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Specific investigations: other studies

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Administrative data

Endpoint:
mechanistic studies
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
not specified
Reliability:
other: not rated acc. to Klimisch
Rationale for reliability incl. deficiencies:
other: Any kind of reliability rating is not considered to be applicable, since the study is not conducted/reported according to a standardised guideline.

Data source

Reference
Reference Type:
publication
Title:
Efflux Transporter ArsK Is Responsible for Bacterial Resistance to Arsenite, Antimonite, Trivalent Roxarsone, and Methylarsenite
Author:
Shi, K., Li, C., Rensing, C., Dai, X., Fan, X., Wang, G.
Year:
2018
Bibliographic source:
Appl Environ Microbiol. 2018 Nov 30;84(24)

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Shi et a. (2018) investigated the phylogeny, regulation, and function of ArsK. Specifically, the role of ArsK in resistance to and efflux of several metalloids, including trivalent inorganic antimonite Sb(III), were investigated. Phylogenetic analyses and reporter gene assays were performed in Agrobacterium tumefaciens wild-type cells. Metalloid resistance and efflux studies were performed in transformed E. coli cells containing the arsK gene.
GLP compliance:
not specified
Remarks:
not specified in the publication
Type of method:
in vitro
Endpoint addressed:
genetic toxicity

Test material

Reference
Name:
Unnamed
Test material form:
not specified
Details on test material:
This test materia identifier is assigned to those studies in which the test material is not clearly specified (such as "trivalent inorganic antimonite", "antimonials"), antimony was measured in biological matrices/environment or the antimony speciation was not clearly specified. In case the species is identified, it will be recorded in the test item description in the study record.
Specific details on test material used for the study:
not specified

Test animals

Species:
other: E. coli; A. tumefaciens
Strain:
not specified
Remarks:
not specified in the publication
Sex:
not specified
Details on test animals and environmental conditions:
NOTE: This is a bacterial culture study conducted with E. coli and A. tumefaciens. Information about the cells used in this study is given below.

CELLS USED
- Strains used: A. tumefaciens GW4 (Wild type, As(III)-oxidizing strain); E. coli DH5alpha (supE44 lacU169(φ 80lacZΔM15) hrdR17 recA1 endA1 gyrA96 thi-1 relA1); E. coli S17-1 (F- RP4-2-Tc::Mu aphA::Tn7 recA λpir lysogen; Smr Tpr); E. coli BL21 (F- ompT hsdSB (rB- mB-) gal dcm me131 (DE3) pLysS (Cmr)); E. coli AW3110 (AW3110 ΔarsRBC::cam F-IN(rrn-rrnE))
- Reference or source: Invitrogen (E. coli DH5alpha; E. coli S17-1; E. coli BL21); * (A. tumefaciens GW4); ** (E. coli AW3110)
- Methods for maintenance in cell culture: E. coli cultures bearing the indicated plasmids were grown aerobically in lysogeny broth (LB) or low-phosphate minimal mannitol (MMNH4) medium at 37°C supplemented with 100 µg/ml ampicillin (Amp) or 50 µg/ml chloramphenicol (Cm), as indicated. MMNH4 medium contains 10 g of D-mannitol, 1 g of K2HPO4, 1 g of KH2PO4, 0.25 g Na2PO4, 0.01 g of FeCl3, 0.25 g of MgCl2, and 0.1 g of CaCl2 per liter.

REFERENCES
*Fan H, Su C, Wang Y, Yao J, Zhao K, Wang Y, Wang G. 2008. Sedimentary arsenite-oxidizing and arsenate-reducing bacteria associated with high arsenic groundwater from Shanyin, Northwestern China. J Appl Microbiol 105:529 –539. https://doi.org/10.1111/j.1365 -2672.2008.03790.x.
**Chen J, Yoshinaga M, Garbinski LD, Rosen BP. 2016. Synergistic interaction of glyceraldehydes-3-phosphate dehydrogenase and ArsJ, a novel organoarsenical efflux permease, confers arsenate resistance. Mol Microbiol 100:945–953. https://doi.org/10.1111/mmi.13371.

