Novel Mechanisms of Selenate and Selenite Reduction in the Obligate Aerobic Bacterium Comamonas Testosteroni S44 T

Journal of Hazardous Materials 359 (2018) 129–138

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Journal of Hazardous Materials

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Novel mechanisms of selenate and selenite reduction in the obligate aerobic bacterium Comamonas testosteroni S44 T

Yuanqing Tana,1, Yuantao Wanga,1, Yu Wanga, Ding Xua, Yeting Huanga, Dan Wanga, ⁎ Gejiao Wanga, Christopher Rensingb, Shixue Zhenga, a State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, 430070, PR China b Institute of Environmental Microbiology, College of Resources and Environment, Fujian Agriculture & Forestry University, Fuzhou, Fujian 350002, PR China

GRAPHICAL ABSTRACT

ARTICLE INFO ABSTRACT

Keywords: Selenium oxyanion reduction is an effective detoxification or/and assimilation processes in organisms, but little Comamonas testosteroni is known the mechanisms in aerobic bacteria. Aerobic Comamonas testosteroni S44 reduces Se(VI)/Se(IV) to less- Se(VI) reduction toxic elemental selenium nanoparticles (SeNPs). For Se(VI) reduction, sulfate and Se(VI) reduction displayed a Sulfate reduction pathway competitive relationship. When essential sulfate reducing genes were respectively disrupted, Se(VI) was not Se(IV) reductase SerT reduced to red-colored SeNPs. Consequently, Se(VI) reduction was catalyzed by enzymes of the sulfate reducing SeNPs pathway. For Se(IV) reduction, one of the potential periplasm molybdenum oxidoreductase named SerT was screened and further used to analyze Se(IV) reduction. Compared to the wild type and the complemented mutant strain, the ability of Se(IV) reduction was reduced 75% in the deletion mutant ΔserT. Moreover, the Se(IV) reduction rate was significantly enhanced when the gene serT was overexpressed in Escherichia coli W3110. In addition, Se(IV) was reduced to SeNPs by the purified SerT with the presence of NADPH as the electron donor in

vitro, showing a Vmax of 61 nmol/min·mg and a Km of 180 μmol/L. A model of Se(VI)/Se(IV) reduction was

Abbreviations: SeNPs, Se(0)-nanoparticles; TEM, transmission electronic microscopy; EDX, energy dispersive X-ray; APSe, adenosine 5’-phosphoselenate; PAPSe, 3’- phosphoadenosine 5’-phosphoselenate ⁎ Corresponding author. E-mail address: [email protected] (S. Zheng). 1 The authors contributed equally. https://doi.org/10.1016/j.jhazmat.2018.07.014 Received 26 February 2018; Received in revised form 2 July 2018; Accepted 3 July 2018 Available online 09 July 2018 0304-3894/ © 2018 Elsevier B.V. All rights reserved. Y. Tan et al. Journal of Hazardous Materials 359 (2018) 129–138

generated in aerobic C. testosteroni S44. This work provides new insights into the molecular mechanisms of Se (VI)/Se(IV) reduction activities in aerobic bacteria.

