Increased unsaturated fatty acid production associated with a suppressor of the fabA6(Ts) mutation in Escherichia coli

Abstract

Plasmids that corrected the temperature-sensitive unsaturated fatty acid auxotrophy of strain M6 [fabA6 (Ts)] were isolated from an Escherichia coli genomic library. Subcloning and physical mapping localized the new gene (called sfa for suppressor of fabA) at 1,070 kb on the E. coli chromosome. DNA sequencing revealed the presence of a 227-bp open reading frame which directed the synthesis of a peptide of approximately 8 kDa, which correlated with the correction of the fabA6(Ts) phenotype. However, the sfa gene was an allele-specific suppressor since plasmids harboring the sfa gene corrected the growth phenotype of fabA6(Ts) mutants but did not correct the growth of fabA2(Ts) or fabB15(Ts) unsaturated fatty acid auxotrophs. Overexpression of the sfa gene in fabA6(Ts) mutants restored unsaturated fatty acid content at 42؇C, and overexpression in wild-type cells resulted in a substantial increase in the unsaturated fatty acid content of the membrane. Thus, the suppression of the fabA6(Ts) mutation by sfa was attributed to its ability to increase the biosynthesis of unsaturated fatty acids.Unsaturated fatty acids are absolutely required for the normal growth of Escherichia coli (9), and the investigation of the enzymes responsible for the formation of these fatty acids and their regulation remains an area of active research (21). In E. coli, there are two gene products known to be required for unsaturated fatty acid synthesis. The product of the fabA gene, ␤-hydroxydecanoyl-acyl carrier protein (ACP) dehydratase, catalyzes the dehydration of ␤-hydroxydecanoyl-ACP to a mixture of trans-2-decenoyl-ACP and cis-3-decenoyl-ACP, thus introducing the cis double bond into the growing fatty acid chain. The trans-2 isomer is the normal intermediate in saturated fatty acid synthesis and is converted to saturated fatty acids following reduction of the double bond by enoyl-ACP reductase (fabI) (14). The double bond in the cis-3 intermediate is preserved, and the 10-carbon intermediate is elongated to form the unsaturated fatty acids. The essential nature of FabA is clear from the isolation of mutants defective in this enzyme activity (26). These mutants cannot make unsaturated fatty acids, although saturated fatty acid synthesis is not affected. The nucleotide sequence of the fabA gene is known (10), and the regulation of fabA expression involves the transcriptional activator, FadR, which also functions as a repressor of fatty acid ␤-oxidation genes (15, 16). The first indication of a second enzyme required for unsaturated fatty acid biosynthesis was that the unsaturated fatty acid auxotrophs could be divided into two complementation groups (8). The fabA gene maps to minute 21.9 on the E. coli chromosome (11), whereas the second mutation, termed fabB, maps to minute 52.6 (5). The second essential enzyme is ␤-ketoacyl-ACP synthase I, the product of the fabB gene (24). This condensing enzyme carries out an essential step in unsaturated fatty acid synthesis, which is most likely the elongation of cis-3-decenoyl-ACP. The fabB gene ...

Overexpression of gnsA , a Multicopy Suppressor of the secG Null Mutation, Increases Acidic Phospholipid Contents by Inhibiting Phosphatidylethanolamine Synthesis at Low Temperatures

“…Although the increases in the unsaturated fatty acid contents were independent of temperature (17,21), acidic phospholipid contents were increased by GnsA overproduction only at low temperatures. The increases in acidic phospholipid contents most likely resulted from inhibition of PE synthesis and not stimulation of PG and CL synthesis.…”

“…Disruption of both gnsA and gnsB affected neither the phospholipid composition nor growth (data not shown). The levels of GnsA and GnsB are under the detection limits in wild-type cells (17,21). More analyses are therefore necessary to reveal the physiological function of GnsA.…”

“…The gnsA gene is one of these suppressors and encodes a hydrophilic protein consisting of 57 amino acids (21). Overproduction of GnsA also corrects the defect in a temperature-sensitive unsaturated fatty acid auxotroph, the fabA6 mutant (17,21), through an unknown mechanism.…”

Overexpression of yccL ( gnsA ) and ydfY ( gnsB ) Increases Levels of Unsaturated Fatty Acids and Suppresses both the Temperature-Sensitive fabA6 Mutation and Cold-Sensitive secG Null Mutation of Escherichia coli

“…The sfa gene has been reported to be a multicopy suppressor for the fabA6 mutation (32). Overexpression of sfa was shown to cause the overproduction of unsaturated fatty acids in the wild-type strain (32).…”

“…E. coli possesses only three major fatty acids, palmitic acid (16:0), palmitoleic acid (16:1), and cisvaccenic acid (18:1). The fabA6 mutant is a temperature-sensitive unsaturated fatty acid auxotroph since the mutated enzyme is unstable at high temperature (32). The sfa gene has been reported to be a multicopy suppressor for the fabA6 mutation (32).…”