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A method for the extraction of cofactor F420 from pure cultures was optimized for the liquid chromatographic separation and analysis of F420 tail length in pure culture and environmental samples.
The cofactor F420 plays a central role as a hydride carrier in the primary and secondary metabolism of many bacterial and archaeal taxa. The cofactor is best known for its role in methanogenesis, where it facilitates thermodynamically difficult reactions. As the polyglutamate tail varies in length between different organisms, length profile analyses might be a powerful tool for distinguishing and characterizing different groups and pathways in various habitats. Here, the protocol describes the extraction and optimization of cofactor F420 detection by applying solid-phase extraction combined with high-performance liquid chromatography analysis independent of cultural or molecular biological approaches. The method was applied to gain additional information on the expression of cofactor F420 from microbial communities in soils, anaerobic sludge, and pure cultures and was evaluated by spiking experiments. Thereby, the study succeeded in generating different F420 tail-length profiles for hydrogenotrophic and acetoclastic methanogens in controlled methanogenic pure cultures as well as from environmental samples such as anaerobic digester sludge and soils.
F420 is a widespread but often neglected cofactor, which functions as an obligate two-electron hydride carrier in primary and secondary metabolic processes of both Archaea and Bacteria1,2. F420 is a 5-deazaflavin and structurally similar to flavins, whereby its chemical and biological properties are more comparable with those of NAD+ or NADP+. Due to the substitution of nitrogen with carbon at position 5 of the isoalloxazine ring, it is a strong reductant, thus exhibiting a low standard redox potential of -340 mV1,3. F420 comprises a 5-deazaflavin ring and a 2-phospho-L-lactate linker (F420-0). An oligoglutamate tail containing n+1 glutamate monomers can be attached to the molecule (F420-n+1)4.
For a long time, the cofactor F420 has been solely associated with Archaea and Actinobacteria. This has largely been overturned. Recent analyses revealed that F420 is distributed among diverse anaerobic and aerobic organisms of the phyla Proteobacteria, Chloroflexi, and potentially Firmicutes inhabiting a myriad of habitats like soils, lakes, and the human gut1,5. In 2019, Braga et al.6, showed that the proteobacterium Paraburkholderia rhizoxinica produces a unique F420 derivative, containing a 3-phosphoglycerate instead of a 2-phospholactate tail, which might be widespread in various habitats. Within the domain Archaea, F420 has been found in several lineages, including methanogenic7, methanotrophic8,9, and sulfate-reducing orders10, and is supposed to be produced in Thaumarchaeota11. F420 is best known as an essential redox coenzyme in hydrogenotrophic and methylotrophic methanogenesis. The reduced form of F420 (F420H2) functions as an electron donor for the reduction of methylenetetrahydromethanopterin (methylene-H4MPT, Mer) and methenyl-H4MPT12,13. It can also be used as an electron carrier in H2-independent electron transport pathways of methanogens containing cytochromes12,14. Moreover, the oxidized form of F420 has a characteristic blue-green fluorescence upon excitation at 420 nm, which facilitates the detection of methanogens microscopically (Figure 1). Due to its low redox potential, F420 facilitates (i) the exogenous reduction of a broad spectrum of otherwise recalcitrant or toxic organic compounds, (ii) synthesis of tetracycline and lincosamide antibiotics or phytotoxins in streptomycetes (phylum Actinobacteria), and (iii) resistance to oxidative or nitrosative stress or other unfavorable conditions in mycobacteria (phylum Actinobacteria)1,5,15,16,17,18,19,20,21,22. Consequently, F420-dependent oxidoreductases are promising biocatalysts for industrial and pharmaceutical purposes as well as for bioremediation of contaminated environments1,23. Despite these recent findings, the exact roles of the cofactor F420 are still marginally known in Actinobacteria or other bacterial phyla.
