Changes in concentrations of coenzyme F420 analogs during batch growth of Methanosarcina barkeri and Methanosarcina mazei

Peck, Michael W.

doi:10.1128/aem.55.4.940-945.1989

Cited by 28 publications

(10 citation statements)

References 32 publications

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“…Thus, the CoM content per cell varied little with the growth phase. The same trend was found for coenzyme F 420 in methanogens (31). We observed that the amount of CoM per milligram of protein varied by up to a factor of 7 among organisms, which was comparable to the factor of 5 associated with the previously described CoM bioassay (5).…”

Section: Resultssupporting

confidence: 87%

Estimation of Methanogen Biomass by Quantitation of Coenzyme M

Elias

Krumholz

Tanner

et al. 1999

Appl Environ Microbiol

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Determination of the role of methanogenic bacteria in an anaerobic ecosystem often requires quantitation of the organisms. Because of the extreme oxygen sensitivity of these organisms and the inherent limitations of cultural techniques, an accurate biomass value is very difficult to obtain. We standardized a simple method for estimating methanogen biomass in a variety of environmental matrices. In this procedure we used the thiol biomarker coenzyme M (CoM) (2-mercaptoethanesulfonic acid), which is known to be present in all methanogenic bacteria. A high-performance liquid chromatography-based method for detecting thiols in pore water (A. Vairavamurthy and M. Mopper, Anal. Chim. Acta 78:363–370, 1990) was modified in order to quantify CoM in pure cultures, sediments, and sewage water samples. The identity of the CoM derivative was verified by using liquid chromatography-mass spectroscopy. The assay was linear for CoM amounts ranging from 2 to 2,000 pmol, and the detection limit was 2 pmol of CoM/ml of sample. CoM was not adsorbed to sediments. The methanogens tested contained an average of 19.5 nmol of CoM/mg of protein and 0.39 ± 0.07 fmol of CoM/cell. Environmental samples contained an average of 0.41 ± 0.17 fmol/cell based on most-probable-number estimates. CoM was extracted by using 1% tri-(N)-butylphosphine in isopropanol. More than 90% of the CoM was recovered from pure cultures and environmental samples. We observed no interference from sediments in the CoM recovery process, and the method could be completed aerobically within 3 h. Freezing sediment samples resulted in 46 to 83% decreases in the amounts of detectable CoM, whereas freezing had no effect on the amounts of CoM determined in pure cultures. The method described here provides a quick and relatively simple way to estimate methanogenic biomass.

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Section: Resultssupporting

confidence: 87%

Estimation of Methanogen Biomass by Quantitation of Coenzyme M

Elias

Krumholz

Tanner

et al. 1999

Appl Environ Microbiol

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“…The reduced cofactor F 420 supplies reduction equivalents for the stepwise covalent binding of the second and third hydrogen atom in methanogenesis (CH–CH 2 –CH 3 ) and is oxidized during the process. The quantification of cofactor F 420 in pure culture extracts has been realized using either HPLC [ 19 , 20 ] or assays with the ADP-linked hydrogenase system of Methanobacterium bryantii M.o.H [ 21 ]. The concentrations measured with either technique varied from 120 to 410 mg kg −1 cell mass (wet) for hydrogenotrophic methanogenic archaea but were considerably lower for organisms conducting non-hydrogenotrophic methanogenesis like Methanosarcina sp.…”

Section: Introductionmentioning

confidence: 99%

Flow cytometric quantification, sorting and sequencing of methanogenic archaea based on F420 autofluorescence

et al. 2017

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BackgroundThe widely established production of CH4 from renewable biomass in industrial scale anaerobic reactors may play a major role in the future energy supply. It relies on methanogenic archaea as key organisms which represent the bottleneck in the process. The quantitative analysis of these organisms can help to maximize process performance, uncover disturbances before failure, and may ultimately lead to community-based process control schemes. Existing qPCR and fluorescence microscopy-based methods are very attractive but can be cost-intensive and laborious.ResultsIn this study we present an autofluorescence-based, flow cytometric method for the fast low-cost quantification of methanogenic archaea in complex microbial communities and crude substrates. The method was applied to a methanogenic enrichment culture (MEC) and digester samples (DS). The methanogenic archaea were quantified using the distinct fluorescence of their cofactor F420 in a range from 3.7 × 108 (± 3.3 × 106) cells mL−1 and 1.8 x 109 (± 1.1 × 108) cells mL−1. We evaluated different fixation methods and tested the sample stability. Stable abundance and fluorescence intensity were recorded up to 26 days during aerobic storage in PBS at 6 °C. The discrimination of the whole microbial community from the ubiquitous particle noise was facilitated by SYBR Green I staining and enabled calculation of relative abundances of methanogenic archaea of up to 9.64 ± 0.23% in the MEC and up to 4.43 ± 0.74% in the DS. The metaprofiling of the mcrA gene reinforced the results.ConclusionsThe presented method allows for fast and reliable quantification of methanogenic archaea in microbial communities under authentic digester conditions and can thus be useful for process monitoring and control in biogas digesters.Electronic supplementary materialThe online version of this article (10.1186/s12934-017-0793-7) contains supplementary material, which is available to authorized users.

