Fatty acid biosynthesis in Pseudomonas aeruginosa is initiated by the FabY class of β-ketoacyl acyl carrier protein synthases

Abstract

The prototypical type II fatty acid synthesis (FAS) pathway in bacteria utilizes two distinct classes of β-ketoacyl synthase (KAS) domains to assemble long-chain fatty acids, the KASIII domain for initiation and the KASI/II domain for elongation. The central role of FAS in bacterial viability and virulence has stimulated significant effort toward developing KAS inhibitors, particularly against the KASIII domain of the β-acetoacetyl-acyl carrier protein (ACP) synthase FabH. Herein, we show that the opportunistic pathogen Pseudomonas aeruginosa does not utilize a FabH ortholog but rather a new class of divergent KAS I/II enzymes to initiate the FAS pathway. When a P. aeruginosa cosmid library was used to rescue growth in a fabH downregulated strain of Escherichia coli, a single unannotated open reading frame, PA5174, complemented fabH depletion. While deletion of all four KASIII domain-encoding genes in the same P. aeruginosa strain resulted in a wild-type growth phenotype, deletion of PA5174 alone specifically attenuated growth due to a defect in de novo FAS. Siderophore secretion and quorum-sensing signaling, particularly in the rhl and Pseudomonas quinolone signal (PQS) systems, was significantly muted in the absence of PA5174. The defect could be repaired by intergeneric complementation with E. coli fabH. Characterization of recombinant PA5174 confirmed a preference for short-chain acyl coenzyme A (acyl-CoA) substrates, supporting the identification of PA5174 as the predominant enzyme catalyzing the condensation of acetyl coenzyme A with malonyl-ACP in P. aeruginosa. The identification of the functional role for PA5174 in FAS defines the new FabY class of β-ketoacyl synthase KASI/II domain condensation enzymes.

Fig 1

Cosmid complementation strategy for identification of FabH-type activity in P. aeruginosa PAO1. The E. coli strain TMY19 has a chloramphenicol cassette (camR) inserted within fabH (b1091) and needs l-Ara supplementation to induce expression of the P_BAD-regulated S. enterica fabH ortholog (inserted within the d-glucitol phosphotransferase operon) in order to sustain wild-type growth rates. The operon is transcriptionally silent under standard culture conditions in the absence of d-glucitol (52). A sheared library of wild-type P. aeruginosa PAO1 genomic DNA was ligated into the pWEB cosmid vector, packaged, and transduced by phage into E. coli TMY19. Transductants were directly selected on unsupplemented LB agar to identify cosmids (pTMYcos1 to pTMYcos17) that complement depletion of FabH.

Fig 2

Fatty acid β-ketoacyl acyl carrier protein (ACP) synthase III (KASIII) domain-containing proteins in P. aeruginosa PAO1. (A) The E. coli K-12 MG1655 FabH sequence (b1091) was used to query the P. aeruginosa PAO1 genome for similar protein sequences using the BLAST search tool. Four candidate P. aeruginosa FabH-encoding open reading frames at 3 distinct genomic loci all containing the KASIII conserved domain were identified: PA3333 (white) (FabH2, E-value = 8e−66), PA3286 (white) (E-value = 3e−25), PA0998 (white) (PqsC, E-value = 3e−17), and PA0999 (gray) (PqsD, E-value = 2e−68). (B) Alignment of putative active site regions with the Cys112-His244-Asn274 catalytic triad of E. coli FabH (FabH Ec) (indicated by asterisks). Identical amino acid residues are shaded with a black background, and partially conserved substitutions are shaded in gray. (C) Growth curves of P. aeruginosa strains with all KASIII candidate genes (wild type [▲]) or with KASIII candidate genes deleted singly (PA0999 [■], PA3286 [□], PA3333 [○], PA0998 [△]) or in combination (TMT16 [●]) in LB broth at 37°C.

