RESUMEN
Siderophores are low-molecular-weight organic bacterial and fungal secondary metabolites that form high affinity complexes with Fe(III). These Fe(III)-siderophore complexes are part of the siderophore-mediated Fe(III) uptake mechanism, which is the most widespread strategy used by microbes to access sufficient iron for growth. Microbial competition for limited iron is met by biosynthetic gene clusters that encode for the biosynthesis of siderophores with variable molecular scaffolds and iron binding motifs. Some classes of siderophores have well understood biosynthetic pathways, which opens opportunities to further expand structural and property diversity using precursor-directed biosynthesis (PDB). PDB involves augmenting culture medium with non-native substrates to compete against native substrates during metabolite assembly. This chapter provides background information and technical details of conducting a PDB experiment towards producing a range of different analogues of the archetypal hydroxamic acid siderophore desferrioxamine B. This includes processes to semi-purify the culture supernatant and the use of liquid chromatography-tandem mass spectrometry for downstream analysis of analogues and groups of constitutional isomers.
Asunto(s)
Sideróforos , Sideróforos/biosíntesis , Sideróforos/química , Sideróforos/metabolismo , Espectrometría de Masas en Tándem/métodos , Deferoxamina/metabolismo , Deferoxamina/química , Cromatografía Liquida/métodos , Vías Biosintéticas , Familia de Multigenes , Hierro/metabolismo , Hierro/química , Medios de Cultivo/química , Medios de Cultivo/metabolismoRESUMEN
To acquire iron essential for growth, the bacterium Shewanella putrefaciens produces the macrocyclic dihydroxamic acid putrebactin (pbH2; [M + H(+)](+), m/zcalc 373.2) as its native siderophore. The assembly of pbH2 requires endogenous 1,4-diaminobutane (DB), which is produced from the ornithine decarboxylase (ODC)-catalyzed decarboxylation of l-ornithine. In this work, levels of endogenous DB were attenuated in S. putrefaciens cultures by augmenting the medium with the ODC inhibitor 1,4-diamino-2-butanone (DBO). The presence in the medium of DBO together with alternative exogenous non-native diamine substrates, (15)N2-1,4-diaminobutane ((15)N2-DB) or 1,4-diamino-2(E)-butene (E-DBE), resulted in the respective biosynthesis of (15)N-labeled pbH2 ((15)N4-pbH2; [M + H(+)](+), m/zcalc 377.2, m/zobs 377.2) or the unsaturated pbH2 variant, named here: E,E-putrebactene (E,E-pbeH2; [M + H(+)](+), m/zcalc 369.2, m/zobs 369.2). In the latter system, remaining endogenous DB resulted in the parallel biosynthesis of the monounsaturated DB-E-DBE hybrid, E-putrebactene (E-pbxH2; [M + H(+)](+), m/zcalc 371.2, m/zobs 371.2). These are the first identified unsaturated macrocyclic dihydroxamic acid siderophores. LC-MS measurements showed 1:1 complexes formed between Fe(III) and pbH2 ([Fe(pb)](+); [M](+), m/zcalc 426.1, m/zobs 426.2), (15)N4-pbH2 ([Fe((15)N4-pb)](+); [M](+), m/zcalc 430.1, m/zobs 430.1), E,E-pbeH2 ([Fe(E,E-pbe)](+); [M](+), m/zcalc 422.1, m/zobs 422.0), or E-pbxH2 ([Fe(E-pbx)](+); [M](+), m/zcalc 424.1, m/zobs 424.2). The order of the gain in siderophore-mediated Fe(III) solubility, as defined by the difference in retention time between the free ligand and the Fe(III)-loaded complex, was pbH2 (ΔtR = 8.77 min) > E-pbxH2 (ΔtR = 6.95 min) > E,E-pbeH2 (ΔtR = 6.16 min), which suggests one possible reason why nature has selected for saturated rather than unsaturated siderophores as Fe(III) solubilization agents. The potential to conduct multiple types of ex situ chemical conversions across the double bond(s) of the unsaturated macrocycles provides a new route to increased molecular diversity in this class of siderophore.
