<p>The symbiotic interaction between rhizobia and host plants takes place inside root nodules. Besides reducing atmospheric nitrogen into ammonium, which is then used by the plant to grow in nitrogen-deficient soils, certain rhizobia produce siderophores which enable iron uptake from the soil. Siderophores are strong iron chelators due to their hydroxamate, catecholate or carboxylate functional groups. In this work, the siderophores of the beta-rhizobial genus <i>Paraburkholderia</i> were analyzed by ultra-high-performance liquid chromatography (UHPLC) coupled to high-resolution mass spectrometry (HRMS). In particular, the production of the novel siderophore phymabactin by <i>Paraburkholderia phymatum</i> was confirmed and the structures of eleven derivatives were elucidated by tandem mass spectrometry (MS/MS) and nuclear magnetic resonance (NMR) spectroscopy. Phymabactins, consisting of a linear tetrapeptide backbone with hydroxamate and hydroxy-carboxylate groups, are structurally closely related to an already described family of siderophores called ornibactins. The acyl chain found in phymabactin derivatives is made up of eight, ten or twelve carbon atoms, contrarily to the more hydrophilic ornibactin derivatives with shorter acyl chains. Interestingly, spontaneous metal complexation of phymabactin derivatives with aluminum was observed. To explore this complexation in more detail, the collision cross sections (CCS) of phymabactins, ornibactins and corresponding metal complexes were determined by trapped ion mobility spectrometry. The CCS values of aluminum-phymabactin complexes were smaller than those of iron-phymabactin complexes, while an inverse relationship was observed for ornibactin complexes. In summary, this study discloses the molecular structures of phymabactins and investigates their metal complex formation.</p>