specific unsaturated fatty acids as the sole lipid supplement. Using the activity of marker enzymes for the outer (kynurenine hydroxylase) and inner (cytochrome c oxidase and oligomycin-sensitive ATPase) mitochondrial membranes, Arrhenius plots have been constructed using both promitochondria and mitochondria obtained from O2-adapting cells in the presence of a second unsaturated fatty acid (i.e. linoleate (N2) to elaidic (O2)). Transition temperatures which reflect the unsaturated fatty acid enrichment of the new membranes reveal interesting features involved in the mechanism of the assembly of these two mitochondrial membranes. This approach was further enforced with both lipid depletion and mitochondrial protein inhibition studies. Kynurenine hydroxylase which does not require fatty acid for its continued synthesis during aerobiosis seems to be incorporated into the preformed linoleate-anaerobic outer membrane. The newly synthesized activities of inner mitochondrial membrane enzymes on the other hand, appear to integrate their activity into newly formed aerobic-elaidic-rich inner membrane. These latter enzymes show a distinct dependence on fatty acid supplement for their continued synthesis during their aerobic phase. This suggests that O2-dependent proteo-lipid precursors are formed before these enzymes are integrated into their membrane mosaic. Two separate models are proposed to explain these results, one for the lipid-rich outer mitochondrial membrane and another for the protein-rich inner mitochondrial membrane.     

Beta-hydroxydecanoyl thio ester dehydrase does not catalyze a rate-limiting step in Escherichia coli unsaturated fatty acid synthesis

The intracellular level of beta-hydroxydecanoyl thio ester dehydrase, the product of the fabA gene of Escherichia coli, was increased by isolation of a putative promotor mutant (termed fabAup) or by molecular cloning of the wild-type fabA gene into plasmid pBR322. The fabAup and plasmid-carrying strains overproduced dehydrase by about 15- and 10-fold, respectively. The phospholipids of all strains that overproduced the dehydrase contained significantly higher levels of saturated fatty acids than isogenic strains producing a normal level of dehydrase. No increased levels of unsaturated fatty acids were observed. This result indicates that, although the dehydrase is required for unsaturated fatty acid synthesis, the level of dehydrase activity in wild-type cells does not limit the rate of unsaturated fatty acid synthesis. The introduction of a plasmid carrying the structural gene for beta-ketoacyl acyl carrier protein synthase I into a fabAup strain overcame the effect of dehydrase overproduction on fatty acid composition.     

内陆土壤冷适应细菌的筛选分类与细胞膜脂肪酸的适冷机制

嗜冷菌及耐冷菌是冷适应酶及生物活性物质的重要资源。本研究从内陆土壤筛得33株冷适应细菌,包括6株革兰氏阳性菌与27株革兰氏阴性菌。通过细胞膜脂肪酸分析表明,革兰氏阳性菌的膜脂肪酸主要为分支脂肪酸,推测分支结构是阳性菌膜脂的主要适冷机制。革兰氏阴性菌呈现了不饱和、分支、短链等多样的膜脂适冷调节方式。根据脂肪酸组分的多样性,选择并鉴定了17株嗜冷及耐冷菌分布在11个属中,细胞膜脂肪酸组成的变化规律与细菌16SrRNA的进化分布高度一致。研究还表明同为不饱和脂肪酸为主的革兰氏阴性菌呈现了不同的适冷机理。相关研究不仅阐述了冷适应细菌的细胞膜脂肪酸的适应机制,而且为相关适冷酶源的开发利用提供了宝贵的资源。     

Studies of energy linked reactions: a cofactor function for unsaturated fatty acids in oxidative phosphorylation; studies with a yeast auxotroph.

Mitochondria from the yeast unsaturated fatty acid auxotroph KD115 when grown with a high unsaturated fatty acid supplement catalyse normal oxidative phosphorylation and dihydrolipate-dependent ATP synthesis in the absence of added cofactors. Mitochondria from unsaturated fatty acid depleted KD115 cells have half the unsaturated fatty acid content of supplemented cells and do not catalyse oxidative phosphorylation and dihydrolipoate dependent ATP synthesis. Dihydrolipoate-dependent ATP synthesis can be restored specifically by addition of cofactor amounts of oleic acid and oleoyl CoA. The results provide further evidence for a cofactor role for an unsaturated fatty acid in oxidative phosphorylation.     

