Biosynthetic pathway of Pseudomonas aeruginosa 4-hydroxy-2-alkylquinolines

Abstract

The role of intercellular communication in the regulation of bacterial multicellular behavior has received widespread attention, and a variety of signal molecules involved in bacterial communication have been discovered. In addition to the N-acyl-homoserine lactones, 4-hydroxy-2-alkylquinolines (HAQs), including the Pseudomonas quinolone signal, have been shown to function as signal molecules in Pseudomonas aeruginosa. In this study we unraveled the biosynthetic pathway of HAQs using feeding experiments with isotope-labeled precursors and analysis of extracted HAQs by gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy. Our results show that the biosynthesis of various HAQ metabolites is directed via a common metabolic pathway involving a "head-to-head" condensation of anthranilic acid and beta-keto fatty acids. Moreover, we provide evidence that the beta-keto-(do)decanoic acids, crucial for the biosynthesis of the heptyl and nonyl derivatives of the 4-hydroxyquinolines in P. aeruginosa, are at least in part derived from a common pool of beta-hydroxy(do)decanoic acids involved in rhamnolipid biosynthesis.

FIG. 1.

(A) Biosynthetic pathway for P. aeruginosa HAQs involving anthranilic acid and activated β-keto fatty acids as direct precursors. Using labeled precursor substrates, the possibility of an alternative pathway via the formation of kynurenic acid as an intermediate, as shown in panel B, was excluded.

FIG. 2.

(Upper panel) Typical gas chromatogram of total trimethylsilylated quinoline derivatives isolated from P. aeruginosa grown with anthranilic acid and acetic acid as the main carbon source. Peak 1, 4-hydroxy-2-heptylquinoline (whose EI mass spectrum is shown in the lower panel); peak 2, 3,4-dihydroxy-2-heptylquinoline; peak 3, 4-hydroxy-2-nonylquinoline; peak 4, 3,4-dihydroxy-2-nonylquinoline; peak 5, 4-hydroxy-2-hydroxynonylquinoline; peak 6, 4-hydroxy-2-undecenylquinoline (the structures of the peak 5 and 6 compounds were only tentatively assigned using their EI mass spectra alone). (Lower panel) EI mass spectrum of 4-trimethylsilyloxy-2-heptylquinoline and inserted fragmentation scheme.

FIG. 3.

Second-order (daughter ion) mass spectra of the monoisotopic molecular ions of the trimethylsilylated derivatives of 4-hydroxy-2-heptylquinoline labeled with four and five ¹³C atoms by addition of [1-¹³C]acetic acid and [2-¹³C]acetic acid, respectively, to the medium in the presence of unlabeled anthranilic acid. The arrows indicate the positions of ¹³C.

FIG. 4.

Comparison of the rhamnolipid constituents and the HAQs present in P. aeruginosa grown on modified Vogel-Bonner medium with d-gluconic acid (upper panel) or acetate (lower panel) as the major carbon source. GC-MS of dichloromethane-extracted and methanolysed samples was performed in triplicate, and representative chromatograms of trimethlysilylated derivatives are shown. Peak 1, β-hydroxydecanoic acid methyl ester; peak 2, rhamnose methylglycoside; peak 3, β-hydroxydodecanoic acid methyl ester; peak 4, 4-hydroxy-2-heptylquinoline; peak 5, 4-hydroxy-2-nonylquinoline. The chromatograms were generated by summation of the most intense fragment ions of the compounds indicated (m/z 204, 231, 259, and 287).