Characterization of bacterial multitask ATPases from Type I ABC transporters

• Autor(a) principal: Sousa, João Pedro Abreu
• Data de Publicação: 2021
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/112288
• topic: Bacillus subtilis; ABC Type I sugar importers; Multitask ATPases; MsmX; NBDs interspecies exchangeability; BACTH system; Importadores de açúcares ABC tipo I; ATPases multitarefa; Permutabilidade interespécie de NBDs; Sistema BACTH; Engenharia e Tecnologia

Bacterial ABC-Type I importers belong to one of the largest and most diverse transporter superfamilies, which make use of ATP binding and hydrolysis to drive transport of several substrates across membranes. These transporters share the same modular architecture of two transmembrane domains (TMDs) and two ATPases or nucleotide-binding domains (NBDs). Additionally, importers rely on an auxiliary domain for binding the substrate and delivery to the TMDs, the substrate-binding protein (SBP). Bacillus subtilis MsmX has been established as a multitask ATPase (NBD), capable of energizing several ABC sugar importers. Most recently, our laboratory has identified several ATPases from bacterial species, the vast majority belonging to the Firmicutes phylum, capable of complementing MsmX function in the AraNPQ and GanSPQ transport systems of B. subtilis. Here, to assess how widespread multitask ATPases are, NBDs from bacteria belonging to other phyla were characterized to determine their multitask capabilities and their interspecies exchangeability in B. subtilis. The results show that Mycobacterium tuberculosis SugC and Thermus thermophilus MalK1 are not capable of energizing either the AraNPQ and MelEDC transport systems, in the absence of MsmX. However, it was found that both proteins do not accumulate in the cells most probably due to differences in the codon usage between B. subtilis and both M. tuberculosis and T. thermophilus. Additionally, we demonstrate that out of the previously identified ATPases, ABC_Bt (Bacillus thuringiensis), MsmK (Streptococcus pneumoniae) and ABC_Cd (Clostridioides difficile) are capable of energizing the MelEDC transporter, whilst YurJ (B. subtilis), ABC_Sa (Staphylococcus aureus) and ABC_Syn (Synechocystis sp.) are not. To map the functional domains of multitask NBDs, a MsmX-MsmK chimeric protein was constructed and characterized. The results revealed that the chimeric protein is unable to energize both the AraNPQ and MelEDC transport systems, but it accumulates in the cell suggesting that the fused domains do not adopt a proper conformation resulting in an inactive protein. Using a bacterial two-hybrid system (BACTH) in Escherichia coli, we additionally show that YurJ is not able to form homodimers nor heterodimers with MsmX in the absence of TMDs. Moreover, the results indicate that MsmX displays a strong interaction with the TMDs AraP, MelD and GanP, while YurJ only interacts with AraP.