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

Abstract

One of the biggest limitations in study and engineering of anaerobic Clostridium organisms is the lack of strong fluorescent reporters capable of strong and real-time fluorescence. Recently, we have 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 strictly anaerobic Clostridium acetobutylicum and C. ljungdahlii. We have also identified a new strong promoter for protein expression in C. acetobutylicum, based on phosphotransacetylase gene (pta) from C. ljungdahlii. Furthermore, the HaloTag and 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. Availability of multiple strong fluorescent reporters is a major addition to the genetic toolkit of Clostridium and other anaerobes, which will lead to better understanding of these unique organisms. IMPORTANCE Up to this point, assays and methods involving fluorescent reporter proteins were unavailable, or limited in Clostridium organisms and other strict anaerobes. GFP, mCherry, and Flavin-binding proteins (and their derivatives) have been used only in a handful of clostridia with limited success and yielded low fluorescence compared to aerobic microbial systems. Recently, we reported a new strong fluorescent reporter system based on the FAST as a first step in expanding the fluorescence-based reporters for Clostridium and other anaerobic microbial platforms. Additional strong orthogonal fluorescent proteins, with distinct emission spectra are needed to allow for: (1) multi-species tracking within the growing field of microbial co-cultures and microbiomes; (2) protein localization and tracking in anaerobes; and (3) identification and development of natural and synthetic promoters, ribosome-binding sites (RBS), and terminators for optimal protein expression in anaerobes. Here we present two new strong fluorescent reporter systems based on the HaloTag and SNAP-tag proteins.

Production of the biocommodities butanol and acetone from methanol with fluorescent FAST-tagged proteins using metabolically engineered strains of Eubacterium limosum

“…Although FbFPs are functional under anoxic conditions, they show a fairly low brightness compared to control strains that do not express the respective fluorescent protein. In contrast, the FAST system was recently established in C. acetobutylicum and C. ljungdahlii and showed a clear and obvious fluorescence [ 31 , 32 , 62 ]. The fluorescence of recombinant E. limosum strains expressing feg constructed in this work is up to 26-fold improved compared to the control strain.…”

“…However, fluorescent reporter systems which are well-established and often used tools in molecular biology to study gene expression [ 23 , 24 ], promoter activities [ 25 , 26 ], or the dynamics in microbial populations and co-cultures [ 27 – 29 ] are still restricted for acetogens, basically due to the lack of proteins that show bright fluorescence under anaerobic conditions. Recently, the fluorescence-activating and absorption shifting tag (FAST) [ 30 ] was established in C. acetobutylicum [ 31 ] and C. ljungdahlii [ 32 ] and opened the door for application in other anaerobic bacteria, since its fluorescence is bright and independent of oxygen. FAST is a small-sized protein with a mass of 14 kDa that only shows fluorescence when it forms a non-covalent reversible complex with a fluorogenic ligand, a so-called fluorogen [ 30 , 33 ].…”

Production of the Biocommodities Butanol and Acetone from Methanol with Fluorescent FAST-tagged Proteins using Metabolically Engineered Strains of Eubacterium Limosum

“…Although FbFPs are functional under anoxic conditions, they show a fairly low brightness compared to control strains that do not express the respective uorescent protein. In contrast, the FAST system was recently established in C. acetobutylicum and C. ljungdahlii and showed a clear and obvious uorescence [32,33,62]. The uorescence of recombinant E. limosum strains expressing feg constructed in this work are up to 26-fold improved compared to the control strain.…”

“…Thus, FAST seems to be perfectly suited as a perfect uorescent reporter for anaerobic bacteria. An alternative to FAST could be the SNAP-and the Halo-Tag, which were shown to be functional in C. acetobutylicum and C. ljungdahlii [33]. Both tags show oxygen-independent bright uorescence when covalently bound to a uorogenic ligand.…”

“…restricted for acetogens, basically due to the lack of proteins that show bright uorescence under anaerobic conditions. Recently, the uorescence-activating and absorption shifting tag (FAST) [31] was established in C. acetobutylicum [32] and C. ljungdahlii [33] and opened the door for application in other anaerobic bacteria, since its uorescence is bright and independent of oxygen. FAST is a small-sized protein with a mass of 14 kDa that only shows uorescence when it forms a non-covalent reversible complex with a uorogenic ligand, a so-called uorogen [31,34].…”