Abstract

Results

T. selenatis Secretes Selenium Nanospheres During Selenate Respiration.

When T. selenatis was grown anaerobically using acetate as the carbon substrate and selenate as the sole electron acceptor, growth was accompanied by the formation of a red precipitate as the culture entered stationary phase. Cell samples were taken at time points selected to represent midexponential phase (t₁ and t₂), late exponential phase (t₃ and t₄), and early (t₅) and late (t₆) stationary phase (Fig. 1A). Samples were analyzed using transmission electron microscopy (TEM) (Fig. 1B and Fig. S1). Cells entering late exponential growth phase (t₄) appear to start to accumulate electron-dense Se particles within the cytoplasmic compartment (Fig. 1B). The particles appear spherical and are approximately 150 nm in diameter, with only one Se particle per cell. It is also evident that cells during this growth phase start to accumulate granules that appear transparent (Fig. 1.B), typical of those normally associated with polyhydroxybutyrate, a product known to form in T. selenatis when growing using acetate as the carbon substrate (5). As the cells enter stationary phase growth (t₅–t₆), Se particles are observed both inside the cell and in the surrounding medium (Fig. 1B and Fig. S1). No evidence for cell lysis or the accumulation of Se in the periplasmic compartment was obtained. Furthermore, micrographs did not show any evidence of distortion or budding of the outer membrane. Centrifugation of the culture, to remove T. selenatis cells and clumps of selenium deposits, liberated a clear supernatant red in color, which, when analyzed by TEM, was shown to contain isolated selenium nanospheres (Fig. 1C), uniform electron-dense spheres of approximately 150 nm diameter without a surrounding membrane (Fig. 1D).

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Fig. 1.

Physiological analysis of Se-nanosphere production. (A) Growth curve of T. selenatis grown on acetate using selenate (10 mM) as the sole electron acceptor (Error bars are SEM; n = 10 cultures). Time points t₁–t₆ indicate the samples used for EM analysis. (B) Transmission electron micrographs of time points from A. Micrographs t₁ and t₂ show midexponential phase, t₃ and t₄ show late exponential phase, and t₅ and t₆ show stationary phase. Scale bar, 200 nm. Selenium deposits are indicated by an arrow. Poly-β-hydroxybutarate granular deposits are indicated by an asterisk. (C and D) Transmission electron micrographs of purified Se nanospheres. (C) Scale bar, 500 nm. (D) Scale bar, 50 nm.

Secreted Protein Profile During Reduction of Selenate and Selenite.

The spent growth medium containing isolated selenium nanospheres was analyzed by SDS-PAGE, to obtain a profile of secreted proteins from T. selenatis when grown using selenate as the electron acceptor (Fig. 2A). A single major protein was observed with an apparent molecular mass of approximately 95 kDa. In order to investigate whether the 95-kDa protein was secreted in response to selenite, cultures were grown anaerobically using nitrate as the sole electron acceptor in the presence of sodium selenite (10 mM) and, again, the 95-kDa protein was detected (Fig. 2A). Cultures grown under anaerobic conditions, using nitrate as the electron acceptor, in the absence of selenate/selenite, produced either very little or failed to secrete detectable levels of the 95-kDa protein. The secretion profile of the 95-kDa protein was further investigated from cells grown under aerobic conditions in the presence/absence of various oxyanions (Fig. 2B). When T. selenatis was grown aerobically on LB medium alone, or in the presence of nitrate, the 95-kDa protein was not detected. Upon the addition of selenate (10 mM), a faint band was resolved at 95 kDa, the amount of which increased when the medium was supplemented with 10 mM selenite. The quantity of protein secreted as a function of time following exposure to selenite was also investigated (Fig. 2C). The amount of the 95-kDa protein secreted increased for 16, 24, and 40 h postincubation with 10 mM selenite. To investigate whether or not a threshold level of selenite concentration was needed to induce secretion of the 95-kDa protein, aerobic cultures of T. selenatis were supplemented with selenite at increasing concentration (0, 0.01, 0.1, 1, and 10 mM) and incubated for 24 h. Culture growth was monitored at OD_600 nm to ensure that the presence of selenite did not have a deleterious effect on cell growth. Red elemental selenium was detected in cultures exposed to > 1 mM selenite (Fig. 2D). Analysis of the secreted protein profile shows that the 95-kDa protein is only detectable from cultures where the selenite concentration was > 1 mM (Fig. 2D). Analysis using inductively coupled plasma atomic emission spectroscopy detected selenium in purified protein samples, giving calculated molar ratios of approximately 3201 selenium to protein.

