Dissecting the role of the TusA protein in the regulation of iron homeostasis and sulfur mobilisation in Escherichia coli

In this work, the role of TusA in the regulation of iron homeostasis and sulfur mobilisation was investigated in Escherichia coli. TusA is the tRNA 2-thiouridine synthesizing protein A and, by interacting with the housekeeping L-cysteine desulfurase IscS, catalyses the biosynthesis of the thio-modified nucleotide mnm5s2U34, present in tRNALys, tRNAGlu and tRNAGln. This modification is important to guarantee a correct translation efficiency for different proteins. TusA also promotes the second step of molybdenum cofactor (Moco) biosynthesis. The deletion of tusA gene has a pleiotropic effect on the E. coli cell, which shows a phenotype characterised by reduced Moco biosynthesis, growth and cell division impairment, lower Fe-S cluster level and iron accumulation. Iron is important for many biological processes, including TCA cycle, DNA biosynthesis, nitrogen fixation, electron transport and respiration. Due to both its essentiality and high reactivity, the intracellular iron concentration must be strictly controlled by the cell. In E. coli, the ferric uptake regulator (Fur) is mainly responsible to fine tune iron homeostasis. This study shows that the deletion of tusA gene reduced and retarded the translation of Fur protein, due to the lack of specific tRNA thiolation. This leads to iron accumulation in the cell and, together with a complex mis-regulation of transcription factors, small RNAs and modulators (as FNR, cAMP-CRP, ErpA, RyhB, FnrS, IscR, CyaY), causes the reduction of Fe-S clusters assembled by the ISC and SUF systems. ISC and SUF systems are the two main Fe-S cluster assembly machineries in E. coli and both systems mobilise the sulfur through L-cysteine desulfurases, IscS and SufS respectively. While SUF system is limited to the production of Fe-S cluster and mainly active under oxidative stress and iron-limiting conditions, ISC system is responsible to mobilise sulfur in different essential biological pathways under normal or iron-rich conditions. The L-cysteine desulfurase IscS has a central role as sulfur donor for the biosynthesis of numerous sulfur-containing biomolecules through interactions with multiple partner proteins, which bind at different sites. In addition to TusA, IscS interacts with IscU, CyaY, Fdx, IscX, involved in iron sulfur (Fe-S) cluster assembly, and with ThiI, required for the biosynthesis of thiamine and s4U8 tRNA modification. Previous studies suggested that IscS can bind only one sulfur acceptor at a time. The preference of IscS for a specific interaction partner, and consequentially the direction taken by the sulfur, is not fully understood. This study shows that TusA has a similar, but lower affinity to IscS than IscU and, under certain conditions, it’s able to partially displace IscU from its binding with IscS. The results indicate the formation of a heterocomplex involving IscS dimer bound to single TusA and IscU molecules. In this complex, TusA affects the L-cysteine desulfurase activity of IscS and re-direct the sulfur flux towards Moco biosynthesis and tRNA thiolation rather than Fe-S cluster assembly. Furthermore, TusA can completely displace CyaY from its complex with IscS. CyaY is the frataxin homolog of E. coli and its negative modulating effect on the formation of Fe-S cluster by ISC system has been extensively studied. In this work, the possibility that the displaced CyaY could interact with the second L-cysteine desulfurase SufS was investigated. The results indicate that CyaY is able to interact with SufS, alone and in complex with SufE, and drastically reduces its activity in vitro. The overexpression of CyaY in vivo has the effect of reducing the L-cysteine desulfurase activity of both IscS and SufS, under different growth conditions. Overall, this work reveals a new role of TusA in the regulation of iron homeostasis and, together with CyaY, in the modulation of L-cysteine desulfurases IscS and SufS.