Sporulation in Bacillus subtilis is independent of membrane fatty acid composition.

Growth and sporulation of a Bacillus subtilis mutant deficient in branched fatty acid synthesis (gene symbol bfmB) were examined. The mutant, which produces an acyl-coenzyme A:acyl carrier protein transacylase with reduced affinity for branched fatty acid primers, could grow in media containing any one of a wide range of low-molecular-weight fatty acids having branched, cyclic, saturated, or unsaturated carbon chains. The fatty acid composition of cellular lipids depended on the compound used to support growth. Cultures of the bfmB mutant grown in the presence of 3-methylcrotonate contained an unusually high fraction (73%) of straight-chain fatty acids in the cellular lipids. The mutant sporulated with any one of the precursors of branched fatty acids in the medium; isolated spores contained mainly this branched fatty acid and only 10% or less straight-chain fatty acids regardless of the straight-chain fatty acid content of vegetative cells. Exceptional were spores grown in the presence of cyclobutane-carboxylic acid, which contained 28% straight-chain fatty acids. The branched fatty acid composition of spores could be modified greatly by changing the supply of precursors in the medium.     

Unsaturated and branched chain-fatty acids in temperature adaptation of Bacillus subtilis and Bacillus megaterium.

The effect of growth temperature on the cellular fatty acid profiles of Bacillus subtilis and Bacillus megaterium was studied over a temperature range from 40 to 10 degrees C. As the growth temperature of B. subtilis was reduced, the lower-melting point anteiso-acids increased, while the higher-melting point iso-acids decreased. Consequently the ratio of branched- to straight-chain acids was unaffected by temperature, although changes in the position of fatty acid branching and the degree of unsaturated branched-chain fatty acids occurred. In B. megaterium a more complicated, biphasic behaviour was observed. Saturated, straight-chain and iso-branched acids decreased only from 40 degrees C down to 20-26 degrees C, and anteiso-acids decreased only from 20-26 degrees C to 10 degrees C, while unsaturated acids increased over the whole temperature range studied. Thus, in B. megaterium total branched-chain acids decreased and straight-chain acids increased as temperature decreased. However, the overall cellular content of lower-melting point fatty acids increased with decreasing temperature in both bacilli, and unsaturated fatty acids appeared to be essential components in the adaptation of the microbes to changes in temperatures. Since changes in the relative amounts of branched- and straight-chain fatty acid biosynthesis are known to reflect differences in fatty acid primers, temperature seems to affect not only the activity of the fatty acid desaturases but also the formation or availability of these primers. The results indicate, however, that notable species-specific regulatory features exist in this genus of bacteria.     

Fatty Acids in Bacillus larvae, Bacillus lentimorbus, and Bacillus popilliae

The types of fatty acids produced by two strains each of Bacillus larvae, B. lentimorbus, and B. popilliae, and their distribution patterns, were studied by gas-liquid chromatography. All six organisms produced eight major fatty acids: six branched (iso-C(14), -C(15), -C(16), and -C(17), and anteiso-C(15) and -C(17)), two normal (n-C(14) and -C(16)), and two minor (n-C(15) and monounsaturated n-C(16)). In addition, some other trace acids were produced. Branched-chain fatty acids accounted for 54 to 85% of the total fatty acids. These compositions are similar to those previously found with 26 strains of 12 species of the genus Bacillus. Thus, an abundance of branched-chain fatty acids seems to be a characteristic of the biochemical nature of the genus Bacillus. It is noteworthy that marked differences between the nutritional requirements of the three insect pathogens used in the present study and those of the other 12 species of the genus Bacillus studied previously are not significantly reflected in their fatty acid composition.     

Cloning and sequence of Bacillus subtilis purA and guaA, involved in the conversion of IMP to AMP and GMP.

Bacillus subtilis genes purA, encoding adenylosuccinate synthetase, and guaA, coding for GMP synthetase, appear to be lethal when cloned in multicopy plasmids in Escherichia coli. The nucleotide sequences of purA and guaA were determined from a series of gene fragments isolated by polymerase chain reaction amplification, library screening, and plasmid rescue techniques. Identifications were based on amino acid sequence alignments with enzymes from other organisms. Comparison of the 5'-flanking regions of purA and guaA with the pur operon suggests similarities in mechanisms for gene regulation. Nucleotide sequences are now available for all genes involved in the 14-step pathway for de novo purine nucleotide synthesis in B. subtilis.     

Iso- and anteiso-fatty acids in bacteria: biosynthesis, function, and taxonomic significance.

