Bifunctional protein ArsRM contributes to arsenite methylation and resistance in Brevundimonas sp. M20

doi:10.21203/rs.3.rs-2610721/v1

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Research Article

Bifunctional protein ArsRM contributes to arsenite methylation and resistance in Brevundimonas sp. M20

https://doi.org/10.21203/rs.3.rs-2610721/v1

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published 17 May, 2023

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Background

Arsenic (As) with various chemical forms is the most prevalent water and environmental toxin. This metalloid occurs worldwide and causes various diseases including cancer, especially arsenite [As(III)]. Organification of arsenite is an effective way for organisms to cope with arsenic toxicity. Microbial communities have been shown to be a vital contributor in the global arsenic biocycle and to be a promising way to reduce arsenite toxicity.

Methods

Brevundimonas sp M20 with arsenite resistance was isolated from aquaculture sewage. arsHRNBC cluster and the metRFHH operon of M20 was identified by sequencing. Gene of ArsR/methyltransferase fusion protein, arsR^M, was amplified and expressed in Escherichia coli BL21 (DE3) and resistance to arsenic was determined in present of 0.25–6 mM As(III), As(V), or Rox(V). Methylation and regulatory details of ArsR^M were analyzed by Discovery Studio 2.0. Then methyltransferase activity analysis and EMSAs confirmed the functions.

Results

MIC of Brevundimonas sp. M20 to arsenite is 4.5 mM. A 3,011-bp arsenite resistant ars cluster arsHRNBC and a 5649-bp methionine biosynthesis met operon were found on the 3.315-Mb chromosome. Transcriptional regulator/methyltransferase fusion protein ArsR^M is a difunctional protein. Expression of ArsR^M in E. coli increased arsenite resistant to 1.5 mM. Arsenite methylation and binding to its own promoter region was confirmed by ArsR^M. The As(III)-binding site (ABS) and SAM-binding motif provide the difunctional characteristic to ArsR^M.

Conclusions

We conclude that ArsR^M promotes the arsenite methylation and binding of to its own promoter region. This difunctional characteristic directly connects methionine and arsenic metabolism. Our findings add important knowledge about microbial arsenic resistance and detoxification. Future work should further explore the regulatory mechanism of ArsR^M on met operon and ars cluster.

Brevundimonas sp.

Arsenic resistance

ars cluster

Bifunctional protein

inorganic arsenic detoxify

Arsenic, classified as a Group 1 human carcinogen by the International Agency for Research on Cancer, is the most prevalent water and environmental toxin [1], and top of the US Priority List of Hazardous Substances. Arsenic compounds enter the biosphere from geochemical sources and anthropogenic sources. Herbicides, growth promoters for farm animals, the semiconductor industry, and other industrial sources contribute the arsenic contamination [2]. Humans are exposed to arsenic daily [3], mostly from food and water supplies, leads to numerous diseases, including cardiovascular and peripheral vascular diseases, neurological disorders, diabetes mellitus, chronic kidney disease [4], and cancer [3, 4]. In addition, low birth rate, fetal death, and delayed infant development are closely associated with arsenic exposure during pregnancy [5].

In the environment, inorganic arsenic exists in various chemical forms, including arsenate [As(V)], arsenite [As(III)], elemental arsenic [As(0)], and arsine [As(− 3)]. As(V) and As(III) are the predominant forms in oxic and reducing environments, respectively [2]. Organic arsenic compounds, such as pentavalent roxarsone [Rox(V)], methylarsenate [MAs(V)], trivalent roxarsone [Rox(III)] [6] and methylarsenite [MAs(III)] [7], were the proven forms. Roxarsone is an extensively used organoarsenic feed additive for poultry, and leads to artificial arsenic pollution of aquatic environments and enrichment of arsenic-tolerant bacteria [8]. As an important part of the ecosystem, microbial communities have been shown to be an vital contributor in the global arsenic biocycle [9].

