Combining modules for versatile and optimal labeling of lactic acid bacteria: Two pMV158-family promiscuous replicons, a pneumococcal system for constitutive or inducible gene expr...

Autores
Garay Novillo, Javier Nicolás; García-Morena, Diego; Ruiz-Masó, José Ángel; Barra, Jose Luis; del Solar, Gloria
Año de publicación
2019
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Labeling of bacterial cells with fluorescent proteins allows tracking the bacteria in competition and interactomic in vivo and in vitro studies. During the last years, a few plasmid vectors have been developed aimed at the fluorescent labeling of specific members of the lactic acid bacteria (LAB), a heterogeneous group that includes microorganisms used in the food industry, as probiotics, or as live vectors for mucosal vaccines. Successful and versatile labeling of a broad range of LAB not only requires a vector containing a promiscuous replicon and a widely recognized expression system for the constitutive or regulated expression of the fluorescence determinant, but also the knowledge of the main features of the entire plasmid/host/fluorescent protein ensemble. By using the LAB model species Lactococcus lactis, we have compared the utility properties of a set of labeling vectors constructed by combining a promiscuous replicon (pMV158 or pSH71) of the pMV158 plasmid family with the gene encoding either the EGFP or the mCherry fluorescent protein placed under control of promoter PX or PM from the pneumococcal mal gene cluster for maltosaccharide uptake and utilization, respectively. Some vectors carrying PM also harbor the malR gene, whose product represses transcription from this promoter, thus enabling maltose-inducible synthesis of the fluorescent proteins. We have determined the plasmid copy number (PCN) and segregational stability of the different constructs, as well as the effect of these features on the fitness and fluorescence intensity of the lactococcal host. Constructs based on the pSH71 replicon had a high copy number (~115) and were segregationally stable. The copy number of vectors based on the pMV158 replicon was lower (~8-45) and varied substantially depending on the genetic context of the plasmid and on the bacterial growth conditions; as a consequence, inheritance of these vectors was less stable. Synthesis of the fluorescent proteins encoded by these plasmids did not significantly decrease the host fitness. By employing inducible expression vectors, the fluorescent proteins were shown to be very stable in this bacterium. Importantly, conditions for accurate quantification of the emitted fluorescence were established based on the maturation times of the fluorescent proteins.
Fil: Garay Novillo, Javier Nicolás. Consejo Superior de Investigaciones Científicas; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina
Fil: García-Morena, Diego. Consejo Superior de Investigaciones Científicas; España
Fil: Ruiz-Masó, José Ángel. Consejo Superior de Investigaciones Científicas; España
Fil: Barra, Jose Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina
Fil: del Solar, Gloria. Consejo Superior de Investigaciones Científicas; España
Materia
FLUORESCENT LABELING VECTORS
FLUORESCENT PROTEIN MATURATION
FLUORESCENT PROTEIN STABILITY
LACTIC ACID BACTERIA
MCHERRY AND EGFP
PLASMID COPY NUMBER
PLASMID FITNESS COST
PLASMID STABILITY
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/128460

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network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Combining modules for versatile and optimal labeling of lactic acid bacteria: Two pMV158-family promiscuous replicons, a pneumococcal system for constitutive or inducible gene expression, and two fluorescent proteinsGaray Novillo, Javier NicolásGarcía-Morena, DiegoRuiz-Masó, José ÁngelBarra, Jose Luisdel Solar, GloriaFLUORESCENT LABELING VECTORSFLUORESCENT PROTEIN MATURATIONFLUORESCENT PROTEIN STABILITYLACTIC ACID BACTERIAMCHERRY AND EGFPPLASMID COPY NUMBERPLASMID FITNESS COSTPLASMID STABILITYhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Labeling of bacterial cells with fluorescent proteins allows tracking the bacteria in competition and interactomic in vivo and in vitro studies. During the last years, a few plasmid vectors have been developed aimed at the fluorescent labeling of specific