Branched-chain fatty acids of the iso and anteiso series occur in many bacteria as the major acyl constituents of membrane lipids. In addition, omega-cyclohexyl and omega-cycloheptyl fatty acids are present in several bacterial species. These two types of fatty acids are synthesized by the repeated condensation of malonyl coenzyme A with one of the branched-chain and cyclic primers by the same enzyme system. The pathway of de novo branched-chain fatty acid synthesis differs only in initial steps of synthesis from that of the common straight-chain fatty acid (palmitic acid) present in most organisms. The cell membranes composed largely of iso-, anteiso-, and omega-alicyclic acids support growth of bacteria, which inhabit normal as well as extreme environments. The occurrence of these types of fatty acids as major cellular fatty acids is an important criterion used to aid identification and classification of bacteria.     

On the question of half- or full-site reactivity of animal fatty acid synthetase

Our model of the animal fatty acid synthetase describes a head-to-tail arrangement of two identical subunits and predicts the presence of two centers for fatty acid synthesis. Current experiments which support this conclusion were conducted using the following approach. The thioesterase component of chicken liver fatty acid synthetase was either inhibited using phenylmethanesulfonyl fluoride or diisopropyl fluorophosphate, or released from the synthetase by limited proteolysis with alpha-chymotrypsin, thus ensuring that the fatty acyl products remain bound to the enzyme. Employing such preparations, the amount of NADPH oxidized in the initial burst of fatty acid synthesis was determined by stopped flow techniques. Gas-liquid chromatography showed that C20:0 and C22:0 constituted 85% of the fatty acids formed de novo, a result that was confirmed using [14C]acetyl-CoA in the reaction. These data showed that 1.0 mol of fatty acyl-enzyme product was formed per mol of phosphopantetheine; in addition, the measured stoichiometry of NADPH oxidation was sufficient to account for de novo fatty acid synthesis. Altogether, these results indicate that the two sites for fatty acid synthesis are active and function simultaneously. They also indicate that the thioesterase plays a key role in determining the chain specificity of fatty acid synthesis.     

Changes in fatty acid branching and unsaturation ofStreptomyces cinnamonensis as a response to NaCl concentration

Three differentStreptomyces cinnamonensis strains (viz. standard, regulatory, and non-differentiating) were grown in a synthetic medium containing 0–4% NaCl and the composition of total cellular fatty acids was analyzed. The increasing salt concentration resulted in an increasing proportion of even-numbered straight-chain fatty acids, mostly at the expense of branched-chain fatty acids. Unsaturated fatty acids, palmitoleic acid in particular, represent the major proportion of straight-chain fatty acids.     

Membrane lipid biosynthesis in Acholeplasma laidlawii B: de novo biosynthesis of saturated fatty acids by growing cells.

The de novo biosynthesis of fatty acids of 12 to 18 carbons from precursors of 5 carbons or fewer has been demonstrated in Acholeplasma laidlawii B. Radiolabeling experiments indicated that the normal primers for the synthesis of the even- and odd-chain fatty acids are acetate and propionate or valerate, respectively. Saturated straight-chain monomethyl-branched fatty acids of up to five carbons were readily utilized as primers, wheras more highly branched species and those possessing halogen substituents or unsaturation were not utilized. At primer concentrations of 1 to 3 mM, up to 80% of the total cellular lipid fatty acids were derived from exogenous primer. The mean chain length of the exogenous primer-derived fatty acids rose with increasing primer incorporation for methyl-branched short-chain fatty acids but was invariant for propionate. The products of de novo biosynthesis varied only slightly with temperature or cholesterol supplementation, suggesting that de novo biosynthesis is not directly influenced by membrane fluidity. Cerulenin inhibited de novo biosynthesis in a fashion that suggests the presence of two beta-ketoacyl thioester synthetases, which differ in substrate chain length specificity and in susceptibility to inhibition by the antibiotic.     