Arsenic resistance (ars) genes have been identified in plasmids or/and chromosomes of various prokaryotes and eukaryotes [10]. Increasingly more genes have been found to confer resistance to arsenic and various organic arsenic compounds [11–14]. ars genes are usually clustered and at least one arsenic resistance system seems to be a necessary composition in prokaryotic species, because canonical arsRBC and its variants appear to be quite common in the bacterial and archaeal species [15]. ArsR, a member of the SmtB/ArsR family of metalloregulatory proteins, is a trans-acting transcriptional repressor protein that controls the transcription of the ars operon [16] by the interaction of ArsR with arsenite dissociates the repressor protein from the DNA [15]. Acr3 and ArsB are widespread in ars operons and are responsible for pumping As(III) from the cytosol across the cytoplasmic membrane into the periplasm or extracellular medium [17]. ArsC, an As(V) reductase, reduces intracellular As(V) to As(III) [18]. In organisms, As(III) tolerance and detoxification could be induced by efflux and methylation [3, 12, 13]. ArsM, a S-adenosylmethionine (SAM) methyltransferase enzymes, catalyzed the methylation of As(III) from SAM in microbes [2]. ArsH is an organoarsenical oxidase that confers resistance to trivalent forms of the monosodium methanearsonate and roxarsone [19, 20].

In this study, a roxarsone-resistant strain, M20, was isolated from aquaculture sewage and identified as Brevundimonas sp. We characterized genes conferring resistance to arsenite in strain M20. An ArsR/methyltransferase fusion protein, ArsR^M, which was encoded in the met operon, was found to be associated with As(III) methylation and binding its own promote, to connect arsenite methylation and methionine metabolism.

Isolation of roxarsone-resistant strainBrevundimonassp. M20

Aquaculture sewage was sampled from the influx of a wastewater treatment facility in Shandong Province, China. After diluted and spread onto Luria–Bertani (LB)-agar plates (0.5% yeast extract, 1% tryptone, 1% sodium chloride, 2% agar) containing 30 µM roxarsone (Sigma Co., Shanghai, China), the sewage samples were then incubated at 28°C for 24 h. All colonies with different phenotypes on the plate were selected and cultivated three to five consecutive times on LB-agar medium with 30 µM roxarsone to obtain pure cultures of single colonies. One colony, named M20 (Table S1), was selected and grown in pure culture for further study.

Whole-genome Sequencing And Genomic Analysis

The genome of strain M20 was sequenced using the Nanopore and BGISEQ-500 platform (BGI, Wuhan, China) and assembled using Unicycler software [21]. Additional genome annotation was performed using the RASTtk server [22] and the Pathosystems Resource Integration Center (PATRIC) server [23]. Multisequence comparison was carried out by using Clustal Omega [24] and ESPript [25]. Phylogenetic affiliation analysis of strain M20 was performed based on its genome sequence, and a whole-genome phylogenetic tree was constructed using the PATRIC server [23].

Bioinformatic Analysis Of Arsenic Resistance Genes And Proteins

The genetic contexts of the arsHRNBC cluster and the metRFHH operon were compared by using BLASTn. Ccomparison of ArsR^M and ArsM orthologs was operated by Blastp, multisequence comparison was carried out by using Clustal Omega and ESPript. Homology models of ArsR^M were constructed by using Discovery Studio 2.0 [26].

Expression And Purification Of Fusion Protein Arsr

The arsR^M gene was amplified by PCR using arsR^M-His-F/R as primers (Table S2) and genome of M20 as template. Purified arsR^M fragment was cloned into pMD18-T and verified by sequencing. NdeI and XhoI were used to digest recombinant plasmid. arsR^M gene fragment was then inserted into digested pET-15b. The resulting plasmid, pET-arsRM, was transformed into Escherichia coli BL21 (DE3), to produce E. coli strain ARM3 (Table S1). 1.0 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) was used to induce the His-tagged ArsR^M fusion protein expression in culture of E. coli ARM3 at OD_{600 nm}= 0.6. The His-tagged ArsR^M was purified using a Ni-NTA-Sefinose column (Sangon) and then analyzed by 10% SDS-PAGE as previously described after incubated at 28°C for 4 h [27].

