Figure 1.
Phosphatidylcholine (PC) degradation pathways in Pseudomonas aeruginosa.
(A) PC is the main component of lung surfactant and can be cleaved by phospholipase C and lipases, producing free fatty acids, glycerol, and phosphorylcholine. Three different pathways then further metabolize each component: the bet pathway for choline head group metabolism, the glp pathway for glycerol metabolism, and the β-oxidation pathway for the degradation of the FAs. (B) The proposed P. aeruginosa FA β-oxidation pathway. Abbreviations: FadA, 3-ketoacyl-CoA thiolase; FadB, cis-Δ3-trans-Δ2-enoyl-CoA isomerase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA epimerase, and 3-hydroxyacyl-CoA dehydrogenase; FadD, fatty acyl-CoA synthetase; FadE, acyl-CoA dehydrogenase; FadL, outer membrane long-chain fatty acid translocase; OM, out membrane; IN, inner membrane.
Figure 2.
Growth analysis of different fadBA mutant combinations on medium (C12∶0) and long chain-length fatty acid (C14∶0, C16∶0 and C18∶1Δ9).
Along with the wildtype PAO1 strain, mutants were grown in 1×M9 minimal medium supplemented with 0.4% different test FAs (A to D) or 1% control casamino acids (CAA, E) as sole carbon sources. Although fadBA mutants showed various defects when grown with FAs of different chain-lengths, no growth defects were observed for any of the mutants when grown with CAA as a control.
Table 1.
Bacterial strains used in this studya.
Figure 3.
Growth analysis on phosphatidylcholine.
(A) Some mutants exhibited growth defects on PC as a sole carbon source. The growth defects were fully recovered in complemented strains, as they had identical growth rates compared to the wildtype PAO1 strain. (B) No growth defects in control LB medium were observed.
Figure 4.
Competition studies of pathway mutants.
(A) In vitro competition studies of the various mutants and their complemented strains in different growth media (n equals the number of independent in vitro competition experiments performed with each carbon source). (B) In vivo lung competition of the various mutants and their complemented strains after 24 h, where n equals the number of mice in each group that were inoculated with a total of 6×106 CFU/mouse. The solid red line indicates the geometric mean of the competitive indices (CI) in each competition group. CI<1 indicates the mutant was less competitive than its complemented strain in various growth media (A) or within the lungs (B). Numbers above the red line represent the average total recovered CFU/mouse for each competition group.
Table 2.
Plasmids used in this studya.
Table 3.
Primers used in this study.