Heavy-metal resistance in Marinobacter aquaeolei 617 insights into copper resistance

Detalhes bibliográficos
Autor(a) principal: Nóbrega, Franklin Luzia de
Data de Publicação: 2009
Tipo de documento: Dissertação
Idioma: eng
Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
Texto Completo: http://hdl.handle.net/10362/5924
Resumo: Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Biotecnologia
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spelling Heavy-metal resistance in Marinobacter aquaeolei 617 insights into copper resistanceDissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em BiotecnologiaHeavy metal resistance in Marinobacter aquaeolei (Ma.aq) 617 in aerobic conditions was studied for three different ions, cadmium, cobalt and copper. The main aim of this work was the study of a putative copper resistance operon, copSRXAB, located in the chromosome of Marinobacter and the biochemical characterization of a unique copper binding protein CopX (proposed designation), associated with the copper resistance system. Growth under heavy metal ion stress was performed for those three heavy metals and the Minimum Inhibitory Concentration (MIC) / Maximum Tolerant Concentration (MTC) was determined using two different approaches, solid artificial sea water (ASW) plates and liquid ASW medium, supplemented with lactate and yeast extract, as carbon sources. The MIC/MTC of cadmium, cobalt and copper ions was found to be 200 μM, 4-6 mM, and 1.6 mM, respectively. These values classify Ma.aq strain 617 as cadmium, cobalt and copper resistant strain. Moreover, during the cobalt resistance studies we observed the production of an unknown protein or compound, which is proposed be a cobalamine containing protein and/or cobalamine itself. Under the scope of copper resistance, preliminary proteomics analysis of the Ma.aq periplasmic fraction was performed. CopX, identified by MALDI TOF-TOF mass spectrometry, was shown to be differentially expressed under copper stress. This demonstrated that the proposed copper operon, copSRXAB, has a role in the Ma.aq copper resistance. CopX was successfully heterologously expressed in Escherichia coli (Es.coli), and purified for the first time using usually two chromatographic steps (anionic exchangeand size exclusion) with a yield of 5.7 mg or 1.8 mg of purified CopX, per L of LB or M9 medium, respectively. Mass spectrometry Electron Spray Ionisation (ESI) and N-terminus analysis revealed that the signal peptide of CopX comprises 21 residues, and is efficiently processed by the Sec system of Es.coli. Biochemical characterization of CopX proved that it is a periplasmic monomeric type 1 copper protein, with a molecular weight of 17253.25 ± 0.30 Da, determined by mass spectrometry (ESI), that binds approximately 1 copper ion per polypeptide chain. The apparent molecular weight of CopX, 20.4 kDa, determined by size-exclusion chromatography does not depend on the ionic strength. Spectroscopic characterization showed that it presents an intense charge transfer (Scys – Cu ion) band at 440 nm and 580 nm and 720 nm. The extinction coefficient at 580 nm was found to be 3.8 mM-1cm-1, according to the copper content. CopX EPR spectrum is axial. The 15N HSQC NMR spectra of CopX confirms that it is folded, with 131 out of 147 backbone amide resonances identified, showing that it is amenable to NMR solution structure determination. CopX presents some unique features, such as, a ratio between A440nm and A580nm of 0.94 and a high hyperfine coupling constant, 170 G. Taking into account the biochemical properties, CopX is proposed to be part of a new class of the type 1 copper proteins, shown preliminarily for the first time to be associated with copper resistance.Faculdade de Ciências e TecnologiaPauleta, SofiaRUNNóbrega, Franklin Luzia de2011-07-13T10:43:07Z20092009-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10362/5924enginfo: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:RCAAP2024-03-11T03:36:51Zoai:run.unl.pt:10362/5924Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T03:16:37.183310Repositório 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 Heavy-metal resistance in Marinobacter aquaeolei 617 insights into copper resistance
title Heavy-metal resistance in Marinobacter aquaeolei 617 insights into copper resistance
spellingShingle Heavy-metal resistance in Marinobacter aquaeolei 617 insights into copper resistance
Nóbrega, Franklin Luzia de
title_short Heavy-metal resistance in Marinobacter aquaeolei 617 insights into copper resistance
title_full Heavy-metal resistance in Marinobacter aquaeolei 617 insights into copper resistance
title_fullStr Heavy-metal resistance in Marinobacter aquaeolei 617 insights into copper resistance
title_full_unstemmed Heavy-metal resistance in Marinobacter aquaeolei 617 insights into copper resistance
title_sort Heavy-metal resistance in Marinobacter aquaeolei 617 insights into copper resistance
author Nóbrega, Franklin Luzia de
author_facet Nóbrega, Franklin Luzia de
author_role author
dc.contributor.none.fl_str_mv Pauleta, Sofia
RUN
dc.contributor.author.fl_str_mv Nóbrega, Franklin Luzia de
description Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Biotecnologia
publishDate 2009
dc.date.none.fl_str_mv 2009
2009-01-01T00:00:00Z
2011-07-13T10:43:07Z
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/10362/5924
url http://hdl.handle.net/10362/5924
dc.language.iso.fl_str_mv eng
language eng
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Faculdade de Ciências e Tecnologia
publisher.none.fl_str_mv Faculdade de Ciências e Tecnologia
dc.source.none.fl_str_mv reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
instacron:RCAAP
instname_str Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
instacron_str RCAAP
institution RCAAP
reponame_str Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
collection Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
repository.name.fl_str_mv Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
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