Skip to main content
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems
  • Log in
  • My alerts
  • My Cart

Main menu

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About AEM
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems

User menu

  • Log in
  • My alerts
  • My Cart

Search

  • Advanced search
Applied and Environmental Microbiology
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About AEM
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
Biotechnology

A Strongly Fluorescing Anaerobic Reporter and Protein-Tagging System for Clostridium Organisms Based on the Fluorescence-Activating and Absorption-Shifting Tag Protein (FAST)

Hannah E. Streett, Katie M. Kalis, Eleftherios T. Papoutsakis
Maia Kivisaar, Editor
Hannah E. Streett
aDepartment of Biological Sciences, University of Delaware, Newark, Delaware, USA
bDelaware Biotechnology Institute, Newark, Delaware, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Katie M. Kalis
bDelaware Biotechnology Institute, Newark, Delaware, USA
cSchool of Marine Science and Policy, University of Delaware, Newark, Delaware, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Eleftherios T. Papoutsakis
aDepartment of Biological Sciences, University of Delaware, Newark, Delaware, USA
bDelaware Biotechnology Institute, Newark, Delaware, USA
dDepartment of Chemical and Biomolecular Engineering, University of Delaware, Newark, Delaware, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Maia Kivisaar
University of Tartu
Roles: Editor
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI: 10.1128/AEM.00622-19
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

ABSTRACT

Visualizing protein localization and characterizing gene expression activity in live Clostridium cells is limited for lack of a real-time, highly fluorescent, oxygen-independent reporter system. Enzymatic reporter systems have been used successfully for many years with Clostridium spp.; however, these assays do not allow for real-time analysis of gene expression activity with flow cytometry or for visualizing protein localization through fusion proteins. Commonly used fluorescent reporter proteins require oxygen for chromophore maturation and cannot be used for most strictly anaerobic Clostridium organisms. Here we show that the fluorescence-activating and absorption-shifting tag protein (FAST), when associated with the fluorogenic ligand 4-hydroxy-3-methylbenzylidene-rhodanine (HMBR; now commercially available) and other commercially available ligands, is highly fluorescent in Clostridium acetobutylicum under anaerobic conditions. Using flow cytometry and a fluorescence microplate reader, we demonstrated FAST as a reporter system by employing the promoters of the C. acetobutylicum thiolase (thl), acetoacetate decarboxylase (adc), and phosphotransbutyrylase (ptb) metabolic genes, as well as a mutant Pthl and modified ribosome binding site (RBS) versions of Padc and Pptb. Flow cytometry-based sorting was efficient and fast in sorting FAST-expressing cells, and positively and negatively sorted cells could be effectively recultured. FAST was also used to tag and examine protein localization of the predicted cell division FtsZ partner protein, ZapA, to visualize the divisome localization in live C. acetobutylicum cells. Our findings suggest that FAST can be used to further investigate Clostridium divisomes and more broadly the localization and expression levels of other proteins in Clostridium organisms, thus enabling cell biology studies with these organisms.

IMPORTANCE FAST in association with the fluorogenic ligand HMBR is characterized as a successful, highly fluorescent reporter system in C. acetobutylicum. FAST can be used to distinguish between promoters in live cells using flow cytometry or a fluorescence microplate reader and can be used to tag and examine protein localization in live, anaerobically grown cells. Given that FAST is highly fluorescent under anaerobic conditions, it can be used in several applications of this and likely many Clostridium organisms and other strict anaerobes, including studies involving cell sorting, sporulation dynamics, and population characterization in pure as well as mixed cultures, such as those in various native or synthetic microbiomes and syntrophic cultures.

FOOTNOTES

    • Received 14 March 2019.
    • Accepted 7 May 2019.
    • Accepted manuscript posted online 10 May 2019.
  • Supplemental material for this article may be found at https://doi.org/10.1128/AEM.00622-19.

  • Copyright © 2019 American Society for Microbiology.

All Rights Reserved.

View Full Text
PreviousNext
Back to top
Download PDF
Citation Tools
A Strongly Fluorescing Anaerobic Reporter and Protein-Tagging System for Clostridium Organisms Based on the Fluorescence-Activating and Absorption-Shifting Tag Protein (FAST)
Hannah E. Streett, Katie M. Kalis, Eleftherios T. Papoutsakis
Applied and Environmental Microbiology Jul 2019, 85 (14) e00622-19; DOI: 10.1128/AEM.00622-19

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Print

Alerts
Sign In to Email Alerts with your Email Address
Email

Thank you for sharing this Applied and Environmental Microbiology article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
A Strongly Fluorescing Anaerobic Reporter and Protein-Tagging System for Clostridium Organisms Based on the Fluorescence-Activating and Absorption-Shifting Tag Protein (FAST)
(Your Name) has forwarded a page to you from Applied and Environmental Microbiology
(Your Name) thought you would be interested in this article in Applied and Environmental Microbiology.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
A Strongly Fluorescing Anaerobic Reporter and Protein-Tagging System for Clostridium Organisms Based on the Fluorescence-Activating and Absorption-Shifting Tag Protein (FAST)
Hannah E. Streett, Katie M. Kalis, Eleftherios T. Papoutsakis
Applied and Environmental Microbiology Jul 2019, 85 (14) e00622-19; DOI: 10.1128/AEM.00622-19
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • INTRODUCTION
    • RESULTS
    • DISCUSSION
    • MATERIALS AND METHODS
    • ACKNOWLEDGMENTS
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

KEYWORDS

anaerobic reporter
Clostridium acetobutylicum
cell biology
cell division
divisome
flow cytometry
fusion protein
population dynamics
protein localization
sporulation

Related Articles

Cited By...

About

  • About AEM
  • Editor in Chief
  • Editorial Board
  • Policies
  • For Reviewers
  • For the Media
  • For Librarians
  • For Advertisers
  • Alerts
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • ASM Author Center
  • Submit a Manuscript
  • Article Types
  • Ethics
  • Contact Us

Follow #AppEnvMicro

@ASMicrobiology

       

ASM Journals

ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology.

About ASM | Contact Us | Press Room

 

ASM is a member of

Scientific Society Publisher Alliance

 

American Society for Microbiology
1752 N St. NW
Washington, DC 20036
Phone: (202) 737-3600

Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback

 

Print ISSN: 0099-2240; Online ISSN: 1098-5336