Name: Extraction of Cofactor F420 for Analysis of Polyglutamate Tail Length from Methanogenic Pure Cultures and Environmental Samples (Video) | JoVE Journal
Uploaded: 2021-10-14T14:00:00.000+00:00
Duration: 4 min 32 s
Description: Learn an optimized extraction method for cofactor F420 analysis using liquid chromatography and solid-phase extraction. This protocol enables accurate measurement of polyglutamate tail length in methanogenic cultures and complex environmental samples.

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5 minutes

We gratefully thank Prof. Colin Jackson for the support with purified cofactor F420. This research was supported by Tyrolean science fund (TWF) and the Universit&#228;t Innsbruck (Publikationsfonds). We greatly acknowledge the support of GPS, HK, SB, GG, and HB.

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
<ol>
	<li>Add up to 5 g of sample to appropriate tubes (e.g., 50 mL conical tubes).</li>
	<li>Add 5 mL of the lysis buffer (2x stock solution, Table 1) to the samples.</li>
	<li>Bring to a final volume of 10 mL with distilled water to reach a final concentration of 0.5 g&#183;mL-1.</li>
	<li>Vortex the diluted samples for 20 s.</li>
	<li>Autoclave for 30 min at 121 &#176;C.</li>
	<li>For dry samples like forest soil, bring to a final volume of 20 mL with distilled water after autoclaving and vortex the diluted sample. 
	&#8203;CAUTION: Temperature increase during autoclaving might cause tubes to burst.</li>
</ol>
2. Pre-purification of the cofactor F 420 via solid-phase extraction (SPE)
NOTE: All steps of SPE are conducted at room temperature
<ol>
	<li>Cool down the samples to room temperature.</li>
	<li>Centrifuge the autoclaved samples for 5 min at 11,000 x g.</li>
	<li>Prepare 5 mL SPE columns filled with 100 mg of weak anion mixed-mode polymeric sorbent.</li>
	<li>Condition the anion exchanger with 3 mL of methanol (condition solution, Table 1).</li>
	<li>Equilibrate the anion exchanger with 3 mL of distilled water (equilibration solution, Table 1).</li>
	<li>Load up to 9.0 mL of the supernatant from the centrifuged lysate onto the SPE column.</li>
	<li>Wash away impurities with 5 mL of 25 mM ammonium acetate (SPE wash solution 1, Table 1).</li>
	<li>Wash away&#160;impurities with 5 mL of methanol (SPE wash solution 2, Table 1).</li>
	<li>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.</li>
</ol>
3. Detection of the cofactor F420
<ol>
	<li>Set the oven to 40 &#176;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&#183;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).
	<ol>
		<li>Filter the eluted samples from the SPE into HPLC vials using a PTFE filter with a pore size of 0.22 &#181;m. 
		NOTE: PTFE filters with a pore size of 0.22 &#181;m are recommended.</li>
		<li>Inject 50 &#181;L 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.</li>
	</ol>
	</li>
</ol>