1. Introduction Escherichia coli strain S17-1 was used as a conjugation donor to in- troduce the transposon for mutagenesis or for complementation of the Selenium is an essential micronutrient for humans, animals and mutants [26]. E. coli BL21 was used to clone and express proteins [24]. microorganisms, particularly because of its incorporation into seleno- E. coli W3110 was used to overexpress serT. Various mutants of C. tes- proteins or enzymes in the form of selenocysteine (Sec) and seleno- tosteroni S44 are also listed. The primers used in gene crossover to get methionine (Se-Met) [1,2]. Selenium is also widely used in the industry mutants are listed in Table S2. including semi-conductors, photoelectric cells, glass and ceramic pig- ments, and catalyst [3]. Despite its essentiality, selenium is considered 2.2. Determination of Se(IV) concentration in broth as an element with high risk because of its bioaccumulation potential in the ecosystem [3]. Selenium toxicity in humans and animals has been Bacterial strains were cultured in liquid medium added with reported in Se excess area such as in the Indian Punjab because of high 0.5 mmol/L sodium selenite at 28 °C for certain time. Selenite con- Se intake (750–4990 μg/person and day) from locally produced foods centrations of cultural supernatant in the reaction mixture were mea- [2,4]. In particular, the oxyanions Se(IV) (selenite) and Se(VI) (sele- sured by HPLC-HG-AFS (Beijing Titan Instruments Co., Ltd., China) nate) that leach from environments and are released by anthropogenic [27]. activities, can be toxic in water, soil and sediment due to the high so- lubility [4]. In microorganisms, dissimilatory reduction of Se oxyanions 2.3. TEM and SeNP analysis with EDX to elemental selenium is known to be an important process for re- moving toxic soluble Se oxyanions and for production of elemental Strain S44 was cultured in 1/5 YTSB medium with 5 mmol/L Se(VI) selenium nanoparticles (SeNPs) in technological applications such as in and LB medium with 5 mmol/L Se(IV), respectively at 28 °C with medicine, bioremediation and production of semiconductors [5–8]. shaking at 180 rpm, harvested at both log phase and stationary phase. A number of bacteria can perform dissimilatory reduction of Se(VI) Cultured samples without Se(IV)/Se(VI) were used as controls. TEM to generate SeNPs either intracellularly or extracellularly under anae- and EDX were performed as described methods [16]. robic conditions [6–12]. However, few investigations have elucidated the mechanisms of Se(VI) reduction. Under anaerobic conditions, Se 2.4. Screening of mutants defective for Se(VI) reduction by transposon (VI) reduction is thought to be divided into two steps, i.e., Se(VI) was mutagenesis shown to be first reduced to Se(IV) and then to SeNPs [7]. The SerABC reductase in the periplasm of Thauera selenatis [10], a membrane-bound E. coli strain S17-1 (pRL27-Cm) was used as the donor strain for SrdBAC of Bacillus selenatarsenatis SF-1 [11], and a proposed reductase transposon Tn5, and strain S44 with rifampin (Rif) resistance was used in the periplasm of Enterobacter cloacae SLD1a-1 catalyzed Se(VI) re- as the recipient. Plasmid pRL27-Cm carrying Tn5 was transferred into duction to Se(IV) [12]. However, it is unclear what the next Se(IV) the recipient strain S44 by conjugation with E. coli strain S17-1 ac- reducing mechanism will be in these Se(VI) reducing anaerobic bacteria cording to the method of Larsen [28]. Selection was carried out on 1/5 [7]. In addition, the mechanism of Se(VI) reduction in aerobic bacteria YTSB agar plates containing 50 μg/mL chloramphenicol (Cm) and remains unknown [7]. 50 μg/mL Rif and 5 mmol/L Se(VI). The mutants were selected when Compared to Se(VI) reducing bacteria, many more bacterial species the colony lost the ability to reduce Se(VI) indicated by formation of red are involved in Se(IV) reduction both under aerobic and anaerobic SeNPs. Plasmid rescue was used according to the method described by conditions [6,7,13–25]. Apart from biogenic glutathione, sulfide and Chen et al [29]. Tn5 insertion sites were analyzed using the NCBI iron siderophore mediated Se(IV) reduction in a non-enzymatical way, BLAST server (http://blast.ncbi.nlm.nih.gov/Blast.cgi) based on the anaerobic respiratory reductases are involved in Se(IV) reduction [7], genome of strain S44 [30]. encompassing sulfite reductase [17], nitrite reductase [18], arsenate reductase [19], hydrogenase I [20] and fumarate reductase [21]. In 2.5. Competitive test of sulfate and Se(VI) reduction in strain S44 aerobic bacteria, glutathione reductase, thioredoxin reductase [22], and NADH: flavin oxidoreductase [23] were predicted to be potentially Competitive test of sulfate and Se(VI) reduction was performed in involved in Se(IV) reduction, but no gene product or enzyme test has chemical defined medium (CDM). The original concentration of sulfate been performed in vivo. Recently, CsrF in Alishewanella [24], and fu- in CDM was 15 mmol/L. Cultured cells of strain S44 in LB broth were marate reductase in Enterobacter cloacae [25] were identified to re- harvested and washed in sterilized water twice. Then 5 μL suspended sponsible for Se(IV) reduction in vivo. In conclusion, reduction of Se(IV) cells were spotted onto the modified CDM plates with sulfate gradients is possible through a variety of diverse mechanisms [1,16]. Therefore, (1, 5, 10 and 15 mmol/L, respectively) and Se(VI) gradients (1 and the mechanisms and characteristics of the Se(IV) reduction still need to 2 mmol/L, respectively). All the plates were cultured at 28 °C for 48 h. be investigated more [6,7]. The red color indicates the presence of SeNPs. Obligate aerobic Comamonas testosteroni S44, reducing both Se(VI) and Se(IV) to SeNPs [13] was used in this study. Molecular character- 2.6. Generation of Se(VI) reduction mutants ization to elucidate both the Se(VI) reducing pathway and Se(IV) reductase was performed, and the location of SeNPs was identified by The suicide allelic exchange vector pCM184-Cm (Table S1) was used TEM and EDX. The present study generated a model of a potential to generate Se(VI) reduction mutants (Table S3). Construction of pathway of Se oxyanion reduction in aerobic C. testosteroni S44. double-crossover mutant was based on the described method [24]. S44- ΔcysI as a single crossover example, the internal region of cysI was 2. Materials and methods amplified by PCR using cysI-SC-F/ cysI-SC-R primer pair (Table S2). Then purified PCR product was cloned into AatII- BsrGI sites of 2.1. Strains, plasmids and primers pCM184-Cm. Next steps were the same as in generating the double crossover. The new plasmid was transformed into E. coli S17-1 and The bacterial strains and plasmids are listed in Table S1. In brief, conjugated with strain S44. Then the single (Cm-resistance, Tet-

