Pyochelin (Pch) and enantio-pyochelin (EPch) are enantiomer siderophores that are produced by Pseudomonas aeruginosa and Pseudomonas fluorescens, respectively, under iron limitation. Pch promotes growth of P. aeruginosa when iron is scarce, and EPch carries out the same biological function in P. fluorescens. However, the two siderophores are unable to promote growth in the heterologous species, indicating that siderophore-mediated iron uptake is highly stereospecific. In the present work, using binding and iron uptake assays, we found that FptA, the Fe-Pch outer membrane transporter of P. aeruginosa, recognized (K d ‫؍‬ 2.5 ؎ 1.1 nM) and transported Fe-Pch but did not interact with Fe-EPch. Likewise, FetA, the Fe-EPch receptor of P. fluorescens, was specific for Fe-EPch (K d ‫؍‬ 3.7 ؎ 2.1 nM) but did not bind and transport Fe-Pch. Growth promotion experiments performed under iron-limiting conditions confirmed that FptA and FetA are highly specific for Pch and EPch, respectively. When fptA and fetA along with adjacent transport genes involved in siderophore uptake were swapped between the two bacterial species, P. aeruginosa became able to utilize Fe-EPch as an iron source, and P. fluorescens was able to grow with Fe-Pch. Docking experiments using the FptA structure and binding assays showed that the stereospecificity of Pch recognition by FptA was mostly due to the configuration of the siderophore chiral centers C4؆ and C2؆ and was only weakly dependent on the configuration of the C4 carbon atom. Together, these findings increase our understanding of the stereospecific interaction between Pch and its outer membrane receptor FptA.To access iron, aerobic bacteria produce siderophores which form complexes with Fe 3ϩ in the environment and deliver it via specific membrane transporters to the bacteria (1). The energy required for this process is provided by the proton motive force of the inner membrane by means of an inner membrane complex comprising TonB, ExbB, and ExbD (2, 3). Pyochelin (Pch), 2 which is the focus of this study, is produced as a secondary siderophore under iron limitation by almost all strains of Pseudomonas aeruginosa and some closely related bacteria (4 -7). In pseudomonads, secondary siderophores (pyochelin, thioquinolobactin, etc.) are usually produced in smaller amounts, demonstrate lower iron affinity, and are rather simple molecules compared with pyoverdin (the main siderophore). It is hypothesized that under certain environmental or physiological conditions, secondary siderophores provide sufficient iron to the cell or fulfill functions other than iron sequestration (8). Pch was isolated for the first time from P. aeruginosa ATCC 15692 by Liu and Shokrani (9), and its structure was established later by Cox et al. (10) as (4ЈR,2ЉR,4ЉR)-2Ј-(2-hydroxyphenyl)-3Љ-methyl-4Ј,5Ј,2Љ,3Љ,4Љ,5Љ-hexahydro-[4Ј,2Љ]bithiazolyl-4Љ-carboxylic acid with three chiral centers located at C4Ј, C2Љ, and C4Љ (see Fig. 1). Pch is synthesized by P. aeruginosa from salicylate and two molecules of cysteine via a thiot...

