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

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Novel Mechanisms of Selenate and Selenite Reduction in the Obligate Aerobic Bacterium Comamonas Testosteroni S44 T Journal of Hazardous Materials 359 (2018) 129–138 Contents lists available at ScienceDirect Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat 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) re- ductase 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.
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