Extraction of Cofactor F<sub>420</sub> for Analysis of Polyglutamate Tail Length from Methanogenic Pure Cultures and Environmental Samples

Rudolf Markt; Mathias Wunderer; Eva Maria Prem; Mira Mutschlechner; Nina Lackner; Andreas  Otto Wagner

doi:10.3791/62737

JoVE Journal > Environment

Environnement

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

Published: October 14, 2021

doi:

10.3791/62737

Rudolf Markt¹, Mathias Wunderer¹, Eva Maria Prem¹, Mira Mutschlechner¹, Nina Lackner², Andreas Otto Wagner¹

¹Department of Microbiology,Universität Innsbruck, ²Department of Hygiene and Medical Microbiology,Medical University of Innsbruck

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 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 F₄₂₀has 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, F₄₂₀has been found in several lineages, including methanogenic⁷, methanotrophic⁸^,⁹, and sulfate-reducing orders¹⁰, and is supposed to be produced in Thaumarchaeota¹¹. F₄₂₀is 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 F₄₂₀has 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 F₄₂₀are 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 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 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).

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 solution, Table 1) to the samples.
Bring to a final volume of 10 mL with distilled water to reach a final concentration of 0.5 g·mL^-1.
Vortex the diluted samples for 20 s.
Autoclave for 30 min at 121 °C.
For dry samples like forest soil, bring to a final volume of 20 mL with distilled water after autoclaving and vortex the diluted sample.
CAUTION: Temperature increase during autoclaving might cause tubes to burst.

2. Pre-purification of the cofactor F ₄₂₀ via solid-phase extraction (SPE)

NOTE: All steps of SPE are conducted at room temperature

Cool down the samples to room temperature.
Centrifuge the autoclaved samples for 5 min at 11,000 x g.
Prepare 5 mL SPE columns filled with 100 mg of weak anion mixed-mode polymeric sorbent.
Condition the anion exchanger with 3 mL of methanol (condition solution, Table 1).
Equilibrate the anion exchanger with 3 mL of distilled water (equilibration solution, Table 1).
Load up to 9.0 mL of the supernatant from the centrifuged lysate onto the SPE column.
Wash away impurities with 5 mL of 25 mM ammonium acetate (SPE wash solution 1, Table 1).
Wash away impurities with 5 mL of methanol (SPE wash solution 2, Table 1).
Elute the cofactor F₄₂₀ in 1.0 mL of elution buffer (Table 1).
NOTE: Prepare fresh elution buffer. Due to the applied vacuum and the high vapor pressure of the elution buffer, elution volumes might differ from sample to sample. In order to secure the same final volume in all the samples, it is recommended to weigh the collection vessels before and after elution and calculate the effective elution volume. Balance the differences by the addition of elution buffer.

3. Detection of the cofactor F₄₂₀

Set the oven to 40 °C and fluorescence detector to 420 nm extinction-wavelength and 470 nm emission-wavelength. Achieve separation via gradient mode using mobile phases A and B (Table 1): 0-3 min: 26% B; 3-24 min: 26%-50% B; 24-25 min: 50% B; 25-27 min: 50%-26% B; 27-35 min: 26% B at a flow rate of 0.75 mL·min^-1.
NOTE: Guarantee equilibration of column conditions prior to injecting samples by flushing the column at least with 3 column volumes of 74% mobile phase A and 26% mobile phase B (Table 1).
1. Filter the eluted samples from the SPE into HPLC vials using a PTFE filter with a pore size of 0.22 µm.
  NOTE: PTFE filters with a pore size of 0.22 µm are recommended.
2. Inject 50 µL of the eluted sample onto the HPLC system to analyze the cofactor F₄₂₀ composition and concentration.
  NOTE: As no quantitative standard is used in this protocol, samples and variants have to be compared by peak area.

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 methane (CH₄) by gas chromatography as described previously³¹. Pure cultures were used for the extraction of cofactor F₄₂₀ according to the presented protocol. In addition, environmental samples including samples from a mesophilic biogas reactor sludge (wastewater treatment plant, Zirl, Austria; for details regarding sludge parameters, please refer to³²), an agriculturally used meadow (Innsbruck, Austria), and forest soil (Lans, Austria) were taken in autumn 2020 for the extraction and analysis of cofactor F₄₂₀.

The growth of pure cultures has been verified by microscopy (Figure 1), with the produced CH₄ analyzed via gas chromatography during 14 days of incubation (data not shown). The efficiency of cofactor F₄₂₀ extraction from pure cultures was tested by applying different disintegration strategies: beat-beating using 0.5-1.0 mm ceramic beats, ultrasonic treatment, and pressure-temperature disintegration using 121°C and 1.2 bar pressure (autoclaving). Maximum extraction efficiency became apparent using pressure-temperature treatment applying a buffer as described in the protocol section and was thus further applied for all subsequent experiments (Figure 2). Extraction efficiency tests were performed via standard addition of different volumes of a well-growing Methanoculleus thermophilus culture. Furthermore, the comparison of different samples and variants was based on peak area from chromatograms.

Subsequently, cell extracts were subjected to a solid-phase extraction (SPE) procedure. For this purpose, different ion exchangers were tested. It turned out that a weak anion mixed-mode polymeric sorbent yielded the highest amount of cofactor F₄₂₀ after elution. In addition, different elution buffers and wash solutions were tested and showed the best results for 25 mM ammonium acetate as a wash buffer and a mixture of NH₃ in methanol as an elution buffer. Methanol from the elution step could be exchanged after elution with water via a vacuum-temperature treatment.

