Biosynthesis of branched-chain fatty acids in Bacillus subtilis. A decarboxylase is essential for branched-chain fatty acid synthetase

Branched long-chain fatty acids of the iso and anteiso series are synthesized in many bacteria from the branched-chain alpha-keto acids of valine, leucine, and isoleucine after their decarboxylation followed by chain elongation. Two distinct branched-chain alpha-keto acid (BCKA) and pyruvate decarboxylases, which are considered to be responsible for primer synthesis, were detected in, and purified in homogenous form from Bacillus subtilis 168 strain by procedures including ammonium sulfate fractionation and chromatography on ion exchange, reversed-phase, and gel absorption columns. The chemical and catalytic properties of the two decarboxylases were studied in detail. The removal of BCKA decarboxylase, using chromatographic fractionation, from the fatty acid synthetase significantly reduced its activity. The synthetase activity was completely lost upon immunoprecipitation of the decarboxylase. The removal of pyruvate decarboxylase by the above two methods, however, did not affect any activity of the fatty acid synthetase. Thus, BCKA decarboxylase, but not pyruvate decarboxylase, is essential for the synthesis of branched-chain fatty acids. The very high affinity of BCKA decarboxylase toward branched-chain alpha-keto acids is responsible for its function in fatty acid synthesis.     

Uptake of branched-chain alpha-keto acids in Bacillus subtilis.

Bacillus subtilis has a constitutive system for the uptake of alpha-keto-beta-methylvalerate, alpha-ketoisovalerate, and (probably) alpha-ketoisocaproate. A mutation, kauA1, which blocks the uptake of alpha-keto-beta-methylvalerate and alpha-ketoisovalerate, is located between metB and citK on the B. subtilis chromosome.     

The effects of temperature and growth rate on the proportion of unsaturated fatty acids in bacterial lipids

The effects of temperature and growth rate on the fatty acid composition of the extractable lipids of four mesophilic and three psychotrophic bacteria were examined. Two of the mesophiles (Escherichia coli and Pseudomonas aeruginosa) increased the proportion of unsaturated fatty acids in their lipids with decreasing temperature over their whole growth temperature range. The other mesophiles (Enterobacter aerogenes and Lactobacillus casei) increased the proportion of unsaturated fatty acids with decreasing temperature only over the lower half of their growth temperature ranges. The psychrotrophs Pseudomonas fluorescens and Enterobacter sp. had a constant proportion of unsaturated acids over the lower half of their growth temperature range, while the psychotrophic Lactobacillus sp. showed no consistent change in its unsaturated fatty acid composition with temperature. All species showed some variation of unsaturated fatty acid composition with growth rate at the highest and lowest growth temperatures, although such variations were small in some species (Ent. aerogenes and Lactobacillus sp.).     

Incorporation of specific exogenous fatty acids into membrane lipids modulates protonophore resistance in Bacillus subtilis.

Attempts to manipulate the level of C16:1 fatty acids in membrane phospholipids were made by using Bacillus subtilis and its protonophore-resistant mutants to test the hypothesis that C16:1 fatty acid levels relate to the bioenergetic properties of the mutant strains. Growth of the three mutants in the presence of palmitoleic acid restored the level of C16:1 fatty acids in the membrane lipids to somewhat above those found in the wild type. The palmitoleic acid was preferentially incorporated into diphosphatidylglycerol (cardiolipin) and phosphatidylethanolamine and was associated with increased levels of these phospholipids. These membrane preparations showed no increase in the levels of free fatty acids. The increase in C16:1 fatty acids achieved by growth in the presence of palmitoleic acid was accompanied by secondary changes in membrane lipids as well as a pronounced diminution in the protonophore resistance of growth and ATP synthesis. Other membrane-associated properties that had been observed in these mutants, e.g., elevated ATPase levels, were not altered coordinately with protonophore resistance and C16:1 fatty acid levels. Growth of the wild type in the presence of palmitic acid caused a modest elevation of the C16:0 of the membrane lipids and a modest increase in the protonophore resistance of growth and ATP synthesis. Growth of the wild type at elevated temperatures, in the absence of fatty acid supplementation, also enhanced its resistance to protonophores. The results support the hypothesis that specific changes in membrane lipid composition underlie the bioenergetic changes associated with protonophore resistance.     

Production of branched-chain volatile fatty acids by certain anaerobic bacteria.

Net production of isobutyric acid, isovaleric acid, and 2-methylbutyric acid by cultures of Bacteroides ruminicola and Megasphaera elsdenii on media that contained Trypticase or casein hydrolysate continued (up to 5 days) after growth had ceased. Only trace quantities of these acids were produced in a medium that contained a mixture of amino acids that did not include the branched-chain amino acids. M. elsdenii produced increased quantities of the branched-chain fatty acids in a medium that contained Trypticase when glucose was reduced or eliminated from the culture medium. However, B. ruminicola produced increased quantities of branched-chain fatty acids and of phenylacetic acid from Trypticase when glucose was supplied at 3 mg/ml rather than at 1 mg/ml. Single strains of Streptococcus bovis, Selenomonas ruminantium, Bacteroides amylophilus, and Butyrivibrio fibrisolvens did not produce branched-chain fatty acids.     

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.