Biosynthesis of a complex yersiniabactin-like natural product via the mic locus in phytopathogen Ralstonia solanacearum

Abstract

A genome mining study in the plant pathogenic bacterium Ralstonia solanacearum GMI1000 unveiled a polyketide synthase/nonribosomal peptide synthetase gene cluster putatively involved in siderophore biosynthesis. Insertional mutagenesis confirmed the respective locus to be operational under iron-deficient conditions and spurred the isolation of the associated natural product. Bioinformatic analyses of the gene cluster facilitated the structural characterization of this compound, which was subsequently identified as the antimycoplasma agent micacocidin. The metal-chelating properties of micacocidin were evaluated in competition experiments, and the cellular uptake of gallium-micacocidin complexes was demonstrated in R. solanacearum GMI1000, indicating a possible siderophore role. Comparative genomics revealed a conservation of the micacocidin gene cluster in defined, but globally dispersed phylotypes of R. solanacearum.

Fig. 1.

Organization of the micacocidin biosynthesis (mic) gene cluster in R. solanacearum GMI1000 and conserved loci in the phytopathogenic strains CMR15 and PSI07 from the R. solanacearum species complex. A rearranged mic cluster was found on plasmid 4 of the Burkholderia gladioli BSR3 genome.

Fig. 2.

Chemical structures of micacocidin (compound 1) and yersiniabactin, metal-chelating agents produced by R. solanacearum GMI1000 and Y. pestis, respectively.

Fig. 3.

Domain architecture of modular biosynthesis enzymes from gene clusters of Y. pestis and R. solanacearum GMI1000. The former system encodes the known siderophore and virulence factor yersiniabactin. Domain notation: A, adenylation; PCP, peptidyl carrier protein; C, condensation; KS, ketosynthase; AT, acyltransferase; MT, SAM-dependent methyltransferase; KR, ketoreductase; TE, thioesterase; Red, thiazolinyl imide reductase. Domains that are unique to one assembly line are drawn on different planes.

Fig. 4.

Metabolic profiles of crude culture extracts from GMI1000 grown under iron-deficient conditions (profile a) and in the presence of 4 μM FeCl₃ (profile b), as well as from the mutant strain RS6 at reduced iron levels (profile c). The peak with m/z 566 [M+H]⁺ is only produced by the wild-type strain under iron-limited conditions.

Fig. 5.

⁶⁸Ga³⁺ uptake of R. solanacearum GMI1000 (A) and the mutant strain RS6 (B) after a 1-h incubation with the ⁶⁸Ga-micacocidin complex (⁶⁸Ga-Mic) and three successive washing steps compared to a micacocidin-free control sample (⁶⁸Ga). Radioactivity is retained in the cell pellet, while the washing fractions are nearly free of activity after two washing steps.