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Fig. 2.

Protein analysis of Se nanospheres from T. selenatis. (A) SDS-PAGE gels stained for secreted proteins from T. selenatis grown under anaerobic conditions. Lane 1, Invitrogen See Blue Plus2 Prestained Standard; lane 2, protein from cells grown on selenate (10 mM); lane 3, protein from cells grown on nitrate (10 mM) plus selenite (10 mM). (B) SDS-PAGE gel stained for total protein secreted in the extracellular medium from cells grown aerobically under different growth conditions. Lane 1, Invitrogen See Blue Plus2 Prestained Standard; lane 2, control (cells grown in LB medium only); lane 3, LB medium containing 10 mM selenite prior to inoculation with T. selenatis; lane 4, cells grown in LB medium supplemented with 10 mM selenate; lane 5, cells grown in LB medium supplemented with 10 mM nitrate; and lane 6, cells grown in LB medium supplemented with 10 mM selenite. (C) Secreted proteins from T. selenatis grown under aerobic conditions on LB medium supplemented with selenite (10 mM) following incubation for 16, 24, and 40 h, respectively. (D) Analysis of protein secretion and regulation upon exposure to increasing concentrations of selenite. (i) Observed selenium precipitation in cultures, (ii) SDS-PAGE analysis of secreted proteins, (iii and iv) end-point RT-PCR of sefA and 16S transcripts, and (v) Northern blot of sefA transcript.

Analysis of the sefA Locus.

Annotation of the ORFs adjacent to SefA (Fig. 3A) revealed that upstream are a putative CHASE2 extracellular sensory domain/guanylate cyclase and a protein containing a tetratricopeptide (TPR) repeat. Downstream is a putative SAM-dependent methyltransferase, a further putative peptide, and a peptide with a domain of unknown function (DUF). A search for putative transcriptional regulator binding sites upstream of the sefA gene was performed using the Prokaryotic Promoter Prediction system (http://bioinformatics.biol.rug.nl/websoftware/ppp/ppp_start.php). This revealed the presence of a putative FNR promoter binding site between positions -87 and -103 relative to the sefA gene start site. FNR is a transcriptional regulator active at low oxygen levels. The motif TGTGATTCCCATCACA falls within the Fnr_Bac/PrfA Group motifs (TGTGA-N₆-TCACA) classified as Crp/FNR factors by Körner et al. (25). Analysis of the intergenic region between sefA and the putative SAM-dependent methyltransferase cannot indentify a known transcriptional regulator binding site. It is thus considered likely that both sefA and the gene encoding the putative methyltransferase are in the same operon. Consequently, we have called the methyltransferase SefB. SefB is a 513 aa protein with a calculated molecular weight of 56.9 kDa. Pfam analysis identifies both putative methyltransferase and regulatory domains. A SefB homologue has also been located in D. indicum strain S5 (Selin_0233).

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Fig. 3.

Analysis of the sef operon. (A) Schematic representation of the sef gene locus. Putative annotations are as follows: CHASE2 extracellular sensory domain and guanylate cyclase (with sec leader peptide in red); SefA, selenium nanosphere assembly protein; SefB, SAM-methyltransferase;?, putative peptide. The (B) nucleotide sequence of the promoter region of sefA. (C) Nucleotide sequence upstream from sefB. A putative Shine–Dalgarno (SD) sequence and putative Fnr_Bac/PrfA binding motif (TGTGA-N₆-TCACA) are located upstream of sefA. No obvious promoter binding sequences are identified between sefA and sefB. A putative SD sequence upstream of sefB is also located.

Expression of SefA in E. coli, in Vivo Localization, and Resistance to Selenite.

