PchC thioesterase optimizes nonribosomal biosynthesis of the peptide siderophore pyochelin in Pseudomonas aeruginosa

Abstract

In Pseudomonas aeruginosa, the antibiotic dihydroaeruginoate (Dha) and the siderophore pyochelin are produced from salicylate and cysteine by a thiotemplate mechanism involving the peptide synthetases PchE and PchF. A thioesterase encoded by the pchC gene was found to be necessary for maximal production of both Dha and pyochelin, but it was not required for Dha release from PchE and could not replace the thioesterase function specified by the C-terminal domain of PchF. In vitro, 2-aminobutyrate, a cysteine analog, was adenylated by purified PchE and PchF proteins. In vivo, this analog strongly interfered with Dha and pyochelin formation in a pchC deletion mutant but affected production of these metabolites only slightly in the wild type. Exogenously supplied cysteine overcame the negative effect of a pchC mutation to a large extent, whereas addition of salicylate did not. These data are in agreement with a role for PchC as an editing enzyme that removes wrongly charged molecules from the peptidyl carrier protein domains of PchE and PchF.

FIG. 1.

Model for PchDEFG-dependent biosynthesis of pyochelin from salicylate in P. aeruginosa (adapted from references , , , and 33). Adenylated forms of salicylate (Sal) and l-cysteine (Cys) are loaded onto the peptide synthetases PchE and PchF via covalent thioester linkages. PchE-dependent condensation of salicylate with l-cysteine, followed by epimerization and cyclization reactions, generates the intermediate HPT, which can be released from PchE to give Dha or can be coupled to PchF-bound l-cysteine to give hydroxyphenyl-bis-thiazoline (HPTT). After reduction of HPTT by PchG and methylation by PchF, pyochelin (Pch) is released by thioester cleavage. Functional domains are indicated for adenylation (A, A1, and A2), cyclization (Cy1 and Cy2), epimerization (E), thiolation (ArCP for the aryl carrier protein domain and PCP1 and PCP2 for peptidyl carrier protein domains), reduction (Red), methyl transfer (MT) and thioesterase (TE) activity.

FIG. 2.

Pyochelin formation by the pyoverdin-negative P. aeruginosa strains PALS128 (▪), PAO6342 (pchC) (•), PALS128-6 (pchA) (□), and PAO6357 (pchA pchC) (○) in GGP medium amended with different amounts of salicylate. The growth conditions, extraction, and analysis of pyochelin were as described in Table 2, footnote a. Means ± standard deviations for three parallel experiments are shown.

FIG. 3.

Effect of cysteine on pyochelin formation by P. aeruginosa strains. In GGP medium amended with different amounts of salicylate, pyochelin formation was measured in strain PALS128-6 (pvdB pchA) grown without cysteine (□) and with 2 mM l-cysteine (▪), as well as in strain PAO6357 (pvdB pchA pchC) grown without cysteine (○) and with 2 mM l-cysteine (•). The growth conditions, extraction, and analysis of pyochelin were as described in Table 2, footnote a. Means ± standard deviations are shown.

FIG. 4.

Relative activation of amino acids by PchE (open bars) and PchF (solid bars). ATP-PP_i exchange activities were measured with proteinogenic amino acids (bars 1 to 20) and nonproteinogenic amino acids (bars 21 to 24) at a concentration of 1 mM. The highest exchange activity measured with l-cysteine was defined as 100%. The standard deviations of values in this assay were ≤20%. Bars 1, l-alanine; bars 2, l-arginine; bars 3, l-asparagine; bars 4, l-aspartate; bars 5, l-cysteine; bars 6, l-glutamate; bars 7, l-glutamine; bars 8, l-glycine; bars 9, l-histidine; bars 10, l-isoleucine; bars 11, l-leucine; bars 12, l-lysine; bars 13, l-methionine; bars 14, l-phenylalanine; bars 15, l-proline; bars 16, l-serine; bars 17, l-threonine; bars 18, l-tryptophan; bars 19, l-tyrosine; bars 20, l-valine; bars 21, l-homoserine; bars 22, l-2-aminobutyrate; bars 23, S-methyl-l-cysteine; bars 24, dl-allylglycine; bars 25, no amino acid added.