Administration / exposure

Route of administration:
other: bacterial culture exposure
Vehicle:
not specified
Remarks:
not specified in the publication
Details on exposure:
please refer to the field "Details on study design" below.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
please refer to the field "Details on study design" below.
Frequency of treatment:
once
Post exposure period:
not specified
Doses / concentrationsopen allclose all
Dose / conc.:
50 other: µM
Remarks:
Metalloid resistance assay, Reporter gene assay
Dose / conc.:
80 other: µM
Remarks:
Metalloid resistance assay
Dose / conc.:
100 other: µM
Remarks:
Metalloid resistance assay
Dose / conc.:
120 other: µM
Remarks:
Metalloid resistance assay
Dose / conc.:
10 other: µM
Remarks:
electrophoretic mobility shift assay
Dose / conc.:
20 other: µM
Remarks:
electrophoretic mobility shift assay
Dose / conc.:
30 other: µM
Remarks:
electrophoretic mobility shift assay
Dose / conc.:
40 other: µM
Remarks:
electrophoretic mobility shift assay
No. of animals per sex per dose:
not applicable
Control animals:
not specified
Details on study design:
PLASMID CONSTRUCTION
For the expression of ArsK in E. coli, the arsK gene was cloned from A. tumefaciens GW4 genomic DNA into the plasmid pUC19 under the control of the lac promoter, creating plasmid pUC19-ArsK. The primers pUC19-ArsK-F and pUC19-ArsK-R were used for cloning arsK. The PCR fragment was gel purified, digested with the indicated restriction enzymes, and ligated into the vector plasmid pUC19, which had been digested with HindIII and BamHI, generating plasmid pUC19-ArsK.

METALLOID RESISTANCE ASSAYS
For the metalloid resistance assay in liquid media, AW3110 competent cells were transformed with the indicated plasmids. Cells were grown overnight with shaking at 37°C in LB with 100 µg/mL Amp and 50 µg/mL Cm. The overnight cultures were diluted 100-fold in MMNH4 medium with 100 µg/mL Amp and 50 µg/mL Cm containing various concentrations of Sb(III) plus 0.3 mM isopropyl-ß-D-thiogalactoside (IPTG) and incubated at 37°C with shaking for another 48 h. The growth was estimated from the absorbance at 600 nm.

DETECTION OF THE AMOUNT OF CELLULAR METALLOIDS
E. coli cells expressing ArsK and with the expression vector only were grown to an optical density at 600 nm (OD600) of 2 at 37°C in LB. The cells were harvested and suspended in buffer A (75 mM HEPES-KOH [pH 7.5], 0.15 M KCl, and 1 mM MgSO4). To initiate the transport reaction, 20 µM Sb(III) was added to a 5-mL cell suspension. Aliquots (1 mL) from the cell suspension were withdrawn at the indicated times, washed twice at room temperature with 1 mL buffer A, and lysed using an ultrasonic cell disruptor (Ningbo Xinzhi Instruments). To monitor the Sb(III) concentration, we used high-performance liquid chromatography with hydride generation-atomic fluorescence spectroscopy (HPLC-HG-AFS) (Beijing Haiguang Instruments).

SITE-DIRECTED MUTAGENESIS
The site-directed mutagenesis of ArsK was generated by the Fast Mutagenesis system (TransGen Biotech). The primers C97-F/C97-R, C183-F/C183-R, and C318-F/C318-R were used for PCR and the construction of the mutational vectors.

REPORTER GENE ASSAYS
The reporter gene assays in this study were evaluated based on ß-galactosidase activity. The promoter region of arsR2 was predicted by BPROM and PCR amplified using primers pLSParsK-F and pLSP-arsK-R. The promoter sequence was inserted into the EcoRI-BamHI sites of pLSP-KT2lacZ, and the resulting plasmids were then introduced into strain GW4 via conjugation. Overnight cultures were inoculated (200 µL) into 100 mL MMNH4 with or without Sb(III) and incubated at 28°C with 100 rpm shaking. During the incubation, ß-galactosidase assays were conducted as described previously.

PLASMID CONSTRUCTION, EXPRESSION, AND PURIFICATION OF ARSR2
The arsR2 gene was PCR cloned as a BamHI-HindIII fragment using primers pET28-arsK-F and pET28-arsK-R into pET-28a(+), resulting in pET-28a-arsR2. BL21 cells containing pET-28a-arsR2 were induced at an OD600 of 0.4 by adding 0.3 mM IPTG and cultivated at 20°C for 12 h. They were then harvested by centrifugation (7000 x g for 10 min at 4°C) and resuspended in borate saline buffer (pH 8.0) with 20 mM imidazole. Unbroken cells and fragments were collected by centrifugation at 7000 rpm for 10 min. The supernatant was mixed with 2 mL Ni-NTA His-Bind resin (7sea Biotech) and gently agitated at 4°C for 1 h to allow the polyhistidine-tagged protein to bind to the resin. The resin was washed with 10 mL borate saline buffer containing 60 mM imidazole and then eluted with 5 mL borate saline buffer containing 300 mM imidazole. Fractions were collected and analyzed by SDS-PAGE, and protein concentrations were determined spectrophotometrically (NanoDrop 2000; Thermo).