<ol><li>Greening, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Physiology%2C+biochemistry%2C+and+applications+of+F420-+and+Fo-dependent+redox+reactions%5BTitle%5D)+AND+%22Microbiology+and+Molecular+Biology+Reviews%3A+MMBR%22%5BJournal%5D)">Physiology, biochemistry, and applications of F420- and Fo-dependent redox reactions.</a> Microbiology and Molecular Biology Reviews: MMBR. 80 (2), 451-493 (2016).</li>
<li>Bashiri, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+revised+biosynthetic+pathway+for+the+cofactor+F420+in+prokaryotes%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">A revised biosynthetic pathway for the cofactor F420 in prokaryotes.</a> Nature Communications. 10 (1), 451(2019).</li>
<li>Grinter, R., Greening, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cofactor+F420%3A+an+expanded+view+of+its+distribution%2C+biosynthesis%2C+and+roles+in+bacteria+and+archaea%5BTitle%5D)+AND+%22FEMS+Microbiology+Reviews%22%5BJournal%5D)">Cofactor F420: an expanded view of its distribution, biosynthesis, and roles in bacteria and archaea.</a> FEMS Microbiology Reviews. , (2021).</li>
<li>Eirich, L. D., Vogels, G. D., Wolfe, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proposed+structure+for+coenzyme+F+420+from+methanobacterium%5BTitle%5D)+AND+%22Biochemistry%22%5BJournal%5D)">Proposed structure for coenzyme F 420 from methanobacterium.</a> Biochemistry. 17 (22), 4583-4593 (1978).</li>
<li>Ney, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+methanogenic+redox+cofactor+F420+is+widely+synthesized+by+aerobic+soil+bacteria%5BTitle%5D)+AND+%22The+ISME+Journal%22%5BJournal%5D)">The methanogenic redox cofactor F420 is widely synthesized by aerobic soil bacteria.</a> The ISME Journal. 11 (1), 125-137 (2017).</li>
<li>Braga, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metabolic+pathway+rerouting+in+Paraburkholderia+rhizoxinica+evolved+long-overlooked+derivatives+of+coenzyme+F420%5BTitle%5D)+AND+%22ACS+Chemical+Biology%22%5BJournal%5D)">Metabolic pathway rerouting in Paraburkholderia rhizoxinica evolved long-overlooked derivatives of coenzyme F420.</a> ACS Chemical Biology. 14 (9), 2088-2094 (2019).</li>
<li>Eirich, L. D., Vogels, G. D., Wolfe, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distribution+of+coenzyme+F420+and+properties+of+its+hydrolytic+fragments%5BTitle%5D)+AND+%22Journal+of+Bacteriology%22%5BJournal%5D)">Distribution of coenzyme F420 and properties of its hydrolytic fragments.</a> Journal of Bacteriology. 140 (1), 20-27 (1979).</li>
<li>Michaelis, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microbial+reefs+in+the+Black+Sea+fueled+by+anaerobic+oxidation+of+methane%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Microbial reefs in the Black Sea fueled by anaerobic oxidation of methane.</a> Science. 297 (5583), 1013-1015 (2002).</li>
<li>Knittel, K., Lösekann, T., Boetius, A., Kort, R., Amann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diversity+and+distribution+of+methanotrophic+archaea+at+cold+seeps%5BTitle%5D)+AND+%22Applied+and+Environmental+Microbiology%22%5BJournal%5D)">Diversity and distribution of methanotrophic archaea at cold seeps.</a> Applied and Environmental Microbiology. 71 (1), 467-479 (2005).</li>
<li>Lin, X. -L., White, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Occurrence+of+Coenzyme+F420+and+Its+y-Monoglutamyl+derivative+in+nonmethanogenic+archaebacteria%5BTitle%5D)+AND+%22Journal+of+Bacteriology%22%5BJournal%5D)">Occurrence of Coenzyme F420 and Its y-Monoglutamyl derivative in nonmethanogenic archaebacteria.</a> Journal of Bacteriology. 168 (1), 444-448 (1986).</li>
<li>Spang, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+genome+of+the+ammonia-oxidizing+Candidatus+Nitrososphaera+gargensis%3A+insights+into+metabolic+versatility+and+environmental+adaptations%5BTitle%5D)+AND+%22Environmental+Microbiology%22%5BJournal%5D)">The genome of the ammonia-oxidizing Candidatus Nitrososphaera gargensis: insights into metabolic versatility and environmental adaptations.</a> Environmental Microbiology. 14 (12), 3122-3145 (2012).</li>
<li>Mand, T. D., Metcalf, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Energy+conservation+and+hydrogenase+function+in+methanogenic+archaea%2C+in+particular+the+genus+Methanosarcina%5BTitle%5D)+AND+%22Microbiology+and+Molecular+Biology+Reviews%3A+MMBR%22%5BJournal%5D)">Energy conservation and hydrogenase function in methanogenic archaea, in particular the genus Methanosarcina.</a> Microbiology and Molecular Biology Reviews: MMBR. 83 (4), (2019).</li>
<li>Lupa, B., Hendrickson, E. L., Leigh, J. A., Whitman, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Formate-dependent+H2+production+by+the+mesophilic+methanogen+Methanococcus+maripaludis%5BTitle%5D)+AND+%22Applied+and+Environmental+Microbiology%22%5BJournal%5D)">Formate-dependent H2 production by the mesophilic methanogen Methanococcus maripaludis.</a> Applied and Environmental Microbiology. 74 (21), 6584-6590 (2008).</li>
<li>Kulkarni, G., Mand, T. D., Metcalf, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Energy+conservation+via+hydrogen+cycling+in+the+methanogenic+archaeon+Methanosarcina+barkeri%5BTitle%5D)+AND+%22mBio%22%5BJournal%5D)">Energy conservation via hydrogen cycling in the methanogenic archaeon Methanosarcina barkeri.</a> mBio. 9 (4), (2018).</li>
<li>Purwantini, E., Gillis, T. P., Daniels, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Presence+of+F420-dependent+glucose-6-phosphate+dehydrogenase+in+Mycobacterium+and+Nocardia+species%2C+but+absence+from+Streptomyces+and+Corynebacterium+species+and+methanogenic+Archaea%5BTitle%5D)+AND+%22FEMS+Microbiology+Letters%22%5BJournal%5D)">Presence of F420-dependent glucose-6-phosphate dehydrogenase in Mycobacterium and Nocardia species, but absence from Streptomyces and Corynebacterium species and methanogenic Archaea.</a> FEMS Microbiology Letters. 146 (1), 129-134 (1997).</li>
<li>Purwantini, E., Daniels, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Purification+of+a+novel+coenzyme+F420-dependent+glucose-6-phosphate+dehydrogenase+from+Mycobacterium+smegmatis%5BTitle%5D)+AND+%22Journal+of+Bacteriology%22%5BJournal%5D)">Purification of a novel coenzyme F420-dependent glucose-6-phosphate dehydrogenase from Mycobacterium smegmatis.</a> Journal of Bacteriology. 178 (10), 2861-2866 (1996).</li>
