Essential fatty acid biosynthetic enzymes of Escherichia coli and Lactococcus lactis subsp. lactis

β-Ketoacyl-acyl carrier protein (ACP) synthase III (KAS III) encoded by the fabH gene is thought to catalyze the first elongation reaction of type II fatty acid synthesis in bacteria and plant plastids. I report the first bacteria strain that lacking KAS III, a fabH mutant constructed in the gram-positive bacterium Lactococcus lactis subsp. lactis IL1403. Upon supplementation of saturated and unsaturated long-chain fatty acids, a L. lactis Δ fabH strain could be obtained through gene replacement. This fabH mutant strain fails to grow without supplementation with exogenous long-chain fatty acids demonstrating that KASIII plays an essential role in cellular metabolism. The fabH deletion mutant of L. lactis requires only long-chain unsaturated fatty acids for growth, indicating existence of another pathway for saturated fatty acid synthesis. Indeed, incorporation of [1-14C]acetate into fatty acids in vivo showed that the fabH mutant retained about 10% of the fatty acid synthetic ability of the wild-type strain and that this residual synthetic capacity was preferentially diverted to the saturated branch of the pathway. Moreover, mass spectrometry showed that the fabH mutant retained low levels of palmitic acid upon fatty acid starvation. I also confirm the essentiality of FabH in Escherichia coli using a plasmid-based gene insertion/deletion system. Together these results provided the first genetic evidence demonstrating that FabH conducts the major condensation reaction in the initiation of type II fatty acid biosynthesis in both gram-positive and gram-negative bacteria.; FabG, β-ketoacyl-ACP reductase, performs the NADPH-dependent reduction of β-ketoacyl-ACP substrates to the β-hydroxyacyl-ACP products. I report the first characterized fabG mutants. By chemical mutagenesis followed by a tritium suicide procedure, I obtained three conditionally-lethal temperature-sensitive fabG (fabGts) mutants. The E. coli mutant has two point mutations: A154T and E233K. In Salmonella enterica Serovar Typhimurium fabGts mutants one strain had a point mutation, S224F whereas the second strain contained two mutations (M125I and A223T). All of the altered residues of the FabG mutant proteins are located on or near the two-fold axes of symmetry at the dimer interfaces in this homotetrameric protein suggesting that the quanternary structures of the mutant FabG proteins may be disrupted at the nonpermissive temperature.