Mutants of Escherichia coli with Temperature-sensitive Malonyl Coenzyme A-Acyl Carrier Protein Transacylase

Mark E Harder,Ruth C Ladenson,Steven D Schimmel,David F Silbert

doi:10.1016/s0021-9258(19)81262-0

Abstract

Abstract We have characterized two mutants of Escherichia coli in which fatty acid biosynthesis is adversely affected by elevated temperature. Under conditions which do not alter the activity of wild type cell-free extracts, virtually all of the malonyl coenzyme A-acyl carrier protein (ACP) transacylase and fatty acid synthetase activities present in mutant extracts are thermolabile. Malonyl transacylase from wild type cells restores normal fatty acid synthetase activity to inactivated mutant extracts. Furthermore, fatty acid synthesis in mutant extracts is corrected by malonyl-ACP. Malonyl transacylase and fatty acid synthetase activities are also restored in revertants which were selected for their ability to grow at elevated temperatures. These experiments furnish evidence that the mutants are altered in the structural gene for malonyl transacylase and that this is their only genetic defect. When these strains, defective in the synthesis of a substrate vital for fatty acid elongation, are grown at elevated temperatures, their phospholipids become deficient in long chain acyl groups. Supplementation of these cultures with long chain fatty acids permits sustained growth and the maintenance of a normal fatty acid composition.

Highlights

Under conditions which do not alter the activity of wild type cell-free extracts, virtually all of the malonyl coenzyme A-acyl carrier protein (ACP) transacylase and fatty acid synthetase activities present in mutant extracts are thermolabile
Malonyl transacylase and fatty acid synthetase activities are restored in revertants which were selected for their ability to grow at elevated temperatures
LA2-89 and LA2-130 are temperature-sensitive mutants isolated by a method employing radiation suicide to enrich for strains defective in lipid biosynthesis

Summary

SUMMARY

We have characterized two mutants of Escherichia coli in which fatty acid biosynthesis is adversely affected by elevated temperature. Malonyl transacylase and fatty acid synthetase activities are restored in revertants which were selected for their ability to grow at elevated temperatures These experiments furnish evidence that the mutants are altered in the structural gene for malonyl transacylase and that this is their only genetic defect. When these strains, defective in the synthesis of a substrate vital for fatty acid elongation, are grown at elevated temperatures, their phospholipids become deficient in long chain acyl groups. Three classes of long chain fatty acids-saturated, unsaturated (or their cyclopropane derivatives), and P-hydroxy acids-are found in Escherichia coli cell envelopes They are the products of two branches from a common biosynthetic pathway that generates D( -)-P-hydroxydecanoyl acyl carrier protein (AC?), the branch point intermediate (I), from acetyl-ACP’ and malo-.

Recipient of Research Career Development

PROCEDURE

RESULTS

DISCUSSION

Methods