Pseudomonas aeruginosa directly shunts β-oxidation degradation intermediates into de novo fatty acid biosynthesis

Abstract

We identified the fatty acid synthesis (FAS) initiation enzyme in Pseudomonas aeruginosa as FabY, a β-ketoacyl synthase KASI/II domain-containing enzyme that condenses acetyl coenzyme A (acetyl-CoA) with malonyl-acyl carrier protein (ACP) to make the FAS primer β-acetoacetyl-ACP in the accompanying article (Y. Yuan, M. Sachdeva, J. A. Leeds, and T. C. Meredith, J. Bacteriol. 194:5171-5184, 2012). Herein, we show that growth defects stemming from deletion of fabY can be suppressed by supplementation of the growth media with exogenous decanoate fatty acid, suggesting a compensatory mechanism. Fatty acids eight carbons or longer rescue growth by generating acyl coenzyme A (acyl-CoA) thioester β-oxidation degradation intermediates that are shunted into FAS downstream of FabY. Using a set of perdeuterated fatty acid feeding experiments, we show that the open reading frame PA3286 in P. aeruginosa PAO1 intercepts C(8)-CoA by condensation with malonyl-ACP to make the FAS intermediate β-keto decanoyl-ACP. This key intermediate can then be extended to supply all of the cellular fatty acid needs, including both unsaturated and saturated fatty acids, along with the 3-hydroxyl fatty acid acyl groups of lipopolysaccharide. Heterologous PA3286 expression in Escherichia coli likewise established the fatty acid shunt, and characterization of recombinant β-keto acyl synthase enzyme activity confirmed in vitro substrate specificity for medium-chain-length acyl CoA thioester acceptors. The potential for the PA3286 shunt in P. aeruginosa to curtail the efficacy of inhibitors targeting FabY, an enzyme required for FAS initiation in the absence of exogenous fatty acids, is discussed.

Fig 1

Fatty acid rescue of the P. aeruginosa ΔfabY mutant. (A) Growth curves in LB medium alone (P. aeruginosa PAO1 [●] and ΔfabY mutant [▲]) or in LB medium supplemented with decanoate (1 μg/ml [○], 10 μg/ml [■], or 100 μg/ml [△]) for the ΔfabY mutant. Growth was measured at 37°C by recording the optical density at 600 nm. (B) The ΔfabY strain was streaked onto LB agar supplemented with the indicated fatty acid (C₂ to C₁₂) at 100 μg/ml. The plates were incubated at 37°C for 16 h before imaging.

Fig 2

Fatty acid composition analysis of P. aeruginosa. (A) Fatty acid methyl esters (FAMEs) prepared from strains grown overnight on LB agar supplemented with either 100 μg/ml of decanoate (C_10:0) or perdeuterated decanoate (d₁₉-C_10:0) were analyzed by gas chromatography with flame ionization detection. FAME peaks that contain deuterated fatty acids analyzed by mass spectrometry in Fig. 3 are indicated with an asterisk. Wt, wild type.

Fig 3

Mass spectra of deuterated FAMEs. Unique FAME peaks appearing after supplementation with perdeuterated decanoate (indicated by asterisks in Fig. 2) were analyzed by mass spectrometry. The parent molecular ions ([M]⁺) and select fragment ions are indicated, including for the McLafferty ion (m/z = 74). The corresponding unlabeled FAME mass spectra are included for comparison in the supplemental information (see Fig. S1 in the supplemental material).

Fig 4

Synthetic lethal analysis for fabY with KASIII domain fabH orthologs and fatty acid rescue. (A) Vectors conferring aacC1-mediated gentamicin (Gm) resistance that targeted the P. aeruginosa fabY gene were designed to either exchange fabH of E. coli (pTMT123 fabH⁺) or to delete fabY (pTMT124). Merodiploid intermediates (after step 1) were confirmed by PCR analysis and passively resolved by outgrowth in LB medium (step 2). Aliquots were plated on LB-sucrose agar to counterselect unresolved clones and to enumerate the sum of A- and B-type recombination events. In parallel, aliquots were plated on LB-sucrose agar plus Gm and on LB-sucrose agar plus Gm with 100 μg/ml of the fatty acid supplement decanoate (FA) to select only the recombinants arising through A-type recombination. (B) The pTMT123/124 vectors were individually introduced into the wild-type (Wt) P. aeruginosa, TMT16 (ΔPA0998 ΔPA0999 ΔPA3333 ΔPA3286), ΔPA3286, and TMT44 (ΔPA3286 attB::PA3286⁺). CFU resulting from resolving either pTMT123 (black bars) or pTMT124 (hatched bars) were determined by counting colonies that appeared after 24 h or 48 h (#) of incubation at 37°C. The selection media and strain background are indicated below the x axis. Data are representative of 3 separate experiments. The results for the ΔPA0998, ΔPA0999, and ΔPA3333 mutant strains are shown in Fig. S2 in the supplemental material. (C) Gas chromatograms with FID detection of FAME extracts obtained from P. aeruginosa strains grown on LB agar with either 100 μg/ml of decanoate (C_10:0) or perdeuterated decanoate (d₁₉-C_10:0). Asterisks indicate FAME peaks unique to d₁₉-C_10:0-fed samples with structures assigned as in Fig. 3.

