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

Streett, Hannah E.; Kalis, Katie M.; Papoutsakis, Eleftherios T.

doi:10.1128/aem.00622-19

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

Journal Article · Fri May 10 00:00:00 EDT 2019 · Applied and Environmental Microbiology

DOI:https://doi.org/10.1128/aem.00622-19· OSTI ID:1613137

Streett, Hannah E. ^[1]; Kalis, Katie M. ^[2]; Papoutsakis, Eleftherios T. ^[3]

Univ. of Delaware, Newark, DE (United States); Delaware Biotechnology Inst., Newark, DE (United States); DOE/OSTI
Delaware Biotechnology Inst., Newark, DE (United States); Univ. of Delaware, Newark, DE (United States)
Univ. of Delaware, Newark, DE (United States); Delaware Biotechnology Inst., Newark, DE (United States)

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 P_thl and modified ribosome binding site (RBS) versions of P_adc and P_ptb. 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.

View Accepted Manuscript (DOE)

Research Organization:: Univ. of Delaware, Newark, DE (United States)

Sponsoring Organization:: Army Research Office (ARO); National Institutes of Health (NIH); National Science Foundation (NSF); USDOE Office of Science (SC)

Grant/Contract Number:: SC0019155

OSTI ID:: 1613137

Journal Information:: Applied and Environmental Microbiology, Journal Name: Applied and Environmental Microbiology Journal Issue: 14 Vol. 85; ISSN 0099-2240

Publisher:: American Society for MicrobiologyCopyright Statement

Country of Publication:: United States

Language:: English

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59 BASIC BIOLOGICAL SCIENCES
Clostridium acetobutylicum
anaerobic reporter
cell biology
cell division
divisome
flow cytometry
fusion protein
population dynamics
protein localization
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A Strongly Fluorescing Anaerobic Reporter and Protein-Tagging System for Clostridium Organisms Based on the Fluorescence-Activating and Absorption-Shifting Tag Protein (FAST)

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References (49)

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