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
- Institución
- Consejo Nacional de Investigaciones Científicas y Técnicas
- OAI Identificador
- oai:ri.conicet.gov.ar:11336/128460
Ver los metadatos del registro completo
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CONICET Digital (CONICET) |
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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 |
_version_ |
1844613865673850880 |
score |
13.070432 |