Fig 5

Fatty acid composition analysis of P. aeruginosa strains fed long-chain perdeuterated fatty acids. (A) Gas chromatograms with FID detection of FAME extracts obtained from stationary-phase cultures of P. aeruginosa grown in liquid LB-BSA medium supplemented with either 100 μg/ml of perdeuterated tetradecanoate (d₂₇-C_14:0) or hexadecanoate (d₃₁-C_16:0). Asterisks indicate deuterated FAMEs of identical structure to those already assigned in Fig. 3 based on time of elution and mass spectra analysis. FAME peaks unique to d₃₁-C_16:0-fed cultures are indicated (○). (B) Mass spectra analysis of the d₃₁-C_16:0-specific FAME with the parent molecular ion ([M]⁺) and diagnostic fragment ions labeled, including for the uniformly deuterium-labeled McLafferty ion (m/z = 77).

Fig 6

Complementation of E. coli fabH by the P. aeruginosa PA3286 gene. (A) The E. coli strain TMT47 [fabH::camR(pET-PA3286)] was grown at 37°C in LB medium alone (▲) or with 1 mM IPTG (□) and compared to the parent Wt strain (●). Growth was monitored by measuring the optical density at 600 nm. (B) The gas chromatogram-FID trace for FAME samples prepared from E. coli strains grown on LB agar with perdeuterated decanoate (d₁₉-C_10:0; 100 μg/ml) and palmitate (C_16:0; 10 μg/ml) for induction of fatty acid degradation genes. The parent wild-type E. coli BW25113 and TMY32 [fabH::camR(pET-PA5174)] strains were included as controls. Asterisks indicate deuterated FAMEs of identical structures to those already assigned in Fig. 3 based on time of elution and mass spectra analysis. Mass spectra and assignment of deuterated FAME peaks unique to E. coli expressing PA3286 (C_14:0, C_14:0 3-OH, C_17:0 cyclo ω7c, and C_19:0 cyclo ω7c) along with the terminally labeled d₇-C16:0 (+) peak present only in the absence of PA3286 are shown in Fig. S3 in the supplemental material. Odd-numbered acyl chain FAMEs with reduced abundance in strain TMT47 are indicated (○).

Fig 7

Acyl-CoA substrate specificity of recombinant PA3286. Reaction products using malonyl-ACP (mACP) and saturated straight-chain acyl-CoAs (C₂ to C₁₆) as potential PA3286 substrates were separated with conformation-sensitive urea-PAGE. The gels were stained with Coomassie blue dye. The position of the malonyl-ACP-only control (malonyl-ACP) is shown to the right of the gel. + AGI, plus E. coli FabAGI coupling enzymes.

Fig 8

The proposed PA3286-mediated fatty acid β-oxidation to synthesis shunt of P. aeruginosa PAO1. After facilitated diffusion across the outer membrane through FadL (not shown), fatty acids are trapped in the cytoplasm by FadD1/FadD2-catalyzed vectorial esterification with CoA (32). Further metabolism depends on acyl-CoA ester chain length. Long-chain fatty acid CoA esters (C₁₆/C₁₈-CoA) can either be recycled en bloc and incorporated into de novo phospholipids by the glycerol-phosphate and acylglycerol-phosphate acyltransferases PlsB/PlsC (not shown) (60), or as with medium-chain acyl CoA esters (C₁₄ to C₁₀-CoA), be further degraded by the β-oxidation pathway (in blue). Once the preferred C₈-CoA substrate chain length is reached, the CoA thioester is intercepted by PA3286 and condensed with malonyl-ACP to form the key uncommitted fatty acid intermediate β-keto-decanoyl-ACP (green). The β-keto-decanoyl-ACP metabolite can be utilized by the anaerobic unsaturated fatty acid (UFA) pathway, the saturated fatty acid (SFA) pathway, and in lipopolysaccharide (LPS) biosynthesis (C_10:0/C_12:0 3-OH). The terminal 7 carbons in β-keto-decanoyl-ACP that remain labeled with deuterium when fed perdeuterated fatty acids are underlined. PA3286 does not significantly contribute to de novo FAS biosynthesis (in red) (56) but becomes essential in the P. aeruginosa ΔfabY background (Fig. 4B) due to a cellular requirement for basal FabH-type acetyl-CoA:malonyl-ACP condensation activity. For clarity, not all putative FAS and β-oxidation isozymes are shown. TCA, tricarboxylic acid cycle.