The gene encoding SefA was cloned and expressed in E. coli (see SI Materials and Methods and Fig. S3). Western blot analysis of soluble cell fractions and extracellular medium are shown in Fig. 4A. After 4 h induction, SefA is detected predominantly in the soluble cell fraction but is also detectable in the extracellular medium. Analysis of the medium by SDS-PAGE indicated that this was not due to cell lysis. At 19 h after induction, the amount of SefA detected in both fractions increased substantially. In the presence of selenite (10 mM), SefA was again detected in both the soluble cell fractions and the extracellular medium at both time points. Samples that were not treated with IPTG failed to produce detectable SefA in either fraction. Further analysis of the selenite-treated samples by TEM revealed that cells not expressing SefA were shown to accumulate Se particles ranging in size from 30–50 nm (Fig. 4B). Cells treated with selenite/IPTG accumulated larger particles (approximately 300 nm) in the cytoplasm (Fig. 4C). No evidence was found for extracellular Se nanospheres. The results show that SefA can be expressed in E. coli and indicate it might be a substrate for protein export. The addition of selenite to the culture had no obvious effect on the level of exported SefA, suggesting that secretion from E. coli was not conditional upon Se binding. These data suggest that although apo-SefA can be secreted from E. coli, a specific export system is required for Se-nanosphere secretion. Analysis of E. coli (pET33b-sefA) to selenite resistance shows that cells in which SefA is expressed are more resistant to selenite concentrations up to 25 mM than those cells without SefA (Fig. 4D).

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Fig. 4.

Expression of SefA in E. coli and in vitro formation of Se nanospheres. (A) Western blot analysis of the localization of SefA expression in E. coli following exposure to selenite. Samples (10 μg) were prepared from cells +/- IPTG and +/- selenite (10 mM). Lanes 2, 4, and 6 represent extracellular protein; lanes 3, 5, and 7 represent soluble cell extracts. Samples were analyzed at both 4 and 19 h after the addition of IPTG and selenite. Lanes 1 and 8 show an 80-kDa marker. (B) Transmission electron micrograph of an E. coli single cell harboring plasmid pET33b-sefA grown in the presence of selenite (10 mM), but not induced with IPTG. (C) Transmission electron micrograph of an E. coli single cell harboring plasmid pET33b-sefA grown in the presence of both selenite (10 mM) and IPTG. Three replicate cells are shown in Fig. S3. Selenium deposits are indicated by an arrow. (D) Growth yield at stationary phase of E. coli cells exposed to increasing selenite concentrations. E. coli harboring plasmid pET33b-sefA in the absence (hashed bars) or presence (solid bars) of IPTG, and E. coli cells harboring plasmid pET33b in the presence of IPTG (open bars). Error bars are SEM (n = 3). ** indicates t test, p < 0.01 compared to the nonrecombinant (pET33b) control. (E) The reaction of GSH (4 mM) with selenite (0.5 mM), either in the presence (○) or absence () of purified rSefA (0.5 μg), monitored at 400 nm. (F and G) Transmission electron micrographs of the reaction product from E, in the absence (F) and in the presence (G) of SefA. Scale bars, 0.5 μm and 200 nm for F and G, respectively.

Discussion

Bacteria that are capable of the respiration of selenium oxyanions have been isolated from a number of selenium-rich environments (26, 27). Using selenium oxides as the sole electron acceptor presents the organism with the challenge of what to do with the elemental Se product following the subsequent reductions. For some bacteria that catalyze the reduction of selenate to Se⁰, Se deposits have been observed to accumulate within the cytoplasmic compartment, whereas in others, they are secreted from the cell (28). For example; Enterobacter cloacae SLD1a-1 (29), R. rubrum (30), and Desulfovibrio desulfuricans (31) have all been found to bioreduce selenite to selenium both inside and outside the cell. E. coli is also able to reduce selenite-forming deposits both in the periplasmic and cytoplasmic compartments (32). However, for a selenate respirer such as T. selenatis, a build-up of Se in the cytoplasm or periplasm might not be sustainable and could lead to necrosis. Consequently, mechanisms have evolved for the secretion of solid Se⁰ precipitates. In the present work, the process by which T. selenatis deposits Se during selenate respiration has been investigated. A secreted protein, SefA, has been identified, and homologues of known function have not been reported previously. Evidence is presented that SefA facilitates Se-nanosphere assembly prior to export from the cell.

