Extraction of Cofactor F420 for Analysis of Polyglutamate Tail Length from Methanogenic Pure Cultures and Environmental Samples

Summary

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.

Abstract

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.

Introduction

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 Bacteria^1,2. 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 mV^1,3. 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 gut^1,5. 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, methanotrophic^8,9, 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-H₄MPT^12,13. It can also be used as an electron carrier in H₂-independent electron transport pathways of methanogens containing cytochromes^12,14. 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 environments^1,23. 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 biosynthesis^2,6,24. 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 F_O-synthase using the substrates tyrosine and 5-amino-6-ribitylamino-2,4(1H, 3H)-pyrimidinedione. The result is the riboflavin level chromophore F_O. 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 F_O 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 numbers^23,25. Different organisms show different patterns in the number of attached glutamate residues, with shorter tails found for methanogens than in mycobacteria^2,25,26. 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 residues^2,25,26,27. 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)-HPLC^27,28. 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).

Protocol

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 Add up to 5 g of sample to appropriate tubes (e.g., 50 mL conical tubes). Add 5 mL of the lysis buffer (2x stock …

Representative Results

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…

Discussion

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…

Materials

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