Characterization of bacterial multitask ATPases from Type I ABC transporters
Autor(a) principal: | |
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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 |
Resumo: | 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. |
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Characterization of bacterial multitask ATPases from Type I ABC transportersBacillus subtilisABC Type I sugar importersMultitask ATPasesMsmXNBDs interspecies exchangeabilityBACTH systemImportadores de açúcares ABC tipo IATPases multitarefaPermutabilidade interespécie de NBDsSistema BACTHEngenharia e TecnologiaBacterial 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.Importadores ABC Tipo I bacterianos pertencem a uma das maiores e mais diversas superfamílias de transportadores, fazendo uso da ligação e hidrólise de ATP para energizar o transporte de vários substratos através de membranas. Estes transportadores partilham a mesma arquitetura modular de dois domínios transmembranares (TMDs) e duas ATPases ou domínio de ligação a nucleótidos (NBDs). Adicionalmente, os importadores dependem de um domínio auxiliar para a ligação do substrato e entrega do mesmo aos TMDs, a proteína de ligação ao substrato (SBP). MsmX de Bacillus subtilis foi estabelecida como uma ATPase (NBD) multitarefa, capaz de energizar vários importadores ABC de açúcares. Mais recentemente, o nosso laboratório identificou várias ATPases de bactérias, a maioria destas pertencentes ao filo Firmicutes capazes de complementar a função de MsmX nos sistemas de transporte AraNPQ e GanSPQ. Para averiguar o quão abrangente é a existência de ATPases multitarefa, NBDs de bactérias pertencentes a outros filos foram caracterizadas com a intenção de determinar a sua capacidade multitarefa e a permutabilidade interespécie em B. subtilis. Os resultados demonstram que SugC de Mycobacterium tubercolosis e MalK1 de Thermus thermophilus não são capazes de energizar os sistemas de transporte AraNPQ e MelEDC, na ausência de MsmX. Contudo, foi verificado que ambas as proteínas não acumulam nas células, muito provavelmente devido a diferenças na utilização de codões entre B. subtilis e M. tuberculosis e T. thermophilus. Adicionalmente, é demonstrado que das ATPases previamente identificadas, ABC_Bt (Bacillus thuringiensis), MsmK (Streptococcus pneumoniae) e ABC_Cd (Clostridioides difficile) são capazes de energizar o transportador MelEDC, enquanto que YurJ (B. subtilis), ABC_Sa (Staphylococcus aureus) e ABC_Syn (Synechocystis sp.) não. Para mapear os domínios funcionais de NBDs multitarefa, uma proteína quimérica MsmXMsmK foi construída e caracterizada. Os resultados demonstram que a quimera é incapaz de energizar os transportadores AraNPQ e MelEDC, mas que acumula na célula, sugerindo que os domínios fundidos não adotam uma conformação adequada resultando numa proteína inativa. Com recurso a um sistema de duplo-híbrido bacteriano (BACTH) em Escherichia coli, é adicionalmente demonstrado que YurJ parece não ser capaz de formar homodímeros nem heterodímeros com MsmX na ausência de TMDs. Além disso, os resultados indicam que MsmX apresenta uma forte interação com os TMDs AraP, MelD e GanP, enquanto que YurJ apenas o estabelece com AraPNogueira, Isabel Maria Godinho de SáRUNSousa, João Pedro Abreu2023-03-26T01:01:11Z2021-01-262021-02-232021-01-26T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10362/112288enginfo:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2024-03-11T04:55:57Zoai:run.unl.pt:10362/112288Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T03:42:09.619832Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
Characterization of bacterial multitask ATPases from Type I ABC transporters |
title |
Characterization of bacterial multitask ATPases from Type I ABC transporters |
spellingShingle |
Characterization of bacterial multitask ATPases from Type I ABC transporters Sousa, João Pedro Abreu 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 |
title_short |
Characterization of bacterial multitask ATPases from Type I ABC transporters |
title_full |
Characterization of bacterial multitask ATPases from Type I ABC transporters |
title_fullStr |
Characterization of bacterial multitask ATPases from Type I ABC transporters |
title_full_unstemmed |
Characterization of bacterial multitask ATPases from Type I ABC transporters |
title_sort |
Characterization of bacterial multitask ATPases from Type I ABC transporters |
author |
Sousa, João Pedro Abreu |
author_facet |
Sousa, João Pedro Abreu |
author_role |
author |
dc.contributor.none.fl_str_mv |
Nogueira, Isabel Maria Godinho de Sá RUN |
dc.contributor.author.fl_str_mv |
Sousa, João Pedro Abreu |
dc.subject.por.fl_str_mv |
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 |
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 |
description |
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. |
publishDate |
2021 |
dc.date.none.fl_str_mv |
2021-01-26 2021-02-23 2021-01-26T00:00:00Z 2023-03-26T01:01:11Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
format |
masterThesis |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/10362/112288 |
url |
http://hdl.handle.net/10362/112288 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
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openAccess |
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application/pdf |
dc.source.none.fl_str_mv |
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Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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RCAAP |
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RCAAP |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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