Application of the Fluorescence-Activating and Absorption-Shifting Tag (FAST) for Flow Cytometry in Methanogenic Archaea

Abstract

Methane-producing archaea play a crucial role in the global carbon cycle and are used for biotechnological fuel production. Methanogenic model organisms such as Methanococcus maripaludis and Methanosarcina acetivorans have been biochemically characterized and can be genetically engineered by using a variety of existing molecular tools. The anaerobic lifestyle and autofluorescence of methanogens, however, restrict the use of common fluorescent reporter proteins (e.g., GFP and derivatives), which require oxygen for chromophore maturation. Recently, the use of a novel oxygen-independent fluorescent activation and absorption-shifting tag (FAST) was demonstrated with M. maripaludis. Similarly, we now describe the use of the tandem activation and absorption-shifting tag protein 2 (tdFAST2), which fluoresces when the cell-permeable fluorescent ligand (fluorogen) 4-hydroxy-3,5-dimethoxybenzylidene rhodanine (HBR-3,5DOM) is present. Expression of tdFAST2 in M. acetivorans and M. maripaludis is noncytotoxic and tdFAST2:HBR-3,5DOM fluorescence is clearly distinguishable from the autofluorescence. In flow cytometry experiments, mixed methanogen cultures can be distinguished, thereby allowing for the possibility of high-throughput investigations of the characteristic dynamics within single and mixed cultures. IMPORTANCE Methane-producing archaea play an essential role in the global carbon cycle and demonstrate great potential for various biotechnological applications, e.g., biofuel production, carbon dioxide capture, and electrochemical systems. Oxygen sensitivity and high autofluorescence hinder the use of common fluorescent proteins for studying methanogens. By using tdFAST2:HBR-3,5DOM fluorescence, which functions under anaerobic conditions and is distinguishable from the autofluorescence, real-time reporter studies and high-throughput investigation of the mixed culture dynamics of methanogens via flow cytometry were made possible. This will further help accelerate the sustainable exploitation of methanogens.

Keywords: FAST; Methanogenic archaea; Methanosarcina; flow cytometry; fluorescence.

FIG 1

tdFAST2-expressing methanogens show a specific fluorescence in the presence of HBR-3,5DOM. Fluorescence of M. acetivorans and M. maripaludis cells is shown. (a) Fluorescence spectrum upon excitation at 515 nm. (b) Fluorescence at 590 nm when different excitation wavelengths are applied. Cells expressing tdFAST2 (tdFAST2) or control cells carrying an empty vector construct (e.v.) were analyzed in presence (w/ fluorogen) or absence (w/o fluorogen) of HBR-3,5DOM. Cells were in the stationary growth phase. Mean values and standard deviation of triplicates are shown.

FIG 2

Wide ranges of cell concentration and fluorogen concentration can be used to measure tdFAST2:HBR-3,5DOM fluorescence in M. acetivorans. M. acetivorans cells expressing tdFAST2 were analyzed in the exponential growth phase. A microplate reader was used to measure OD(600), tdFAST2:HBR-3,5DOM fluorescence (λ_Ex = 515 nm/λ_Em = 590 nm), and autofluorescence (λ_Ex = 420 nm/λ_Em = 480 nm). (a) Correlation of fluorescence and cell-density when the fluorogen concentration (5 μM) is constant. (b) Influence of the fluorogen concentration on the fluorescence when the cell concentration is constant (1 OD unit/mL). Note that, due to different light path lengths, the OD(600) determined by the microplate reader (upper panel) is smaller than the actual cell concentration (OD units/mL) which was determined using a standard spectrophotometer. Values of duplicates are shown.

FIG 3

M. acetivorans fluorescence changes at different growth phases. M. acetivorans cells expressing tdFAST2 (+) or harboring an empty vector construct (–) were analyzed at exponential (Exp; OD[600] 0.3–0.6), late exponential (Late Exp; 0.8 to 1.1), and stationary growth (Stat; >1.1). tdFAST2:HBR-3,5DOM fluorescence (λ_Ex = 515 nm/λ_Em = 590 nm), and autofluorescence (λ_Ex = 420 nm/λ_Em = 480 nm) was measured. Technical duplicates are shown.

FIG 4

tdFAST2 is not toxic for methanogens. Growth of M. acetivorans and M. maripaludis expressing eighter tdFAST2 or harboring an empty vector construct (e.v.) was analyzed. Mean values and standard deviation of triplicates are shown.

FIG 5

Flow cytometry allows visualization of tdFAST2 expression and separation of populations. Histograms of (a) M. acetivorans and (b) M. maripaludis cells expressing tdFAST2 (tdFAST2) or control cells carrying an empty vector construct (e.v.) in the presence of increasing fluorogen (HBR-3,5DOM) concentrations. (c and d) Mixtures of tdFAST2-expressing cells and non tdFAST2-expressing cells were analyzed in the presence of 5 mM HBR-3,5DOM. M. acetivorans cultures were mixed in a 2 to 1 ratio (excess of e.v.), while M. maripaludis cultures were mixed in a 1 to 1 ratio.

FIG 6

Golden Gate cloning vectors to construct tdFAST2-tagged proteins. The plasmids pMaFAST(C) and pMaFAST(N) encode the tdFAST2 gene which is codon-optimized for expression in Methanosarcina. Golden Gate cloning (BsaI) can be used to replace the lacZ cassette with a protein-encoding sequence of interest leading to a C- or N-terminal fusion to the tdFAST2. Nucleotide sequence of the cloning sites are shown below. The BsaI sites, ribosome-binding site (rbs) and the tdFAST2 open-reading frame is indicated.