A sweet new set of inducible and constitutive promoters in Haloferax volcanii
- Autores
- Rados, Theopi; Andre, Katherine; Cerletti, Micaela; Bisson, Alex
- Año de publicación
- 2023
- Idioma
- inglés
- Tipo de recurso
- artículo
- Estado
- versión publicada
- Descripción
- Inducible promoters are one of cellular and molecular biology’s most important technical tools. The ability to deplete, replete, and overexpress genes on demand is the foundation of most functional studies. Here, we developed and characterized a new xylose-responsive promoter (Pxyl), the second inducible promoter system for the model haloarcheon Haloferax volcanii. Generating RNA-seq datasets from cultures in the presence of four historically used inducers (arabinose, xylose, maltose, and IPTG), we mapped upregulated genomic regions primarily repressed in the absence of the above inducers. We found a highly upregulated promoter that controls the expression of the xacEA (HVO_B0027-28) operon in the pHV3 chromosome. To characterize this promoter region, we cloned msfGFP (monomeric superfold green fluorescent protein) under the control of two upstream regions into a modified pTA962 vector: the first 250 bp (P250) and the whole 750 bp intergenic fragments (P750). The P250 sequence drove the expression of msfGFP constitutively, and its expression did not respond to the presence or absence of xylose. However, the P750 promoter showed not only to be repressed in the absence of xylose but also expressed higher levels of msfGFP than the previously described inducible promoter PtnaA in the presence of the inducer. Finally, we validated the inducible Pxyl promoter by reproducing morphological phenotypes already described in the literature. By overexpressing the tubulin-like FtsZ1 and FtsZ2, we observed similar but slightly more pronounced morphological defects than the tryptophan-inducible promoter PtnaA. FtsZ1 overexpression created larger, deformed cells, whereas cells overexpressing FtsZ2 were smaller but mostly retained their shape. In summary, this work contributes a new xylose-inducible promoter that could be used simultaneously with the well-established PtnaA in functional studies in H. volcanii in the future.
Fil: Rados, Theopi. Brandeis University; Estados Unidos
Fil: Andre, Katherine. Brandeis University; Estados Unidos
Fil: Cerletti, Micaela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina. Brandeis University; Estados Unidos
Fil: Bisson, Alex. Brandeis University; Estados Unidos - Materia
-
ARCHAEA
CONSTITUTIVE PROMOTERS
HALOARCHAEA
HALOFERAX VOLCANII
INDUCIBLE PROMOTERS
XYLOSE-INDUCIBLE PROMOTER - Nivel de accesibilidad
- acceso abierto
- Condiciones de uso
- https://creativecommons.org/licenses/by/2.5/ar/
- Repositorio
- Institución
- Consejo Nacional de Investigaciones Científicas y Técnicas
- OAI Identificador
- oai:ri.conicet.gov.ar:11336/216989
Ver los metadatos del registro completo
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CONICETDig_ede3b51f6661bfa8ac2376ee01298c38 |
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oai:ri.conicet.gov.ar:11336/216989 |
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3498 |
network_name_str |
CONICET Digital (CONICET) |
spelling |
A sweet new set of inducible and constitutive promoters in Haloferax volcaniiRados, TheopiAndre, KatherineCerletti, MicaelaBisson, AlexARCHAEACONSTITUTIVE PROMOTERSHALOARCHAEAHALOFERAX VOLCANIIINDUCIBLE PROMOTERSXYLOSE-INDUCIBLE PROMOTERhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Inducible promoters are one of cellular and molecular biology’s most important technical tools. The ability to deplete, replete, and overexpress genes on demand is the foundation of most functional studies. Here, we developed and characterized a new xylose-responsive promoter (Pxyl), the second inducible promoter system for the model haloarcheon Haloferax volcanii. Generating RNA-seq datasets from cultures in the presence of four historically used inducers (arabinose, xylose, maltose, and IPTG), we mapped upregulated genomic regions primarily repressed in the absence of the above inducers. We found a highly upregulated promoter that controls the expression of the xacEA (HVO_B0027-28) operon in the pHV3 chromosome. To characterize this promoter region, we cloned msfGFP (monomeric superfold green fluorescent protein) under the control of two upstream regions into a modified pTA962 vector: the first 250 bp (P250) and the whole 750 bp intergenic fragments (P750). The P250 sequence