Total fatty acid content of the plasma membrane of Saccharomyces cerevisiae is more responsible for ethanol tolerance than the degree of unsaturation

The effect of change in unsaturated fatty acid composition on ethanol tolerance in Saccharomyces cerevisiae overexpressing ScOLE1 (?9 fatty acid desaturase gene of S. cerevisiae), CaFAD2 (?12 fatty acid desaturase gene of Candida albicans), or CaFAD3 (ω3 fatty acid desaturase gene of C. albicans) was examined. ScOLE1 over-expression increased the total unsaturated fatty acid content and enhanced ethanol tolerance, compared with a control strain. In contrast, overexpression of CaFAD2 and CaFAD3, which led to production of linoleic acid (18:2) and α-linolenic acid (18:3), respectively, neither changed total unsaturated fatty acids nor enhanced ethanol tolerance. The total unsaturated fatty acid content rather than the degree of unsaturation is thus an important factor for ethanol tolerance.     

Fine structure of the arg-7 cistron in Chlamydomonas reinhardi

Summary In Chlamydomonas reinhardi, the arg-7 cistron is the structural gene for the enzyme argininosuccinate lyase which catalyzes the last reaction in the biosynthesis of arginine.Fourteen mutants (nine previously analyzed and five new mutants) defective in the lyase have been investigated so far: they all map within a cistron (length: 1.0–1.6 recombination units) of the linkage group I and fall within six groups of complementation. The enzyme activity found in the diploids formed by intragenic complementation was always lower than in wild-type haploid or diploid strains. The study of the denaturation curves obtained by heat treatment of the lyase indicates that in some diploids, several enzyme varieties can be present.These results and those previously obtained with diploids formed by intragenic and intergenic complementation (Matagne and Loppes, 1972; Matagne, 1976) are discussed in relation to the recent data showing that the argininosuccinate lyase is a multimeric enzyme probably composed of five identical polypeptide chains (Matagne and Schlösser, 1977)     

Manipulation of fatty acid composition of membrane phospholipid and its effects on cell growth in mouse LM cells

Fatty acid composition of the phospholipids of mouse LM cells grown in suspension culture in serum-free chemically defined medium was modified by supplementing the medium with various fatty acids bound to bovine serum albumin.Following supplementation with saturated fatty acids of longer than 15 carbons (100 μM) profound inhibition of cell growth occurred; this inhibitory effect was completely abolished when unsaturated fatty acids were added at the same concentration. Supplementing with unsaturated fatty acids such as linoleic acid, linolenic acid or arachidonic acid had no effect on the cell growth.Fatty acid composition of membrane phospholipids could be manipulated by addition of different fatty acids. The normal percentage of unsaturated fatty acids in LM cell membrane phospholipids (63%) was reduced to 35–41% following incorporation of saturated fatty acids longer than 15 carbon atoms and increased to 72–82% after addition of unsaturated fatty acids.A good correlation was found between the unsaturated fatty acid content of membrane phospholipids and cell growth. When incorporated saturated fatty acids reduced the percentage of unsaturated fatty acids in membrane phospholipids to less than 50%, severe inhibition of the cell growth was found. Simultaneous addition of an unsaturated fatty acid completely abolished this effect of saturated fatty acids.     

Ethanol-induced changes in lipid composition of Escherichia coli: Inhibition of saturated fatty acid synthesis in vitro

Growth of Escherichia coli in the presence of ethanol results in the synthesis of lipids containing elevated proportions of unsaturated fatty acids. Previous in vivo experiments indicated that the ethanol-induced changes in fatty acid composition result from a preferential inhibition of saturated fatty acid synthesis. In this study, the inhibition of saturated fatty acid synthesis by ethanol was confirmed in vitro. This inhibition was not membrane mediated and resulted from a direct action of ethanol on the soluble enzymes of fatty acid synthesis. The addition of ethanol resulted in a decrease in chain length of both saturated and unsaturated acyl products in vitro. Experiments with enzymes prepared from several fatty acid synthesis mutants of E. coli indicate that β-hydroxydecanoyl-acyl carrier protein dehydrase is not the site of the ethanol inhibition of saturated fatty acid synthesis. The two condensing enzymes are the probable sites for inhibition by ethanol.