members of the lactic acid bacteria (LAB), a heterogeneous group that includes microorganisms used in the food industry, as probiotics, or as live vectors for mucosal vaccines. Successful and versatile labeling of a broad range of LAB not only requires a vector containing a promiscuous replicon and a widely recognized expression system for the constitutive or regulated expression of the fluorescence determinant, but also the knowledge of the main features of the entire plasmid/host/fluorescent protein ensemble. By using the LAB model species Lactococcus lactis, we have compared the utility properties of a set of labeling vectors constructed by combining a promiscuous replicon (pMV158 or pSH71) of the pMV158 plasmid family with the gene encoding either the EGFP or the mCherry fluorescent protein placed under control of promoter PX or PM from the pneumococcal mal gene cluster for maltosaccharide uptake and utilization, respectively. Some vectors carrying PM also harbor the malR gene, whose product represses transcription from this promoter, thus enabling maltose-inducible synthesis of the fluorescent proteins. We have determined the plasmid copy number (PCN) and segregational stability of the different constructs, as well as the effect of these features on the fitness and fluorescence intensity of the lactococcal host. Constructs based on the pSH71 replicon had a high copy number (~115) and were segregationally stable. The copy number of vectors based on the pMV158 replicon was lower (~8-45) and varied substantially depending on the genetic context of the plasmid and on the bacterial growth conditions; as a consequence, inheritance of these vectors was less stable. Synthesis of the fluorescent proteins encoded by these plasmids did not significantly decrease the host fitness. By employing inducible expression vectors, the fluorescent proteins were shown to be very stable in this bacterium. Importantly, conditions for accurate quantification of the emitted fluorescence were established based on the maturation times of the fluorescent proteins.Fil: Garay Novillo, Javier Nicolás. Consejo Superior de Investigaciones Científicas; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: García-Morena, Diego. Consejo Superior de Investigaciones Científicas; EspañaFil: Ruiz-Masó, José Ángel. Consejo Superior de Investigaciones Científicas; EspañaFil: Barra, Jose Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: del Solar, Gloria. Consejo Superior de Investigaciones Científicas; EspañaFrontiers Media S.A.2019-06info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/128460Garay Novillo, Javier Nicolás; García-Morena, Diego; Ruiz-Masó, José Ángel; Barra, Jose Luis; del Solar, Gloria; Combining modules for versatile and optimal labeling of lactic acid bacteria: Two pMV158-family promiscuous replicons, a pneumococcal system for constitutive or inducible gene expression, and two fluorescent proteins; Frontiers Media S.A.; Frontiers in Microbiology; 10; 6-2019; 1-221664-302XCONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://www.frontiersin.org/article/10.3389/fmicb.2019.01431/fullinfo:eu-repo/semantics/altIdentifier/doi/10.3389/fmicb.2019.01431info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T10:04:08Zoai:ri.conicet.gov.ar:11336/128460instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-29 10:04:08.991CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Combining modules for versatile and optimal labeling of lactic acid bacteria: Two pMV158-family promiscuous replicons, a pneumococcal system for constitutive or inducible gene expression, and two fluorescent proteins
title Combining modules for versatile and optimal labeling of lactic acid bacteria: Two pMV158-family promiscuous replicons, a pneumococcal system for constitutive or inducible gene expression, and two fluorescent proteins
spellingShingle Combining modules for versatile and optimal labeling of lactic acid bacteria: Two pMV158-family promiscuous replicons, a pneumococcal system for constitutive or inducible gene expression, and two fluorescent proteins
Garay Novillo, Javier Nicolás
FLUORESCENT LABELING VECTORS
FLUORESCENT PROTEIN MATURATION
FLUORESCENT PROTEIN STABILITY
LACTIC ACID BACTERIA
MCHERRY AND EGFP
PLASMID COPY NUMBER
PLASMID FITNESS COST
PLASMID STABILITY
