Figure 1.
P. aeruginosa fatty acid degradation pathway (FA degradation).
(A) P. aeruginosa FA degradation model was based on the E. coli β-oxidation pathway. Known P. aeruginosa FA degradation enzyme homologues are indicated by numbers: FadD1 (PA3299), FadD2 (PA3300), FadD3 (PA3860), FadD4 (PA1617), FadD5 (PA2893), FadD6 (PA3924), FadAB1 (PA1736–PA1737), and FadBA5 (PA3013–PA3014). 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, outer membrane; IN, inner membrane. (B) Alignment of FadD homologues motifs with E. coli FadD motifs. Amino acids with similar properties are assigned the same colors using CLC Sequence Viewer 6 software (www.clcbio.com).
Table 1.
Single copy complementation of the E.coli fadD mutant with P. aeruginosa fadD homologues.
Table 2.
Strains utilized in this study.
Table 3.
Growth of various P. aeruginisa fadD mutants on FAs after 24 h.
Table 4.
Growth of various P. aeruginosa fadD mutants on FAs after 96 h.
Figure 2.
fadD mutants and growth on FAs.
Various strains were grown on glucose (A), C6∶0 (B), C10∶0 (C), C14∶0 (D), and C18∶1Δ9 (E) to investigate further the role of fadD4 in FA degradation in comparison to rest of homologues. These growth curves demonstrate the hierarchical dominance of fadD1, fadD2 and fadD4 over other fadDs. Growth experiments were performed twice and representative curves are shown.
Figure 3.
Growth phenotypes of various fadD homologues mutants on acyclic terpenes.
Strains were grown in liquid 1x M9 medium +1% (w/v) Brij-58 supplemented with 20 mM glucose, 0.1% (w/v) of citronellic acid, or 0.1% (w/v) geranic acid at 30°C. Optical densities (ODs) of cultures were measured and compared to PAO1 at day one (A, C, and E). Growth of ΔfadD4 mutant and ΔfadD4/attB::fadD4 complement strain in different carbon source were compared to PAO1 and ODs from day six are presented (B, D, and F). Results shown are from representative experiments that were performed twice by measuring triplicate cultures.
Figure 4.
Growth characteristics on PC and competition studies of fadD sextuple mutant.
(A) PAO1 and several mutant strains were individually grown on PC. Growth curves were performed twice and representative results are shown. (B) In vitro competition between ΔfadD1D2D3D4D5D6 and its competitor, ΔfadD1D2D3D4D5D6/complement (P1021), in different growth media after 24 h. (C) In vivo competition between ΔfadD1D2D3D4D5D6/mucA− (P973) and its competitor, ΔfadD1D2D3D4D5D6/complement/mucA− (P1028), in BALB/c mice lungs. Seven mice for each time point were inoculated with 6 x106 CFU/mouse. The geometric mean of competitive indices (CI) from each group is marked by red line. Mutant strain is less competitive than complement when CI<1. Total average lung CFU recovered form mice in each group are indicated above red line. * P<0.05 based on one sample t test.
Table 5.
Plasmids used in this study.
Table 6.
Oligonucleotides primers utilized in this study.