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A novel pair of inducible expression vectors for use in Methylobacterium extorquens

BACKGROUND: Due to the ever increasing use of diverse microbial taxa in basic research and industrial settings, there is a growing need for genetic tools to alter the physiology of these organisms. In particular, there is a dearth of inducible expression systems available for bacteria outside common...

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Autores principales: Chubiz, Lon M, Purswani, Jessica, Carroll, Sean Michael, Marx, Chistopher J
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3694467/
https://www.ncbi.nlm.nih.gov/pubmed/23648175
http://dx.doi.org/10.1186/1756-0500-6-183
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author Chubiz, Lon M
Purswani, Jessica
Carroll, Sean Michael
Marx, Chistopher J
author_facet Chubiz, Lon M
Purswani, Jessica
Carroll, Sean Michael
Marx, Chistopher J
author_sort Chubiz, Lon M
collection PubMed
description BACKGROUND: Due to the ever increasing use of diverse microbial taxa in basic research and industrial settings, there is a growing need for genetic tools to alter the physiology of these organisms. In particular, there is a dearth of inducible expression systems available for bacteria outside commonly used γ-proteobacteria, such as Escherichia coli or Pseudomonas species. To this end, we have sought to develop a pair of inducible expression vectors for use in the α-proteobacterium Methylobacterium extorquens, a model methylotroph. FINDINGS: We found that the P (R) promoter from rhizobial phage 16-3 was active in M. extorquens and engineered the promoter to be inducible by either p-isopropyl benzoate (cumate) or anhydrotetracycline. These hybrid promoters, P (R/cmtO) and P (R/tetO), were found to have high levels of expression in M. extorquens with a regulatory range of 10-fold and 30-fold, respectively. Compared to an existing cumate-inducible (10-fold range), high-level expression system for M. extorquens, P (R/cmtO) and P (R/tetO) have 33% of the maximal activity but were able to repress gene expression 3 and 8-fold greater, respectively. Both promoters were observed to exhibit homogeneous, titratable activation dynamics rather than on-off, switch-like behavior. The utility of these promoters was further demonstrated by complementing loss of function of ftfL - essential for growth on methanol - where we show P (R/tetO) is capable of not only fully complementing function but also producing a conditional null phenotype. These promoters have been incorporated into a broad-host-range backbone allowing for potential use in a variety of bacterial hosts. CONCLUSIONS: We have developed two novel expression systems for use in M. extorquens. The expression range of these vectors should allow for increased ability to explore cellular physiology in M. extorquens. Further, the P (R/tetO) promoter is capable of producing conditional null phenotypes, previously unattainable in M. extorquens. As both expression systems rely on the use of membrane permeable inducers, we suspect these expression vectors will be useful for ectopic gene expression in numerous proteobacteria.
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spelling pubmed-36944672013-06-28 A novel pair of inducible expression vectors for use in Methylobacterium extorquens Chubiz, Lon M Purswani, Jessica Carroll, Sean Michael Marx, Chistopher J BMC Res Notes Technical Note BACKGROUND: Due to the ever increasing use of diverse microbial taxa in basic research and industrial settings, there is a growing need for genetic tools to alter the physiology of these organisms. In particular, there is a dearth of inducible expression systems available for bacteria outside commonly used γ-proteobacteria, such as Escherichia coli or Pseudomonas species. To this end, we have sought to develop a pair of inducible expression vectors for use in the α-proteobacterium Methylobacterium extorquens, a model methylotroph. FINDINGS: We found that the P (R) promoter from rhizobial phage 16-3 was active in M. extorquens and engineered the promoter to be inducible by either p-isopropyl benzoate (cumate) or anhydrotetracycline. These hybrid promoters, P (R/cmtO) and P (R/tetO), were found to have high levels of expression in M. extorquens with a regulatory range of 10-fold and 30-fold, respectively. Compared to an existing cumate-inducible (10-fold range), high-level expression system for M. extorquens, P (R/cmtO) and P (R/tetO) have 33% of the maximal activity but were able to repress gene expression 3 and 8-fold greater, respectively. Both promoters were observed to exhibit homogeneous, titratable activation dynamics rather than on-off, switch-like behavior. The utility of these promoters was further demonstrated by complementing loss of function of ftfL - essential for growth on methanol - where we show P (R/tetO) is capable of not only fully complementing function but also producing a conditional null phenotype. These promoters have been incorporated into a broad-host-range backbone allowing for potential use in a variety of bacterial hosts. CONCLUSIONS: We have developed two novel expression systems for use in M. extorquens. The expression range of these vectors should allow for increased ability to explore cellular physiology in M. extorquens. Further, the P (R/tetO) promoter is capable of producing conditional null phenotypes, previously unattainable in M. extorquens. As both expression systems rely on the use of membrane permeable inducers, we suspect these expression vectors will be useful for ectopic gene expression in numerous proteobacteria. BioMed Central 2013-05-06 /pmc/articles/PMC3694467/ /pubmed/23648175 http://dx.doi.org/10.1186/1756-0500-6-183 Text en Copyright © 2013 Chubiz et al.; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Technical Note
Chubiz, Lon M
Purswani, Jessica
Carroll, Sean Michael
Marx, Chistopher J
A novel pair of inducible expression vectors for use in Methylobacterium extorquens
title A novel pair of inducible expression vectors for use in Methylobacterium extorquens
title_full A novel pair of inducible expression vectors for use in Methylobacterium extorquens
title_fullStr A novel pair of inducible expression vectors for use in Methylobacterium extorquens
title_full_unstemmed A novel pair of inducible expression vectors for use in Methylobacterium extorquens
title_short A novel pair of inducible expression vectors for use in Methylobacterium extorquens
title_sort novel pair of inducible expression vectors for use in methylobacterium extorquens
topic Technical Note
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3694467/
https://www.ncbi.nlm.nih.gov/pubmed/23648175
http://dx.doi.org/10.1186/1756-0500-6-183
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