Project description:Biogenic methane formation, methanogenesis, a key process in the global carbon cycle is the only energy metabolism known to sustain growth of the microorganisms employing it, the methanogenic archaea. All known methanogenic pathways converge at the methane-liberating step where also the terminal electron acceptor of methanogenic respiration, the heterodisulfide of coenzyme M and coenzyme B is formed. Carbon monoxide (CO) utilization of Methanosarcina acetivorans is unique in that the organism can shift from methanogenesis towards acetogenesis. Here, we show that M. acetivorans can dispense of methanogenesis for energy conservation completely. By disrupting the methanogenic pathway through targeted mutagenesis, followed by adaptive evolution, a strain capable of sustained growth by CO-dependent acetogenesis was created. Still, a minute flux through the methane-liberating reaction remained essential, which was attributed to the involvement of the heterodisulfide in at least one essential anabolic reaction. Genomic and proteomic analysis showed that substantial metabolic rewiring had occurred in the strain. Most notably, heterodisulfide reductase, the terminal respiratory oxidoreductase was eliminated to funnel the heterodisulfide towards anabolism. These results suggest that the metabolic flexibility of “methanogenic” archaea is much greater than anticipated and open avenues for probing the mechanism of energetic coupling and the crosstalk between catabolism and anabolism.
Project description:Population dynamics of methanogenic genera was investigated in pilot anaerobic digesters. Cattle manure and two-phase olive mill wastes were codigested at a 3:1 ratio in two reactors operated at 37 ï¾°C and 55 ï¾°C. Other two reactors were run with either residue at 37 ï¾°C. Sludge DNA extracted from samples taken from all four reactors on days 4, 14 and 28 of digestion was used for hybridisation with the AnaeroChip, an oligonucleotide microarray targeting those groups of methanogenic archaea that are commonly found under mesophilic and thermophilic conditions (Franke-Whittle et al. 2009, in press, doi:10.1016/j.mimet.2009.09.017).
Project description:Identification of the peptides composing the enriched multisubunit enzymes natively purified from the microbial enrichment, based on gel bands obtained by native electrophoresis. The anaerobic oxidation of alkanes is a microbial process occurring in deep-sea hydrocarbon seeps that plays a key ecological role in these exotic niches. The metabolic capacity of anaerobic ethane oxidation, involving uncharted biochemistry, was reported in two archaeal species depending on sulfate-reducing partner bacteria. This study deciphers the molecular basis of the CO2-generating steps of ethanotrophy by characterising the native archaeal enzymes isolated from a thermophilic enrichment. While other microorganisms couple these steps to ferredoxin reduction, we found that the CO-dehydrogenase and the formylmethanofuran-dehydrogenase are bound to F420-reductase modules. The crystal structures of these multi-metalloenzyme complexes revealed electronic bridges coupling C1-oxidation to F420-reduction. Accordingly, both systems exhibit robust F420-reductase activities, which are not detected in methanogenic or methanotrophic relative organisms. We speculate that the whole catabolism of these archaea is reoriented towards F420-reduction, which facilitates the electron transfer to the sulfate-reducing partner, therefore representing the driving force of ethanotrophy.
Project description:Phosphofructokinase (PFK) is a key enzyme of the glycolytic pathway in all domains of life. Two related PFKs, ATP-dependent and PP(i)-dependent PFK, have been distinguished in bacteria and eucarya, as well as in some archaea. Hyperthermophilic archaea of the order Thermococcales, including Pyrococcus and Thermococcus spp., have recently been demonstrated to possess a unique ADP-dependent PFK (ADP-PFK) that appears to be phylogenetically distinct. Here, we report the presence of ADP-PFKs in glycogen-producing members of the orders Methanococcales and Methanosarcinales, including both mesophilic and thermophilic representatives. To verify the substrate specificities of the methanogenic kinases, the gene encoding the ADP-PFK from Methanococcus jannaschii was functionally expressed in Escherichia coli, and the produced enzyme was purified and characterized in detail. Compared to its counterparts from the two members of the order Thermococcales, the M. jannaschii ADP-PFK has an extremely low K(m) for fructose 6-phosphate (9.6 microM), and it accepts both ADP and acetyl-phosphate as phosphoryl donors. Phylogenetic analysis of the ADP-PFK reveals it to be a key enzyme of the modified Embden-Meyerhof pathway of heterotrophic and chemolithoautotrophic archaea. Interestingly, uncharacterized homologs of this unusual kinase are present in several eucarya.
Project description:Proteomic quantitative analysis of the methanogenic archaea in the experimental group and the control group,each group was repeated three times.For each 6-plex TMT,control samples were labelled with TMT tags 126,127,128,and experimental samples were labelled with TMT tags 129,130,and 131,respectively
Project description:In this study, we used multiple meta-omic approaches to characterize the microbial community and the active metabolic pathways of a stable industrial biogas reactor operating at thermophilic temperatures (60°C) and elevated levels of free ammonia (367 mg NH3-N/L).