Name: extraction of cofactor f420 for analysis of polyglutamate tail length from methanogenic pure cultures and environmental samples
Uploaded: 2021-10-14T14:00:00
Description: Watch this Scientific Journal Video about Extraction of Cofactor F420 for Analysis of Polyglutamate Tail Length from Methanogenic Pure Cultures and Environmental Samples at JoVE.com

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 ...

<ol>
	<li>Greening, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology,+biochemistry,+and+applications+of+F420-+and+Fo-dependent+redox+reactions.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+revised+biosynthetic+pathway+for+the+cofactor+F420+in+prokaryotes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cofactor+F420:+an+expanded+view+of+its+distribution,+biosynthesis,+and+roles+in+bacteria+and+archaea.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proposed+structure+for+coenzyme+F+420+from+methanobacterium.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+methanogenic+redox+cofactor+F420+is+widely+synthesized+by+aerobic+soil+bacteria.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+pathway+rerouting+in+Paraburkholderia+rhizoxinica+evolved+long-overlooked+derivatives+of+coenzyme+F420.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+coenzyme+F420+and+properties+of+its+hydrolytic+fragments.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+reefs+in+the+Black+Sea+fueled+by+anaerobic+oxidation+of+methane.">Microbial reefs in the Black Sea fueled by anaerobic oxidation of methane.</a> Science. 297 (5583), 1013-1015 (2002).</li><li>Knittel, K., L&#246;sekann, T., Boetius, A., Kort, R., Amann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+and+distribution+of+methanotrophic+archaea+at+cold+seeps.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Occurrence+of+Coenzyme+F420+and+Its+y-Monoglutamyl+derivative+in+nonmethanogenic+archaebacteria.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genome+of+the+ammonia-oxidizing+Candidatus+Nitrososphaera+gargensis:+insights+into+metabolic+versatility+and+environmental+adaptations.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+conservation+and+hydrogenase+function+in+methanogenic+archaea,+in+particular+the+genus+Methanosarcina.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formate-dependent+H2+production+by+the+mesophilic+methanogen+Methanococcus+maripaludis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+conservation+via+hydrogen+cycling+in+the+methanogenic+archaeon+Methanosarcina+barkeri.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presence+of+F420-dependent+glucose-6-phosphate+dehydrogenase+in+Mycobacterium+and+Nocardia+species,+but+absence+from+Streptomyces+and+Corynebacterium+species+and+methanogenic+Archaea.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+a+novel+coenzyme+F420-dependent+glucose-6-phosphate+dehydrogenase+from+Mycobacterium+smegmatis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identity+of+cosynthetic+factor+I+of+Streptomyces+aureofaciens+and+fragment+FO+from+coenzyme+F420+of+Methanobacterium+species.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery,+production,+and+biological+assay+of+an+unusual+flavenoid+cofactor+involved+in+lincomycin+biosynthesis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+the+Streptomyces+scabies+coronafacoyl+phytotoxins+involves+a+novel+biosynthetic+pathway+with+an+F420+-dependent+oxidoreductase+and+a+short-chain+dehydrogenase/reductase.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+F(420)+-dependent+anti-oxidant+mechanism+protects+Mycobacterium+tuberculosis+against+oxidative+stress+and+bactericidal+agents.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycobacterial+F420H2-dependent+reductases+promiscuously+reduce+diverse+compounds+through+a+common+mechanism.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enantio-+and+regioselective+ene-reductions+using+F420H2-dependent+enzymes.">Enantio- and regioselective ene-reductions using F420H2-dependent enzymes.</a> Chemical Communications. 54 (79), 11208-11211 (2018).</li><li>Ney, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cofactor+tail+length+modulates+catalysis+of+bacterial+F420-dependent+oxidoreductases.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+structural+basis+of+synthesis+of+the+unique+intermediate+dehydro-F420-0+in+mycobacteria.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+concentrations+of+coenzyme+F420+analogs+during+batch+growth+of+Methanosarcina+barkeri+and+Methanosarcina+mazei.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cofactor+contents+of+methanogenic+bacteria+reviewed.">Cofactor contents of methanogenic bacteria reviewed.</a> BioFactors. 4 (3-4), 139-145 (1994).</li><li>Bair, T. B., Isabelle, D. W., Daniels, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structures+of+coenzyme+F(420)+in+Mycobacterium+species.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moonmilk+deposits+originate+from+specific+bacterial+communities+in+Altamira+Cave+(Spain).">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medium+preparation+for+the+cultivation+of+microorganisms+under+strictly+anaerobic/anoxic+conditions.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogenotrophic+methanogenesis+and+autotrophic+growth+of+Methanosarcina+thermophila.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+different+acetate:+Propionate+ratios+on+the+methanogenic+community+during+thermophilic+anaerobic+digestion+in+batch+experiments.">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><li>Wagner, A. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+preparation,+preservation,+and+storage+for+volatile+fatty+acid+quantification+in+biogas+plants.">Sample preparation, preservation, and storage for volatile fatty acid quantification in biogas plants.</a> Engineering in Life Sciences. 17 (2), 132-139 (2017).</li><li>Bashiri, G., Rehan, A. M., Greenwood, D. R., Dickson, J. M. J., Baker, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+engineering+of+cofactor+F420+production+in+Mycobacterium+smegmatis.">Metabolic engineering of cofactor F420 production in Mycobacterium smegmatis.</a> PloS one. 5 (12), 15803 (2010).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#8220;Falcon&#8221; tubes (11.000 rcf) and appropriate tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC Column: Gemini NX C18, 5 &#956;m, 150 x 3 mm</td>
			<td>Phenomenex</td>
			<td></td>
			<td>HPLC column</td>
		</tr>
		<tr>
			<td>PTFE filter (pore size 0.22 &#181;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 &#956;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

