A bacterial process for selenium nanosphere assembly Charles M. Debieuxa, Elizabeth J. Dridgea,1, Claudia M. Muellera,1, Peter Splatta, Konrad Paszkiewicza, Iona Knighta, Hannah Florancea, John Lovea, Richard W. Titballa, Richard J. Lewisb, David J. Richardsonc, and Clive S. Butlera,2 aBiosciences, College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter EX4 4QD, United Kingdom; bInstitute for Cell and Molecular Biosciences, University of Newcastle, Newcastle upon Tyne NE2 4HH, United Kingdom; and cSchool of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom Edited by Dianne K. Newman, California Institute of Technology/Howard Hughes Medical Institute, Pasadena, CA, and accepted by the Editorial Board July 1, 2011 (received for review April 13, 2011) Thauera selenatis 2− − þ 2− During selenate respiration by , the reduction of SeO4 þ 2e þ 2H ⇆ SeO3 þ H2O [1] selenate results in the formation of intracellular selenium (Se) deposits that are ultimately secreted as Se nanospheres of approxi- and mately 150 nm in diameter. We report that the Se nanospheres 2− − þ 0 SeO þ 4e þ 6H ⇆ Se þ 3H2O: [2] are associated with a protein of approximately 95 kDa. Subsequent 3 experiments to investigate the expression and secretion profile of Thauera selenatis (a β-proteobacterium) is by far the best-studied this protein have demonstrated that it is up-regulated and secreted selenate respiring bacterium (5–8). The selenate reductase in response to increasing selenite concentrations. The protein was (SerABC) isolated from T. selenatis (6) is a soluble periplasmic purified from Se nanospheres, and peptide fragments from a tryp- enzyme. The enzyme is a type II molybdoenzyme that comprises tic digest were used to identify the gene in the draft T. selenatis three subunits, SerA (96 kDa), SerB (40 kDa), and SerC genome. A matched open reading frame was located, encoding a (23 kDa), and coordinates molybdenum, heme (b-type), and protein with a calculated mass of 94.5 kDa. N-terminal sequence numerous [Fe-S] centers as prosthetic constituents (9). SerABC analysis of the mature protein revealed no cleavable signal pep- contributes to proton-motive force generation by accepting elec- tide, suggesting that the protein is exported directly from the trons from a diheme c-type cytochrome (cytc4), which mediates cytoplasm. The protein has been called Se factor A (SefA), and electron flux from either a quinol-cytochrome c oxidoreductase homologues of known function have not been reported previously. (QCR) or quinol dehydrogenase. The use of QCR ensures that The sefA gene was cloned and expressed in Escherichia coli, and selenate reduction is coupled to the Q-cycle mechanism provid- the recombinant His-tagged SefA purified. In vivo experiments de- þ − ing a minimum net gain of 2q ∕2e of proton electrochemical monstrate that SefA forms larger (approximately 300 nm) Se nano- gradient (10). spheres in E. coli when treated with selenite, and these are retained 2− The resultant product from SerABC is selenite (SeO3 ). The within the cell. In vitro assays demonstrate that the formation of reduction of selenite in T. selenatis does not support growth and Se nanospheres upon the reduction of selenite by glutathione are is not a respiratory substrate. There has been much debate re- stabilized by the presence of SefA. The role of SefA in selenium garding the mechanisms by which selenite is reduced to selenium nanosphere assembly has potential for exploitation in bionanoma- in bacterial cells. Early reports by Macy and coworkers (11) terial fabrication. implicated a nitrite reductase in the process of selenite reduction, T. selenatis ∣ ∣ by virtue that a nonspecific mutant strain of , which was nanoparticles biomineralization anaerobic respiration deficient in nitrite reductase activity, also failed to produce de- tectable Se0 upon growth in selenate-rich medium. The authors he utilization of oxygen or nitrogen oxyanions (nitrate and speculated that it was probably a periplasmic nitrite reductase Tnitrite) as respiratory substrates presents a fortuitous advan- that was responsible for selenite reduction. Selenite reacts readily tage to organisms, because their respiratory products are either with thiols following the reactions described by Painter (12). Glu- aqueous or gaseous and simply diffuse away from the cell. How- tathione (GSH) is the primary reduced thiol in Escherichia coli, ever, this is not always the case. Some microorganisms that live and it is now widely believed that it is the prime candidate for in niche environments have adapted to utilize more unusual sub- bacterial intracellular selenite reduction. Bacteria belonging to strates for energy conservation, such as metal ions or chalcogen the α, β, and γ groups of the proteobacteria are all abundant in oxides (1). Often, the reduction of these compounds can result in GSH (13), so the utilization of GSH for the reduction of selenite the precipitation of insoluble products that ultimately accumulate during