HPLC analysis of cofactor F₄₂₀ was tested with different C18 columns with the best results for the system configuration achieved during the presented study with an NX C18 column. A standard containing a known distribution of F₄₂₀ derivates with varying glutamate tail length was used for reference purposes. This standard was kindly provided by Prof. Colin Jackson from the Australian National University. Analysis of glutamate tail length revealed differences in the overall concentration of cofactor F₄₂₀ and the distribution of F₄₂₀ tail length of methanogenic pure cultures and environmental samples (Figure 3).

Buffer	Composition
Lysis buffer (2x stock solution)	200 mM potassium dihydrogen phosphate (KH₂PO₄) 50 mM ethylenediaminetetraacetic acid (EDTA) 1% (w/v) polysorbate 80 (Tween 80) adjusted to pH 7.0 with 5 M sodium hydroxide solution
SPE conditioning solution	Methanol (HPLC grade)
SPE equilibration solution	Distilled water 0.2 µm filtered
SPE wash solution 1	25 mM ammonium acetate
SPE wash solution 2	Methanol (HPLC grade)
SPE elution buffer	2% (v/v) ammonia in methanol by diluting 20%–25% aqueous ammonia solution in methanol
HPLC mobile phase A	10 mM tetrabutylammonium hydroxide (TBAH) 20 mM di-ammonium hydrogen phosphate ((NH₄)₂HPO₄) adjusted to pH 7.0 with 85% phosphoric acid
HPLC mobile phase B	Acetonitrile (HPLC grade)

Table 1: Buffer and mobile phase composition for solid-phase extraction (SPE) and HPLC analysis.

Figure 1: Fluorescent methanogenic pure culture. Visualization of Methanosarcina thermophila via (A) phase-contrast microscopy and via (B) fluorescence microscopy when cofactor F₄₂₀ is excited with UV light (excitation at 395-440 nm and emission at 475-495 nm). Scale bar: 10 µm. Please click here to view a larger version of this figure.

Figure 2: Standard addition. Peak area of recovered cofactor F₄₂₀ after SPE from 1.0 g of matrix spiked with different volumes of M. thermophilus cultures. Matrix was amended with 0 µL, 250 µL, 500 µL, 750 µL, and 1000 µL of culture and subjected to different disintegration strategies: beat-beating, ultrasonic treatment, and pressure-temperature disintegration (autoclaving). Please click here to view a larger version of this figure.

Figure 3: Glutamate tail length distribution. Cofactor F₄₂₀ tail length distribution of pure cultures and environmental samples. From top to bottom: agriculturally used meadow (soil), forest (soil), mesophilic biogas reactor, pure culture of M. thermophilus, and pure culture of M. thermophila. Relative absorbance was calculated by normalization on the highest peak within the shown chromatogram. Please click here to view a larger version of this figure.

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 subsequent fluorometric analysis using HPLC, as described previously⁵, can not only provide information on the overall concentration of cofactor F₄₂₀ but also on polygutamate tail length distribution.

For extraction of cofactor F₄₂₀, a pressure-temperature treatment was shown to be highly effective (Figure 2) and is in accordance with the previous findings⁵^,²⁷^,³³. Via this method and applying a phosphate buffer lysis system including EDTA and polysorbate, the highest concentrations of cofactor F₄₂₀ were obtained from methanogenic pure cultures containing high concentrations of the factor. Moreover (and in comparison with the other tested cell disruption methods), the pressure-temperature treatment is easily applicable and material-saving.

SPE was performed to enable a downstream HPLC analysis aiming at the determination of cofactor F₄₂₀ polyglutamate tail length distribution within a sample. Among various ion exchangers, a weak anion mixed-mode polymeric sorbent showed the best performance and allows effective binding of the cofactor F₄₂₀ to the matrix for washing purposes as well as its subsequent removal from the extraction matrix after washing away undesired by-products. For this purpose, basic methanol proved best.

Via the presented method, various pure cultures and environmental samples could be analyzed reproducibly regarding cofactor F₄₂₀ (Figure 3). Even samples such as soils or sludges containing high proportions of undesired by-products could be analyzed by the presented procedure. Therefore, downstream analysis via HPLC was successfully implemented for analyzing the total concentration of F₄₂₀and the length distribution of the polyglutamate tails of F₄₂₀ derivatives. The detection of high levels of F₄₂₀ in soil and other samples supports Ney et el.⁵, who proposed that the cofactor is widespread in aerobic soil bacteria based on genomic and metagenomics analysis.

To sum up, this is the first protocol aiming at extracting and analyzing cofactor F₄₂₀ not only from pure cultures but also from environmental samples like soil or sludge. The most critical step in extracting F₄₂₀ from environmental samples is the SPE needed for the pre-clean-up of lysates for subsequent HPLC analysis. The presented protocol will be helpful for future projects to unveil the role of F₄₂₀ in various environments and bioprocesses.

Divulgations

The authors have nothing to disclose.

Acknowledgements

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.

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

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Markt, R., Wunderer, M., Prem, E. M., Mutschlechner, M., Lackner, N., Wagner, A. O. Extraction of Cofactor F₄₂₀ for Analysis of Polyglutamate Tail Length from Methanogenic Pure Cultures and Environmental Samples. J. Vis. Exp. (176), e62737, doi:10.3791/62737 (2021).