Characterization of the fatty acid synthetase system of Curtobacterium pusillum

Curtobacterium pusillum contains 11-cyclohexylundecanoic acid as a major component of cellular fatty acids. A trace amount of 13-cyclohexyltridecanoic acid is also present. Fatty acids other than omega-cyclohexyl fatty acids present are 13-methyltetradecanoic, 12-methyltetradecanoic, n-pentadecanoic, 14-methylpentadecanoic, 13-methylpentadecanoic, n-hexadecanoic, 15-methylhexadecanoic, 14-methylhexadecanoic, and n-heptadecanoic acids. The fatty acid synthetase system of this bacterium was studied. Various 14C-labeled precursors were added to the growth medium and the incorporation of radioactivity into cellular fatty acids was analyzed. Sodium [14C]acetate and [14C]glucose were incorporated into almost all species of cellular fatty acids, the incorporation into 11-cyclohexylundecanoic acid being predominant. [14C]Isoleucine was incorporated into 12-methyltetradecanoic and 14-methylhexadecanoic acids: [14C]leucine into 13-methyltetradecanoic and 15-methylhexadecanoic acids; and [14C]valine into 14-methylpentadecanoic acid. [14C]-Shikimic acid was incorporated almost exclusively into omega-cyclohexyl fatty acids. The fatty acid synthetase activity of the crude enzyme preparation of C. pusillum was reconstituted on the addition of acyl carrier protein. This synthetase system required NADPH and preferentially utilized cyclohexanecarbonyl-CoA as a primer. The system was also able to use branched- and straight-chain acyl-CoAs with 4 to 6 carbon atoms effectively as primers but was unable to use acetyl-CoA. However, if acetyl acyl carrier protein was used as the priming substrate, the system produced straight-chain fatty acids. The results imply that the specificity of the initial acyl-CoA:acyl carrier protein acyltransferase dictates the structure of fatty acids synthesized and that the enzymes catalyzing the subsequent chain-elongation reactions do not have the same specificity restriction.     

Contributions of fatty acid and sterol synthesis to triglyceride and cholesterol secretion by the perfused rat liver in genetic hyperlipemia and obesity

The relative importance of fatty acid synthesis in triglyceride secretion by perfused livers from lean (normal control) and obese Zucker rats was investigated. Livers from fed animals were perfused in a recirculating system with tritiated water and a constant infusion of oleic acid. Triglyceride secretion was 5 times greater and cholesterol secretion was 35% greater in the obese rat livers. The very-low-density lipoprotein hypersecreted by perfused livers from obese rats contained more apolipoprotein B and exhibited an increased B-48/B-100 ratio. Apo-B was also elevated in the hypertriglyceridemic plasma of obese rats in both fed and fasting states. The very-low-density lipoprotein isolated therefrom was likewise characterized by an increased B-48/B-100 ratio. Ketogenesis was depressed 40% in the obese rat livers and increased hepatic malonyl-CoA was implicated in this alteration. The de novo synthesis and secretion of newly synthesized cholesterol was moderately increased in the perfused livers from obese rats. Tritium incorporation into fatty acids was 15 times greater in the obese genotype. Most of the synthesized fatty acids remained in the liver and were recovered after perfusion in triglyceride and phospholipids. Newly synthesized fatty acids accounted for only 3 and 15% of the triglyceride secreted by the lean and obese rat livers, respectively. A large portion of the secreted triglyceride fatty acids was derived from endogenous liver lipids. When the turnover of newly synthesized fatty acids in these pools was considered, the contribution of de novo fatty acid synthesis to triglyceride secretion was estimated to be 9% in the lean and 44% in the obese rat livers. Therefore, the altered partition of free fatty acids (Fukuda, N., Azain, M. J., and Ontko, J. A. (1982) J. Biol. Chem. 257, 14066-14072) and increased fatty acid synthesis are both major determinants of the hypersecretion of triglyceride-rich lipoproteins by the liver in the genetically obese Zucker rat.     

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.     

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.     

Inhibition of brain prostaglandin D synthetase and prostaglandin D2 dehydrogenase by some saturated and unsaturated fatty acids

The activities of rat brain prostaglandin D synthetase and swine brain prostaglandin D2 dehydrogenase were inhibited by some saturated and unsaturated fatty acids. Myristic acid was most potent among saturated straight-chain fatty acids so far tested. The IC50 values of this acid were 80 microM for prostaglandin D synthetase and 7 microM for prostaglandin D2 dehydrogenase, respectively. Little inhibition was found with methyl myristate and myristyl alcohol. The IC50 values of these derivatives were more than 200 microM for both enzymes, suggesting that the free carboxyl group was essential for the inhibition. The effects of cis double bond structure of fatty acids on the inhibition potency were examined by the use of the carbon 18 and 20 fatty acids. The inhibition potencies for both enzymes increased with the number of cis double bonds; the IC50 values of stearic, oleic, linoleic and linolenic acid were, respectively, more than 200, 60, 30 and 30 microM for prostaglandin D synthetase, and 20, 10, 8.5 and 7 microM for prostaglandin D2 dehydrogenase. Arachidonic acid also inhibited the activities of both enzymes with respective IC50 values of 40 microM for prostaglandin D synthetase and 3.9 microM for prostaglandin D2 dehydrogenase, while arachidic acid showed little inhibition. The kinetic studies with myristic acid and arachidonic acid demonstrated that the inhibition by these fatty acids was competitive and reversible for both enzymes. Myristic acid and other fatty acids also inhibited the activities of several enzymes in prostaglandin metabolism, although to a lesser extent. The IC50 values of myristic acid for prostaglandin E isomerase, thromboxane synthetase and NAD-linked prostaglandin dehydrogenase (type I) were 200, 700 and 100 microM, respectively. However, this fatty acid showed little inhibition on fatty acid cyclooxygenase (20% at 800 microM), glutathione-requiring prostaglandin D synthetase from rat spleen (20% at 800 microM), and NADP-linked prostaglandin dehydrogenase (type II) (no inhibition at 200 microM).     

