The two-component system of a novel copper resistant operon of Marinobacter hydrocarbonoclasticus

Detalhes bibliográficos
Autor(a) principal: Pinto, Cecilia de Agrela
Data de Publicação: 2012
Tipo de documento: Dissertação
Idioma: eng
Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
Texto Completo: http://hdl.handle.net/10362/10062
Resumo: Dissertation for the Master’s Degree in Structural and Functional Biochemistry
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spelling The two-component system of a novel copper resistant operon of Marinobacter hydrocarbonoclasticusCopper toleranceResponse regulatorHistidine kinaseTwo-component systemMarinobacterDissertation for the Master’s Degree in Structural and Functional BiochemistryThe majority of bacterial heavy metal resistance systems are regulated by twocomponent signal transduction systems. Stimuli from the environment interact with the histidine kinase, which in turn activates the response regulator by phosphorylation. The effector domain of the response regulator then binds to DNA, eliciting the specific response. Analysis of the Marinobacter hydrocarbonoclasticus genome revealed the presence of genes, copXAB, that code for proteins associated with copper response. The biochemical characterization of the two-component signal transduction system, copSR, is of interest due to the vital role it plays in the regulation of expression of the copXAB operon. The genes that encode for the CopR and CopS_C (cytosolic sensor domain of CopS) proteins were heterologously expressed in E.coli and expression was optimized for the production of soluble protein using LB medium. Due to solubility problems, the genes that code for these proteins were cloned as hexahistidine or glutathione S-transferase fusion proteins. CopR and its domains were optimally expressed at 16°C for 16 and 3 h after induction, respectively, whilst CopS_C was expressed at 37°C during 3 h after induction. Proteins were purified using different chromatographic strategies, most of them using affinity chromatography. The yields of pure protein per liter of growth culture obtained after complete purification from the soluble cellular extract were: 0.14 to 0.23 mg/L for CopR; 0.42 mg/L, CopR_NHis6; for the CopR_CHis6 it was 0.16 mg/L and 4.2 mg/L of CopS_C. The molecular mass of each protein was determined by gel filtration, 31 kDa for CopR, 17.5 kDa for CopR_NHis6, 15.1 kDa for CopR_CHis6 and 38.2 kDa for CopS_C. In the case of CopS_C there is the possibility that a dimer is formed, which should be evaluated. From the evaluation of disulfide bonds, using SDS PAGE and PAGE gels, all proteins or protein domains appeared to be monomers when in the presence of β-MEtOH. Circular dichroism evaluated the state of folding of the CopS_C and CopR proteins, which were shown to be folded in which the α-helix structures predominate. A model structure for CopR was also determined which agrees with this analysis. However, in the case of the CopR domains, the data obtained merely indicate folding, due to the low concentrations of the proteins. Phosphorylation and electrophoresis mobility shift assays of the CopR protein were, for the most part, inconclusive. However, in the absence of BSA, formation of the CopR:DNA complex in a gel filtration column is observed, though requires additional evaluation.Faculdade de Ciências e TecnologiaPauleta, SofiaRUNPinto, Cecilia de Agrela2013-07-09T10:12:08Z20122012-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10362/10062enginfo: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:RCAAP2024-03-11T03:43:31Zoai:run.unl.pt:10362/10062Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T03:19:12.518466Repositório 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 The two-component system of a novel copper resistant operon of Marinobacter hydrocarbonoclasticus
title The two-component system of a novel copper resistant operon of Marinobacter hydrocarbonoclasticus
spellingShingle The two-component system of a novel copper resistant operon of Marinobacter hydrocarbonoclasticus
Pinto, Cecilia de Agrela
Copper tolerance
Response regulator
Histidine kinase
Two-component system
Marinobacter
title_short The two-component system of a novel copper resistant operon of Marinobacter hydrocarbonoclasticus
title_full The two-component system of a novel copper resistant operon of Marinobacter hydrocarbonoclasticus
title_fullStr The two-component system of a novel copper resistant operon of Marinobacter hydrocarbonoclasticus
title_full_unstemmed The two-component system of a novel copper resistant operon of Marinobacter hydrocarbonoclasticus
title_sort The two-component system of a novel copper resistant operon of Marinobacter hydrocarbonoclasticus
author Pinto, Cecilia de Agrela
author_facet Pinto, Cecilia de Agrela
author_role author
dc.contributor.none.fl_str_mv Pauleta, Sofia
RUN
dc.contributor.author.fl_str_mv Pinto, Cecilia de Agrela
dc.subject.por.fl_str_mv Copper tolerance
Response regulator
Histidine kinase
Two-component system
Marinobacter
topic Copper tolerance
Response regulator
Histidine kinase
Two-component system
Marinobacter
description Dissertation for the Master’s Degree in Structural and Functional Biochemistry
publishDate 2012
dc.date.none.fl_str_mv 2012
2012-01-01T00:00:00Z
2013-07-09T10:12:08Z
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/10362/10062
url http://hdl.handle.net/10362/10062
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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