Asunto(s)
Ácidos Hidroxámicos/química , Compuestos Macrocíclicos/química , Shewanella putrefaciens/química , Shewanella putrefaciens/genética , Sideróforos/biosíntesis , Sideróforos/química , Cromatografía Liquida , Espectrometría de Masas , Modelos Moleculares , Estructura Molecular , Péptidos Cíclicos/química , Putrescina/análogos & derivados , Putrescina/química , Shewanella putrefaciens/metabolismo , Succinatos/químicaRESUMEN
To manage iron acquisition in an oxic environment, Shewanella putrefaciens produces the macrocyclic dihydroxamic acid putrebactin (PB) as its native siderophore. In this work, we have established the siderophore profile of S. putrefaciens in cultures augmented with the native PB precursor putrescine and in putrescine-depleted cultures. Compared to base medium, PB increased by two-fold in cultures of S. putrefaciens with 10 mM NaCl and 20 mM exogenous putrescine. In cultures augmented with 1,4-diaminobutan-2-one (DAB), PB decreased with only 0.02-fold PB detectable at 10 mM DAB. As an ornithine decarboxylase (ODC) inhibitor, DAB depleted levels of endogenous putrescine which attenuated downstream PB assembly. Under putrescine-depleted conditions, S. putrefaciens produced as its replacement siderophore the cadaverine-based desferrioxamine B (DFO-B), as characterised by ESI-MS of the Fe(III)-loaded form (m/z(obs) 614.13; m/z(calc) 614.27). A third siderophore, independent of DAB, was observed in low levels. LC/MS Analysis of the Fe(III)-loaded extract gave m/z(obs) 440.93, which, formulated as a 1:1 Fe(III) complex with a macrocyclic dihydroxamic acid, comprising one putrescine- and one cadaverine-based precursor (m/z(calc) 440.14). These results show that the production of native PB or non-native DFO-B by S. putrefaciens can be directed though upstream inhibition of ODC. This approach could be used to increase the molecular diversity of siderophores produced by S. putrefaciens and to map alternative diamine-dependent metabolites.
Asunto(s)
Deferoxamina/metabolismo , Inhibidores de la Ornitina Descarboxilasa , Putrescina/análogos & derivados , Shewanella putrefaciens/metabolismo , Deferoxamina/farmacología , Inhibidores Enzimáticos/farmacología , Estructura Molecular , Putrescina/biosíntesis , Putrescina/metabolismo , Putrescina/farmacología , Shewanella putrefaciens/efectos de los fármacos , Shewanella putrefaciens/enzimología , Sideróforos/biosíntesis , Sideróforos/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Succinatos/metabolismoRESUMEN
Iron(III)-uptake mechanisms in bacteria indigenous to the Antarctic, which is the most Fe-deficient continent on Earth, have not been extensively studied. The cold-adapted, Antarctic bacterium, Shewanella gelidimarina, does not produce detectable levels of the siderophore, putrebactin, in the supernatant of Fe(III)-deprived cultures. This is distinct from the putrebactin-producing bacterium from the same genus, Shewanella putrefaciens, which is adapted to middle-range temperatures. The production of putrebactin by S. putrefaciens is optimal, when the pH value of the medium is 7.0. According to the strong positive response from whole cells in the Chrome Azurol S (CAS) agar diffusion assay, Shewanella gelidimarina appears to produce cell-associated siderophores. In the RP-HPLC trace of an Fe(III)-loaded extract from the cell-associated components of S. gelidimarina cultured in media with [Fe(III)] ca. 0 microM, a peak appears at [MeCN] ca. 77%, which decreases in intensity in a parallel experiment in which [Fe(III)] ca. 5 microM, and is barely detectable in Fe(III)-replete media ([Fe(III)] ca. 20 microM). The Fe(III)-dependence of this peak suggests that the attendant species, which is significantly more hydrophobic than putrebactin (RP-HPLC elution: [MeCN] ca. 14%), is associated with Fe(III)-management in S. gelidimarina. This study highlights the diversity in Fe(III)-uptake mechanisms in Shewanella species adapted to different environmental and thermal niches.