Cloning and heterologous overexpression of three gap genes encoding different glyceraldehyde-3-phosphate dehydrogenases from the plant pathogenic bacterium Pseudomonas syringae pv. tomato strain DC3000

The gammaproteobacterium Pseudomonas syringae pv. tomato DC3000 is the causal agent of bacterial speck, a common disease of tomato. The mode of infection of this pathogen is not well understood, but according to molecular biological, genomic and proteomic data it produces a number of proteins that m...

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Autores: Elkhalfi, Bouchra, Araya, José Miguel, Rodríguez Castro, Jorge, Rey, Manuel, Soukri, Abdelaziz, Serrano Delgado, Aurelio
Tipo de recurso: artículo
Estado:Versión aceptada para publicación
Fecha de publicación:2013
País:España
Institución:Universidad de Sevilla (US)
Repositorio:idUS. Depósito de Investigación de la Universidad de Sevilla
OAI Identifier:oai:idus.us.es:11441/85064
Acceso en línea:https://hdl.handle.net/11441/85064
https://doi.org/10.1016/j.pep.2013.02.005
Access Level:acceso abierto
Palabra clave:Pseudomonas syringae pv tomato DC3000
Glyceraldehyde-3-phosphate dehydrogenase
GAPDH
Paralogous gapgenes
Heterologous protein
Optimized overexpression
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spelling Cloning and heterologous overexpression of three gap genes encoding different glyceraldehyde-3-phosphate dehydrogenases from the plant pathogenic bacterium Pseudomonas syringae pv. tomato strain DC3000Elkhalfi, BouchraAraya, José MiguelRodríguez Castro, JorgeRey, ManuelSoukri, AbdelazizSerrano Delgado, AurelioPseudomonas syringae pv tomato DC3000Glyceraldehyde-3-phosphate dehydrogenaseGAPDHParalogous gapgenesHeterologous proteinOptimized overexpressionThe gammaproteobacterium Pseudomonas syringae pv. tomato DC3000 is the causal agent of bacterial speck, a common disease of tomato. The mode of infection of this pathogen is not well understood, but according to molecular biological, genomic and proteomic data it produces a number of proteins that may promote infection and draw nutrients from the plant. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a major enzyme of carbon metabolism that was reported to be a surface antigen and virulence factor in other pathogenic microorganisms, but its possible role in the infection process of P. syringae has so far not been studied. Whole-genome sequence analyses revealed the occurrence in this phytopathogenic bacterium of three paralogous gap genes encoding distinct GAPDHs, namely two class I enzymes having different molecular mass subunits and one class III bifunctional D-erythrose-4-phosphate dehydrogenase/GAPDH enzyme. By using genome bioinformatics data, as well as alignments of both DNA and deduced protein sequences, the three gap genes of P. syringae were one-step cloned with a His-Tag in pET21a vector using a PCR-based strategy, and its expression optimized in E. coli BL21 to achieve high yield of the heterologous proteins. In accordance with their distinct molecular phylogenies, these bacterial gap genes encode functional GAPDHs of diverse molecular masses and nicotinamide-coenzyme specificities, suggesting specific metabolic and/or cellular roles.Agencia Española de Cooperación Internacional y Desarrollo (MAEC) A1/043076/11 A/030965/10Junta de Andalucía BIO-261Academic PressBioquímica Vegetal y Biología Molecular2013info:eu-repo/semantics/articleinfo:eu-repo/semantics/acceptedVersionapplication/pdfapplication/pdfhttps://hdl.handle.net/11441/85064https://doi.org/10.1016/j.pep.2013.02.005reponame:idUS. Depósito de Investigación de la Universidad de Sevillainstname:Universidad de Sevilla (US)InglésProtein Expression and Purification, 89, 146-155.A1/043076/11A/030965/10BIO-261http://dx.doi.org/10.1016/j.pep.2013.02.005info:eu-repo/semantics/openAccessoai:idus.us.es:11441/850642026-06-17T12:51:07Z
dc.title.none.fl_str_mv Cloning and heterologous overexpression of three gap genes encoding different glyceraldehyde-3-phosphate dehydrogenases from the plant pathogenic bacterium Pseudomonas syringae pv. tomato strain DC3000
title Cloning and heterologous overexpression of three gap genes encoding different glyceraldehyde-3-phosphate dehydrogenases from the plant pathogenic bacterium Pseudomonas syringae pv. tomato strain DC3000
spellingShingle Cloning and heterologous overexpression of three gap genes encoding different glyceraldehyde-3-phosphate dehydrogenases from the plant pathogenic bacterium Pseudomonas syringae pv. tomato strain DC3000
Elkhalfi, Bouchra
Pseudomonas syringae pv tomato DC3000
Glyceraldehyde-3-phosphate dehydrogenase
GAPDH
Paralogous gapgenes
Heterologous protein
Optimized overexpression
title_short Cloning and heterologous overexpression of three gap genes encoding different glyceraldehyde-3-phosphate dehydrogenases from the plant pathogenic bacterium Pseudomonas syringae pv. tomato strain DC3000
title_full Cloning and heterologous overexpression of three gap genes encoding different glyceraldehyde-3-phosphate dehydrogenases from the plant pathogenic bacterium Pseudomonas syringae pv. tomato strain DC3000
title_fullStr Cloning and heterologous overexpression of three gap genes encoding different glyceraldehyde-3-phosphate dehydrogenases from the plant pathogenic bacterium Pseudomonas syringae pv. tomato strain DC3000
title_full_unstemmed Cloning and heterologous overexpression of three gap genes encoding different glyceraldehyde-3-phosphate dehydrogenases from the plant pathogenic bacterium Pseudomonas syringae pv. tomato strain DC3000
title_sort Cloning and heterologous overexpression of three gap genes encoding different glyceraldehyde-3-phosphate dehydrogenases from the plant pathogenic bacterium Pseudomonas syringae pv. tomato strain DC3000
dc.creator.none.fl_str_mv Elkhalfi, Bouchra
Araya, José Miguel
Rodríguez Castro, Jorge
Rey, Manuel
Soukri, Abdelaziz
Serrano Delgado, Aurelio
author Elkhalfi, Bouchra
author_facet Elkhalfi, Bouchra
Araya, José Miguel
Rodríguez Castro, Jorge
Rey, Manuel
Soukri, Abdelaziz
Serrano Delgado, Aurelio
author_role author
author2 Araya, José Miguel
Rodríguez Castro, Jorge
Rey, Manuel
Soukri, Abdelaziz
Serrano Delgado, Aurelio
author2_role author
author
author
author
author
dc.contributor.none.fl_str_mv Bioquímica Vegetal y Biología Molecular
dc.subject.none.fl_str_mv Pseudomonas syringae pv tomato DC3000
Glyceraldehyde-3-phosphate dehydrogenase
GAPDH
Paralogous gapgenes
Heterologous protein
Optimized overexpression
topic Pseudomonas syringae pv tomato DC3000
Glyceraldehyde-3-phosphate dehydrogenase
GAPDH
Paralogous gapgenes
Heterologous protein
Optimized overexpression
description The gammaproteobacterium Pseudomonas syringae pv. tomato DC3000 is the causal agent of bacterial speck, a common disease of tomato. The mode of infection of this pathogen is not well understood, but according to molecular biological, genomic and proteomic data it produces a number of proteins that may promote infection and draw nutrients from the plant. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a major enzyme of carbon metabolism that was reported to be a surface antigen and virulence factor in other pathogenic microorganisms, but its possible role in the infection process of P. syringae has so far not been studied. Whole-genome sequence analyses revealed the occurrence in this phytopathogenic bacterium of three paralogous gap genes encoding distinct GAPDHs, namely two class I enzymes having different molecular mass subunits and one class III bifunctional D-erythrose-4-phosphate dehydrogenase/GAPDH enzyme. By using genome bioinformatics data, as well as alignments of both DNA and deduced protein sequences, the three gap genes of P. syringae were one-step cloned with a His-Tag in pET21a vector using a PCR-based strategy, and its expression optimized in E. coli BL21 to achieve high yield of the heterologous proteins. In accordance with their distinct molecular phylogenies, these bacterial gap genes encode functional GAPDHs of diverse molecular masses and nicotinamide-coenzyme specificities, suggesting specific metabolic and/or cellular roles.
publishDate 2013
dc.date.none.fl_str_mv 2013
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/acceptedVersion
format article
status_str acceptedVersion
dc.identifier.none.fl_str_mv https://hdl.handle.net/11441/85064
https://doi.org/10.1016/j.pep.2013.02.005
url https://hdl.handle.net/11441/85064
https://doi.org/10.1016/j.pep.2013.02.005
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv Protein Expression and Purification, 89, 146-155.
A1/043076/11
A/030965/10
BIO-261
http://dx.doi.org/10.1016/j.pep.2013.02.005
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.publisher.none.fl_str_mv Academic Press
publisher.none.fl_str_mv Academic Press
dc.source.none.fl_str_mv reponame:idUS. Depósito de Investigación de la Universidad de Sevilla
instname:Universidad de Sevilla (US)
instname_str Universidad de Sevilla (US)
reponame_str idUS. Depósito de Investigación de la Universidad de Sevilla
collection idUS. Depósito de Investigación de la Universidad de Sevilla
repository.name.fl_str_mv
repository.mail.fl_str_mv
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