<li>McCormick, J. R. D., Morton, G. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identity+of+cosynthetic+factor+I+of+Streptomyces+aureofaciens+and+fragment+FO+from+coenzyme+F420+of+Methanobacterium+species%5BTitle%5D)+AND+%22Journal+of+the+American+Chemical+Society%22%5BJournal%5D)">Identity of cosynthetic factor I of Streptomyces aureofaciens and fragment FO from coenzyme F420 of Methanobacterium species.</a> Journal of the American Chemical Society. 104 (14), 4014-4015 (1982).</li>
<li>Coats, J. H., Li, G. P., Kuo, M. -S. T., Yurek, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Discovery%2C+production%2C+and+biological+assay+of+an+unusual+flavenoid+cofactor+involved+in+lincomycin+biosynthesis%5BTitle%5D)+AND+%22The+Journal+of+Antibiotics%22%5BJournal%5D)">Discovery, production, and biological assay of an unusual flavenoid cofactor involved in lincomycin biosynthesis.</a> The Journal of Antibiotics. 42 (3), 472-474 (1989).</li>
<li>Bown, L., Altowairish, M. S., Fyans, J. K., Bignell, D. R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Production+of+the+Streptomyces+scabies+coronafacoyl+phytotoxins+involves+a+novel+biosynthetic+pathway+with+an+F420+-dependent+oxidoreductase+and+a+short-chain+dehydrogenase%2Freductase%5BTitle%5D)+AND+%22Molecular+Microbiology%22%5BJournal%5D)">Production of the Streptomyces scabies coronafacoyl phytotoxins involves a novel biosynthetic pathway with an F420 -dependent oxidoreductase and a short-chain dehydrogenase/reductase.</a> Molecular Microbiology. 101 (1), 122-135 (2016).</li>
<li>Gurumurthy, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+F%28420%29+-dependent+anti-oxidant+mechanism+protects+Mycobacterium+tuberculosis+against+oxidative+stress+and+bactericidal+agents%5BTitle%5D)+AND+%22Molecular+microbiology%22%5BJournal%5D)">A novel F(420) -dependent anti-oxidant mechanism protects Mycobacterium tuberculosis against oxidative stress and bactericidal agents.</a> Molecular microbiology. 87 (4), 744-755 (2013).</li>
<li>Greening, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mycobacterial+F420H2-dependent+reductases+promiscuously+reduce+diverse+compounds+through+a+common+mechanism%5BTitle%5D)+AND+%22Frontiers+in+Microbiology%22%5BJournal%5D)">Mycobacterial F420H2-dependent reductases promiscuously reduce diverse compounds through a common mechanism.</a> Frontiers in Microbiology. 8, 1000(2017).</li>
<li>Mathew, S., Trajkovic, M., Kumar, H., Nguyen, Q. -T., Fraaije, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enantio-+and+regioselective+ene-reductions+using+F420H2-dependent+enzymes%5BTitle%5D)+AND+%22Chemical+Communications%22%5BJournal%5D)">Enantio- and regioselective ene-reductions using F420H2-dependent enzymes.</a> Chemical Communications. 54 (79), Cambridge, England. 11208-11211 (2018).</li>
<li>Ney, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cofactor+tail+length+modulates+catalysis+of+bacterial+F420-dependent+oxidoreductases%5BTitle%5D)+AND+%22Frontiers+in+Microbiology%22%5BJournal%5D)">Cofactor tail length modulates catalysis of bacterial F420-dependent oxidoreductases.</a> Frontiers in Microbiology. 8, 1902(2017).</li>
<li>Grinter, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellular+and+structural+basis+of+synthesis+of+the+unique+intermediate+dehydro-F420-0+in+mycobacteria%5BTitle%5D)+AND+%22mSystems%22%5BJournal%5D)">Cellular and structural basis of synthesis of the unique intermediate dehydro-F420-0 in mycobacteria.</a> mSystems. 5 (3), (2020).</li>
<li>Peck, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Changes+in+concentrations+of+coenzyme+F420+analogs+during+batch+growth+of+Methanosarcina+barkeri+and+Methanosarcina+mazei%5BTitle%5D)+AND+%22Applied+and+Environmental+Microbiology%22%5BJournal%5D)">Changes in concentrations of coenzyme F420 analogs during batch growth of Methanosarcina barkeri and Methanosarcina mazei.</a> Applied and Environmental Microbiology. 55 (4), (1989).</li>
<li>Gorris, L. G., vander Drift, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cofactor+contents+of+methanogenic+bacteria+reviewed%5BTitle%5D)+AND+%22BioFactors%22%5BJournal%5D)">Cofactor contents of methanogenic bacteria reviewed.</a> BioFactors. 4 (3-4), Oxford, England. 139-145 (1994).</li>
<li>Bair, T. B., Isabelle, D. W., Daniels, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structures+of+coenzyme+F%28420%29+in+Mycobacterium+species%5BTitle%5D)+AND+%22Archives+of+Microbiology%22%5BJournal%5D)">Structures of coenzyme F(420) in Mycobacterium species.</a> Archives of Microbiology. 176 (1-2), 37-43 (2001).</li>
<li>Portillo, M. C., Gonzalez, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Moonmilk+deposits+originate+from+specific+bacterial+communities+in+Altamira+Cave+%28Spain%29%5BTitle%5D)+AND+%22Microbial+Ecology%22%5BJournal%5D)">Moonmilk deposits originate from specific bacterial communities in Altamira Cave (Spain).</a> Microbial Ecology. 61, (2011).</li>
<li>Wagner, A. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Medium+preparation+for+the+cultivation+of+microorganisms+under+strictly+anaerobic%2Fanoxic+conditions%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%3A+JoVE%22%5BJournal%5D)">Medium preparation for the cultivation of microorganisms under strictly anaerobic/anoxic conditions.</a> Journal of Visualized Experiments: JoVE. (150), e60155(2019).</li>
<li>Lackner, N., Hintersonnleitner, A., Wagner, A. O., Illmer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hydrogenotrophic+methanogenesis+and+autotrophic+growth+of+Methanosarcina+thermophila%5BTitle%5D)+AND+%22Archaea%22%5BJournal%5D)">Hydrogenotrophic methanogenesis and autotrophic growth of Methanosarcina thermophila.</a> Archaea. 2018 (5), 1-7 (2018).</li>
<li>Wagner, A. O., Reitschuler, C., Illmer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+different+acetate%3A+Propionate+ratios+on+the+methanogenic+community+during+thermophilic+anaerobic+digestion+in+batch+experiments%5BTitle%5D)+AND+%22Biochemical+Engineering+Journal%22%5BJournal%5D)">Effect of different acetate: Propionate ratios on the methanogenic community during thermophilic anaerobic digestion in batch experiments.</a> Biochemical Engineering Journal. 90, 154-161 (2014).</li>
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</ol>