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...

Watch this Scientific Journal Video about Extraction of Cofactor F420 for Analysis of Polyglutamate Tail Length from Methanogenic Pure Cultures and Environmental Samples at JoVE.com

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

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.

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 ...

The cofactor F420 plays an important role in the primary and secondary metabolism of many prokaryotes. Measuring this molecule in environmental samples enables a deeper understanding of its prevalence and function. This technique enables the analysis of the cofactor in difficult sample materials such sludge and soil.Thereby the lysis of the material and pre-purification before analysis are the central steps. The protocol includes different steps during the sample preparation. These steps must be planned and prepared very well before starting with the sample processing.For example, the preparation of fresh buffers is important. To begin the sample lysis, place five grams of the sample in a 50-milliliter conical tube. Then add five milliliters of 2X lysis buffer to the samples, and bring the volume to 10 milliliters with distilled water to reach a final concentration of 0.5 grams per milliliter.Vortex the diluted samples for 20 seconds followed by autoclaving for 30 minutes at 121 degrees Celsius. For dry samples like forest soil, bring to a final volume of 20 milliliters with distilled water after autoclaving, and vortex the diluted sample again for 20 seconds as demonstrated. Once the samples cool down, centrifuge the sample lysate for five minutes at 11, 000 x g, and collect the supernatant.Prepare a five-milliliter solid-phase extraction or SPE column filled with 100 milligrams of weak anion mixed-mode polymeric sorbent conditioning the anion exchanger with three milliliters of methanol. Then, equilibrate the anion exchanger with three milliliters of distilled water. Load up to nine milliliters of the supernatant from the centrifuged lysate onto the SPE column.Sequentially wash the impurities from the column with five milliliters of 25-millimolar ammonia acetate and five milliliters of methanol. After washing, elute the cofactor F420 with one milliliter of elution buffer. Preset the oven to 40 degrees Celsius, and set the fluorescence detector to 420 nanometers extinction wavelength and 470 nanometers emission wavelength.Next, filter the eluted sample from the SPE into HPLC vials using a PTFE filter with a pore size of 0.22 micron. Inject 50 microliters of the filtered sample into the HPLC system, and separate cofactor F420 via gradient mode using a combination of mobile phases as described in the manuscript. Analyze the cofactor F420 composition and concentration.The growth of pure cultures of methanogens was verified by microscopy. In phase contrast microscopy, agglomerates of methanogens are visible, which emit a bluish light when cofactor F420 is excited with ultraviolet light. The peak area of recovered cofactor F420 after SPE with different volumes of the Methanoculleus thermophilus cultures was analyzed.The autoclaving disintegration strategy achieved the highest peak area, concluding maximum extraction efficiency using a pressure-temperature treatment. Analysis of the tail length distribution revealed the differences in the overall concentration of cofactor F420 and the distribution of F420 tail length of the pure methanogenic cultures in environmental samples. When applying this procedure, be aware to completely collect total volume of the elution buffer or record the exact volume of flow-through.This technique paves the way for the exploration of cofactor F420 in more complex samples like soils and sludges and enables deeper insights into the ecological impact of this molecule.

Research

JoVE Journal

Environment

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

Use of Chironomidae (Diptera) Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Use of Chironomidae Diptera Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. ...

Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA ...

Physical Isolation of Endospores from Environmental Samples by Targeted Lysis of Vegetative Cells

Endospore formation is a survival strategy found among some bacteria from the phylum Firmicutes. During endospore formation, these bacteria enter a ...

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and ...

Preparation and Testing of Impedance-based Fluidic Biochips with RTgill-W1 Cells for Rapid Evaluation of Drinking Water Samples for Toxicity

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial (RTgill-W1) cells for use in a field portable water toxicity ...

Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about ...

Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies ...

A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment ...