Development of Strong Anaerobic Fluorescent Reporters for Clostridium acetobutylicum and Clostridium ljungdahlii Using HaloTag and SNAP-tag Proteins

Charubin, Kamil; Streett, Hannah; Papoutsakis, Eleftherios Terry

doi:10.1128/aem.01271-20

Development of Strong Anaerobic Fluorescent Reporters for Clostridium acetobutylicum and Clostridium ljungdahlii Using HaloTag and SNAP-tag Proteins

Journal Article · Thu Oct 01 00:00:00 EDT 2020 · Applied and Environmental Microbiology

DOI:https://doi.org/10.1128/aem.01271-20· OSTI ID:1852895

Charubin, Kamil ^[1]; Streett, Hannah ^[2]; ^[2]

Univ. of Delaware, Newark, DE (United States); OSTI
Univ. of Delaware, Newark, DE (United States)

One of the biggest limitations in the study and engineering of anaerobic Clostridium organisms is the lack of strong fluorescent reporters capable of strong and real-time fluorescence. Recently, we developed a strong fluorescent reporter system for Clostridium organisms based on the FAST protein. Here, we report the development of two new strong fluorescent reporter systems for Clostridium organisms based on the HaloTag and SNAP-tag proteins, which produce strong fluorescent signals when covalently bound to fluorogenic ligands. These new fluorescent reporters are orthogonal to the FAST ligands and to each other, allowing for simultaneous labeling and visualization. We used HaloTag and SNAP-tag to label the strictly anaerobic organisms Clostridium acetobutylicum and Clostridium ljungdahlii. We have also identified a new strong promoter for protein expression in C. acetobutylicum, based on the phosphotransacetylase gene (pta) from C. ljungdahlii. Furthermore, the HaloTag and the SNAP-tag, in combination with the previously described FAST system, were successfully used to measure cell populations in bacterial mixed cultures and showed the simultaneous orthogonal labeling of HaloTag and SNAP-tag together with the FAST protein reporter. Finally, we show the expression of recombinant fusion protein of FAST and the ZapA division protein (from C. acetobutylicum) in C. ljungdahlii. The availability of multiple strong fluorescent reporters is a major addition to the genetic toolkit of Here, Clostridium and other anaerobes that will lead to better understanding of these unique organisms.

View Accepted Manuscript (DOE)

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

Sponsoring Organization:: USDOE Office of Science (SC)

Grant/Contract Number:: SC0019155

OSTI ID:: 1852895

Journal Information:: Applied and Environmental Microbiology, Journal Name: Applied and Environmental Microbiology Journal Issue: 20 Vol. 86; ISSN 0099-2240

Publisher:: American Society for MicrobiologyCopyright Statement

Country of Publication:: United States

Language:: English

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59 BASIC BIOLOGICAL SCIENCES
Biotechnology & Applied Microbiology
Clostridium
Clostridium acetobutylicum
Clostridium ljungdahlii
FAST
HaloTag
Microbiology
SNAP-tag
anaerobic fluorescent reporters
flow cytometry
fluorescent protein
super-resolution microscopy

Development of Strong Anaerobic Fluorescent Reporters for Clostridium acetobutylicum and Clostridium ljungdahlii Using HaloTag and SNAP-tag Proteins

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