The authors have nothing to disclose.

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...

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.&#160;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&#39;-guanosine by 2-phospho-L-lactate guanylyltransferase (CofC). In the next step, L-lactyl-2-diphospho-5&#39;-guanosine is linked to FO by a 2-phospho-L-lactate transferase (CofD) to form F420-02. Finally, the enzyme F420-0:&#611;-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).

<table><tbody><tr><td>Autoclave</td><td></td><td></td><td></td></tr><tr><td>Biocompatible HPLC system equipped with gradient modul, oven and fluorescence detector</td><td>Shimadzu</td><td></td><td>HPLC system</td></tr><tr><td>Centrifuge and rotor for 50 mL &amp;ldquo;Falcon&amp;rdquo; tubes (11.000 rcf) and appropriate tubes</td><td></td><td></td><td></td></tr><tr><td>HPLC Column: Gemini NX C18, 5 &amp;mu;m, 150 x 3 mm</td><td>Phenomenex</td><td></td><td>HPLC column</td></tr><tr><td>PTFE filter (pore size 0.22 &amp;micro;m) to remove particulate matter prior HPLC analysis</td><td></td><td></td><td></td></tr><tr><td>Resin for SPE: Strata-X-AW 33 &amp;mu;m as weak anion mixed-mode polymeric sorbent</td><td>Phenomenex</td><td></td><td>weak anion mixed-mode polymeric sorbent</td></tr><tr><td>Vacuum manifold for SPE and appropriate collection tubes</td><td></td><td></td><td>SPE equipment</td></tr><tr><td>Vortex mixer</td><td></td><td></td><td></td></tr></tbody></table>

extraction of cofactor f420 for analysis of polyglutamate tail length from methanogenic pure cultures and environmental samples

Extraction of Cofactor F420 for Analysis of Polyglutamate Tail Length from Methanogenic Pure Cultures 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.

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...

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 ...

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Learn an optimized extraction method for cofactor F420 analysis using liquid chromatography and solid-phase extraction. This protocol enables accurate measurement of polyglutamate tail length in methanogenic cultures and complex environmental samples.

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