skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

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}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Save / Share:
  • Colonization of plant root surfaces by Pseudomonas putida may require mechanisms that protect this bacterium against superoxide anion and hydrogen peroxide produced by the root. Catalase and superoxide dismutase may be important in this bacterial defense system. Stationary-phase cells of P. putida were not killed by hydrogen peroxide (H{sub 2}O{sub 2}) at concentrations up to 10 mM, and extracts from these cells possessed three isozymic bands (A, B, and C) of catalase activity in native polyacrylamide gel electrophoresis. Logarithmic-phase cells exposed directly to hydrogen peroxide concentrations above 1 mM were killed. Extracts of logarithmic-phase cells displayed only band A catalasemore » activity. Protection against 5 mM H{sub 2}O{sub 2} was apparent after previous exposure of the logarithmic-phase cells to nonlethal concentrations (30 to 300 {mu}M) of H{sub 2}O{sub 2}. Extracts of these protected cells possessed enhanced catalase activity of band A and small amounts of bands B and C. A single form of superoxide dismutase and isoforms of catalase were apparent in extracts from a foliar intercellular pathogen, Pseudomonas syringae pv. phaseolicola. The mobilities of these P. syringae enzymes were distinct from those of enzymes in P. putida extracts.« less
  • Methane is produced by anaerobic archaebacteria known as methanogens. Currently the only available plasmid from a methanogen is pME2001. The authors incorporated pME2001 into plasmids which should be capable of replication in a range of microbial host species. Plasmid pET2411, a recombinant plasmid formed by joining pBR322 to pME2001, directs the synthesis of pME2001 encoded polypeptides in Escherichia coli but cannot replicate in E. coli in the absence of E. coli DNA polymerase I. 23 references, 3 figures, 1 table.
  • While several thermus genes have been cloned and T. thermophilus has been shown to be transformable, molecular genetic studies of these thermophiles have been hampered by the absence of selectable cloning vectors. The authors have constructed a selectable plasmid by random insertion of a heterologous gene encoding a thermostable kanamycin nucleotidyltransferase activity into a cryptic, multicopy plasmid from T. thermophilus HB8. This plasmid should serve as a suitable starting point for the development of a gene expression system for T. thermophilus.
  • Highlights: ► A new therapeutic strategy based on tumstatin in vivo overexpression is proposed. ► pVAX1©–tumstatin electrotransfer in muscle mediates protein expression in muscle. ► A substantial expression of tumstatin is detected in the serum of electrotransfected mice. ► Tumstatin overexpression decreases tumor growth and increases mouse survival. -- Abstract: The NC1 domains from the different α(IV) collagen chains were found to exert anti-tumorigenic and/or anti-angiogenic activities. A limitation to the therapeutic use of these matrikines is the large amount of purified recombinant proteins, in the milligram range in mice that should be administered daily throughout the experimental procedures. Inmore » the current study, we developed a new therapeutic approach based on tumstatin (NC1α3(IV)) overexpression in vivo in a mouse melanoma model. Gene electrotransfer of naked plasmid DNA (pDNA) is particularly attractive because of its simplicity, its lack of immune responsiveness and its safety. The pDNA electrotransfer in muscle mediates a substantial gene expression that lasts several months. A pVAX1© vector containing the tumstatin cDNA was injected into the legs of C57BL/6 mice and submitted to electrotranfer. Sera were collected at different times and tumstatin was quantified by ELISA. Tumstatin secretion reached a plateau at day 21 with an expression level of 12 μg/mL. For testing the effects of tumstatin expression on tumor growth in vivo, B16F1 melanoma cells were subcutaneously injected in mice 7 days after empty pVAX1© (Mock) or pVAX1©–tumstatin electrotransfer. Tumstatin expression triggered a large decrease in tumor growth and an increase in mouse survival. This new therapeutic approach seems promising to inhibit tumor progression in vivo.« less