Methyl sterol and cyclopropane fatty acid composition of Methylococcus capsulatus grown at low oxygen tensions.

Methylococcus capsulatus contained extensive intracytoplasmic membranes when grown in fed-batch cultures over a wide range of oxygen tensions (0.1 to 10.6%, vol/vol) and at a constant methane level. Although the biomass decreased as oxygen levels were lowered, consistently high amounts of phospholipid and methyl sterol were synthesized. The greatest amounts of sterol and phospholipid were found in cells grown between 0.5 and 1.1% oxygen (7.2 and 203 mumol/g [dry weight], respectively). While sterol was still synthesized in significant amounts in cells grown at 0.1% oxygen, the major sterol product was the dimethyl form. Analysis by capillary gas chromatography-mass spectrophotometry showed that the phospholipid esterified fatty acids were predominantly 16:0 and 16:1 and that the hexadecenoates consisted of cis delta 9, delta 10, and delta 11 isomers. At low oxygen tensions, the presence of large amounts (25%) of cyclopropane fatty acids (cy 17:0) with the methylene groups at the delta 9, delta 10, and delta 11 positions was detected. Although the delta 9 monoenoic isomer was predominant, growth at low oxygen levels enhanced the synthesis of the delta 10 isomers of 16:1 and cy 17:0. As the oxygen level was increased, the amount of cyclopropanes decreased, such that only a trace of cy 17:0 could be detected in cells grown at 10.6% oxygen. Although M. capsulatus grew at very low oxygen tensions, this growth was accompanied by changes in the membrane lipids.

Characterization of Escherichia coli mutants completely defective in synthesis of cyclopropane fatty acids.

The synthesis of cyclopropane fatty acids (CFA) in bacteria represents a biochemically and physiologically unique membrane modification whose importance for the cell remains unknown, despite extensive study of a Cfa- mutant of Escherichia coli and of the cloned cfa gene. Recently we reported the isolation of new Cfa- mutants (D. W. Grogan and J. E. Cronan, Jr., Mol. Gen. Genet. 196:367-372, 1984). Molecular-genetic and biochemical analysis indicated that these were null mutants of the E. coli cfa locus which were formed by inversions of a chromosomal segment. Isogenic Cfa+ and Cfa- strains were constructed from one such mutant and subjected to various stress conditions. In nearly all cases, both strains responded equally, but certain treatments, such as repeated freezing and thawing, favored the survival of Cfa+ strains over Cfa- strains. Though not essential, CFA thus appeared to play some beneficial role (or roles) in the bacterial cell.

Penitricin, a new class of antibiotic produced by Penicillium aculeatum. IV. Biosynthesis.

Bioconversion experiments using washed mycelia of Penicillium aculeatum NR 5165 and NR 6216 revealed that penitricin (Ro 09-0804, hydroxymethylcyclopropenone) was biosynthesized from trans-2-butene-1,4-diol via 4-hydroxycrotonaldehyde.

Penitricin, a new class of antibiotic produced by Penicillium aculeatum. I. Taxonomy of the producer strain and fermentation.

A novel antibiotic, penitricin (Ro 09-0804) was discovered in the culture filtrate of a fungal strain NR 5165. Taxonomic studies of the producing organism resulted in its assignment to Penicillium aculeatum. Further examination on penitricin production by other strains of this species and related taxa revealed that penitricin was produced by several other strains of P. aculeatum, but not by any available strains of the closely similar species, P. verruculosum. It was also found that copper ion was essential for production of penitricin.

Cloning and manipulation of the Escherichia coli cyclopropane fatty acid synthase gene: physiological aspects of enzyme overproduction.

Like many other eubacteria, cultures of Escherichia coli accumulate cyclopropane fatty acids (CFAs) at a well-defined stage of growth, due to the action of the cytoplasmic enzyme CFA synthase. We report the isolation of the putative structural gene, cfa, for this enzyme on an E. coli-ColE1 chimeric plasmid by the use of an autoradiographic colony screening technique. When introduced into a variety of E. coli strains, this plasmid, pLC18-11, induced corresponding increases in CFA content and CFA synthase activity. Subsequent manipulation of the cfa locus, facilitated by the insertion of pLC18-11 into a bacteriophage lambda vector, allowed genetic and physiological studies of CFA synthase in E. coli. Overproduction of this enzyme via multicopy cfa plasmids caused abnormally high levels of CFA in membrane phospholipid but no discernable growth perturbation. Infection with phage lambda derivatives bearing cfa caused transient overproduction of the enzyme, although pL-mediated expression of cfa could not be demonstrated in plasmids derived from such phages. CFA synthase specific activities could be raised to very high levels by using cfa runaway-replication plasmids. A variety of physiological factors were found to modulate the levels of CFA synthase in normal and gene-amplified cultures. These studies argue against several possible mechanisms for the temporal regulation of CFA formation.

Studies on cyclopropane fatty acid synthesis. Correlation between the state of reduction of respiratory components and the accumulation of methylene hexadecanoic acid by Pseudomonas denitrificans.

A delay in the onset of accumulation of methylene hexadecanoic acid could be engendered in Pseudomonas denitrificans growing under limited oxygen conditions when the concentration of citrate but not the concentration of succinate in the medium was increased from 0.1 to 0.5%. Ascorbate, which specifically reduced a cytochrome component possessing a maximum absorbance at 551 nm, partially inhibited the accumulation of methylene hexadecanoic acid under conditions which otherwise led to maximal production. Limiting terminal cytochrome oxidase activity by controlling the oxygen supply, or by the use of low concentrations of the oxidase inhibitors cyanide or azide also prevented the accumulation of the fatty acid regardless of the nature or concentration of carbon source in the medium. Measurement of the levels of ATP, NAD and NADH as well as the steady state of reduction of respiratory components in vivo showed that the onset of accumulation of methylene hexadecanoic acid could be specifically correlated with the state of reduction of respiratory components. The uniqueness of succinate respiration in promoting the synthesis of cyclopropane synthetase (unsaturated-phospholipid methyltransferase (EC 2.1.1.16) under limited oxygen conditions could therefore be assigned to the high degree of oxidation of respiratory components observed under this condition.

Effect of biotin on fatty acids and phospholipids of biotin-sensitive strains of Rhizobium japonicum.

The effect of biotin on fatty acids and intact lipids was studied by comparing a biotin-requiring, a biotin-inhibited, and a biotin-indifferent strain of Rhizobium japonicum. These organisms were grown in a defined medium with added levels of 0, 0.3, and 0.5 mug of biotin per liter, and were analyzed for fatty acids and lipid components. Myristic, palmitic, and octadecenoic acids were found to be the major fatty acids in these strains. The indifferent strain also contained large amounts of C(19) cyclopropane acid and small amounts of a C(17) cyclopropane acid. Several unidentified acids were present in the other two strains. The percentages of fatty acids showed statistically significant changes corresponding with changes in level of biotin in the medium. When biotin concentration was increased in the medium, the C(18) monoenoic acids of the biotin-requiring strain increased significantly, and those of the biotin-inhibited and biotin-indifferent strains decreased significantly. Palmitic acid showed a statistically significant increase in the indifferent strain with increasing biotin concentration. The principal intact lipid components in these strains are phospholipids. The major phospholipids are phosphatidylserine, phosphatidylcholine, phosphatitidylethanolamine, and cardiolipin. These phospholipids were not affected by biotin level and were independent of medium composition.