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

Abstract

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.

Afrikaans: 'nTermofiliese bakteriëwas geïsoleer in 1999 deur Kieft en mede-werkers van grondwater op' n diepte van 3,2 kmbls in Mponeng, Republiek van Suid-Afrika, die bakterië was later geïdentifiseer as Thermus scotoducts SA-01. T. scotoductus SA-01 het die vermoë getoon om sekere metale onder groei en niegroei kondisies te reduseer. Die metale sluit in Mn (IV), Co (III)-EDTA, Cr (VI), en selfsuraan(VI). T. scotoductus SA-01 het gegroei in die teenwoordigheid van tot 1,25 mM uraan(VI), die spesifieke groeitempo het afgeneem met ‘n toename inuraan konsentrasies. In vergelyking met die kontrole was daar 'n dramatiese afname in groei tempo te sien na 0.5 mM. TEM en EDS analise, na afloop van die groei van T. scotoductus in uraan-bevattende middel, toon duidelike uraan “clusters” geassosieer met die selle extraselluler. Uraan reduksie was waargeneem met die Bromo-PADAP tegniek. Resultatehet aangedui dat T. scotoductus SA-01 het die vermoë om 0.25 mM uraan te reduseer binne 20 uur. Die heel sel reduksie onder nie-groei kondisiesblyk om optimal by ‘n temperatuur van 65-70 °C en'n pH van 7-8 plaas te vind. Ongelukkig het 2D elektroforese, gedoen op kulture gekweek in die afwesigheid en teenwoordigheid van uraan(VI), geen dramatiese verskuiwings in proteïen uitdrukkingin die teenwoordigheid van uraan getoon nie. Dit, asook die feit dat uraan geen metaboliese funksie het nie, impliseer dat die proteïen wat betrokke is by uraan reduksie meer as waarskynlik 'n proteïen met' n ander funksie is, maar wat ook uraan kan reduseer. Dit sal dus ook nie vervaardig word indien stresvolle toestande soos die teenwoordigheid van uraan bestaan nie. Ondersoek na uraan(VI) reduksie aktiwiteit in die subsellulere fraksies van die selle, naamlik in die periplasmiese, sitoplasmiese en membraan fraksies het gelei tot die ontdekking dat aktiwiteit teenwoordig is in die periplasmiese en membraan fraksies. Vir verdere skeiding van hierdie fraksies was chromatografiese metodes aangewend en deur 'n kombinasie van die anioon en kation uitruilerkolomme was 'n proteïen geïdentifiseer wat dalk verantwoordelik kan wees vir die uraan reduksie aktiwiteit. Hierdie proteïene is gestuur vir MS/MS en N-terminale volgorde bepaling en beide van hierdie metodes het dieproteïen geïdentifiseer as 'n peptied ABC vervoerder, wat ‘n peptied bindende proteïen. Die bevinding is teenstreidig met die meeste literatuur verslae wat daarop dui dat sitochroom c-tipe proteiene uraan reduseer. Die vermoë van die geisoleerde ABC peptide om goud te kan (Van Marwijk, 2010), impliseer dat die proteien die vermoë het om as ‘n “reduktase” te kan funksioneer. Die proteïen van belang was bevestig in Escherichia coli, gesuiwer deur gebruik te maak van nikkel affiniteitschromatografiewaarna uraan(VI) reduksie karakteristieke bepaal was. Deur ondersoek in te stel met betrekking tot die struktuur van die proteïen was ‘n di-sulfide verbinding geidentifiseer wat moontlik verantwoordelik kan wees vir die uraan(VI) reduksie aktiwiteit. Voordaturaan(VI) reduksie in aanvang geneem het, was die di-sulfide verbinding van die proteïen gereduseer met behulp van β-mercaptoethanol. Optimale eksperimentele parameters vir uraan(VI)reduksie met die betrokke proteien was waargeneem tussen65-70 °C enpH gebied tussen 7-8. Die doel van hierdie studie, naamlik om 'n proteïen wat betrokke is by uraan(VI) reduksie in die termofiliese bakteriêT. scotoductus SA-01 te identifiseer was suksesvol voltooi.