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A sweet new set of inducible and constitutive promoters in Haloferax volcanii

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 indu...

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Autores principales: Rados, Theopi, Andre, Katherine, Cerletti, Micaela, Bisson, Alex
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10448506/
https://www.ncbi.nlm.nih.gov/pubmed/37637112
http://dx.doi.org/10.3389/fmicb.2023.1204876
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author Rados, Theopi
Andre, Katherine
Cerletti, Micaela
Bisson, Alex
author_facet Rados, Theopi
Andre, Katherine
Cerletti, Micaela
Bisson, Alex
author_sort Rados, Theopi
collection PubMed
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.
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spelling pubmed-104485062023-08-25 A sweet new set of inducible and constitutive promoters in Haloferax volcanii Rados, Theopi Andre, Katherine Cerletti, Micaela Bisson, Alex Front Microbiol Microbiology 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. Frontiers Media S.A. 2023-08-10 /pmc/articles/PMC10448506/ /pubmed/37637112 http://dx.doi.org/10.3389/fmicb.2023.1204876 Text en Copyright © 2023 Rados, Andre, Cerletti and Bisson. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Microbiology
Rados, Theopi
Andre, Katherine
Cerletti, Micaela
Bisson, Alex
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
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_short A sweet new set of inducible and constitutive promoters in Haloferax volcanii
title_sort sweet new set of inducible and constitutive promoters in haloferax volcanii
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10448506/
https://www.ncbi.nlm.nih.gov/pubmed/37637112
http://dx.doi.org/10.3389/fmicb.2023.1204876
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