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Title: A Molecular Biology Tool Kit for the Phototrophic Firmicute Heliobacterium modesticaldum

Abstract

The heliobacteria are members of the bacterial orderClostridialesand form the only group of phototrophs in the phylumFirmicutes. Several physiological and metabolic characteristics make them an interesting subject of investigation, including their minimalist photosynthetic system, nitrogen fixation abilities, and ability to reduce toxic metals. Although the speciesHeliobacterium modesticaldumis an excellent candidate as a model system for the familyHeliobacteriaceae, since an annotated genome and transcriptomes are available, studies in this organism have been hampered by the lack of genetic tools. We adapted techniques for genetic manipulation of related clostridial species for use withH. modesticaldum. Five heliobacterial DNA methyltransferase genes were expressed in anEscherichia colistrain engineered as a conjugative plasmid donor for broad-host-range plasmids. Premethylation of the shuttle vectors before conjugation intoH. modesticaldumis absolutely required for production of transconjugant colonies. The introduced shuttle vectors are maintained stably and can be recovered using a modified minipreparation procedure developed to inhibit endogenous DNase activity. Moreover, we describe the formulation of various growth media, including a defined medium for metabolic studies and isolation of auxotrophic mutants. Heliobacteria are anoxygenic phototrophic bacteria with the simplest known photosynthetic apparatus. They are unique in using bacteriochlorophyllgas their main pigment and lacking a peripheral antenna system. Until now, research onmore » this organism has been hampered by the lack of a genetic transformation system. Without such a system, gene knockouts, site-directed mutations, and gene expression studies cannot be performed to help us further understand or manipulate the organism. Here we report the genetic transformation of a heliobacterium, which should enable future genetic studies in this unique phototrophic organism.« less

Authors:
 [1]; ORCiD logo [1];  [2];  [1];  [1];  [3]; ORCiD logo [1]
  1. Arizona State Univ., Tempe, AZ (United States)
  2. Arizona State Univ., Tempe, AZ (United States); Univ. of Peshawar (Pakistan)
  3. Arizona State Univ., Tempe, AZ (United States); Marana High School, Tucson, Arizona (United States)
Publication Date:
Research Org.:
Arizona State Univ., Tempe, AZ (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES). Chemical Sciences, Geosciences, and Biosciences Division
OSTI Identifier:
1631806
Grant/Contract Number:  
SC0010575
Resource Type:
Accepted Manuscript
Journal Name:
Applied and Environmental Microbiology
Additional Journal Information:
Journal Volume: 85; Journal Issue: 19; Journal ID: ISSN 0099-2240
Publisher:
American Society for Microbiology
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; DNA methylation; heliobacteria; conjugation; restriction

Citation Formats

Baker, Patricia L., Orf, Gregory S., Khan, Zahid, Espinoza, Levi, Leung, Sabrina, Kevershan, Kimberly, and Redding, Kevin E. A Molecular Biology Tool Kit for the Phototrophic Firmicute Heliobacterium modesticaldum. United States: N. p., 2019. Web. https://doi.org/10.1128/AEM.01287-19.
Baker, Patricia L., Orf, Gregory S., Khan, Zahid, Espinoza, Levi, Leung, Sabrina, Kevershan, Kimberly, & Redding, Kevin E. A Molecular Biology Tool Kit for the Phototrophic Firmicute Heliobacterium modesticaldum. United States. https://doi.org/10.1128/AEM.01287-19
Baker, Patricia L., Orf, Gregory S., Khan, Zahid, Espinoza, Levi, Leung, Sabrina, Kevershan, Kimberly, and Redding, Kevin E. Tue . "A Molecular Biology Tool Kit for the Phototrophic Firmicute Heliobacterium modesticaldum". United States. https://doi.org/10.1128/AEM.01287-19. https://www.osti.gov/servlets/purl/1631806.
@article{osti_1631806,
title = {A Molecular Biology Tool Kit for the Phototrophic Firmicute Heliobacterium modesticaldum},
author = {Baker, Patricia L. and Orf, Gregory S. and Khan, Zahid and Espinoza, Levi and Leung, Sabrina and Kevershan, Kimberly and Redding, Kevin E.},
abstractNote = {The heliobacteria are members of the bacterial orderClostridialesand form the only group of phototrophs in the phylumFirmicutes. Several physiological and metabolic characteristics make them an interesting subject of investigation, including their minimalist photosynthetic system, nitrogen fixation abilities, and ability to reduce toxic metals. Although the speciesHeliobacterium modesticaldumis an excellent candidate as a model system for the familyHeliobacteriaceae, since an annotated genome and transcriptomes are available, studies in this organism have been hampered by the lack of genetic tools. We adapted techniques for genetic manipulation of related clostridial species for use withH. modesticaldum. Five heliobacterial DNA methyltransferase genes were expressed in anEscherichia colistrain engineered as a conjugative plasmid donor for broad-host-range plasmids. Premethylation of the shuttle vectors before conjugation intoH. modesticaldumis absolutely required for production of transconjugant colonies. The introduced shuttle vectors are maintained stably and can be recovered using a modified minipreparation procedure developed to inhibit endogenous DNase activity. Moreover, we describe the formulation of various growth media, including a defined medium for metabolic studies and isolation of auxotrophic mutants. Heliobacteria are anoxygenic phototrophic bacteria with the simplest known photosynthetic apparatus. They are unique in using bacteriochlorophyllgas their main pigment and lacking a peripheral antenna system. Until now, research on this organism has been hampered by the lack of a genetic transformation system. Without such a system, gene knockouts, site-directed mutations, and gene expression studies cannot be performed to help us further understand or manipulate the organism. Here we report the genetic transformation of a heliobacterium, which should enable future genetic studies in this unique phototrophic organism.},
doi = {10.1128/AEM.01287-19},
journal = {Applied and Environmental Microbiology},
number = 19,
volume = 85,
place = {United States},
year = {2019},
month = {9}
}

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    Works referencing / citing this record:

    Expression and purification of affinity-tagged variants of the photochemical reaction center from Heliobacterium modesticaldum
    journal, September 2019


    Using the Endogenous CRISPR-Cas System of Heliobacterium modesticaldum To Delete the Photochemical Reaction Center Core Subunit Gene
    journal, September 2019

    • Baker, Patricia L.; Orf, Gregory S.; Kevershan, Kimberly
    • Applied and Environmental Microbiology, Vol. 85, Issue 23
    • DOI: 10.1128/aem.01644-19