De novo fatty acid synthesis in developing rat lung

The rate of de novo fatty acid synthesis in developing rat lung was measured by the rate of incorporation of 3H from 3H2O into fatty acids in lung slices and by the activity of acetyl-CoA carboxylase in fetal, neonatal and adult lung. Both tritium incorporation and acetyl-CoA carboxylase activity increased sharply during late gestation, peaked on the last fetal day, and declined by 50% 1 day after birth. In the adult, values were only one-half the peak fetal rates. In vitro regulation of acetyl-CoA carboxylase activity in fetal lung was similar to that described in adult non-pulmonary tissues: activation by citrate and inhibition by palmitoyl-CoA. Similarly, incubation conditions that favored enzyme phosphorylation inhibited acetyl-CoA carboxylase activity in lung while dephosphorylating conditions stimulated activity. Incorporation of [U-14 C]glucose into lung lipids during development was influenced heavily by incorporation into fatty acids, which generally paralleled the rate of tritium incorporation into fatty acids. The relative utilization of acetyl units from exogenous glucose for overall fatty acid synthesis was greater in adult lung than in fetal or neonatal lung, suggesting that other substrates may be important for fatty acid synthesis in developing lung. In fetal lung explants, de novo fatty acid synthesis was inhibited by exogenous palmitate. Taken together, these data suggest that de novo synthesis may be an important source of saturated fatty acids in fetal lung but of lesser importance in the neonatal period. Furthermore, the regulation of acetyl-CoA carboxylase activity and fatty acid synthesis in lung may be similar to non-pulmonary tissues.     

Fatty acid elongation activity in fibroblasts from patients with adrenoleukodystrophy (ALD)

The activities of microsomal fatty acid elongation and cytoplasmic de novo fatty acid synthesis were measured in human cultured skin fibroblasts. Both activities in fibroblasts from normal controls and patients with adrenoleukodystrophy (ALD) were compared and slight but a significant increase of elongation activities in ALD fibroblasts was demonstrated. On the other hand, there were no significant differences in the fatty acid synthetase activities. In this study, we measured microsomal fatty acid elongation activities in the presence of N-ethylmaleimide, which completely inhibited the activity of contaminating fatty acid synthetase and also the degradation of fatty acids, and made accurate determination of the elongation activities possible.     

Lipid compositional manipulation in Acholeplasma laidlawii B. Effect of exogenous fatty acids on fatty acid composition and cell growth when endogenous fatty acid production is inhibited

A variety of potential inhibitors of de novo fatty acid biosynthesis have been tested for activity in Acholeplasma laidlawii B. Two compounds, avidin and N,N-dimethyl-4-oxo-2trans-dodecenamide (CM-55), an antimicrobial fatty amide, strongly inhibit de novo biosynthesis without nonspecific toxic effects at moderate dosages. Avidin is the more potent inhibitor, abolishing de novo fatty acid synthesis and greatly reducing the chain elongation of exogenous fatty acids at level of 25 U/l. CM-55 gives complete inhibition of de novo biosynthesis only at low temperatures and inhibits exogenous fatty acid elongation to a variable extent. However, CM-55 is still a more potent antilipogenic agent in this organism than is the fungal antibiotic cerulenin. Cells cultured with avidin grow only when one or more exogenous medium- or long-chain fatty acids are added to the growth medium. The extent of cell growth under these conditions depends primarily on the physical properties of the exogenous fatty acid(s). In general, fatty acids giving diacylglycerolipids of very high or very low fluidity are unsuitable growth substrates, while those whose diacylglycerol derivatives are of intermediate fluidity support fair to good cell growth.     

Triacylglycerol synthesis in goat mammary gland. The effect of ATP, Mg2+ and glycerol 3-phosphate on the esterification of fatty acids synthesized de novo.