Determination Of Minimum Inhibitory Concentrations (Mics)

LB-agar plates were used for MIC tests of Rox(V), NaH₂AsO₃ [As(III)], and NaH₂AsO₄ [As(V)]. First, strain M20 was activated on an LB-agar plate and single colonies were individually transferred into LB medium and cultured overnight at 28°C with shaking at 180 rpm. OD_{600 nm} of the culture was adjusted to 0.1, and then it was diluted 100-fold with LB medium. Diluted culture was streaked on LB-agar plates containing 0.25–6 mM (0.25-mM increment) of filtered As(III), As(V), or Rox(V). The plates were inoculated at 28°C for 3 days. MICs toward E. coli ARM3 were tested in the presence and absence of IPTG (0.5mM). E. coli BL21 (DE3) harboring vector pET-15b in the presence of IPTG was used in negative controls.

Molecular Simulation And Electromobility Shift Assays (Emsas)

The interactions between ArsR^M and its substrates As(III) and SAM were analyzed using the CDOCKER protocol of Discovery Studio 2.0 [26]. Molecular docking between ArsR^M and its target gene was analyzed by using the ZDOCKER protocol of Discovery Studio 2.0 with the arsR^M promoter region as the ligand. The interaction between ArsR^M and its gene target was verified by EMSAs. The promoter region of the metRFHH operon was divided into 80- to 120-bp fragments, which were used to design three short oligonucleotide probes (Table S2). Biotin-labeled oligonucleotide probes were synthesized by Sangon. EMSAs were conducted as previously described [27, 28].

Methyltransferase Activity Analysis Of Arsr

The capacity of ArsR^M for methylation of As(III) was determined in vitro by using a Methyltransferase Activity Assay Kit (Abcam, UK). Briefly, ArsR^M, SAM, and arsenious acid were combined; the mixture was adjusted to 50 µL with MT Assay Buffer. Sample Reaction Mix (50 µL) was prepared and added. The absorbance was measured at 570 nm every 30 s for at least 45 min at 37°C. One unit of methyltransferase activity was defined as the amount of enzyme that generated 1.0 µmol of S-adenosyl homocysteine per min at 37°C.

Isolation and identification of arsenic-resistant strain M20

Strain M20 was isolated from aquaculture sewage under the pressure of 30 µM roxarsone, an organic compound of arsenic. MIC analysis revealed that strain M20 was also resistant to inorganic arsenic compounds, such as NaH₂AsO₃ and NaH₂AsO₄ (Table S3). On the basis of phylogenetic affiliation analysis, strain M20 was identified as Brevundimonas sp. (Fig. 1A). Brevundimonas spp. are opportunistic pathogens [29]. Brevundimonas sp. M20 harbors a 3.315-Mb chromosome (67.56% G + C mol%) (Fig. 1B). The genome annotation included 1,208 hypothetical proteins and 2,052 proteins with functional assignments. The proteins with functional assignments included 758 proteins with Enzyme Commission (EC) numbers, 648 with Gene Ontology (GO) assignments, and 567 proteins that were mapped to Kyoto Encyclopedia of Genes and Genomes pathways (Table S4). Subsystem analysis of PATRIC annotation indicted that strain M20 contains 97 stress response, defense, and virulence genes, and 53 membrane transport-related genes (Fig. 1C).

Identification of an ars cluster and a met operon

Genome annotation of Brevundimonas sp. M20 revealed a novel ars cluster, which contained putative arsenic resistance genes arsH, arsR, arsN, arsB, and arsC (Fig. 2). In this 3,011-bp cluster, arsH is an organoarsenical oxidase-encoding gene, which was considered to confer resistance to roxarsone and methylarsenite [19]. arsR, encoding a regulatory protein, is responsible for regulating ars cluster gene expression. arsB and arsC are arsenic detoxification genes; arsenate is reduced to arsenite by arsenate reductase (ArsC), followed by efflux of arsenite by the arsenite transporter ArsB [7]. ArsN detoxifies arsenate by acetylation of the α-amino group of arsinothricin [9]. BLAST analysis of the ars cluster revealed high similarity with the ars cluster in Caulobacterales spp. (JAFLCT010000083), Brevundimonas sp. Bin7 (JACVCC01000001), and B. nasdae strain Au-Bre29 (CP080034), although the ars cluster in the latter does not contain arsN (Fig. 2A).