130 Y. Tan et al. Journal of Hazardous Materials 359 (2018) 129–138 resistance) cross over event leading to cysI mutants were screened using 2.7. Native-PAGE of cellular proteins and Se(IV) reduction test in vitro 50 μg/mL Rif, 50 μg/mL Cm and 25 μg/mL Tet resistance experiments. The mutant inserting the entire plasmid pCM184-Cm-cysI into cysI was The cells in stationary phase of strain S44 growing in LB broth with obtained. All the mutants are shown in Table S3. 1 mmol/L Se(IV) were collected using a centrifuge at 4 °C

Fig. 1. TEM micrographs and EDX spectra of SeNPs for Se(VI) and Se(IV) reduction in C. testosteroni S44. SeNPs located in cytoplasm for Se(VI) reduction (C & D) and EDX spectra (E). SeNPs located in periplasm for Se(IV) reduction (F & G), and EDX spectra (H). The A and B are controls without Se(VI)/Se(IV).

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(8000 rpm × 10 min). The cells were then rinsed with a precooled PBS obtained. buffer (pH7.2), and broken in a French Press (4 °C, 100 MPa-150 MPa). 1.0 mL supernatant was collected and separated using 10% non-dena- 2.11. The heterologous over-expression of SerT in E. coli W3110 turing polyacrylamide gel electrophoresis (7–8 h under 80 V) [23]. One lane of the gel was soaked in 20 mL 50 mmol/L HEPES buffer con- The transgenic strain W3110 (pCPP30-serT) was obtained by taining 50 mmol/L Se(IV) and 200 μmol/L NADPH. Another lane was transformation of E. coli W3110 with pCPP30-serT. Then the generated the same condition except for NADPH being changed to NADH. After strain W3110 (pCPP30-serT) and wild strain W3110 were inoculated incubation at 28 °C for 2 h, a band of red color indicated the presence of and grown in 5 mL LB medium, respectively. Their respective growth SeNPs. The gel lane of control was stained using coomassie brilliant and Se(IV) reduction were measured in 100 mL CDM medium at 37 °C. blue. 0.5 mmol/L Se(IV) was added into the medium after 16 h (OD600 = 0.5- 0.6). 2.8. Mass spectrometry analysis of the proteins involved in Se(IV) reduction in vitro 2.12. Expression and purification of SerT