Goat mammary-gland microsomal fraction by itself induces synthesis of medium-chain-length fatty acids by goat mammary fatty acid synthetase and incorporates short- and medium-chain fatty acids into triacylglycerol. Addition of ATP in the absence or presence of Mg2+ totally inhibits triacylglycerol synthesis from short- and medium-chain fatty acids, and severely inhibits synthesis de novo of medium-chain fatty acids. The inhibition by ATP of fatty acid synthesis and triacylglycerol synthesis de novo can be relieved by glycerol 3-phosphate. The effect of ATP could not be mimicked by the non-hydrolysable ATP analogue, adenosine 5'-[beta,gamma-methylene]triphosphate and could not be shown to be caused by inhibition of the diacylglycerol acyltransferase by a phosphorylation reaction. Possible explanations for the mechanism of the inhibition by ATP are discussed, and a hypothetical model for its action is outlined.     

Mutants of Bacillus species isolated on the basis of protonophore resistance are deficient in fatty acid desaturase activity.

The fatty acid desaturase activity in cell extracts of Bacillus subtilis was characterized and found to be O2 dependent, NADH dependent, and cyanide sensitive. In cell fractionation studies, only 10% of the desaturase activity was recovered in the membrane fraction; the addition of cytosolic factors, which by themselves were devoid of activity, restored membrane activity to the level found in the unfractionated cell extracts. NADH was preferred over NADPH as an electron donor, and palmitoyl-coenzyme A was used preferentially over stearoyl-coenzyme A as the straight-chain fatty acid substrate. An increase in desaturase activity was observed when either the growth or the assay temperature was lowered from 37 to 20 degrees C, although the assay temperature appeared to be the more important parameter. Three protonophore-resistant mutants of B. subtilis and a comparable mutant of Bacillus megaterium had been found to possess reduced levels of unsaturated fatty acids in their membrane phospholipids; their protonophore resistance was abolished when grown in the presence of an unsaturated fatty acid supplement. All of these strains were found to be either significantly deficient in or totally lacking desaturase activity in comparison with their wild-type parent strains. Full, protonophore-sensitive revertants of the mutants had levels of desaturase activity comparable to those of the wild-type. Temperature-sensitive revertants of two of the mutants, which grew at 32 degrees C but not at 26 degrees C in the presence of protonophore, exhibited desaturase activity comparable to that of the wild-type at 26 degrees C but lacked activity at 32 degrees C. These results indicate that the biochemical basis for protonophore resistance in these Bacillus mutants is a fatty acid desaturase deficiency.     

De novo fatty acid synthesis by freshly isolated alveolar type II epithelial cells

Fatty acid synthesis was studied in freshly isolated type II pneumocytes from rabbits by 3H2O and (U-14C)-labeled glucose, lactate and pyruvate incorporation and the activity of acetyl-CoA carboxylase. The rate of lactate incorporation into fatty acids was 3-fold greater than glucose incorporation; lactate incorporation into the glycerol portion of lipids was very low but glucose incorporation into this fraction was approximately equal to incorporation into fatty acids. The highest rate of de novo fatty acid synthesis (3H2O incorporation) required both glucose and lactate. Under these circumstances lactate provided 81.5% of the acetyl units while glucose provided 5.6%. Incubations with glucose plus pyruvate had a significantly lower rate of fatty acid synthesis than glucose plus lactate. The availability of exogenous palmitate decreased de novo fatty acid synthesis by 80% in the isolated cells. In a cell-free supernatant, acetyl-CoA carboxylase activity was almost completely inhibited by palmitoyl-CoA; citrate blunted this inhibition. These data indicate that the type II pneumocyte is capable of a high rate of de novo fatty acid synthesis and that lactate is a preferred source of acetyl units. The type II pneumocyte can rapidly decrease the rate of fatty acid synthesis, probably by allosteric inhibition of acetyl-CoA carboxylase, if exogenous fatty acids are available.     

Mammalian fatty acid synthetase. III. Characterization of human liver synthetase products and kinetics of methylmalonyl-CoA inhibition.

When propionyl-CoA was substituted for either acetyl-CoA or butyryl-CoA in the presence of [14C]malonyl-CoA and NADPH, the pure human liver fatty acids synthetase complex synthesized only straight-chain, saturated, 15- and 17-carbon radioactive fatty acids. At optimal concentrations, propionyl-CoA was a better primer of fatty acid synthesis than acetyl-CoA. Methylmalonyl-CoA inhibited the synthetase competitively with respect to malonyl-CoA. The Ki was calculated to be 8.4 muM. These findings provide an in vitro model and offer a direct explanation at the molecular level for some of the abnormal manifestations observed in diseases characterized by increased cellular concentrations of propionyl-CoA and methylmalonyl-CoA.