23Iron, an essential element for all organisms, acts as a cofactor of enzymes in bacterial 24 degradation of recalcitrant aromatic compounds. The bacterial family, Sphingomonadaceae 25 comprises various degraders of recalcitrant aromatic compounds; however, little is known 26 about their iron acquisition system. Here, we investigated the iron acquisition system in a 27 model bacterium capable of degrading lignin-derived aromatics, Sphingobium sp. strain 28 SYK-6. Analyses of SYK-6 mutants revealed that FiuA (SLG_34550), a TonB-dependent 29 receptor (TBDR), was the major outer membrane iron transporter. Three other TBDRs 30 encoded by SLG_04340, SLG_04380, and SLG_10860 also participated in iron uptake, and 31 tonB2 (SLG_34550), one of the six tonB comprising the Ton complex which enables 32 TBDR-mediated transport was critical for iron uptake. The ferrous iron transporter FeoB 33 (SLG_36840) played an important role in iron uptake across the inner membrane. The 34 promoter activities of most of the iron uptake genes were induced under iron-limited 35 conditions, and their regulation is controlled by SLG_29410 encoding the ferric uptake 36 regulator, Fur. Although feoB, among all the iron uptake genes identified is highly conserved 37 in Sphingomonad strains, the outer membrane transporters seem to be diversified. Elucidation 38 of the iron acquisition system promises better understanding of the bacterial degradation 39 mechanisms of aromatic compounds.40 41 42Iron is an essential nutrient utilised as a cofactor for enzymes that control various life 43 phenomena such as respiration, dissimilation, and stress response 1 . Iron exists mainly in 44 ferrous and ferric forms. Ferrous iron is soluble and highly bioavailable; however, the 45 predominant form of iron in the environment is an insoluble ferric form 2 . Ferric iron in soil 46 and water is solubilised by forming complexes with organic ligands such as siderophores, 47 humic acid, lipids, proteins, and polysaccharides 3,4 . Many bacteria secrete specific 48 high-affinity siderophores which form complexes with ferric iron and then uptake these 49 ferric-siderophore complexes to enhance iron acquisition over competitors [5][6][7] . Besides, 50 pathogenic bacteria can acquire haem and transferrin specifically from their host cells 8,9 . 51 Gram-negative bacteria need to transport iron through both the outer and inner 52 membranes. TonB-dependent receptors (TBDRs) mediate transport of ferric complexes (e.g. 53 siderophore, haem, and transferrin) across the outer membrane 1,10,11 . TBDRs utilize energy 54 derived from the proton motive force transmitted by TonB-ExbB-ExbD complex (Ton 55 complex) localised in the inner membrane (Ton system) 12 . Beside siderophores, the Ton 56 system is involved in the uptake of vitamin B12, saccharide, aromatic compounds, and metals 57 such as nickel, copper, and lanthanoid [13][14][15][16][17][18] . The uptake of the ferric complex across the inner 58 membrane is mainly achieved by ATP-binding cassette (ABC) transporters 11...