The importance of bifunctional enzymes for U(VI) reduction in Thermus scotoductus SA-01

English: A thermophilic bacterium was isolated in 1999 by Kieft and co-workers from groundwater sampled at a depth of 3.2 kmbls in Mponeng, Republic of South Africa, which was later identified asThermus scotoducts SA-01. T. scotoductus SA-01 has shown the ability to reduce certain metals under growth and nongrowth conditions, including Mn(IV), Co(III)-EDTA, Cr(VI)and even uranium(VI). T. scotoductus SA-01 was grown in the presence of up to 1.25 mM uranium(VI), the specific growth rate decreased with the increasing uranium concentrations. A dramatic decline is seen at 0.5 mM, when compared to the control, where an apparent lag phase was observed. TEM and EDS analyses, subsequent to growth of T. scotoductus in uranium-containing medium, clearly show uranium metal clusters associated extracellularly with the cells. Uranium reduction assays with the Br-PADAP complexing agent, showed that T. scotoductus SA-01 has the ability to reduce 0.25 mM of uranium in under 20 hours. The whole cell reduction under non-growth conditions exhibited an optimum temperature of 65-70 °C and an optimum pH of 7-8. Unfortunatly 2D electrophoresis done on cultures grown in the absence and presence of uranium(VI) showed no dramatic shifts in protein expression. This, as well as the fact that uranium has no metabolic function, led us to believe that the protein involved in uranium reduction was more than likely a protein with another function but which can also reduce uranium and will not be expressed due to stressful conditions such as the presence of uranium. Screening for uranium(VI) reduction activity in the subcellulare fractions of cells, namely the periplasmic, cytoplasmic and membrane fractions, led to the discovery that activity is present in the periplasmic and membrane fractions. For further separation of these fractions chromatographic methods were applied and through a combination of anion and cation exchange columns a protein was identified which might be responsible for the uranium reduction activity. This protein was sent for MS/MS and N-terminal sequence determination and both of these methods identified the protein in question as a peptide ABC transporter, peptide binding protein. This was quite an interesting find since all work done in literature has pointed to cytochromes c-type proteins as the “uranium reductases” but work done in our lab has previously shown this protein to be capable of reducing gold, thus we know it can function as a “reductase”. The protein in question was expressed in Escherichia coli and purified using nickel affinity chromatography and uranium(VI) reduction activity was determined. Through structure modelling a disulphide bond believed to be responsible for uranium(VI) reduction was identified. Before uranium(VI) reduction was monitored over time, the disulfide moiety of the protein was reduced using β-mercaptoethanol. Just like the paramaters observed for whole cell reduction, the protein reduced uranium(VI) at an optimum of 65-70 °C and an optimum pH of 7-8. The aim of this study, namely to identify a protein involved in uranium(VI) reduction from Thermus scotoductus SA-01 was successfully completed.