selenate respiration would seem plausible. Selenite reacts within the cell (2). If such compounds are to be used as respira- readily with GSH producing selenodiglutathione (GS–Se–SG). tory substrates, mechanisms for the disposal of the insoluble pro- GS–Se–SG is a good substrate for GSH reductase and is subse- ducts are essential. A number of systems exist in Gram-negative bacteria for secretion out of the cell and are commonly referred to as the type 1–6 secretion systems (TxSS). A further mechanism Author contributions: R.J.L. D.J.R., and C.S.B. designed research; C.M.D., E.J.D., C.M.M., P.S., I.K., and C.S.B. performed research; K.P. and H.F. contributed new reagents/analytic tools; for secretion of both soluble and insoluble material is the J.L., R.W.T., D.J.R., and C.S.B. analyzed data; and C.S.B. wrote the paper. process of outer membrane vesiculation. In response to stress, The authors declare no conflict of interest. a section of the outer membrane forms a distinct spherical vesicle, This article is a PNAS Direct Submission. D.K.N. is a guest editor invited by the Editorial composed of a lipid bilayer and encloses material exclusively from Board. the periplasm (3, 4). 2− Data deposition: The sefA gene sequence and the Thauera selenatis AX draft genome In the present work, the process of bacterial selenate (SeO4 ) sequence have been deposited in the NCBI GenBank database, www.ncbi.nlm.nih.gov/ respiration has been used to investigate the mechanism of Se genbank (accession nos. HQ380173 and PRJNA53521, respectively). precipitation and secretion. The reduction of selenate follows 1E.J.D. and C.M.M. contributed equally to this work. a sequential series of reductive steps ultimately leading to the 2To whom correspondence should be addressed. E-mail: [email protected]. 0 1 2 generation of elemental selenium (Se ). Eqs. and summarize This article contains supporting information online at www.pnas.org/lookup/suppl/ the overall reactions: doi:10.1073/pnas.1105959108/-/DCSupplemental. 13480–13485 ∣ PNAS ∣ August 16, 2011 ∣ vol. 108 ∣ no. 33 www.pnas.org/cgi/doi/10.1073/pnas.1105959108 Downloaded by guest on September 23, 2021 quently reduced to form a selenopersulfide of GSH (GS–Se−). transmission electron microscopy (TEM) (Fig. 1B and Fig. S1). − 0 GS–Se is unstable and dismutates into elemental Se (Se ) and Cells entering late exponential growth phase (t4) appear to start reduced GSH. The reaction has been studied for the phototrophic to accumulate electron-dense Se particles within the cytoplasmic α-proteobacterium, Rhodospirillum rubrum, where the reduction compartment (Fig. 1B). The particles appear spherical and are of selenite by GSH results in the accumulation of Se particles in approximately 150 nm in diameter, with only one Se particle the cytoplasm (13). Selenite can also be detoxified by methylation, per cell. It is also evident that cells during this growth phase start liberating volatile selenium compounds dimethylselenide and to accumulate granules that appear transparent (Fig. 1.B), typical dimethyldiselenide (14, 15). Recently, the S-adenosyl-L-methio- of those normally associated with polyhydroxybutyrate, a product nine (SAM)-dependent methyltransferase (TehB) from E. coli, known to form in T. selenatis when growing using acetate as the which is involved in tellurite resistance, has been shown to be carbon substrate (5). As the cells enter stationary phase growth effective in selenium methylation in vitro (16). It is therefore (t5–t6), Se particles are observed both inside the cell and in the of interest to investigate the mechanisms by which selenite is de- surrounding medium (Fig. 1B and Fig. S1). No evidence for cell toxified in a true selenate respiring organism. If T. selenatis utilizes lysis or the accumulation of Se in the periplasmic compartment an intracellular reductant to detoxify selenite during selenate was obtained. Furthermore, micrographs did not show any evi- respiration, then elemental Se would inevitably accumulate within dence of distortion or budding of the outer membrane. Centrifu- the cell. Consequently, it is considered likely that an export system gation of the culture, to remove T. selenatis cells and clumps of is required for the secretion of the Se0 deposits in order to sustain selenium deposits, liberated a clear supernatant red in color, the use of selenate as the sole respiratory substrate. The aim of the which, when analyzed by TEM, was shown to contain isolated present work was to resolve the mechanism by which T. selenatis selenium nanospheres (Fig. 1C), uniform electron-dense spheres deposits Se during selenate respiration, which has led to the iden- of approximately 150 nm diameter without a surrounding mem- tification of a Se-nanosphere assembly protein. brane (Fig. 1D). Results Secreted Protein Profile During Reduction of Selenate and Selenite. T. selenatis Secretes Selenium Nanospheres During Selenate Respira- The spent growth medium containing isolated selenium nano- tion.