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Methanobrevibacter boviskoreani JH1T growth on alcohols allows development of a high throughput bioassay to detect methanogen inhibition

ABSTRACT: Highlights • Methanobrevibacter boviskoreani JHIT, a rumen methanogen, can grow without pressurised hydrogen, using a variety of short chain alcohols.• Results demonstrate that in the absence of hydrogen, JHIT can use ethanol, propanol and butanol but not methanol, as a source of reducing potential for methanogenesis.• The ability of JHIT to use ethanol to drive methane production makes it a model rumen methanogen to use in high throughput culture-based bioassays.• Results demonstrate for the first time a direct inhibitory effect of the bacteriocin nisin on a rumen methanogen.• Methanobrevibacter boviskoreani JHIT is a useful methanogen culture for researchers looking to conduct high-throughput experiments with rumen methanogens. Rumen methanogenic archaea use by-products of fermentation to carry out methanogenesis for energy generation. A key fermentation by-product is hydrogen (H2), which acts as the source of reducing potential for methane (CH4) formation in hydrogenotrophic methanogens. The in vitro cultivation of hydrogenotrophic rumen methanogens requires pressurised H2 which limits the ability to conduct high-throughput screening experiments with these organisms. The genome of the hydrogenotrophic methanogen Methanobrevibacter boviskoreani JH1T harbors genes encoding an NADP-dependent alcohol dehydrogenase and a F420-dependent NADP reductase, which may facilitate the transfer of reducing potential from ethanol to F420 via NADP. The aim of this study was to explore the anaerobic culturing of JH1T without pressurised H2, using a variety of short chain alcohols. The results demonstrate that in the absence of H2, JHIT can use ethanol, 1-propanol, and 1-butanol but not methanol, as a source of reducing potential for methanogenesis. The ability to use ethanol to drive CH4 formation in JH1T makes it possible to develop a high throughput culture-based bioassay enabling screening of potential anti-methanogen compounds. The development of this resource will help researchers globally to accelerate the search for methane mitigation technologies for ruminant animals. Global emissions pathways that are consistent with the temperature goal of the Paris Agreement, rely on substantial reductions of agricultural greenhouse gasses, particularly from ruminant animals. Graphical abstract Image, graphical abstract

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PROVIDER: S-EPMC10139955 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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Project description:BackgroundSchistosomiasis, caused by infection with the blood fluke Schistosoma, is responsible for greater than 200,000 human deaths per annum. Objective high-throughput screens for detecting novel anti-schistosomal targets will drive 'genome to drug' lead translational science at an unprecedented rate. Current methods for detecting schistosome viability rely on qualitative microscopic criteria, which require an understanding of parasite morphology, and most importantly, must be subjectively interpreted. These limitations, in the current state of the art, have significantly impeded progress into whole schistosome screening for next generation chemotherapies.Methodology/principal findingsWe present here a microtiter plate-based method for reproducibly detecting schistosomula viability that takes advantage of the differential uptake of fluorophores (propidium iodide and fluorescein diacetate) by living organisms. We validate this high-throughput system in detecting schistosomula viability using auranofin (a known inhibitor of thioredoxin glutathione reductase), praziquantel and a range of small compounds with previously-described (gambogic acid, sodium salinomycin, ethinyl estradiol, fluoxetidine hydrochloride, miconazole nitrate, chlorpromazine hydrochloride, amphotericin b, niclosamide) or suggested (bepridil, ciclopirox, rescinnamine, flucytosine, vinblastine and carbidopa) anti-schistosomal activities. This developed method is sensitive (200 schistosomula/well can be assayed), relevant to industrial (384-well microtiter plate compatibility) and academic (96-well microtiter plate compatibility) settings, translatable to functional genomics screens and drug assays, does not require a priori knowledge of schistosome biology and is quantitative.Conclusions/significanceThe wide-scale application of this fluorescence-based bioassay will greatly accelerate the objective identification of novel therapeutic lead targets/compounds to combat schistosomiasis. Adapting this bioassay for use with other parasitic worm species further offers an opportunity for great strides to be made against additional neglected tropical diseases of biomedical and veterinary importance.

Project description:BackgroundMethane (CH(4)) is a potent greenhouse gas (GHG), having a global warming potential 21 times that of carbon dioxide (CO(2)). Methane emissions from agriculture represent around 40% of the emissions produced by human-related activities, the single largest source being enteric fermentation, mainly in ruminant livestock. Technologies to reduce these emissions are lacking. Ruminant methane is formed by the action of methanogenic archaea typified by Methanobrevibacter ruminantium, which is present in ruminants fed a wide variety of diets worldwide. To gain more insight into the lifestyle of a rumen methanogen, and to identify genes and proteins that can be targeted to reduce methane production, we have sequenced the 2.93 Mb genome of M. ruminantium M1, the first rumen methanogen genome to be completed.Methodology/principal findingsThe M1 genome was sequenced, annotated and subjected to comparative genomic and metabolic pathway analyses. Conserved and methanogen-specific gene sets suitable as targets for vaccine development or chemogenomic-based inhibition of rumen methanogens were identified. The feasibility of using a synthetic peptide-directed vaccinology approach to target epitopes of methanogen surface proteins was demonstrated. A prophage genome was described and its lytic enzyme, endoisopeptidase PeiR, was shown to lyse M1 cells in pure culture. A predicted stimulation of M1 growth by alcohols was demonstrated and microarray analyses indicated up-regulation of methanogenesis genes during co-culture with a hydrogen (H(2)) producing rumen bacterium. We also report the discovery of non-ribosomal peptide synthetases in M. ruminantium M1, the first reported in archaeal species.Conclusions/significanceThe M1 genome sequence provides new insights into the lifestyle and cellular processes of this important rumen methanogen. It also defines vaccine and chemogenomic targets for broad inhibition of rumen methanogens and represents a significant contribution to worldwide efforts to mitigate ruminant methane emissions and reduce production of anthropogenic greenhouse gases.

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Methanobrevibacter boviskoreani JH1T growth on alcohols allows development of a high throughput bioassay to detect methanogen inhibition

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