Abstract
Multicopy plasmids bearing the structural gene (fabB) for beta-ketoacyl-acyl carrier protein (ACP) synthase I were constructed in vitro and transformed into various Escherichia coli strains. Introduction of these plasmids into fabB strains resulted in a fabB+ phenotype and a large (8- to 10-fold) overproduction of synthase I activity. Strains carrying these plasmids were also unusually resistant to cerulenin (an antibiotic that specifically inhibits beta-ketoacyl-ACP synthase activity) and overproduced cis-vaccenic acid. Strains (fabF-) lacking beta-ketoacyl-ACP synthase II are deficient in both cis-vaccenic acid synthesis and thermal regulation. Introduction of the fabB plasmids into these strains resulted in the restoration of cis-vaccenic acid synthesis. However, the plasmid-engendered cis-vaccenic acid synthesis of these strains was unaffected by temperature. These results demonstrate that synthase II, the product of the fabF gene, is the sole enzyme regulating the temperature-dependent composition of the membrane phospholipid acyl chains.
Highlights
Strains lacking P-ketoacyl-acyl carrier protein (ACP) synthase I1 our resultsindicated a key role for synthase I1 in are deficient in both cis-vaccenic acid synthesis and thermal regulation
This proposal could most clearly be tested in a fabF- strain in order thatregulation of fatty acid synthesis would not complicate the results.,since fabF- strains lackcis-vaccenicacid, the key fatty acidin thermal regulationa, physiologically relevantexperiment In Escherichia coli the chain elongation step of fatty acid could not be done
Molecular cloning of the structuralgenefor P-ketoacyl-ACP synthase I resulted in an appreciable overproduction of the enzyme in vivo (Table I)
Summary
Fluidity in Escherichia coli of E. coli changes as a function of growth temperature, the proportion of unsaturated fatty acidincreasingwith lower. Multicopy plasmids bearing the structural gene (fabB) for P-ketoacyl-acyl carrier protein (ACP) synthase I were constructed in vitro and transformed into various Escherichia coli strains. The plasmid-engendered cis-vaccenic acid synthesis of these strains was unaffected by temperature These results demonstrate that synthase 11, the product of the f a b F gene, is the sole enzyme regulating the temperature-dependent composition of the membrane phospholipid acyl chains. In unsaturated fatty acid constructed by cotransduction of fabFl with the closely linked TnlO synthesis, synthaseI is required for an early step (probably thelongation of cis-3-decenoyl-ACP) whereas synthase I1 is required for t h e last elongation, that of palmitoleoyl-ACP to the keto precursofr cis-vaccenoyl-ACP transposon of strain CY288 (14).
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