The variable hydroxamic acid siderophore metabolome of the marine actinomycete Salinispora tropica CNB-440

Abstract

The recently sequenced genome of the marine actinomycete Salinispora tropica CNB-440 revealed a high frequency of gene clusters which code for the biosynthesis of known and novel secondary metabolites. Of these metabolites, bioinformatics analysis predicted that S. tropica CNB-440 could potentially biosynthesize, as high affinity Fe(III) ligands, siderophores from the hydroxamic acid desferrioxamine class (sid1 gene cluster) and the phenolate-thia(oxa)zoli(di)ne class (sid2 and sid4 gene clusters). In this work, we have used Ni(II)-based immobilized metal ion affinity chromatography (IMAC) to pre-fractionate the hydroxamic acid siderophore metabolome of S. tropica CNB-440 from the secondary metabolome, to reveal low abundance siderophores. LC-MS measurements and electronic absorption spectra from purified extracts incubated with exogenous Fe(III) revealed eight siderophores from the desferrioxamine class (DFOA₂, DFOA_1a, DFOA_1b, DFOB, DFON, DFOD₂, DFOE, DFOD₁), which included two constitutional isomers (DFOA_1a, DFOA_1b), and one new siderophore (DFON), the latter which would require assembly from a combination of 1,5-diaminopentane and 1,6-diaminohexane as diamine substrates. Three additional species (m/z_obs 496.14, 792.34 and 804.34) with electronic absorption spectra characteristic of complexes formed between Fe(III) and hydroxamic acid-type siderophores were evident under some conditions. The signal at m/z_obs 792.34 eluted in the hydrophobic region of the reverse-phase LC and correlated with a DFOD₁ analogue with a C-terminal branched chain fatty acid ([M + K⁺]⁺m/z_calc 792.35), which has been previously identified from marine sediment dwelling Micrococcus luteus KLE1011. The S. tropica CNB-440 hydroxamic acid siderophore metabolome was modulated by culture conditions (pH 7, 22 °C; pH 7, 28 °C; pH 9, 28 °C) designed to simulate the variable marine environment. An increase in temperature at constant pH value showed increased levels of DFOA₂ and DFOA₁, and decreased levels of DFOB and DFOE. An increase in pH value at constant temperature showed decreased levels of DFOA₂ and DFOA₁, and increased levels of DFOB, DFON and DFOE. These results indicate that the marine adaptation of S. tropica CNB-440 could involve its ability to select a suite of siderophores from a large number of candidates, which are optimized for the iron microenvironment.