drove the expression of msfGFP constitutively, and its expression did not respond to the presence or absence of xylose. However, the P750 promoter showed not only to be repressed in the absence of xylose but also expressed higher levels of msfGFP than the previously described inducible promoter PtnaA in the presence of the inducer. Finally, we validated the inducible Pxyl promoter by reproducing morphological phenotypes already described in the literature. By overexpressing the tubulin-like FtsZ1 and FtsZ2, we observed similar but slightly more pronounced morphological defects than the tryptophan-inducible promoter PtnaA. FtsZ1 overexpression created larger, deformed cells, whereas cells overexpressing FtsZ2 were smaller but mostly retained their shape. In summary, this work contributes a new xylose-inducible promoter that could be used simultaneously with the well-established PtnaA in functional studies in H. volcanii in the future.Fil: Rados, Theopi. Brandeis University; Estados UnidosFil: Andre, Katherine. Brandeis University; Estados UnidosFil: Cerletti, Micaela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina. Brandeis University; Estados UnidosFil: Bisson, Alex. Brandeis University; Estados UnidosFrontiers Media2023-08info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/216989Rados, Theopi; Andre, Katherine; Cerletti, Micaela; Bisson, Alex; A sweet new set of inducible and constitutive promoters in Haloferax volcanii; Frontiers Media; Frontiers in Microbiology; 14; 8-2023; 1-91664-302XCONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://www.frontiersin.org/articles/10.3389/fmicb.2023.1204876/fullinfo:eu-repo/semantics/altIdentifier/doi/10.3389/fmicb.2023.1204876info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T10:22:22Zoai:ri.conicet.gov.ar:11336/216989instacron: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 10:22:22.646CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse |
dc.title.none.fl_str_mv |
A sweet new set of inducible and constitutive promoters in Haloferax volcanii |
title |
A sweet new set of inducible and constitutive promoters in Haloferax volcanii |
spellingShingle |
A sweet new set of inducible and constitutive promoters in Haloferax volcanii Rados, Theopi ARCHAEA CONSTITUTIVE PROMOTERS HALOARCHAEA HALOFERAX VOLCANII INDUCIBLE PROMOTERS XYLOSE-INDUCIBLE PROMOTER |
title_short |
A sweet new set of inducible and constitutive promoters in Haloferax volcanii |
title_full |
A sweet new set of inducible and constitutive promoters in Haloferax volcanii |
title_fullStr |
A sweet new set of inducible and constitutive promoters in Haloferax volcanii |
title_full_unstemmed |
A sweet new set of inducible and constitutive promoters in Haloferax volcanii |
title_sort |
A sweet new set of inducible and constitutive promoters in Haloferax volcanii |
dc.creator.none.fl_str_mv |
Rados, Theopi Andre, Katherine Cerletti, Micaela Bisson, Alex |
author |
Rados, Theopi |
author_facet |
Rados, Theopi Andre, Katherine Cerletti, Micaela Bisson, Alex |
author_role |
author |
author2 |
Andre, Katherine Cerletti, Micaela Bisson, Alex |
author2_role |
author author author |
dc.subject.none.fl_str_mv |
ARCHAEA CONSTITUTIVE PROMOTERS HALOARCHAEA HALOFERAX VOLCANII INDUCIBLE PROMOTERS XYLOSE-INDUCIBLE PROMOTER |
topic |
ARCHAEA CONSTITUTIVE PROMOTERS HALOARCHAEA HALOFERAX VOLCANII INDUCIBLE PROMOTERS XYLOSE-INDUCIBLE PROMOTER |
purl_subject.fl_str_mv |
https://purl.org/becyt/ford/1.6 https://purl.org/becyt/ford/1 |
dc.description.none.fl_txt_mv |
Inducible promoters are one of cellular and molecular biology’s most important technical tools. The ability to deplete, replete, and overexpress genes on demand is the foundation of most functional studies. Here, we developed and characterized a new xylose-responsive promoter (Pxyl), the second inducible promoter system for the model haloarcheon Haloferax volcanii. Generating RNA-seq datasets from cultures in the presence of four historically used inducers (arabinose, xylose, maltose, and IPTG), we mapped upregulated genomic regions primarily repressed in the absence of the above inducers. We found a highly upregulated promoter that controls the expression of the xacEA (HVO_B0027-28) operon in the pHV3 chromosome. To characterize this promoter region, we cloned msfGFP (monomeric superfold green fluorescent protein) under the control of two upstream regions into a modified pTA962 vector: the first 250 bp (P250) and the whole 750 bp intergenic fragments (P750). The P250 sequence drove the expression of msfGFP constitutively, and its expression did not respond to the presence or absence of xylose. However, the P750 promoter showed not only to be repressed in the absence of xylose but also expressed higher levels of msfGFP than the previously described inducible promoter PtnaA in the presence of the inducer. Finally, we validated the inducible Pxyl promoter by reproducing morphological phenotypes already described in the literature. By overexpressing the tubulin-like FtsZ1 and FtsZ2, we observed similar but slightly more pronounced morphological defects than the tryptophan-inducible promoter PtnaA. FtsZ1 overexpression created larger, deformed cells, whereas cells overexpressing FtsZ2 were smaller but mostly retained their shape. In summary, this work contributes a new xylose-inducible promoter that could be used simultaneously with the well-established PtnaA in functional studies in H. volcanii in the future. Fil: Rados, Theopi. Brandeis University; Estados Unidos Fil: Andre, Katherine. Brandeis University; Estados Unidos Fil: Cerletti, Micaela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina. Brandeis University; Estados Unidos Fil: Bisson, Alex. Brandeis University; Estados Unidos |
description |
Inducible promoters are one of cellular and molecular biology’s most important technical tools. The ability to deplete, replete, and overexpress genes on demand is the foundation of most functional studies. Here, we developed and characterized a new xylose-responsive promoter (Pxyl), the second inducible promoter system for the model haloarcheon Haloferax volcanii. Generating RNA-seq datasets from cultures in the presence of four historically used inducers (arabinose, xylose, maltose, and IPTG), we mapped upregulated genomic regions primarily repressed in the absence of the above inducers. We found a highly upregulated promoter that controls the expression of the xacEA (HVO_B0027-28) operon in the pHV3 chromosome. To characterize this promoter region, we cloned msfGFP (monomeric superfold green fluorescent protein) under the control of two upstream regions into a modified pTA962 vector: the first 250 bp (P250) and the whole 750 bp intergenic fragments (P750). The P250 sequence drove the expression of msfGFP constitutively, and its expression did not respond to the presence or absence of xylose. However, the P750 promoter showed not only to be repressed in the absence of xylose but also expressed higher levels of msfGFP than the previously described inducible promoter PtnaA in the presence of the inducer. Finally, we validated the inducible Pxyl promoter by reproducing morphological phenotypes already described in the literature. By overexpressing the tubulin-like FtsZ1 and FtsZ2, we observed similar but slightly more pronounced morphological defects than the tryptophan-inducible promoter PtnaA. FtsZ1 overexpression created larger, deformed cells, whereas cells overexpressing FtsZ2 were smaller but mostly retained their shape. In summary, this work contributes a new xylose-inducible promoter that could be used simultaneously with the well-established PtnaA in functional studies in H. volcanii in the future. |
publishDate |
2023 |
dc.date.none.fl_str_mv |
2023-08 |
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/11336/216989 Rados, Theopi; Andre, Katherine; Cerletti, Micaela; Bisson, Alex; A sweet new set of inducible and constitutive promoters in Haloferax volcanii; Frontiers Media; Frontiers in Microbiology; 14; 8-2023; 1-9 1664-302X CONICET Digital CONICET |
url |
http://hdl.handle.net/11336/216989 |
identifier_str_mv |
Rados, Theopi; Andre, Katherine; Cerletti, Micaela; Bisson, Alex; A sweet new set of inducible and constitutive promoters in Haloferax volcanii; Frontiers Media; Frontiers in Microbiology; 14; 8-2023; 1-9 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/articles/10.3389/fmicb.2023.1204876/full info:eu-repo/semantics/altIdentifier/doi/10.3389/fmicb.2023.1204876 |
dc.rights.none.fl_str_mv |
info:eu-repo/semantics/openAccess https://creativecommons.org/licenses/by/2.5/ar/ |
eu_rights_str_mv |
openAccess |
rights_invalid_str_mv |
https://creativecommons.org/licenses/by/2.5/ar/ |
dc.format.none.fl_str_mv |
application/pdf application/pdf |
dc.publisher.none.fl_str_mv |
Frontiers Media |
publisher.none.fl_str_mv |
Frontiers Media |
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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score |
13.070432 |