NemA Catalyzes Trivalent Organoarsenical Oxidation and Is Regulated by the Trivalent Organoarsenical-Selective Transcriptional Repressor NemR.

Synthetic aromatic arsenicals such as roxarsone (Rox(V)) and nitarsone (Nit(V)) have been used as animal growth enhancers and herbicides. Microbes contribute to redox cycling between the relatively less toxic pentavalent and highly toxic trivalent arsenicals. In this study, we report the identification of nemRA operon from Enterobacter sp. Z1 and show that it is involved in trivalent organoarsenical oxidation. Expression of nemA is induced by chromate (Cr(VI)), Rox(III), and Nit(III). Heterologous expression of NemA in Escherichia coli confers resistance to Cr(VI), methylarsenite (MAs(III)), Rox(III), and Nit(III). Purified NemA catalyzes simultaneous Cr(VI) reduction and MAs(III)/Rox(III)/Nit(III) oxidation, and oxidation was enhanced in the presence of Cr(VI). The results of electrophoretic mobility shift assays and fluorescence assays demonstrate that the transcriptional repressor, NemR, binds to either Rox(III) or Nit(III). NemR has three conserved cysteine residues, Cys21, Cys106, and Cys116. Mutation of any of the three resulted in loss of response to Rox(III)/Nit(III), indicating that they form an Rox(III)/Nit(III) binding site. These results show that NemA is a novel trivalent organoarsenical oxidase that is regulated by the trivalent organoarsenical-selective repressor NemR. This discovery expands our knowledge of the molecular mechanisms of organoarsenical oxidation and provides a basis for studying the redox coupling of environmental toxic compounds.

Simultaneous removal of chromate and arsenite by the immobilized Enterobacter bacterium in combination with chemical reagents.

Simultaneous chromate [Cr(VI)] reduction and arsenite [As(III)] oxidation is a promising pretreatment process for Cr and As removal. Here, a facultative anaerobic bacterium, Enterobacter sp. Z1, presented capacities of simultaneous Cr(VI) reduction and As(III) oxidation during anoxic cultivation in a wild range of temperature (20-45 °C) and pH (Cerkez et al., 2015; Chen et al., 2015; China Environmental Prote, 1996; Fan et al., 2008, 2019) conditions. Strikingly, strain Z1 could simultaneously contribute up to 92.8% of the reduction of Cr(VI) and 45.8% of the oxidation of As(III) in wastewater. The cells of strain Z1 were embedded with sodium alginate to produce biobeads, and the biobeads exhibited stable ratio of Cr(VI) reduction (91.8%) and As(III) oxidation (29.6%) even in the 5 continuous cycles of wastewater treatment. Moreover, in a process pretreated with the Z1 biobeads followed a precipitation with Ca(OH)2 and FeCl3, the removal efficiencies in wastewater were 98.9% and 98.3% for total Cr and As, respectively, which were 44.1% and 9.8% higher than those of using Ca(OH)2 and FeCl3, only. The residual amounts of Cr and As met the national standard levels of wastewater discharge. Proteomics analysis showed that cysteine, sulfur and methionine metabolisms, As resistance and oxidoreductase (CysH, CysI, CysJ, NemA and HemF) were induced by Cr(VI) and As(III). Moreover, the addition of cysteine to the medium also significantly improved bacterial Cr(VI) reduction rate. Our results provide a novel microbial pretreatment approach for enhancing remediation of Cr(VI) and As(III) pollution in wastewater, and reveal the evident that cysteine, sulfur and methionine metabolisms, As resistance and oxidoreductases are associated with the redox conversion of Cr(VI) and As(III).

Paenibacillus flagellatus sp. nov., isolated from selenium mineral soil.

Strain DXL2T, a Gram-stain-negative, rod-shaped, endospore-forming, motile, aerobic bacterium, was isolated from selenium mineral soil. DXL2T had the highest 16S rRNA gene sequence similarities with those of Paenibacillus ginsengarviGsoil 139T (96.8 %), Paenibacillushemerocallicola DLE-12T (95.5 %) and Paenibacillus hodogayensisSGT (95.4 %). The genome size of DXL2T was 7.24 Mb, containing 6243 predicted protein-coding genes, with a DNA G+C content of 60.2 mol%. DXL2T contained meso-diaminopimelic acid in the cell-wall peptidoglycan. The major cellular fatty acids were anteiso-C15 : 0, iso-C16 : 0 and iso-C15 : 0. The major quinone was menaquinone 7. The polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, two aminophospholipids, an unidentified aminolipid, phosphatidylmethylethanolamine, an unidentified glycolipid and an unidentified phospholipid. Compared with the other strains, DXL2T had a specific phospholipid and a specific aminolipid, it hydrolyzed Tween 40 and could not assimilate potassium gluconate. On the basis of the phenotypic, chemotaxonomic and phylogenetic results, strain DXL2T represents a novel species within the genus Paenibacillus, for which the name Paenibacillusflagellatus sp. nov. is proposed. The type strain is DXL2T (=KCTC 33976T=CCTCC AB 2018054T).

Efflux Transporter ArsK Is Responsible for Bacterial Resistance to Arsenite, Antimonite, Trivalent Roxarsone, and Methylarsenite.

Arsenic-resistant bacteria have evolved various efflux systems for arsenic resistance. Five arsenic efflux proteins, ArsB, Acr3, ArsP, ArsJ, and MSF1, have been reported. In this study, comprehensive analyses were performed to study the function of a putative major facilitator superfamily gene, arsK, and the regulation of arsK transcriptional expression in Agrobacterium tumefaciens GW4. We found that (i) arsK is located on an arsenic gene island in strain GW4. ArsK orthologs are widely distributed in arsenic-resistant bacteria and are phylogenetically divergent from the five reported arsenic efflux proteins, indicating that it may be a novel arsenic efflux transporter. (ii) Reporter gene assays showed that the expression of arsK was induced by arsenite [As(III)], antimonite [Sb(III)], trivalent roxarsone [Rox(III)], methylarsenite [MAs(III)], and arsenate [As(V)]. (iii) Heterologous expression of ArsK in an arsenic-hypersensitive Escherichia coli strain showed that ArsK was essential for resistance to As(III), Sb(III), Rox(III), and MAs(III) but not to As(V), dimethylarsenite [dimethyl-As(III)], or Cd(II). (iv) ArsK reduced the cellular accumulation of As(III), Sb(III), Rox(III), and MAs(III) but not to As(V) or dimethyl-As(III). (v) A putative arsenic regulator gene arsR2 was cotranscribed with arsK, and (vi) ArsR2 interacted with the arsR2-arsK promoter region without metalloids and was derepressed by As(III), Sb(III), Rox(III), and MAs(III), indicating the repression activity of ArsR2 for the transcription of arsK These results demonstrate that ArsK is a novel arsenic efflux protein for As(III), Sb(III), Rox(III), and MAs(III) and is regulated by ArsR2. Bacteria use the arsR2-arsK operon for resistance to several trivalent arsenicals or antimonials.IMPORTANCE The metalloid extrusion systems are very important bacterial resistance mechanisms. Each of the previously reported ArsB, Acr3, ArsP, ArsJ, and MSF1 transport proteins conferred only inorganic or organic arsenic/antimony resistance. In contrast, ArsK confers resistance to several inorganic and organic trivalent arsenicals and antimonials. The identification of the novel efflux transporter ArsK enriches our understanding of bacterial resistance to trivalent arsenite [As(III)], antimonite [Sb(III)], trivalent roxarsone [Rox(III)], and methylarsenite [MAs(III)].