A red protein band in the native-PAGE possessing Se(IV) reductase Plasmid pET-28a and E. coli BL21 are used to clone and express serT activity was obtained and analyzed by LCeMS. The sequence of these [24]. The serT gene of C. testosteroni strain S44 without sequence en- peptides was then compared with the annotated strain S44 protein se- coding 33 signal peptides was amplified by PCR using primers serT(BD)- quence from NCBI database (https://www.ncbi.nlm.nih.gov/Traces/ F/serT(BD)-R (Table S2) and the restriction sites BamHI and HindIII wgs/?val=ADVQ01) to determine the protein corresponding to the were introduced. peptide segment (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The results The purification of the protein SerT was performed by the modified of mass spectrometry analysis are listed in Table S4. method [24]. The cells were collected by centrifugation (4 °C, 8000 rpm for 6 min). Buffer A (50 mmol/L Tris−HCl, pH 8.0, 300 mmol/L NaCl) 2.9. Construction of serT mutant, complementation and strains was used to wash the cells twice and then the cells were broken by overexpressing serT in Se(IV) reduction pressure breaker until the suspension were transparent. The upper cleaning liquid (4 °C, 8000 rpm for 20 min) and the final concentration A serT deletion was obtained using pCM184-Cm plasmid to the of 20 mmol/L imidazole were mixed with the pretreated nickel purified strain S44 as Se(VI) reduction. The plasmid pCPP30 was used in com- resin. Rotating shake was performed slowly at 4 °C and 15 rpm for 1 h to plementation and overexpression experiments. After the deleted gene ensure the protein linking with His labels combined fully with the resin. fragment was replaced by the chloramphenicol gene, the ΔserT deletion Two columns of Buffer B (50 mmol/L Tris−HCl, pH 8.0, 300 mmol/L strain contained both rifampin and chloramphenicol resistance. The NaCl, 20, 40 and 60 mmol/L imidazole) were used to wash off the gene serT was cloned into the expression plasmid pCPP30 and the impurity proteins. The target protein was eluted for three times using plasmid pCPP30-serT was generated. Then pCPP30-serT was transferred 1mLBuffer C (50 mmol/L Tris−HCl, pH 8.0, 300 mmol/L NaCl, with into E. coli S17-1. The cross of two-parental conjugation was performed 80, 100, 120 and 200 mmol/L imidazole). 12% SDS-PAGE was used for among strain E. coli S17-1 (pCPP30-serT), strain S44-ΔserT and wild the protein detection. 2.0 L buffer A (50 mmol/L Tris−HCl, pH8.0, strain S44. The complemented strain S44-ΔserT-C and the over- 300 mmol/L NaCl) was used for the removal of imidazole by ice dia- expression strain S44::serT containing an additional tetracycline re- lysis. The final concentration of 10% glycerin was added into the pro- sistance were generated. tein solutions for conservation at −80 °C.

2.10. Homology and functional prediction of SerT 2.13. Optimization and in vitro activity test of Se(IV) reductase SerT

The functional prediction of SerT were obtained using the HHpred The concentration of puriﬁed protein was determined by the fol- server [31]. Amino acid sequences were obtained from the NCBI da- lowing methods. A protein of 50 μg/mL solution was added to 1.0 mL of tabase. Multiple alignments were performed using CLUSTAL X software 50 mL Tris−HCl (pH8.0) reaction system contained 2.0 mmol/L [32] and Espript 3.0 [33]. Highly similar sequences and some reported NADPH and gradient concentrations of Se(IV) (50, 100, 200, 300, 400, gene sequences encoding putative selenite reductases were selected for 600 and 800 μmol/L). The reaction systems were immediately boiled further analysis. By comparing the protein sequences in NCBI and after reaction at 28 °C for 20 min. The mechanical curve of enzyme Uniprot protein databases, the functional domains of SerT could be activity was calculated by the determination of the decrease of Se(IV).

Fig. 2. Competitive test of Se(VI) and sulfate metabolism of C. testosteroni S44. All plates were incubated on CDM plates at 28 °C for 48 h. Red color indicates the presence of SeNPs. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.)

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Fig. 3. Plate assay for Se(VI) reduction. C. testosteroni S44 strain and mutants were streaked onto LB agar plates containing 10.0 mmol/L Se(VI) (left) or 1.0 mmol/L Se(IV) (right). All plates were incubated at 28 °C for 60 h. The red color indicates the presence of SeNPs. WT, wild type of strain S44. cysA, cysN and cysI are sulfate reducing genes involved in encoding sulfate transporter, sulfate reductase and sulﬁte reductase, respectively. The genes of ssu and sse are involved in alkanesulfonate and thiosulfate utilization, respectively. cysB encodes transcriptional regulator of sulfate reduction. cbl, cysB-like gene. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.)

The gradients of temperature (4–60 °C) and pH (3.0–12.0) were re- L, more Se(VI) was reduced with 2 mmol/L Se(VI) than with 1 mmol/L spectively set up as a single variable. The optimal temperature and pH Se(VI). These results clearly indicated that sulfate and Se(VI) reduction of the protein SerT were determined by using the above reaction system displayed a competitive relationship, suggesting Se(VI) was reduced to determine the decrease of Se(IV) concentrations. through the sulfate reduction pathway.

