Heterologous Expression and Maturation of an NADP-Dependent [NiFe]-Hydrogenase: A Key Enzyme in Biofuel Production

Hydrogen gas is a major biofuel and is metabolized by a wide range of microorganisms. Microbial hydrogen production is catalyzed by hydrogenase, an extremely complex, air-sensitive enzyme that utilizes a binuclear nickel-iron [NiFe] catalytic site. Production and engineering of recombinant [NiFe]-hydrogenases in a genetically-tractable organism, as with metalloprotein complexes in general, has met with limited success due to the elaborate maturation process that is required, primarily in the absence of oxygen, to assemble the catalytic center and functional enzyme. We report here the successful production in Escherichia coli of the recombinant form of a cytoplasmic, NADP-dependent hydrogenase from Pyrococcus furiosus, an anaerobic hyperthermophile. This was achieved using novel expression vectors for the co-expression of thirteen P. furiosus genes (four structural genes encoding the hydrogenase and nine encoding maturation proteins). Remarkably, the native E. coli maturation machinery will also generate a functional hydrogenase when provided with only the genes encoding the hydrogenase subunits and a single protease from P. furiosus. Another novel feature is that their expression was induced by anaerobic conditions, whereby E. coli was grown aerobically and production of recombinant hydrogenase was achieved by simply changing the gas feed from air to an inert gas (N2). The recombinant enzyme was purified and shown to be functionally similar to the native enzyme purified from P. furiosus. The methodology to generate this key hydrogen-producing enzyme has dramatic implications for the production of hydrogen and NADPH as vehicles for energy storage and transport, for engineering hydrogenase to optimize production and catalysis, as well as for the general production of complex, oxygen-sensitive metalloproteins

Tick paralysis in spectacled flying-foxes (Pteropus conspicillatus) in North Queensland, Australia: impact of a ground-dwelling ectoparasite finding an arboreal host

When a parasite finds a new wildlife host, impacts can be significant. In the late 1980s populations of Spectacled Flying-foxes (SFF) (Pteropus conspicillatus), a species confined, in Australia, to north Queensland became infected by paralysis tick (Ixodes holocyclus), resulting in mortality. This Pteropus-tick relationship was new to Australia. Curiously, the relationship was confined to several camps on the Atherton Tableland, north Queensland. It was hypothesised that an introduced plant, wild tobacco (Solanum mauritianum), had facilitated this new host-tick interaction. This study quantifies the impact of tick paralysis on SFF and investigates the relationship with climate. Retrospective analysis was carried out on records from the Tolga Bat Hospital for 1998–2010. Juvenile mortality rates were correlated to climate data using vector auto-regression. Mortality rates due to tick paralysis ranged between 11.6 per 10,000 bats in 2003 and 102.5 in 2009; more female than male adult bats were affected. Juvenile mortality rates were negatively correlated with the total rainfall in January to March and July to September of the same year while a positive correlation of these quarterly total rainfalls existed with the total population. All tick affected camps of SFF were located in the 80% core range of S. mauritianum. This initial analysis justifies further exploration of how an exotic plant might alter the relationship between a formerly ground-dwelling parasite and an arboreal host