title_short Combining modules for versatile and optimal labeling of lactic acid bacteria: Two pMV158-family promiscuous replicons, a pneumococcal system for constitutive or inducible gene expression, and two fluorescent proteins
title_full Combining modules for versatile and optimal labeling of lactic acid bacteria: Two pMV158-family promiscuous replicons, a pneumococcal system for constitutive or inducible gene expression, and two fluorescent proteins
title_fullStr Combining modules for versatile and optimal labeling of lactic acid bacteria: Two pMV158-family promiscuous replicons, a pneumococcal system for constitutive or inducible gene expression, and two fluorescent proteins
title_full_unstemmed Combining modules for versatile and optimal labeling of lactic acid bacteria: Two pMV158-family promiscuous replicons, a pneumococcal system for constitutive or inducible gene expression, and two fluorescent proteins
title_sort Combining modules for versatile and optimal labeling of lactic acid bacteria: Two pMV158-family promiscuous replicons, a pneumococcal system for constitutive or inducible gene expression, and two fluorescent proteins
dc.creator.none.fl_str_mv Garay Novillo, Javier Nicolás
García-Morena, Diego
Ruiz-Masó, José Ángel
Barra, Jose Luis
del Solar, Gloria
author Garay Novillo, Javier Nicolás
author_facet Garay Novillo, Javier Nicolás
García-Morena, Diego
Ruiz-Masó, José Ángel
Barra, Jose Luis
del Solar, Gloria
author_role author
author2 García-Morena, Diego
Ruiz-Masó, José Ángel
Barra, Jose Luis
del Solar, Gloria
author2_role author
author
author
author
dc.subject.none.fl_str_mv FLUORESCENT LABELING VECTORS
FLUORESCENT PROTEIN MATURATION
FLUORESCENT PROTEIN STABILITY
LACTIC ACID BACTERIA
MCHERRY AND EGFP
PLASMID COPY NUMBER
PLASMID FITNESS COST
PLASMID STABILITY
topic FLUORESCENT LABELING VECTORS
FLUORESCENT PROTEIN MATURATION
FLUORESCENT PROTEIN STABILITY
LACTIC ACID BACTERIA
MCHERRY AND EGFP
PLASMID COPY NUMBER
PLASMID FITNESS COST
PLASMID STABILITY
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Labeling of bacterial cells with fluorescent proteins allows tracking the bacteria in competition and interactomic in vivo and in vitro studies. During the last years, a few plasmid vectors have been developed aimed at the fluorescent labeling of specific members of the lactic acid bacteria (LAB), a heterogeneous group that includes microorganisms used in the food industry, as probiotics, or as live vectors for mucosal vaccines. Successful and versatile labeling of a broad range of LAB not only requires a vector containing a promiscuous replicon and a widely recognized expression system for the constitutive or regulated expression of the fluorescence determinant, but also the knowledge of the main features of the entire plasmid/host/fluorescent protein ensemble. By using the LAB model species Lactococcus lactis, we have compared the utility properties of a set of labeling vectors constructed by combining a promiscuous replicon (pMV158 or pSH71) of the pMV158 plasmid family with the gene encoding either the EGFP or the mCherry fluorescent protein placed under control of promoter PX or PM from the pneumococcal mal gene cluster for maltosaccharide uptake and utilization, respectively. Some vectors carrying PM also harbor the malR gene, whose product represses transcription from this promoter, thus enabling maltose-inducible synthesis of the fluorescent proteins. We have determined the plasmid copy number (PCN) and segregational stability of the different constructs, as well as the effect of these features on the fitness and fluorescence intensity of the lactococcal host. Constructs based on the pSH71 replicon had a high copy number (~115) and were segregationally stable. The copy number of vectors based on the pMV158 replicon was lower (~8-45) and varied substantially depending on the genetic context of the plasmid and on the bacterial growth conditions; as a consequence, inheritance of these vectors was less stable. Synthesis of the fluorescent proteins encoded by these plasmids did not significantly decrease the host fitness. By employing inducible expression vectors, the fluorescent proteins were shown to be very stable in this bacterium. Importantly, conditions for accurate quantification of the emitted fluorescence were established based on the maturation times of the fluorescent proteins.
