Synopsis

Desulfovibrio sps are obligatory anaerobic sulfate reducing bacteria. These bacteria hold important place in biodegradation as well as occasional pathogens and also in biotechnology, as rich source of biotechnologically important metalloprotiens. It is established that multiple hydrogenases and sulfate reducing enzymes (four in number) are integral for deriving cellular bioenergetics. The hydrogenases are iron sulfur cluster periplasmic enzymes, which are highly sensitive to oxygen. The catalytic active site of such enzymes is often associated with transition metals like Nickel and Iron. The structurally wellcharacterized Nickel Iron [NiFe] Hydrogenase undergoes N-terminal and C-terminal processing before getting directed to periplasm by twin-arginine pathway. The complex and conserved maturation process of these enzymes does not allow simple expression of these proteins into E. coli, thus making it difficult to control the expression and manipulation of active site. Working on these lines, a new periplasmic hydrogenases that included a heterodimeric Nickel Iron selenium [NiFeSe] and a membrane associated multimeric Ech hydrogenase were sequenced from the genomic library of Desulfovibrio vulgaris Miyazaki F by means of Southern hybridization using digoxygenin labeled homologous probes. The [NiFeSe] hydrogenase genes were located only about 1.5 kb upstream of the previously recognized [NiFe] Hydrogenase. Known to be more active and less oxygen sensitive, the expression of [NiFeSe] hydrogenase was obtained as a dominant hydrogenase by adding 1M selenium and 1M Nickel in growth medium. Further, five hydrogenase pleiotropic genes were elucidated. Various cloning strategies were used to obtain expression and purification of these hydrogenase maturation proteins in E. coli.. Additionally, two proteins of sulfate metabolism (APS reductase and Desulfoviridin) were purified and crystallized. The efforts continued at co-expression of hydrogenase along with maturation machinery, aiming at developing a system for site directed mutagenesis of hydrogenase and future application into a proposed biofuel cell along with some photosynthetic proteins. In conclusion of this work, the obtained information could form a basis to generate various hydrogenase deletion mutants and compare growth kinetics by means of molecular and biochemical characterizations. As the role of selenium in the regulation of hydrogenase expression has been figured out, additional factors can be identified to control the hydrogen metabolism of the cell in a desirable manner. The [NiFeSe] hydrogenase of DvMF has been reported for the first time, a viable protocol for the copious purification of this detergent soluble hydrogenase needs to be procured for subsequent structural, biochemical and spectroscopic characterizations. This may be also helpful to ascertain the types of bonding present in the iron sulfur clusters of the small subunit. The integration of the selenocysteine, when expressed in E. coli, is another interesting topic to study. The [NiFe] hydrogenase of DvMF, when expressed from a plasmid can be now

isolated from non-native hosts by means of a strep-tag, in a highly pure form. This could be very helpful while employing some of the closely related heterologous host strains and even when expressed homologously, especially for carrying mutagenesis studies. The experiments to try new antibiotic markers in Desulfovibrio sps are on the way and also the logistics for co-expressing the structural together with the maturation genes sequenced for DvMF in this work, using E. coli as host appear to be feasible. As the [NiFe] - and [NiFeSe] hydrogenases are quite comparable structurally and biochemically, similar trials can be extended to the [NiFeSe] hydrogenase of DvMF that are epitope tagging and co-expression with the maturation machinery.