Monday, July 25, 2011
Grand Ballroom, 5th fl (Sheraton New Orleans)
Clostridium thermocellum is a thermophilic bacterium that can degrade cellulosic biomass using a complex extracellular cellulolytic organelle, cellulosome. During cellulose fermentation, the bacterium produces copious amounts of H2, catalyzed by the hydrogenase enzymes. Analysis of the genome sequence of C. thermocellum reveals the presence of at least three putative FeFe-hydrogenases (CtHydA1, CtHydA2 and CtHydA3) and one putative NiFe-hydrogenase. The versatility implies the significance of H2 metabolism during cellulose fermentation, yet little is known about the physiological functions of these hydrogenases. We employ two approaches to elucidate their physiological roles. In the first approach, we determine expression profiles of the four hydrogenases in C. thermocellum. q-RTPCR and protein immunoblots reveal the expression of some but not all of the hydrogenases when cultured in cellobiose. Analysis of both the DNA and transcript sequences encountered a mutation in CtHydA3 yielding a truncated and inactive protein. More detailed study will reveal the impact of this mutation on overall H2 production, the cellular metabolic profile, and its underlying regulatory mechanism. In the second approach, we developed a heterologous system in E. coli expressing the wild-type CtHydA3 along with three maturation proteins (CtHydE, CtHydF, and CtHydG). Protein immunoblots confirmed the expression of the His-tagged CtHydA3, which is corroborated by a two-fold increase in hydrogenase activity over a high background from native E. coli hydrogenases. Affinity purification of the His-tagged hydrogenase yielded an active protein, albeit with low yield. Work is underway to improve heterologous expression for the characterization of the CtHydA3 hydrogenase.