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Title: A New Suite of Plasmid Vectors for Fluorescence-Based Imaging of Root Colonizing Pseudomonads

Abstract

In the terrestrial ecosystem, plant-microbe symbiotic associations are ecologically and economically important processes. To better understand these associations at structural and functional levels, different molecular and biochemical tools are applied. In this study, we have constructed a suite of vectors that incorporates several new elements into the rhizosphere stable, broad-host vector pME6031. The new vectors are useful for studies requiring multi-color tagging and visualization of plant-associated, Gram negative bacterial strains such as Pseudomonas plant growth promotion and biocontrol strains. A number of genetic elements, including constitutive promoters and signal peptides that target secretion to the periplasm, have been evaluated. Several next generation fluorescent proteins, namely mTurquoise2, mNeonGreen, mRuby2, DsRed-Express2 and E2-Crimson have been incorporated into the vectors for whole cell labeling or protein tagging. Secretion of mTurquoise2 and mNeonGreen into the periplasm of Pseudomonas fluorescens SBW25 has also been demonstrated, providing a vehicle for tagging proteins in the periplasmic compartment. A higher copy number version of select plasmids has been produced by introduction of a previously described repA mutation, affording an increase in protein expression levels. The utility of these plasmids for fluorescence-based imaging is demonstrated by root colonization of Solanum lycopersicum seedlings by P. fluorescens SBW25 in a hydroponicmore » growth system. As a result, the plasmids are stably maintained during root colonization in the absence of selective pressure for more than two weeks.« less

Authors:
 [1];  [1];  [1];  [2];  [1];  [1];  [1]
  1. Argonne National Lab. (ANL), Argonne, IL (United States)
  2. Argonne National Lab. (ANL), Argonne, IL (United States); Illinois Institute of Technology, Chicago, IL (United States)
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
OSTI Identifier:
1421946
Grant/Contract Number:  
AC02-06CH11357
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Frontiers in Plant Science
Additional Journal Information:
Journal Volume: 8; Journal ID: ISSN 1664-462X
Publisher:
Frontiers Research Foundation
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; broad-host vector; fluorescent protein; rhizosphere; Pseudomonas; pVS1; plant growth promotion; biocontrol strain

Citation Formats

Wilton, Rosemarie, Ahrendt, Angela J., Shinde, Shalaka, Sholto-Douglas, Deirdre J., Johnson, Jessica L., Brennan, Melissa B., and Kemner, Kenneth M.. A New Suite of Plasmid Vectors for Fluorescence-Based Imaging of Root Colonizing Pseudomonads. United States: N. p., 2018. Web. doi:10.3389/fpls.2017.02242.
Wilton, Rosemarie, Ahrendt, Angela J., Shinde, Shalaka, Sholto-Douglas, Deirdre J., Johnson, Jessica L., Brennan, Melissa B., & Kemner, Kenneth M.. A New Suite of Plasmid Vectors for Fluorescence-Based Imaging of Root Colonizing Pseudomonads. United States. doi:10.3389/fpls.2017.02242.
Wilton, Rosemarie, Ahrendt, Angela J., Shinde, Shalaka, Sholto-Douglas, Deirdre J., Johnson, Jessica L., Brennan, Melissa B., and Kemner, Kenneth M.. Thu . "A New Suite of Plasmid Vectors for Fluorescence-Based Imaging of Root Colonizing Pseudomonads". United States. doi:10.3389/fpls.2017.02242. https://www.osti.gov/servlets/purl/1421946.
@article{osti_1421946,
title = {A New Suite of Plasmid Vectors for Fluorescence-Based Imaging of Root Colonizing Pseudomonads},
author = {Wilton, Rosemarie and Ahrendt, Angela J. and Shinde, Shalaka and Sholto-Douglas, Deirdre J. and Johnson, Jessica L. and Brennan, Melissa B. and Kemner, Kenneth M.},
abstractNote = {In the terrestrial ecosystem, plant-microbe symbiotic associations are ecologically and economically important processes. To better understand these associations at structural and functional levels, different molecular and biochemical tools are applied. In this study, we have constructed a suite of vectors that incorporates several new elements into the rhizosphere stable, broad-host vector pME6031. The new vectors are useful for studies requiring multi-color tagging and visualization of plant-associated, Gram negative bacterial strains such as Pseudomonas plant growth promotion and biocontrol strains. A number of genetic elements, including constitutive promoters and signal peptides that target secretion to the periplasm, have been evaluated. Several next generation fluorescent proteins, namely mTurquoise2, mNeonGreen, mRuby2, DsRed-Express2 and E2-Crimson have been incorporated into the vectors for whole cell labeling or protein tagging. Secretion of mTurquoise2 and mNeonGreen into the periplasm of Pseudomonas fluorescens SBW25 has also been demonstrated, providing a vehicle for tagging proteins in the periplasmic compartment. A higher copy number version of select plasmids has been produced by introduction of a previously described repA mutation, affording an increase in protein expression levels. The utility of these plasmids for fluorescence-based imaging is demonstrated by root colonization of Solanum lycopersicum seedlings by P. fluorescens SBW25 in a hydroponic growth system. As a result, the plasmids are stably maintained during root colonization in the absence of selective pressure for more than two weeks.},
doi = {10.3389/fpls.2017.02242},
journal = {Frontiers in Plant Science},
number = ,
volume = 8,
place = {United States},
year = {Thu Feb 01 00:00:00 EST 2018},
month = {Thu Feb 01 00:00:00 EST 2018}
}

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Works referenced in this record:

Automated design of synthetic ribosome binding sites to control protein expression
journal, October 2009

  • Salis, Howard M.; Mirsky, Ethan A.; Voigt, Christopher A.
  • Nature Biotechnology, Vol. 27, Issue 10, p. 946-950
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