Fil: Garay Novillo, Javier Nicolás. Consejo Superior de Investigaciones Científicas; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina
Fil: García-Morena, Diego. Consejo Superior de Investigaciones Científicas; España
Fil: Ruiz-Masó, José Ángel. Consejo Superior de Investigaciones Científicas; España
Fil: Barra, Jose Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina
Fil: del Solar, Gloria. Consejo Superior de Investigaciones Científicas; España
description Labeling of bacterial cells with fluorescent proteins allows tracking the bacteria in competition and interactomic in vivo and in vitro studies. During the last years, a few plasmid vectors have been developed aimed at the fluorescent labeling of specific members of the lactic acid bacteria (LAB), a heterogeneous group that includes microorganisms used in the food industry, as probiotics, or as live vectors for mucosal vaccines. Successful and versatile labeling of a broad range of LAB not only requires a vector containing a promiscuous replicon and a widely recognized expression system for the constitutive or regulated expression of the fluorescence determinant, but also the knowledge of the main features of the entire plasmid/host/fluorescent protein ensemble. By using the LAB model species Lactococcus lactis, we have compared the utility properties of a set of labeling vectors constructed by combining a promiscuous replicon (pMV158 or pSH71) of the pMV158 plasmid family with the gene encoding either the EGFP or the mCherry fluorescent protein placed under control of promoter PX or PM from the pneumococcal mal gene cluster for maltosaccharide uptake and utilization, respectively. Some vectors carrying PM also harbor the malR gene, whose product represses transcription from this promoter, thus enabling maltose-inducible synthesis of the fluorescent proteins. We have determined the plasmid copy number (PCN) and segregational stability of the different constructs, as well as the effect of these features on the fitness and fluorescence intensity of the lactococcal host. Constructs based on the pSH71 replicon had a high copy number (~115) and were segregationally stable. The copy number of vectors based on the pMV158 replicon was lower (~8-45) and varied substantially depending on the genetic context of the plasmid and on the bacterial growth conditions; as a consequence, inheritance of these vectors was less stable. Synthesis of the fluorescent proteins encoded by these plasmids did not significantly decrease the host fitness. By employing inducible expression vectors, the fluorescent proteins were shown to be very stable in this bacterium. Importantly, conditions for accurate quantification of the emitted fluorescence were established based on the maturation times of the fluorescent proteins.
publishDate 2019
dc.date.none.fl_str_mv 2019-06
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/128460
Garay Novillo, Javier Nicolás; García-Morena, Diego; Ruiz-Masó, José Ángel; Barra, Jose Luis; del Solar, Gloria; Combining modules for versatile and optimal labeling of lactic acid bacteria: Two pMV158-family promiscuous replicons, a pneumococcal system for constitutive or inducible gene expression, and two fluorescent proteins; Frontiers Media S.A.; Frontiers in Microbiology; 10; 6-2019; 1-22
1664-302X
CONICET Digital
CONICET
url http://hdl.handle.net/11336/128460
identifier_str_mv Garay Novillo, Javier Nicolás; García-Morena, Diego; Ruiz-Masó, José Ángel; Barra, Jose Luis; del Solar, Gloria; Combining modules for versatile and optimal labeling of lactic acid bacteria: Two pMV158-family promiscuous replicons, a pneumococcal system for constitutive or inducible gene expression, and two fluorescent proteins; Frontiers Media S.A.; Frontiers in Microbiology; 10; 6-2019; 1-22
1664-302X
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/https://www.frontiersin.org/article/10.3389/fmicb.2019.01431/full
info:eu-repo/semantics/altIdentifier/doi/10.3389/fmicb.2019.01431
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.publisher.none.fl_str_mv Frontiers Media S.A.
publisher.none.fl_str_mv Frontiers Media S.A.
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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