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:To test the effects of hypoxia on transcription in Caulobacter crescentus, we cultured cells in a New Brunswick bioreactor under controlled conditions. Prior to innoculation, the medium was bubbled with laboratory air at maximum flow and stirred at 300 rpm for 2 hours. After this period, the medium was considered saturated with air and the oxygen probe was set to 100%. Untreated cultures were grown in air-saturated complex medium at 30 degrees C to OD660=0.5 at pH=7 (continuous air-bubbling; 300 rpm stirring). At cell harvest in aerated culture, the dissolved oxygen probe remained above 98%. To subject cells to hypoxia, culture at OD660=0.5, pH=7 was sparged continuously with nitrogen gas; the dissolved oxygen level as measured by the gas probe dropped from 100% to 0% over the course of 5 minutes under this condition. Hypoxic cultures were continually stirred and bubbled with nitrogen for another 20 minutes after the dissolved gas probe read 0%. Hypoxic cells were then harvested for RNA isolation.
Project description:We present a comparative analysis of two genome fragments isolated from a diverse and widely distributed group of uncultured euryarchaea from deep-sea hydrothermal vents. The optimal activity and thermostability of a DNA polymerase predicted in one fragment were close to that of the thermophilic archaeon Thermoplasma acidophilum, providing evidence for a thermophilic way of life of this group of uncultured archaea.