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“…C oenzyme F 420 , a deazaflavin derivative with two to eight glutamate residues in the side chain ( Fig. 1A) (1)(2)(3)(4), is found in a wide range of archaea and bacteria, including methanogens, where it serves major roles in catabolism (5,6). In mycobacteria, it facilitates redox transformations of cell wall lipids, neutralization of oxidative and nitrosative stress, and degradation of aromatic compounds (7)(8)(9)(10)(11)(12)(13)(14).…”

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confidence: 99%

“…Our report concerns the reaction that incorporates glutamate residues in the side chain of F 420 . We describe the product of this reaction as F 420 -n, where n is the number of glutamate residues that are linked almost exclusively via ␥-linkages (1)(2)(3)(4). In mycobacteria and late-evolving methanogenic archaea, such as Methanosarcina species, the value of n is two to eight (1).…”

mentioning

confidence: 99%

“…Such variations in the number of glutamate residues in the side chain of F 420 have physiological implications. For example, in Methanosarcina species, the intracellular levels of F 420 -2 through F 420 -5 and FO vary with the growth phase and energy substrate being used (4). For F 420 -dependent glucose-6-phosphate dehydrogenase (Fgd) of Mycobacterium smegmatis, the K d values of F 420 -5 through F 420 -6 are 6.5-fold lower than that observed with F 420 -2, and this change cause a 1.5-fold drop in the apparent K m value for glucose-6-phosphate (29).…”

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confidence: 99%

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Coenzyme F ₄₂₀ -Dependent Glucose-6-Phosphate Dehydrogenase-Coupled Polyglutamylation of Coenzyme F ₄₂₀ in Mycobacteria

2018

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Coenzyme F420 plays a key role in the redox metabolisms of various archaea and bacteria, including Mycobacterium tuberculosis. In M. tuberculosis, F420-dependent reactions have been linked to several virulence factors. F420 carries multiple glutamate residues in the side chain, forming F420-n species (n, number of glutamate residues), and the length of this side chain impacts cellular physiology. M. tuberculosis strains with F420 species carrying shorter side chains exhibit resistance to delamanid and pretomanid, two new tuberculosis (TB) drugs. Thus, the process of polyglutamylation of F420 is of great interest. It has been known from genetic analysis that in mycobacteria an F420-0 γ-glutamyl ligase (FbiB) introduces up to seven glutamate residues into F420. However, purified FbiB of M. tuberculosis (MtbFbiB) is either inefficient or incapable of incorporating more than two glutamates. We found that, in vitro, MtbFbiB synthesized side chains containing up to seven glutamate residues if F420 was presented to the enzyme in a two-electron reduced state (F420H2). Our genetic analysis in Mycobacterium bovis BCG and Mycobacterium smegmatis and an analysis of literature data on M. tuberculosis revealed that in these mycobacteria the polyglutamylation process requires the assistance of F420-dependent glucose-6-phosphate dehydrogenase (Fgd) which reduces F420 to F420H2. We hypothesize that, starting with F420-0H2, the amino-terminal domain of FbiB builds F420-2H2, which is then transferred to the carboxy-terminal domain for further glutamylation; F420-2H2 modifies the carboxy-terminal domain structurally to accommodate longer glutamyl chains. This system is analogous to folylpolyglutamate synthase, which introduces more than one glutamate residue into folate only after this vitamin is reduced to tetrahydrofolate. IMPORTANCE Coenzyme F420-dependent reactions of Mycobacterium tuberculosis, which causes tuberculosis, potentially contributes to the virulence of this bacterium. The coenzyme carries a glutamic acid-derived tail, the length of which influences the metabolism of M. tuberculosis. Mutations that eliminate the production of F420 with longer tails make M. tuberculosis resistant to two new tuberculosis drugs. This report describes that the synthesis of longer glutamyl tails of F420 requires concerted actions of two enzymes, one of which reduces the coenzyme prior to the action of the other, which catalyzes polyglutamylation. This knowledge will help to develop more effective tuberculosis (TB) drugs. Remarkably, the introduction of multiple glutamate residues into the sidechain of folate (vitamin B9) requires similar concerted actions, where one enzyme reduces the vitamin to tetrahydrofolate and the other catalyzes polyglutamylation; folate is required for DNA and amino acid synthesis. Thus, the reported research has also revealed a key similarity between two important cellular systems.

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Changes in concentrations of coenzyme F420 analogs during batch growth of Methanosarcina barkeri and Methanosarcina mazei

Cited by 28 publications

References 32 publications

Estimation of Methanogen Biomass by Quantitation of Coenzyme M

Estimation of Methanogen Biomass by Quantitation of Coenzyme M

Flow cytometric quantification, sorting and sequencing of methanogenic archaea based on F420 autofluorescence

Coenzyme F ₄₂₀ -Dependent Glucose-6-Phosphate Dehydrogenase-Coupled Polyglutamylation of Coenzyme F ₄₂₀ in Mycobacteria

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Changes in concentrations of coenzyme F420 analogs during batch growth of Methanosarcina barkeri and Methanosarcina mazei

Cited by 28 publications

References 32 publications

Estimation of Methanogen Biomass by Quantitation of Coenzyme M

Estimation of Methanogen Biomass by Quantitation of Coenzyme M

Flow cytometric quantification, sorting and sequencing of methanogenic archaea based on F420 autofluorescence

Coenzyme F 420 -Dependent Glucose-6-Phosphate Dehydrogenase-Coupled Polyglutamylation of Coenzyme F 420 in Mycobacteria

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Coenzyme F ₄₂₀ -Dependent Glucose-6-Phosphate Dehydrogenase-Coupled Polyglutamylation of Coenzyme F ₄₂₀ in Mycobacteria