Optimization of fluorescent tools for cell biology studies in Gram-positive bacteria.
Autor(a) principal: | |
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Data de Publicação: | 2014 |
Outros Autores: | , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/10400.18/3158 |
Resumo: | The understanding of how Gram-positive bacteria divide and ensure the correct localization of different molecular machineries, such as those involved in the synthesis of the bacterial cell surface, is crucial to design strategies to fight bacterial infections. In order to determine the correct subcellular localization of fluorescent proteins in Streptococcus pneumoniae, we have previously described tools to express derivatives of four fluorescent proteins, mCherry, Citrine, CFP and GFP, to levels that allow visualization by fluorescence microscopy, by fusing the first ten amino acids of the S. pneumoniae protein Wze (the i-tag), upstream of the fluorescent protein. Here, we report that these tools can also be used in other Gram-positive bacteria, namely Lactococcus lactis, Staphylococcus aureus and Bacillus subtilis, possibly due to optimized translation rates. Additionally, we have optimized the i-tag by testing the effect of the first ten amino acids of other pneumococcal proteins in the increased expression of the fluorescent protein Citrine. We found that manipulating the structure and stability of the 59 end of the mRNA molecule, which may influence the accessibility of the ribosome, is determinant to ensure the expression of a strong fluorescent signal. Introduction |
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Optimization of fluorescent tools for cell biology studies in Gram-positive bacteria.Gram-PositiveStreptococcus PneumoniaemCherryCitrineGFPThe understanding of how Gram-positive bacteria divide and ensure the correct localization of different molecular machineries, such as those involved in the synthesis of the bacterial cell surface, is crucial to design strategies to fight bacterial infections. In order to determine the correct subcellular localization of fluorescent proteins in Streptococcus pneumoniae, we have previously described tools to express derivatives of four fluorescent proteins, mCherry, Citrine, CFP and GFP, to levels that allow visualization by fluorescence microscopy, by fusing the first ten amino acids of the S. pneumoniae protein Wze (the i-tag), upstream of the fluorescent protein. Here, we report that these tools can also be used in other Gram-positive bacteria, namely Lactococcus lactis, Staphylococcus aureus and Bacillus subtilis, possibly due to optimized translation rates. Additionally, we have optimized the i-tag by testing the effect of the first ten amino acids of other pneumococcal proteins in the increased expression of the fluorescent protein Citrine. We found that manipulating the structure and stability of the 59 end of the mRNA molecule, which may influence the accessibility of the ribosome, is determinant to ensure the expression of a strong fluorescent signal. IntroductionThis study was funded by Fundação para a Ciência e Tecnologia (FCT), Lisbon, Portugal, through research grant PTDC/BIA-MIC/111817/2009 awarded to SRF. The work performed at Instituto de Tecnologia Quı´mica e Biolo´gica was supported additionally by FCT through grant PEstOE/EQB/LA0004/2011. MXH and MJC were supported by FCT fellowships SFRH/BD/43797/2008 and SFRH/BPD/77758/2011, respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Public Library of ScienceRepositório Científico do Instituto Nacional de SaúdeCatalão, MJFigueiredo, JHenriques, MXGomes, João PauloFilipe, SR2015-09-24T15:06:30Z2014-12-022014-12-02T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/10400.18/3158engPLoS One. 2014 Dec 2;9(12):e113796. doi: 10.1371/journal.pone.0113796. eCollection 20141932-620310.1371/journal.pone.0113796info: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:RCAAP2023-07-20T15:39:39Zoai:repositorio.insa.pt:10400.18/3158Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T18:38:05.702445Repositório 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 |
Optimization of fluorescent tools for cell biology studies in Gram-positive bacteria. |
title |
Optimization of fluorescent tools for cell biology studies in Gram-positive bacteria. |
spellingShingle |
Optimization of fluorescent tools for cell biology studies in Gram-positive bacteria. Catalão, MJ Gram-Positive Streptococcus Pneumoniae mCherry Citrine GFP |
title_short |
Optimization of fluorescent tools for cell biology studies in Gram-positive bacteria. |
title_full |
Optimization of fluorescent tools for cell biology studies in Gram-positive bacteria. |
title_fullStr |
Optimization of fluorescent tools for cell biology studies in Gram-positive bacteria. |
title_full_unstemmed |
Optimization of fluorescent tools for cell biology studies in Gram-positive bacteria. |
title_sort |
Optimization of fluorescent tools for cell biology studies in Gram-positive bacteria. |
author |
Catalão, MJ |
author_facet |
Catalão, MJ Figueiredo, J Henriques, MX Gomes, João Paulo Filipe, SR |
author_role |
author |
author2 |
Figueiredo, J Henriques, MX Gomes, João Paulo Filipe, SR |
author2_role |
author author author author |
dc.contributor.none.fl_str_mv |
Repositório Científico do Instituto Nacional de Saúde |
dc.contributor.author.fl_str_mv |
Catalão, MJ Figueiredo, J Henriques, MX Gomes, João Paulo Filipe, SR |
dc.subject.por.fl_str_mv |
Gram-Positive Streptococcus Pneumoniae mCherry Citrine GFP |
topic |
Gram-Positive Streptococcus Pneumoniae mCherry Citrine GFP |
description |
The understanding of how Gram-positive bacteria divide and ensure the correct localization of different molecular machineries, such as those involved in the synthesis of the bacterial cell surface, is crucial to design strategies to fight bacterial infections. In order to determine the correct subcellular localization of fluorescent proteins in Streptococcus pneumoniae, we have previously described tools to express derivatives of four fluorescent proteins, mCherry, Citrine, CFP and GFP, to levels that allow visualization by fluorescence microscopy, by fusing the first ten amino acids of the S. pneumoniae protein Wze (the i-tag), upstream of the fluorescent protein. Here, we report that these tools can also be used in other Gram-positive bacteria, namely Lactococcus lactis, Staphylococcus aureus and Bacillus subtilis, possibly due to optimized translation rates. Additionally, we have optimized the i-tag by testing the effect of the first ten amino acids of other pneumococcal proteins in the increased expression of the fluorescent protein Citrine. We found that manipulating the structure and stability of the 59 end of the mRNA molecule, which may influence the accessibility of the ribosome, is determinant to ensure the expression of a strong fluorescent signal. Introduction |
publishDate |
2014 |
dc.date.none.fl_str_mv |
2014-12-02 2014-12-02T00:00:00Z 2015-09-24T15:06:30Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/10400.18/3158 |
url |
http://hdl.handle.net/10400.18/3158 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
PLoS One. 2014 Dec 2;9(12):e113796. doi: 10.1371/journal.pone.0113796. eCollection 2014 1932-6203 10.1371/journal.pone.0113796 |
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 |
Public Library of Science |
publisher.none.fl_str_mv |
Public Library of Science |
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 |
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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 |
reponame_str |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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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 |
repository.mail.fl_str_mv |
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1817552666615611392 |