3. Results 3.4. Genetic evidences indicated that Se(VI) reduction via the sulfate reducing pathway 3.1. SeNPs reduced from Se(VI) and Se(IV) occurred in different cellular locations Besides of sulfate assimilation, there are other sulfur metabolic pathways present in strain S44. The ssu operon (responsible for alka- Strain S44 reduced Se(VI) and Se(IV) to red-colored SeNPs under nesulfonate utilization), sse operon (responsible for thiosulfate utiliza- aerobic conditions [13]. TEM of ultra-thin sections revealed the pre- tion), and cbl (cysB-like encoding sulfonate utilization transcriptional sence of intracellular SeNPs when bacterial cells were grown in medium regulator) may be associated with Se(VI) reduction. Therefore, the with added 5 mmol/L Se(VI) (Fig. 1C and D) and 5 mmol/L Se(IV) three genes were disrupted respectively. (Fig. 1F and G), respectively. The sizes of intracellular SeNPs varied When cysA encoding a sulfate uptake transporter, cysN encoding an from 100 to 200 nm in length. Dark, fine-grained nanoparticles were enzyme that catalyzes sulfate conversion to APS, cysI encoding a sulfite observed by EDX spectra which indicated that these nanoparticles were reductase, and cysB as mentioned above were interrupted, Se(VI) was composed entirely of selenium since the expected emission peaks for not reduced to red-colored SeNPs (Fig. 3A), but Se(IV) was still reduced selenium at 1.37, 11.22 and 12.49 keV (Fig. 1E and H) could be de- (Fig. 3B). In contrast, when other sulfur metabolic pathways were in- tected corresponding to the SeLα, SeKα, and SeKβ transitions, respec- terrupted by disrupting ssuD, sseA and cbl, both Se(VI) and Se(IV) re- tively. duction showed no difference to the control (Fig. 3). Based on genome It is interesting that the location of SeNPs in cells of Se(VI) reduction analysis in strain S44, CysA, CysN and CysI are predicted to be involved differed from the Se(IV) reduction (Fig. 1C, D, F and G). TEM images in sulfate reduction/assimilation to cysteine, which is regulated by showed that SeNPs reduced by Se(VI) occurred in the center of cells. In CysB. Therefore, Se(VI) reduction followed the pathway of sulfate re- contrast, the most SeNPs reduced Se(IV) were present at the border of duction, while other sulfur utilization pathways are not responsible for cells. Moreover, the number of SeNPs reduced by Se(VI) was less than Se(VI) reduction. that reduced by Se(IV). 3.5. Screening putative Se(IV) reducing proteins by Native-PAGE of cellular 3.2. Isolation of Se(VI) reduction defective mutants by Tn5 transposon proteins and Se(IV) reduction test

Eleven mutants showing absence of Se(VI) reduction ability (red- In order to identify the protein responsible for Se(IV) reduction, the color) were obtained from approximately 4000 transposon mutants. total protein of cells was extracted and ran on a non-denatured poly- The genomic regions flanking the transposon insertion of these 11 acrylamide gel electrophoresis (native-PAGE) (Fig. 4). The slightly red sensitive mutants were sequenced and analyzed by BlastX in the band of SeNPs was observed when the gel was incubated in a Se(IV) GenBank database. The Tn-insertion site in 7 mutants was the gene solution amended with NADPH as an electron donor (Fig. 4A). How- encoding the transcriptional regulator CysB involved in sulfur meta- ever, no red band could be observed with NADH as an electron donor. bolism. CysB is responsible for regulating cysteine anabolism. We fur- This indicated that the protein visualized in the red active band, ther confirmed that the cysB mutants did not reduce Se(VI) to SeNPs. mediated Se(IV) reduction. Accordingly, the protein band was cut out and used for component analysis. 3.3. Sulfate and Se(VI) reduction displayed a competitive relationship The protein band previously cut out contained more than twenty proteins as identified by mass spectrometry analysis (Table S4). Based An experiment looking at the competitive relationship between on the annotated functioning of these proteins, two oxidoreductases sulfate and Se(VI) was first conducted (Fig. 2). The ability of Se(VI) were identified that could be involved in Se(IV) reduction, corre- reduction indicated by red-colored SeNPs in strain S44 decreased with sponding to ethanol dehydrogenase gene 3396 and molybdenum oxi- increasing sulfate concentrations. The Se(VI) reduction of 1 mmol/L doreductase gene 1962. Consequently, these two genes were disrupted was nearly completely inhibited when the concentration of sulfate respectively and tested for their ability to reduce Se(IV). reached 15 mmol/L. Under the same sulfate concentration of 15 mmol/ To assess Se(IV) reduction capabilities, single crossover mutants