The observation that SefA has no detectable selenite reductase activity would suggest that SefA functions only to bind to, or to stabilize, Se⁰. Because selenate reduction in T. selenatis is periplasmic, the location of the selenite reductive pathway remains unclear. A previous study has suggested that a periplasmic nitrite reductase could be responsible for selenite reduction (11); however, selenite reductase activity in periplasmic fractions, using reduced methyl viologen as the electron donor, has not been detected. Based upon the present data, it is suggested that selenite reduction occurs in the cytoplasm. The reduction to Se⁰ could be due to interaction with thiols, possibly GSH, because GSH is abundant in both the β-proteobacteria (T. selenatis) and γ-proteobacteria (E. coli) (12, 13). GSH is synthesized by sequential actions of γ-glutamylcysteine synthetase (GshA) and GSH synthetase (GshB), and homologues for both GshA and GshB have been identified in the draft T. selenatis genome. The reaction of selenite with GSH can result in the generation of , which is detoxified in part by cytochrome c (13). Interestingly, we have previously identified a c-type cytochrome (cytc-Ts7) that is up-regulated during selenate respiration but is not involved in electron transfer to SerABC (10). It is possible that the function of cytc-Ts7 is to help detoxify any generated.

The diagram presented in Fig. 5 shows the model for the selenate respiration pathway in T. selenatis. The reduction of selenate draws electrons from the membrane-bound QCR, which concomitantly provides a net gain of 2q⁺/2e^- of proton electrochemical gradient (10). Although the utilization of a QCR is unusual for a periplasmic molybdoenzyme, the additional 2H⁺ translocated via the link to the Q-cycle could provide the driving force for the translocation of selenite across the cytoplasmic membrane. In this case, energy would also need to be conserved by the utilization of an electrogenic primary dehydrogenase. The exact nature of the selenite transporter in T. selenatis is unknown, but in E. coli it has been suggested that selenite crosses the cytoplasmic membrane via the sulphate transporter (33). Once in the cytoplasm, selenite could be reduced by GSH (or other reductants), and the resultant Se⁰ binds to SefA, forming a nanosphere inside the cell. In addition, it is likely that the putative SAM-dependent methyltransferase (SefB), downstream from SefA, might function to generate methyl selenite or other volatile selenium compounds. The involvement of the tellurite resistance SAM-dependent methyltransferase (TehB) has been shown recently to be effective in the methylation of both tellurite and selenite (16). Interestingly, although E. coli TehB has been identified as responsible for the methylation of tellurite in vitro (16), in vivo assays have not established the production of volatile methylated tellurium, yet precipitation of tellurium within cells is clearly seen as black deposits (34). The identification of SefA and SefB in the same operon, suggests that in T. selenatis there could be a link between both reductive and methylation-dependent selenite detoxification.

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Fig. 5.

Schematic diagram showing the proposed pathway of selenium oxyanion reduction and Se-nanosphere assembly in T. selenatis. The reduction of selenate draws electrons from the membrane-bound QCR, generating a net gain of 2q⁺/2e^- of proton electrochemical gradient, which could provide the driving force for the translocation of selenite across the cytoplasmic membrane. Once in the cytoplasm, selenite reduction occurs, and the resultant Se⁰ binds to SefA, forming a Se nanosphere prior to export from the cell. The process by which SefA–Se is exported remains unknown. The identification of a gene (sefB) encoding a putative SAM-dependent methyltransferase might also provide a mechanism for selenite detoxification via volatilization to methylated selenides (R–Se–R). OM, outer membrane; IM, cytoplasmic inner membrane.

