Relationship Between The Activation Of Exogenous Fatty Acids And The Initiation Of Fatty Acid Synthesis In Pseudomonas Aeruginosa

Pseudomonas aeruginosa is a human opportunistic pathogentic bacteria that can cause hospital-acquired infections.The prevention and treatment of P.aeruginosa is a hot topic in medical research.Fatty acids are important component of cell membranes and an essential substance for bacterial growth.In bacterial,each step of the fatty acid synthesis has a specific enzyme to catalysis,of which the 3-ketoacyl-ACP synthase Ⅲ is a key enzyme in the initial reaction of fatty acid synthesis.There are two 3-ketoacyl-ACP synthetases Ⅲ :PA3286 and PA5174 in P.aeruginosa.Studies have shown that PA5174 is responsible for the de novo synthesis of fatty acids,its substrate are short-chain acyl-CoAs.PA3286 starts the synthesis of long-chain fatty acids with medium-chain acyl-CoAs,it is a special 3-ketoacyl-ACP synthetase.Acyl-CoA synthetase is a key enzyme for the activation of exogenous fatty acids,which can convert fatty acids to acyl-CoA for β-oxidation and other metabolism.The P.aeruginosa genome contains six genes encoding acyl-CoA synthetase.Early studies showed that FadD1 and FadD2 are key enzymes for the activation of exogenous fatty acids,but the relationship between these two proteins and the initiation of fatty acid synthesis is not clear.In this study,the complementary analysis of fadD1 and fadD2 from P.aeruginosa on the Escherichia coli fadD mutant strain JW1794-1 showed that the proteins encoded by fadD1 and fadD2 both have acyl-CoA synthetase activity.Then we obtain the fadD1 and fadD2 knockout mutants by homologous recombination.The plate assay showed that the ability of ΔfadD1 to utilize long-chain fatty acids was significantly reduced,while ΔfadD2 was basically consistent with the wild-type PAO1,indicating that FadD1 was the main enzyme that activates exogenous fatty acids in P.aeruginosa,while FadD2 only plays a supporting role.Then mutation studies on PA5174 and PA3286 showed that the growth of PA5174 knockout mutant lags behind of wild type in LB,but after the addition of medium chain fatty acids,the growth of the mutants can be restored;whereas the mutant of PA3286 has no special requirement for the use of exogenous fatty acids.These results are also consistent with previous studies.Double mutant strains of acyl-CoA synthetase(FadD1 and FadD2)and 3-ketoacyl ACP synthetase Ⅲ(PA5174 and PA3286)were further constructed.It was found that the fadD1 and fadD2 mutations had no effect on the growth of the PA3286 mutant strain,and fadD2 mutation had no effect on the growth of the PA5174 mutant strain,but the PA5174 mutant strain could not restore normal growth with the mutation of fadD1 when added exogenously caprylic acid or lauric acid.This indicates that the exogenous fatty acids require the activation of FadD1 to be used to initial synthesize fatty acids.