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Fig. 4. Screening candidates involved in Se(IV) reduction. (A) Native-PAGE of cellular proteins. M: Protein marker; 1, Stained by R-250; 2/3, NADH/NADPH as electron donor. The red color indicates the presence of SeNPs. (B) Gene cluster of candidate gene serT. (C) Amino acid sequence alignment of SerT with relative proteins. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) were generated and strains Δ3396 and Δ1962 were tested in CDM 3.6. Protein SerT belonged to sulfur metabolic family by bioinformatic medium (Fig. S1). The Se(IV) reduction ability of mutant strain Δ3396 analysis did not change compared to the wild type strain. In contrast, the Se(IV) reduction of mutant strain Δ1962 was significantly decreased, sug- The genome sequence of strain S44 was published previously gesting gene 1962 encoded a Se(IV) reductase, named SerT. Further (GenBank ID, ADVQ00000000)[30]. The arrangement of serT on the bioinformatic analysis was performed. genome is shown in Fig. 4B. The sequence homology of SerT was similar to several sulfite oxidases (Fig. 4C). The four most similar homologous proteins in comparison to SerT in structure were protein SoxCD [34], SorT [35], CSO [36] and SDH [37], and showed the

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3.7. SerT mediated Se(IV) reduction

To identify whether Se(IV) reduction is mediated by SerT in strain S44, a serT deletion, complemented and serT overexpressing strains were constructed. The growth shown by measuring cellular proteins showed no difference between all strains (Fig. 5A). In contrast, Se(IV) reduction in deletion strain S44-ΔserT was reduced by 75% compared to wild type strain S44. Se(IV) reduction in the complemented strain S44- ΔserT-C was comparable to S44 (Fig. 5B and C). This result indicated that SerT mediated the reduction of Se(IV) with some other processes resulting also being responsible for the remaining 25% Se(IV) reduction in strain S44-ΔserT. However, Se(IV) reduction activity in the strain overexpressing SerT did not improve compared to the strain S44. In CDM medium with added 0.5 mmol/L Se(IV), the heterologous overexpression strain E. coli W3110::serT displayed a deeper red col- oring than strain W3110 at 48 h (Fig. 6), and the Se(IV) reduction rate of E. coli W3110::serT was increased by 40% compared to E. coli W3110 at 48 h with no difference in growth (Fig. 6A and B). These results confirmed that SerT was responsible for the reduction of Se(IV).

3.8. SerT catalyzed Se(IV) reduction using NADPH as electron donor in vitro

SerT was expressed in E. coli BL21 (pET-28a-serT) and puriﬁed. According to the protein sequence analysis and SDS-PAGE, the mole- cule weight of SerT was determined as 42.46 kDa (Fig. 7A), and the PI value was 9.15. Puriﬁed SerT reduced Se(IV) to Se(0) and generated red SeNP precipitation at the bottom of the vial in a 10 mmol/L Se(IV) solution with 2.0 mmol/L NADPH as electron donor (Fig. 7B). In contrast, no Se(IV) was reduced in controls without NADPH or with NADPH but in absence of SerT (Fig. S3). This result indicated that SerT catalyzed Se(IV) reduction using NADPH as electron donor in vitro. Using Se(IV) as substrates, the activities of SerT were investigated in the temperature range of 4–60 °C and pH 4.0–9.0 (Fig. 7C, D). The activity of SerT disappeared when temperature reached 50 °C. The optimum pH and temperature were pH 8 and 28 °C, respectively. Dynamic curve of enzyme activity was calculated in Fig. 7E, showing180 ± 5 μmol/L of

Km, and 61 ± 2 nmol/min·mg of Vmax.