Fig 3

Complementation of FabH depletion in E. coli K-12 by a P. aeruginosa cosmid library. (A) The plasmid-genomic DNA insert junction sites of cosmids that rescued growth of the FabH-depleted E. coli strain TMY19. All 14 sites were located in the immediate 5′ intergenic region of the unknown open reading frame PA5174 and contained 24 to 42 kb of downstream DNA. All inserts except for pTMYcos13 (asterisk) placed PA5174 proximal to the T7 promoter on the pWEB vector backbone. The number of base pairs intervening between the respective cosmid-junction site and the start codon of PA5174 is indicated in parentheses. Cosmids selected for further growth analysis are underlined. (B) Growth curves of E. coli strain TMY19 grown in various conditions at 37°C. E. coli TMY19 was grown in LB only (▲) or in LB plus 0.2% l-arabinose inducer (△). E. coli TMY19 was grown in LB plus 0.2% l-arabinose inducer with cosmid pTMY2 (●), cosmid pTMY10 (■), cosmid pTMY17 (○) or plasmid pWEB-PA5174 (□). (C) Growth curves of the IPTG-inducible PA5174 E. coli strain TMY32 with fabH deleted in LB (in the absence of IPTG [■] or in the presence of 1 mM IPTG [△]) were measured at 37°C in LB and compared to that of the parent wild-type strain BW25113 (●).

Fig 4

Growth characteristics and complementation of the P. aeruginosa ΔPA5174 strain. (A) Growth of the P. aeruginosa PAO1 parent strain (●), ΔPA5174 strain (■), PA5174 back-complemented with plasmid (TMT41 [△]), and the E. coli FabH-complemented strain (TMT38 [□]) in LB at 37°C. Basal expression of PA5174 on the plasmid is sufficient for full complementation in the absence of IPTG induction. (B) The rate of radiolabeled precursor incorporation by FAS ([³H]acetate [black bars]) and RNA ([³H]uridine [white bars]) biosynthetic pathways was measured after a 30-min incubation period in supplemented M9 minimal medium. The percent incorporated label was normalized to the value of the wild-type (Wt) P. aeruginosa PAO1 control. Error bars represent the standard deviations from the means calculated from three independent experiments.

Fig 5

Characterization of rhl quorum sensing and dependent exoproducts produced in P. aeruginosa ΔPA5174 strain. (A) The acylated HSL fraction was extracted from the supernatants of stationary-phase cultures grown in LB broth at 37°C for 24 h and separated by reverse-phase TLC. The levels of acylated HSL were estimated using a soft agar overlay of the C. violaceum reporter strain TMT45 and compared to standards (C₄-HSL [20 nmol] and C₆-HSL [0.2 nmol]). The cvi QS system of C. violaceum detects C₆-HSL with the highest sensitivity (51), limiting quantitative comparisons to within the same HSL acyl chain length. (B) Cultures of P. aeruginosa were spotted into wells cut into the agar of SW plates. Blue halos indicate zones of ion-pairing complexation between the methylene blue dye, cetyltrimethylammonium bromide, and secreted rhamnolipids. (C) Rhamnose levels in stationary-phase culture supernatants were measured using the colorimetric anthrone assay. (D) Swarming motility was evaluated on M8 agar plates supplemented with Casamino Acids and glucose after overnight incubation at 37°C.

Fig 6

Characterization of las and PQS-dependent quorum sensing in the P. aeruginosa ΔPA5174 strain. (A) The 3-oxo-HSL produced by the las quorum-sensing system was extracted from the supernatants of stationary-phase cultures grown in LB broth at 37°C for 24 h and separated by reverse-phase TLC. The levels of 3-oxo-HSL were estimated using a soft agar overlay of the A. tumefaciens NTL4(pZLR4) β-galactosidase reporter strain and compared to standards ([3-oxo-C₆-HSL [1 pmol], 3-oxo-C₈-HSL [0.2 pmol], 3-oxo-C₁₀-HSL [20 pmol], and 3-oxo-C₁₂-HSL [100 pmol]). The tra QS system of A. tumefaciens detects 3-oxo-C₈-HSL with the highest sensitivity, limiting quantitative comparisons to within the same HSL acyl chain length (74). (B) The elastase activity (LasB) in culture supernatants was measured using a Congo red-elastin dye release assay. (C) The amount of PQS was compared by direct UV illumination of culture supernatant extracts separated by normal-phase TLC on silica 60 F₂₅₄ plates. Synthetic PQS standard (20 nmol) and P. aeruginosa TMT15 (ΔpqsC) were used as the positive and negative controls, respectively. (D) Pyocyanin was extracted from supernatants obtained from cultures grown in glycerol alanine minimal media for 42 h at 37°C, acidified, and quantified by absorbance at 520 nm. Values (in μg/ml of supernatant) were determined using a standard curve generated with purified pyocyanin.