There are at least three pathways for F420 biosynthesis2,6,24. In the beginning, the biosynthesis pathway is split into a 5-deazaflavin biosynthesis and a 2-phospholactate metabolism branch. The reactive part of the F420 molecule is synthesized via FO-synthase using the substrates tyrosine and 5-amino-6-ribitylamino-2,4(1H, 3H)-pyrimidinedione. The result is the riboflavin level chromophore FO. Within the currently accepted lactate metabolism branch, L-lactate is phosphorylated to 2-phospho-L-lactate by an L-lactate kinase (CofB); 2-phospho-L-lactate, in turn, is guanylated to L-lactyl-2-diphospho-5'-guanosine by 2-phospho-L-lactate guanylyltransferase (CofC). In the next step, L-lactyl-2-diphospho-5'-guanosine is linked to FO by a 2-phospho-L-lactate transferase (CofD) to form F420-02. Finally, the enzyme F420-0:ɣ-glutamyl ligase (CofE) ligates glutamate monomers to F420-0, forming the final cofactor6 in varying numbers23,25. Different organisms show different patterns in the number of attached glutamate residues, with shorter tails found for methanogens than in mycobacteria2,25,26. Generally, methanogens show tail lengths from two to three, with up to five in the acetoclastic methanogen, Methanosarcina sp., while tail lengths found in Mycobacterium sp. ranged from five to seven glutamate residues2,25,26,27. However, recent findings showed that long-chain F420 binds to F420-dependent oxidoreductases with a higher affinity than short-chain F420; moreover, bound long-chain F420 increases the substrate affinity but decreases the turnover rate of respective enzymes23.
Detection of cofactor F420 is often based on its fluorescence. Thereby, its oligo glutamate derivates were separated using reversed-phase (RP)-HPLC27,28. Recently, Ney et al. used tetrabutylammonium hydroxide as an ion-pairing reagent for the negatively charged glutamate tail to enhance separation on RP-HLPC successfully5. Here, we present a method for the preparation of samples, subsequent lysis, extraction, purification, separation, and quantification of cofactor F420 not only from pure cultures but also from different environmental samples (i.e., soils and digester sludge).
NOTE: Extraction and analysis of cofactor F420 is a three-step process including sample lysis, cofactor pre-purification via solid-phase extraction (SPE), and cofactor detection via ion-paired-RP-HPLC (IP-RP-HPLC) with fluorescence detection. Prior to starting, prepare the materials and reagents as stated in Table 1.
1. Sample lysis
2. Pre-purification of the cofactor F 420 via solid-phase extraction (SPE)
NOTE: All steps of SPE are conducted at room temperature
3. Detection of the cofactor F420
Pure cultures of Methanosarcina thermophila and Methanoculleus thermophilus, both thermophilic methanogenic Archaea, were grown in suitable media as described previously29,30. For Methanosarcina thermophila, methanol was used as an energy source, whereas Methanoculleus thermophilus was grown on H2/CO2. Growth was checked by microscopic evaluation, while activity was examined via measurement of met...
For the evaluation of cofactor F420 from methanogenic pure cultures, a microscopic evaluation can be performed to visualize the growth and activity (fluorescence microscopy) of the involved microorganisms (Figure 1). For samples deriving from natural environments, the use of microscopy to detect or quantify F420 is limited due to interferences with other fluorescent microorganisms, organic and inorganic particles. In this context, extraction of F420 and subse...
The authors have nothing to disclose.
We gratefully thank Prof. Colin Jackson for the support with purified cofactor F420. This research was supported by Tyrolean science fund (TWF) and the Universität Innsbruck (Publikationsfonds). We greatly acknowledge the support of GPS, HK, SB, GG, and HB.
Name | Company | Catalog Number | Comments |
Autoclave | |||
Biocompatible HPLC system equipped with gradient modul, oven and fluorescence detector | Shimadzu | HPLC system | |
Centrifuge and rotor for 50 mL “Falcon” tubes (11.000 rcf) and appropriate tubes | |||
HPLC Column: Gemini NX C18, 5 μm, 150 x 3 mm | Phenomenex | HPLC column | |
PTFE filter (pore size 0.22 µm) to remove particulate matter prior HPLC analysis | |||
Resin for SPE: Strata-X-AW 33 μm as weak anion mixed-mode polymeric sorbent | Phenomenex | weak anion mixed-mode polymeric sorbent | |
Vacuum manifold for SPE and appropriate collection tubes | SPE equipment | ||
Vortex mixer |
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