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

Abstract

A multicopy suppressor of the cold-sensitive secG null mutation was isolated. The suppressor contained sfa and yccL, the former of which has been reported to be a multicopy suppressor of the fabA6 mutation carried by a temperature-sensitive unsaturated fatty acid auxotroph. Subcloning of the suppressor gene revealed that yccL, renamed gnsA (secG null mutant suppressor), was responsible for the suppression of both the secG null mutation and the fabA6 mutation. In contrast, the sfa gene did not suppress the fabA6 mutation. The ydfY (gnsB) gene, encoding a protein which is highly similar to GnsA, also suppressed both the secG null mutation and the fabA6 mutation. Although both gnsA and gnsB are linked to cold shock genes, the levels of GnsA and GnsB did not exhibit a cold shock response. A gnsA-gnsB double null mutant grew normally under all conditions examined; thus, the in vivo functions of gnsA and gnsB remain unresolved. However, overexpression of gnsA and gnsB stimulated proOmpA translocation of the secG null mutant at low temperature and caused a significant increase in the unsaturated fatty acid content of phospholipids. Taken together, these results suggest that an increase in membrane fluidity due to the increase in unsaturated fatty acids compensates for the absence of the SecG function, especially at low temperature.

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.…”

“…The increases in acidic phospholipid contents most likely resulted from inhibition of PE synthesis and not stimulation of PG and CL synthesis. However, it is not clear at present why the overproduction of GnsA, a small cytosolic protein possessing a helix-turn-helix motif (21), has such drastic and pleiotropic effects on the compositions of both fatty acids and phospholipids.…”

“…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.…”

“…The secG null mutation significantly retards protein translocation (4,22). All multicopy suppressors of the secG null mutation have been found to be involved in lipid synthesis (7,19,(21)(22)(23). The gnsA gene is one of these suppressors and encodes a hydrophilic protein consisting of 57 amino acids (21).…”

“…Cells were cultivated in the presence of [ 32 P]orthophosphoric acid (0.37 MBq/ml; NEN Life Science Products) at 37°C to an OD 660 of 0.8 or at 20°C for 24 h. The OD of the culture reached ϳ1.0 after 24 h of incubation at 20°C. Phospholipids were extracted and analyzed by thin-layer chromatography (Silicagel 60; Merck) as described previously (21). The level of incorporation of radioactivity into phospholipids was determined at least twice throughout this study, and experimental error was within 5%.…”