4. Discussion

4.1. Sulfate reducing pathway could be a general mechanism of Se(VI) reduction in aerobic organisms

In C. testosteroni S44, disruption of key enzymes of the sulfate assimilation pathway led to the inability to reduce Se(VI) to SeNPs (Fig. 3A). The results indicated that Se(VI) was reduced to SeNPs through the sulfate assimilation pathway, which is further supported by other evidences as listed below, (i) competitive relationship between sulfate and Se(VI) reduction (Fig. 2); (ii) morphological evidence showed that formation of SeNPs through Se(VI) reduction occurred in Fig. 5. Effect of enzyme SerT on Se(IV) reduction in C. testosteroni S44. The the cytoplasm (Fig. 1C and D); (iii) in vitro reaction showing ATP sul- growth curves (A) and Se(IV) reducing curves (B) of strain S44, deleted mutant 35 75 furylase incorporating radio-labeled Na2 SO4/Na2 SeO4 into APS/ Δ Δ S44- serT, complementary strain S44- serT-C and overexpressed strain APSe [38]. Consequently, a model of Se(VI) reduction was developed in S44::serT under 0.5 mmol/L Se(IV) in CDM medium. (C) Phenotype of Se(IV) aerobic C. testosteroni S44 (Fig. 8). In detail, CysPUWA is predicted to be reduction. responsible for Se(VI) uptake into cytoplasm. Then, Se(VI) is converted to APSe and PAPSe by CysND and CysC, respectively, and CysH is sequence similarity of 30%, 60%, 31% and 33% by NCBI blast re- predicted to catalyze PAPSe to Se(IV). Finally, sulfite reductase (CysIJ spectively. in this case) catalyzes the reduction of Se(IV) to Se(0). The structure and function of protein SerT was also analyzed (Fig. It is clear that Se(VI) reduction to Se(0) is a very complicated pro- S2). SerT belonged to the family of sulfite oxidase and contained a cess, sharing the pathway of sulfate assimilation to cysteine [36]. This signal peptide, a molybdenum cofactor and a dimer domain. The pro- process is different from previous studies that showed Se(VI) reduction teins of this family are generally located in the periplasmic space, which to Se(IV) occurring in the periplasm or SeNPs on the surfaces of cells in were composed of cytochrome proteins that transmit electrons to a those anaerobic bacteria [7,10–12]. Moreover, to our knowledge, this is specific electron (https://toolkit.tuebingen.mpg.de). the first report to reveal the next Se(IV) reducing mechanism in these Se (VI) reducing bacteria.

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Fig. 6. Heterologous overexpression of SerT in E. coli W3110 under 0.5 mmol/L Se(IV) in CDM medium. (A) Growth curve. (B) Se(IV) reducing curve. (C) Phenotype of Se(IV) reduction. W3110::serT, overexpressed strain in E. coli W3110.

Fig. 7. Determination of enzyme SerT activity in vitro. (A) Purification of enzyme SerT. (B) Se (IV) reducing test, ck-control, tube 1 and 2- SerT plus Se(IV) and NADPH, red colored precipitation indicates the presence of SeNPs. (C) and (D) Effects of temperature and pH on enzyme activity. (E) Dynamics of enzyme activity. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

In addition, the involvement of the sulfate assimilation pathway in (IV) to SeNPs in vitro [13]. Previous studies have shown similar results synthesis of organic Se such as selenoproteins is a general mechanism in in that Se(IV) reduction was not entirely inhibited in a Se(IV) reductase aerobic mammals, yeasts and plants [38–42]. Therefore, the sulfate mutant [21,24,25]. In vitro enzyme test also showed the presence of two assimilation pathway could represent a general mechanism of Se(VI) selenite reductases in a bacterial strain [22,23]. Therefore, it could be a reduction in aerobic organisms. This provides new insights into the more general occurrence that multiple pathways are responsible for Se mechanisms of the selenium bio-cycle under various aerobic conditions (IV) reduction both in aerobic and in anaerobic bacteria. It suggests that such as in soil and water. bacteria have evolved various pathways to promote cell survival under environmental stress such as Se pollution. 4.2. Multiple pathways of Se(IV) reduction in aerobic and anaerobic In anaerobic bacteria, Se(IV)/Se(VI) reduction is able to occur not bacteria only in cells but also on the cellular surface where selenite acts as final electron acceptor linked to energy generation [7,14]. In contrast, in In C. testosteroni S44, disruption of SerT did not totally inhibit Se(IV) aerobic bacteria, it would be difficult to reduce Se(IV) on the cellular reduction (Fig. 5). This residual activity could be catalyzed by sulfite surface due to oxygen accepting the electrons prior to Se(IV) [16]. reductase. Se(IV) reduction can be generated through two pathways: (i) Therefore, Se(IV) reduction has to occur in the reducing environment of Se(IV) is primarily reduced by SerT in the periplasm; (ii) a minor intracellular spaces in aerobic bacteria, which is further supported by fraction of Se(IV) transported from the periplasm, and the Se(IV) re- the presence of intracellular SeNPs [7,14–16,24,25]. However, this duced from Se(VI) were reduced by sulfite reductase in the cytoplasm would make a further secretion of intracellular SeNPs difficult. Possible (Fig. 8). This is supported by morphological evidence showing a minor mechanisms are thought to be cell lysis and/or efflux via a vesicular fraction SeNPs were generated in the cytoplasm when only Se(IV) was secretion system [7]. Another alternative pathway would be efflux of Se added to the medium (Fig. 1E and F). This is consistent with the pre- (0) and subsequent SeNPs accumulation outside of cells [13]. Apart vious result that both cytoplasmic and periplasmic fractions reduced Se from Se(IV) reduction in cells, a further investigation is needed to