L-2-Amino-4-methoxy-trans-3-butenoic acid (AMB) is a potent antibiotic and toxin produced by Pseudomonas aeruginosa. Using a novel biochemical assay combined with site-directed mutagenesis in strain PAO1, we have identified a five-gene cluster specifying AMB biosynthesis, probably involving a thiotemplate mechanism. Overexpression of this cluster in strain PA7, a natural AMB-negative isolate, led to AMB overproduction.The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen that causes a wide range of human infections and is considered the main pathogen responsible for chronic pneumonia in cystic fibrosis patients (7, 23). P. aeruginosa also infects other organisms, such as insects (4), nematodes (6), plants (18), and amoebae (20). Its ability to thrive as a pathogen and to compete in aquatic and soil environments can be partly attributed to the production and interplay of secreted virulence factors and secondary metabolites. While the importance of many of these exoproducts has been studied, the antimetabolite L-2-amino-4-methoxy-trans-3-butenoic acid (AMB; methoxyvinylglycine) ( Fig. 1) has received only limited attention. Identified during a search for new antibiotics, AMB was found to reversibly inhibit the growth of Bacillus spp. (26) and Escherichia coli (25) and was later shown to inhibit the growth and metabolism of cultured Walker carcinosarcoma cells (28). AMB is a ␥-substituted vinylglycine, a naturally occurring amino acid with a ␤,␥-CAC double bond. Other members of this family are aminoethoxyvinylglycine from Streptomyces spp. (19) and rhizobitoxine, made by Bradyrhizobium japonicum (16) and Pseudomonas andropogonis (15) (Fig. 1). As inhibitors of pyridoxal phosphate-dependent enzymes (13, 17, 21, 22), ␥-substituted vinylglycines have multiple targets in bacteria, animals, and plants (3,5,10,21,22,29). However, the importance of AMB as a toxin in biological interactions with P. aeruginosa has not been addressed, as AMB biosynthesis and the genes involved have not been elucidated.Identification of the ambABCDE biosynthesis cluster. To identify the AMB biosynthetic genes, we searched a Tn5Gm mutant library of P. aeruginosa PAO (9) for mutants that lacked the ability to produce AMB using a bioassay based on growth inhibition of E. coli K-12 (25). Bacteria were spotted onto minimal medium E (MME) (30) plates amended with 0.5% glucose as a carbon source and 1 mM threonine and grown at 37°C for 14 h. The bacteria were then killed by a 5-min UV exposure, and the plates were overlaid with a mixture of 3 ml 0.5% bacteriological agar no. 1 (Oxoid) and 0.3 ml of an E. coli K-12 culture grown overnight in MME and adjusted to an optical density at 600 nm (OD 600 ) of 0.5 with 0.9% NaCl. The plates were incubated at 37°C and were scored for zones of clearance after 1 day. As shown in Fig. 2 (left panel), growth inhibition of the E. coli indicator resulted in a clearing zone around P. aeruginosa PAO1 as well as around a filter disk soaked with chemically synthesized AMB (2). In both cases,...

The Pseudomonas aeruginosa toxin L-2-amino-4-methoxy-trans-3-butenoic acid (AMB) is a non-proteinogenic amino acid which is toxic for prokaryotes and eukaryotes. Production of AMB requires a five-gene cluster encoding a putative LysE-type transporter (AmbA), two non-ribosomal peptide synthetases (AmbB and AmbE), and two iron(II)/α-ketoglutarate-dependent oxygenases (AmbC and AmbD). Bioinformatics analysis predicts one thiolation (T) domain for AmbB and two T domains (T1 and T2) for AmbE, suggesting that AMB is generated by a processing step from a precursor tripeptide assembled on a thiotemplate. Using a combination of ATP-PPi exchange assays, aminoacylation assays, and mass spectrometry-based analysis of enzyme-bound substrates and pathway intermediates, the AmbB substrate was identified to be L-alanine (L-Ala), while the T1 and T2 domains of AmbE were loaded with L-glutamate (L-Glu) and L-Ala, respectively. Loading of L-Ala at T2 of AmbE occurred only in the presence of AmbB, indicative of a trans loading mechanism. In vitro assays performed with AmbB and AmbE revealed the dipeptide L-Glu-L-Ala at T1 and the tripeptide L-Ala-L-Glu-L-Ala attached at T2. When AmbC and AmbD were included in the assay, these peptides were no longer detected. Instead, an L-Ala-AMB-L-Ala tripeptide was found at T2. These data are in agreement with a biosynthetic model in which L-Glu is converted into AMB by the action of AmbC, AmbD, and tailoring domains of AmbE. The importance of the flanking L-Ala residues in the precursor tripeptide is discussed.

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The Pseudomonas aeruginosa antimetabolite L-2-amino-4-methoxy-trans-3-butenoic acid (AMB) shares biological activities with 4-formylaminooxyvinylglycine, a related molecule produced by Pseudomonas fluorescens WH6. We found that culture filtrates of a P. aeruginosa strain overproducing AMB weakly interfered with seed germination of the grassy weed Poa annua and strongly inhibited growth of Erwinia amylovora, the causal agent of the devastating orchard crop disease known as fire blight. AMB was active against a 4-formylaminooxyvinylglycine-resistant isolate of E. amylovora, suggesting that the molecular targets of the two oxyvinylglycines in Erwinia do not, or not entirely, overlap. The AMB biosynthesis and transport genes were shown to be organized in two separate transcriptional units, ambA and ambBCDE, which were successfully expressed from IPTG-inducible tac promoters in the heterologous host P. fluorescens CHA0. Engineered AMB production enabled this model biocontrol strain to become inhibitory against E. amylovora and to weakly interfere with the germination of several graminaceous seeds. We conclude that AMB production requires no additional genes besides ambABCDE and we speculate that their expression in marketed fire blight biocontrol strains could potentially contribute to disease control.