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,....

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

1. Add up to 5 g of sample to appropriate tubes (e.g., 50 mL conical tubes).
2. Add 5 mL of the lysis buffer (2x stock solution, Table 1) to the samples.
3. Bring to a final volume of 10 mL with distilled water to reach a final concentration of ....

Results

Pure cultures of Methanosarcina thermophila and Methanoculleus thermophilus, both thermophilic methanogenic Archaea, were grown in suitable media as described previously^29,30. 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

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