During selenate respiration by Thauera selenatis, the reduction of selenate results in the formation of intracellular selenium (Se) deposits that are ultimately secreted as Se nanospheres of approximately 150 nm in diameter. We report that the Se nanospheres are associated with a protein of approximately 95 kDa. Subsequent experiments to investigate the expression and secretion profile of this protein have demonstrated that it is up-regulated and secreted in response to increasing selenite concentrations. The protein was purified from Se nanospheres, and peptide fragments from a tryptic digest were used to identify the gene in the draft T. selenatis genome. A matched open reading frame was located, encoding a protein with a calculated mass of 94.5 kDa. N-terminal sequence analysis of the mature protein revealed no cleavable signal peptide, suggesting that the protein is exported directly from the cytoplasm. The protein has been called Se factor A (SefA), and homologues of known function have not been reported previously. The sefA gene was cloned and expressed in Escherichia coli, and the recombinant His-tagged SefA purified. In vivo experiments demonstrate that SefA forms larger (approximately 300 nm) Se nanospheres in E. coli when treated with selenite, and these are retained within the cell. In vitro assays demonstrate that the formation of Se nanospheres upon the reduction of selenite by glutathione are stabilized by the presence of SefA. The role of SefA in selenium nanosphere assembly has potential for exploitation in bionanomaterial fabrication.nanoparticles | biomineralization | anaerobic respiration

Bacterial anaerobic respiration using selenium oxyanions as the sole electron acceptor primarily result in the precipitation of selenium biominerals observed as either intracellular or extracellular selenium deposits. Although a better understanding of the enzymology of bacterial selenate reduction is emerging, the processes by which the selenium nanospheres are constructed, and in some cases secreted, has remained poorly studied. Thauera selenatis is a Gram-negative betaproteobacterium that is capable of respiring selenate due to the presence of a periplasmic selenate reductase (SerABC). SerABC is a molybdoenzyme that catalyses the reduction of selenate to selenite by accepting electrons from the Q-pool via a dihaem c-type cytochrome (cytc4). The product selenite is presumed to be reduced in the cytoplasm, forming intracellular selenium nanospheres that are ultimately secreted into the surrounding medium. The secretion of the selenium nanospheres is accompanied by the export of a ~95 kDa protein SefA (selenium factor A). SefA has no cleavable signal peptide, suggesting that it is also exported directly for the cytoplasmic compartment. It has been suggested that SefA functions to stabilize the formation of the selenium nanospheres before secretion, possibly providing reaction sites for selenium nanosphere creation or providing a shell to prevent subsequent selenium aggregation. The present paper draws on our current knowledge of selenate respiration and selenium biomineralization in T. selenatis and other analogous systems, and extends the application of nanoparticle tracking analysis to determine the size distribution profile of the selenium nanospheres secreted.

Selenate reductase (SER) from Thauera selenatis is a periplasmic enzyme that has been classified as a type II molybdoenzyme. The enzyme comprises three subunits SerABC, where SerC is an unusual b-heme cytochrome. In the present work the spectropotentiometric characterization of the SerC component and the identification of redox partners to SER are reported. The mid-point redox potential of the b-heme was determined by optical titration (E m ؉ 234 ؎ 10 mV). A profile of periplasmic c-type cytochromes expressed in T. selenatis under selenate respiring conditions was undertaken. Two c-type cytochromes were purified (ϳ24 and ϳ6 kDa), and the 24-kDa protein (cytc-Ts4) was shown to donate electrons to SerABC in vitro. Protein sequence of cytc-Ts4 was obtained by N-terminal sequencing and liquid chromatographytandem mass spectrometry analysis, and based upon sequence similarities, was assigned as a member of cytochrome c 4 family. Redox potentiometry, combined with UV-visible spectroscopy, showed that cytc-Ts4 is a diheme cytochrome with a redox potential of ؉282 ؎ 10 mV, and both hemes are predicted to have His-Met ligation. To identify the membrane-bound electron donors to cytcTs4, growth of T. selenatis in the presence of respiratory inhibitors was monitored. The specific quinol-cytochrome c oxidoreductase (QCR) inhibitors myxothiazol and antimycin A partially inhibited selenate respiration, demonstrating that some electron flux is via the QCR. Electron transfer via a QCR and a diheme cytochrome c 4 is a novel route for a member of the DMSO reductase family of molybdoenzymes.Within the DMSO reductase family of type II molybdoenzymes (1) there is a distinct clade of enzymes that are translocated to the periplasm using the twin arginine translocation (TAT) 4 pathway (2, 3) and possess a monomeric b-type heme-containing ␥-subunit (1). The enzymes within this clade function as either dehydrogenases (e.g. ethylbenzene dehydrogenase (EBDH) from Aromatoleum aromaticum (4) and dimethylsulfide dehydrogenase from Rhodovulum sulfidophilum (1, 5)) or reductases (e.g. selenate reductase from Thauera selenatis (6, 7) and chlorate reductase from Ideonella dechloratans (8, 9)) and catalyze either hydride or oxygen transfer as generalized by Reaction 1.These soluble enzymes consist of three subunits and in addition to the b-heme cytochrome (␥-subunit), they comprise an ironsulfur protein (␤-subunit) coordinating 1 ϫ [3Fe-4S] cluster and 3 ϫ [4Fe-4S] clusters, and a catalytic component (␣-subunit) that coordinates a [4Fe-4S] cluster and the active site molybdopterin guanine dinucleotide cofactor (10, 11) (Fig. 1). The reductases play a pivotal function, coupling the reduction of substrates to the generation of the proton-motive force (PMF). Identifying the route by which electrons are transferred to these reductases is vital to understanding their bioenergetics (12). How periplasmic substrate reduction can generate a PMF, which is sufficient to support growth, is of considerable interest. The use of selenate and selenite as bacterial...