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Fig. 8. Schematic model of Se(VI) and Se(IV) reduction in C. testosteroni S44. determine whether Se(IV) is reduced outside of cells. generate more cells and so can provide an optimal outcome within a short time period and under less stringent culture conditions [8,25]. As 4.3. Assimilation, dissimilation or detoxification of Se Oxyanions far as biological and industrial properties are concerned, SeNPs have fi reduction? wide applications in the elds of medicine, microelectronic, agriculture and animal husbandry [5,6,8]. SeNP synthesis via aerobic bacteria is a ffi It has been shown that dissimilatory reduction of Se-oxyanions to safe, eco-friendly and e cient procedure. SeNPs is an effective detoxification process [2]. At the same time, low In many cases, bioremediation processes such as water treatment concentrations of selenium such as 0.1 μmol/L Se could match the de- and soil remediation occur under aerobic conditions. Strain C. testos- mand for assimilatory reduction of Se(VI) for synthesis of organic Se in teroni S44 has the ability to resist multiple metal(loid)s including As, all organisms despite Se being thought as useless for plants [39–42]. Cu, Zn and Se [13,30]. Consequently, aerobic bacteria such as strain Several genes such as selD and trxB indicated the existence of a Se as- S44 have distinct advantages for bioremediation under aerobic condi- similatory pathway in bacteria [43,44]. In strain S44, selD encoding a tions. Furthermore, it is helpful to monitor and identify functional selenophophate synthetase, selA encoding a selenocysteine synthase, microbiota responsible for Se oxyanion reduction to understand the and trxB encoding a thioredoxin reductase catalyzing the production of molecular mechanisms of Se(VI) and Se(IV) reduction in the environ- selenopersulfide were found on the genome. Therefore, C. testosteroni ment. For example, the abundance of Se(VI) reductase gene serA cor- fi S44 may display an assimilatory reduction under lower concentrations related well to selenate removal in bio lms [45]. It is possible that the – of Se-oxyanions. In contrast, detoxification occurred when the con- genes encoding selenite reductase [22 25] and serT of this case could be centrations of Se oxyanions reached a certain threshold level. More- used to identify selenite reducing microbiota and subsequent selenite over, SeNPs would be reduced to Se(-II) for assimilation. It is interesting removal. In addition, the selenite reductase CsrF was expressed in Es- that SeNPs were only observed outside of cells under 1.0 mmol/L Se cherichia coli to remove selenite and the reduced product SeNPs were (IV), possibly due to Se(0) efflux (one of the detoxification processes) of further used in removal of dyes in water [46]. strain C. testosteroni S44 [13]. However, SeNPs occurred in cells when Se(IV) concentration reached 5 mmol/L in this case, possibly due to 5. Conclusions limited efflux ability and subsequently Se(0) was aggregated into SeNPs. These results indicated that assimilation and dissimilation of Se Biosynthesis of SeNPs is a safe and eco-friendly procedure compared oxyanion reduction are not two completely exclusive processes. Bac- to a chemically-synthesized process under environmentally harmful teria have evolved a very sophisticated homeostatic mechanism to conditions. Diverse aerobic bacteria are able to reduce toxic Se oxya- adapt to the various levels of environmental Se. nions to non/low toxic Se(0) and generate SeNPs, showing rapid growth and optimal outcomes such as SeNPs under less stringent culture con- 4.4. Application potential of aerobic bacteria in green bio-synthesis of ditions. Thus, aerobic Se(VI)/Se(IV)-reducing bacteria such as C. tes- SeNPs and Se bioremediation tosteroni S44 may be used to develop a bioreactor for biosynthesis of SeNPs and used for bioremediation in Se polluted soil or water. Recently, a diverse array of aerobic bacterial strains have been de- Elucidation of various molecular mechanisms of Se(VI)/Se(IV) reduc- monstrated to reduce toxic Se(VI)/Se(IV) to non or low toxic Se(0) and tion is essential to construct powerful, engineered strain to be used in then generate SeNPs [7,13–16,22–25]. Compared to anaerobic bacteria, biosynthesis of SeNPs and bioremediation. It is also helpful to monitor aerobic Se-reducing bacteria possess the ability to rapidly grow and and identify functional microbiota responsible for Se(VI)/Se(IV)

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