Fig 7

Siderophore secretion in P. aeruginosa ΔPA5174. The wild-type P. aeruginosa PAO1 (Wt), TMT39 (ΔPA5174), ΔPA5174(pZEN-PA5174) (TMT41), and ΔpqsD PQS⁻ (TMT02) strains were streaked onto LB agar plates containing CAS and incubated at 37°C for 24 h. Zones of yellow-orange clearing indicate sequestration of Fe³⁺ away from CAS- Fe³⁺ complexes by secreted siderophores.

Fig 8

Enzymatic activity and acyl-CoA acceptor substrate specificity of recombinant PA5174. (A) The continuous FabG coupled assay was initiated with 15 nM FabH (●) or PA5174 (○), and the reaction mixture was incubated at 30°C. Reaction progress was monitored by UV absorbance at 340 nm to assay NADPH consumption. Background absorbance (no enzyme addition) was subtracted from each curve before plotting. (B) Discontinuous conformation-sensitive urea-PAGE was employed to separate acyl-ACP products with incorporated [2-³H]malonyl-ACP substrate. Gels were sequentially imaged using Coomassie blue staining and autoradiography. All lanes contained [2-³H]malonyl-ACP and acetyl-CoA substrates. Lanes: 1, substrates only; 2, E. coli FabH; 3, PA5174; 4, E. coli FabH plus FabG/FabA/FabI; 5, PA5174 plus FabG/FabA/FabI. The positions of malonyl-ACP (malonyl), β-acetoacetyl-ACP or acetyl-ACP (β-acac/ac), and β-butyryl-ACP (β-butyryl) are labeled. (C) The acyl-CoA substrate specificity of PA5174 was assayed using [2-³H]malonyl-ACP substrate alone (control [Con]) or with various acyl-CoA acceptors (C₂ to C₁₆). Reaction products were separated by urea-PAGE, stained, and sequentially imaged. Under these assay conditions, the [2-³H]β-acetoacetyl-ACP reaction product using C₂-CoA as the acceptor is not adequately separated from the [2-³H]acetyl-ACP side product produced by decarboxylation of [2-³H]malonyl-ACP unless further reduced by FabG/FabA/FabI as in lanes 4 and 5 of panel B.

Fig 9

Phylogenetic tree and distribution of KASI/II domain-containing proteins among pseudomonads. All open reading frames within each genome having significant similarity to P. aeruginosa PAO1 KASI/II proteins (E-value < 1e−10) and that are not a subdomain of a polyketide synthase multidomain protein were included in the analysis. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The tree is drawn to scale, with branch lengths in the units of number of amino acid substitutions per site. Analyses were conducted by using MEGA5 (82). The three main KASI/II groups (FabB, FabF, and FabY) are indicated, and all members within each cluster share at least partial genomic synteny. Locus tag abbreviations: PA, P. aeruginosa PAO1 (orange); PSPA7, P. aeruginosa PA7 (orange); PA14, P. aeruginosa UCBPP-PA14 (orange); PLES, P. aeruginosa LESB58 (orange); Pmen, P. mendocina ymp (blue); MDS, P. mendocina NK-01 (blue); Psefu, P. fulva 12-X (red), PST, P. stutzeri A1501 (black), PputGB1, P. putida GB-1 (black); Psyr, P. syringae pv. syringae B728a (black); PFLU, P. fluorescens SBW25 (black); Avin, Azotobacter vinelandii DJ (purple); b, E. coli (green).

Fig 10

Cellular products dependent on FabY (i.e., FabH-type) activity in P. aeruginosa PAO1. The β-acetoacetyl-ACP product of FabY is used as a primer for elongation by the FAS elongation machinery, whose products are directly incorporated into a multitude of essential cell components, cofactors, and quorum sensing (QS) signaling molecules. Some exoproducts, including rhamnolipids, are dependent on fatty acid precursors as well as being subject to transcriptional QS regulation. Molecular fragments originating from FabY enzymatic activity are shown in bold type (blue, red, or green). The depicted biotin pathway in P. aeruginosa PAO1 is based on the proposed bioC and/or bioH pathway of E. coli (16). E2, (α)-ketoacid dehydrogenase subunit E2-containing lipoylation domain.