Growth of ∆ strains is restored by increased saturated fatty acid synthesis.

The acyl-acyl carrier protein (ACP) phosphate acyltransferase PlsX plays an important role in phospholipid synthesis and exogenous fatty acid incorporation. Loss of almost completely blocks growth by decreasing phospholipid synthesis, which leads to abnormally long-chain acyl chains in the cell membrane phospholipids. The ∆ strain failed to grow without supplementation with an appropriate exogenous fatty acid. Introduction of a ∆ mutation into the ∆ strain to increase fatty acid synthesis allowed very weak growth. The ∆ strain accumulated suppressor mutants. One of these encoded a truncated β-ketoacyl-ACP synthase II (FabO) which restored normal growth and restored phospholipid acyl chain synthesis by increasing saturated acyl-ACP synthesis. Saturated acyl-ACPs are cleaved by a thioesterase to provide free fatty acids for conversion to acyl-phosphates by the FakAB system. The acyl-phosphates are incorporated into position 1 of the phospholipids by PlsY. We report the gene encodes a thioesterase that can provide free fatty acids. However, we were unable to delete the chromosomal gene to confirm that it is the responsible enzyme. TesE readily cleaves unsaturated acyl-ACPs, whereas saturated acyl-ACPs are cleaved much more slowly. Overexpression of an enoyl-ACP reductase either FabK or FabI which results in high levels of saturated fatty acid synthesis also restored the growth of the ∆ strain. The ∆ strain grew faster in the presence of palmitic acid than in the presence of oleic acid with improvement in phospholipid acyl chain synthesis. Positional analysis of the acyl chain distribution in the phospholipids showed that saturated acyl chains dominate the 1-position indicating a preference for saturated fatty acids at this position. High-level production of saturated acyl-ACPs is required to offset the marked preference of the TesE thioesterase for unsaturated acyl-ACPs and allow the initiation of phospholipid synthesis.