Manipulation of the DNA coding for the desulphurizing activity in a new isolate of Arthrobacter sp.

A new bacterial strain able to cleave C-S bonds from organosulphur heterocyclic compounds through the 4-S pathway and tentatively classified as Arthrobacter sp. was recently isolated. In the present short article we describe the cloning and the characterization of the DNA encoding the enzymes responsible for desulphurization in this microorganism, referred to as Arthrobacter sp. DS7. The desulphurization operon was found to be located in a large plasmid that also bears the genes conferring cadmium and arsenic resistance. By shortening this plasmid, a new cloning vector was prepared and used to obtain a recombinant derivative strain that desulphurizes dibenzothiophene despite of the presence of inorganic sulphur in the growth medium.

The [Ni-Fe] hydrogenase from the thermophilic bacterium Acetomicrobium flavidum.

Biochemical analysis of the soluble hydrogenase from the thermophilic organism Acetomicrobium flavidum revealed that the enzyme is an alpha 2 beta 2 tetramer, with the alpha and beta subunits having a molecular mass of 50 kDa and 25 kDa, respectively. The most important biochemical properties of the enzyme are a specific activity of 77 mumol min-1 (mg protein)-1, a Km for methylviologen of 0.2 mM, a pH optimum of 7.5 and a T50 of about 70 degrees C. In addition, the enzyme is remarkably stable to oxygen inactivation, retaining full activity after 24 h exposure to air. By using oligodeoxynucleotides designed on the basis of the N-terminal sequences of the two subunits, the corresponding genes have been isolated and sequenced. When compared to the other hydrogenases so far characterized, the A. flavidum hydrogenase appears to be a typical [Ni-Fe] enzyme. The hydrogenase was expressed in Escherichia coli at high levels in a soluble form but it was not active. The analysis of the recombinant large subunit showed that it was not post-translationally processed at its C-terminus.