How does the Se nanosphere get out of the cell? SefA does not have an N-terminal signal sequence for targeting to the periplamsic compartment. Furthermore, analysis of neighboring genes on the chromosome does not identify any likely TAT substrate candidates that might “carry” SefA to the periplasm. The size of the particles is consistent with those seen during outer membrane vesiculation (20–250 nm) (4), but by TEM we see no evidence of outer membrane distortion or bulging. Furthermore, outer-membrane vesicles contain exclusively material from the periplasm, whereas the Se particles are not associated with other periplasmic proteins (such as SerABC) and from the EM images SefA–Se nanospheres would appear to be translocated directly from the cytoplasm. Analysis of the T. selenatis draft genome assembly using an Interpro scan of all ORFs > 50 amino acids revealed the presence of all but Type V secretion systems. SefA could thus be exported via the T6SS because other proteins secreted by this system also lack an N-terminal signal sequence and are not first translocated to the periplasmic compartment. S-layer proteins (which show some sequence similarity to SefA) are typically substrates for the T1SS. Most notably, T1SS substrates share a common distinctive glycine-rich repeat (GGXGXDXXX) and contain very few or no cysteine residues (35, 36), both features of SefA. These characteristics might explain why SefA was detected in the extracellular medium when expressed in E. coli. However, it would appear that in T. selenatis the SefA–Se complex is assembled in the cell and only exported once at an optimum size. T. selenatis is somehow capable of sensing the size of the internal particles and selectively only secretes Se nanospheres of approximately 150 nm diameter. Evidently, E. coli lacks this capability and accumulated much larger Se–SefA particles, suggesting that the Se-nanosphere export system is specific to T. selenatis. The mechanism of how the Se nanospheres as a whole are assembled and secreted across the inner and outer membranes remains unknown.

The identification of another SefA family member in Nitrosomonas sp. (AL212 NAL212_3002) is interesting and deserves comment. The strain AL212 was isolated from cultures that could grow in up to 10.7 mM (NH₄)SO₄ and along with strain JL21 (grown in 3.57 mM) were called the “sensitive” strains (21). It had been noted in previous studies that some of the other strains isolated from treatment plants and laboratory sludges, once enriched in culture with (NH₄)SO₄, showed the formation of “particles” in the cytoplasm (37), but not in strain JL21. Curiously, the EM images of strain JL21 (and to a lesser extent AL212) reported in Suwa et al. (21) show evidence of extracellular spherical particles reminiscent of those created by SefA. It would seem likely that in this case, these particles could be based upon the chalcogen sulfur rather than selenium.

Various proteins that bind Se have been reported. Rhodanese (a sulfur transferase) (38), cysteine/selenocysteine lyases (39), SeBP (40) from Methanococcus vannielii, and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (41) can all bind selenium at a reactive cysteine residue. Analysis of the amino acid sequence of SefA reveals no cysteine residues are present, indicating that the Se–SefA interaction is not via a direct thiol ligand. BSA has also been shown to stabilize nano-Se following reduction by GSH. Because BSA is (moderately) soluble in salt solutions, it serves well as a carrier for molecules of low water solubility. The nonspecific interaction with BSA and Se is thought to stabilize the Se nanosphere by allowing interactions of its functional groups with water and by sterically avoiding Se aggregation in aqueous solution (42), thus capping the particle surface. It would seem likely that, in the absence of any thiol ligands, SefA would also function as a capping agent, providing reaction sites for the creation of Se nanospheres and providing a shell to prevent aggregation of the secreted particles.

The involvement of SefA in Se-nanoshpere assembly might have applications pertinent to nanotechnology. In particular, selenium nanoparticles have excellent bioavailability, high biological activity, and relatively low toxicity. A number of methods have been developed to generate Se nanoparticles, nanorods, and nanowires (43), but most require the use of hydrazine, glycol, surfactant, or high temperature. A number of studies have also taken advantage of biomolecules to generate Se nanoparticles in vitro. For example, Abdelouas et al. (44) have used reduced cytochrome c to synthesize Se nanowires at room temperature. More recently, Zhang et al. (45) synthesized t-Se nanowires using β-carotene as an in situ soft template. Having identified a protein that can stabilize Se nanospheres secreted from T. selenatis, this could, through molecular engineering, enable particles to be produced with structural arrangements that are not only unique, but also have yet to be reproduced by conventional chemical synthesis. The extracellular secretion of such nanoparticles from T. selenatis also has distinct benefits when compared to those bacterial systems that display intracellular accumulation, and as such the current work could present an opportunity for the synthesis of secreted Se nanomaterials.

Materials and Methods

See SI Materials and Methods for details of growth of T. selenatis, isolation of SefA, identification of the sefA gene, generation of recombinant SefA in E. coli, assays for in vitro selenium nanosphere formation, imaging T. selenatis cells and Se nanospheres by TEM, RT-PCR, and Northern blotting reactions and protocols.

Supplementary Material

Acknowledgments.

Footnotes

References