ELECTROPHORETIC MOBILITY SHIFT ASSAYS
The DNA fragment of the arsR2 regulatory region was amplified using the primers arsK-EMSA-F and arsK-EMSA-R. The forward primer was labeled with the fluorophore 6-carboxyfluorescein (FAM). All reaction mixtures with or without Sb(III) were incubated at 28°C for 30 min in binding buffer (20 mM Tris-HCl [pH 7.0], 50 mM NaCl, 1 mM dithiothreitol [DTT], 10 mM MgCl2, 100 µg/mL bovine serum albumin [BSA]). The binding solution was then loaded onto a 6% native PAGE gel. After 3 h of running at 80 V in 1x Tris-glycine-EDTA (TGE) buffer (120 mM Tris, 950 mM glycine, 5 mM EDTA), the gels were exposed in a phosphor imaging system (Fujifilm FLA-5100).

Examinations

Examinations:
please refer to the field "Details on study design" above.
Positive control:
not specified

Results and discussion

Details on results:
GENOMIC AND PHYLOGENETIC ANALYSES
Genome analysis identified a putative gene, which we named arsK, in the arsenic gene island of A. tumefaciens GW4. The arsK homologs were also closely related to other arsenic-resistant bacteria, such as A. tumefaciens 5A, Rhizobium sp. strain NT-26, Ensifer adhaerens, Mesorhizobium sp. strain YR577, Pseudochrobactrum sp. strain B5, Rhizobium nepotum, and Sinorhizobium sp. strain NFACC03. Besides, the ArsK orthologs were found in bacteria (including Cyanobacteria), but not in archaea, fungi, plants, or animals, revealing that ArsK may be widely distributed in bacteria only or that the similar function proteins are much less homologous in archaea or eukaryotes. The arsK gene product belongs to the MFS superfamily, and BLAST and conserved domains analyses showed that ArsK has the MFS_1 domain and the predicted arabinose efflux permease domain. In addition, a genome analysis showed that there are four arsR genes in the genomes of strains A. tumefaciens GW4 and 5A, and they have been named arsR1 to arsR4 (8). The arsR2 is present upstream of arsK in strains GW4 and 5A (Fig. 1). Interestingly, the most closely related arsR2-like genes are all located adjacent to arsK, indicating that ArsR2 may regulate the expression of arsK. ArsK, ArsJ, and MFS1 sequences also formed distinct groups together, but they were clearly divergent from each other in the subgroups. The multiple sequence alignments and phylogenetic analysis revealed that ArsK may be a novel arsenic efflux protein that is clearly divergent from ArsB, Acr3, ArsP, ArsJ, and MFS1.

RESISTANCE ASSAY
E. coli containing the pUC-19-ArsK plasmid showed significantly higher growth rates at concentrations of 50, 80, and 100 µM. At a Sb(III) concentration of 120 µM the growth rates were in both strains markedly and comparably depressed. These results indicate that strain AW3110 cells harboring the plasmid pUC19-ArsK were resistant to up to 100 µM Sb(III).

EFFLUX ASSAY
E. coli expressing arsK accumulated less Sb(III) than cells lacking the arsK gene. In addition, compared to that in strain AW3110(pUC19), the intracellular accumulation of Sb(III) in strain AW3110(pUC19-ArsK) was reduced by approximately 50% in 15 min. These results indicated that ArsK can export Sb(III) out of cells.

REPORTER GENE ASSAYS
As expected, arsK::lacZ expression was significantly induced Sb(III) in A. tumefaciens wild-type GW4, which was consistent with the resistance and efflux phenotype of ArsK to Sb(III). Sb(III) induced the expression of arsK within 1 h. These results indicate that ArsK can be induced by Sb(III), which then extrudes Sb(III) out of the cells.

ELECTROPHORETIC MOBILITY SHIFT ASSAYS
With the addition of increasing amounts Sb(III) the electrophoretic shifts of the FAM-labeled arsR2-arsK promoter gradually disappeared This result indicates that Sb(III) binds ArsR2, affecting a change in the conformation of ArsR2. ArsR2 then regulates the expression of arsK, and the ArsK transporter excretes Sb(III) out of the cells

Applicant's summary and conclusion

Conclusions:
In this study, it was shown 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.