No arsenic methylation gene was found in the ars cluster of Brevundimonas sp. M20. Downstream of the ars cluster, a 5649-bp met operon related to methionine biosynthesis was identified. The met operon was located 21.5 kb downstream of the ars cluster and contained four genes, arsR^M (encoding a transcriptional regulator fused with a methyltransferase), metF (methylenetetrahydrofolate reductase), HMT-1 (5-methyltetrahydrofolate-homocysteine methyltransferase), and metH (methionine synthase) (Fig. 2B). The DNA sequence of the arsR fragment in arsR^M showed very low similarity with the arsR gene in the ars cluster, although the similarity of the amino acid sequence was 21.78%. The met operon showed high similarity with that in B. nasdae Au-Bre29, which was isolated in Fujian Province, China, in 2022 [12]. However, the gap between the ars cluster and the met operon in strains M20 and Au-Bre29 was 21.5 kb and 2.1 Mb, respectively. The function of ArsR^M has not yet been explained.

ArsR ^M increased the arsenite resistance of recombinant E. coli

No arsenic methylation gene was found in the ars cluster of Brevundimonas sp. M20, while ArsR^M, encoded by arsR^M of the met operon, was identified as a transcriptional regulator fused with a methyltransferase. To analyze the functions of ArsR^M, the complete arsR^M gene (960 bp) was cloned and inserted into pET-15b (Fig. 3A). The resulting plasmid was transferred into E. coli BL21 (DE3), and the resulting strain E. coli ARM3 was verified by PCR (Fig. 3B).

After induction by IPTG, the MIC of NaH₂AsO₃ toward E. coli ARM3 was increased to 1.5 mM compared with that in the uninduced control (Table S3). This enhancement indicated that ArsR^M likely contributes to arsenite resistance in strain M20. On the basis of similarities with various arsenite methyltransferases in different species (Figure S1), the arsenite resistance mediated by ArsR^M may be due to arsenite methylation.

ArsR^M is a bifunctional fusion protein

To further investigate its function, the protein structure of ArsR^M was homology modeled. ArsR^M from Brevundimonas sp. M20 formed a dimer (Fig. 4A). The two monomers adopt different orientations (Fig. 4B). Each monomer contains a transcriptional regulator domain (TRD; α1–α5 and β1–β2), a methyltransferase domain (MTD; α6–α9 and β3–β7), and a 11-amino acid-residue loop linker (residues E111–A121) (Fig. 4C).

Unlike the typical ArsR [Protein Data Bank Code (PDB): 1R1T], which contains five α-helixes and two antiparallel β-sheets [30, 31], the TRD of ArsR^M is composed of six short α-helixes and two antiparallel β-sheets. The DNA-binding domain (DBD) in the TRD (TRD has a diameter of approximately 36.6 Å) is composed of three helices (α1–α3) that form the core of the TRD and a C-terminal β-hairpin (β1–β2) (Fig. 4D). Compared with the tight domain in ArsR, these shortened α-helices in the DBD of ArsR^M produce a flexible structure that may lead to a different regulatory mechanism.

The MTD of ArsR^M has a mixed structure consisting of α-helices (α6–α9) and β-strands (β3–β7). This domain (residues 122–320) was compared with arsenite methyltransferases in bacteria (including Thermosediminibacter oceani, Streptomyces barringtoniae, Chloroflexi spp., Alteripontixanthobacter maritimus, and Hymenobacter roseosalivarius); Homo sapiens (residues 1–279); and the unicellular red alga Cyanidioschyzon sp. (complete protein). A glycine-rich sequence, “DLGTGSG,” is conserved as the hallmark of the SAM-binding motif [30] (Figure S1). This glycine-rich motif presented a circular, open shape and was located among α1, α2, β3, β4, and β5 (Fig. 4E). The As(III)-binding site (ABS) has three modular components in ArsR (PDB: 1R1T) [30], while arsenite methyltransferases in different species are variable (Figure S1). The ABS of ArsR^M, adjacent to the SAM-binding motif, was composed of residue C134 (equivalent to C72 in PDB: 1R1T) and residues in the region Q224-L229 (Fig. 4F).

ArsR^M binds its own promoter region

Molecular docking analysis showed that the TRD of the fusion protein ArsR^M from Brevundimonas sp. M20 could bind to its own gene promoter region (P_arsRM). Similar to the DNA binding in ArsR [31], the dimer formed wing regions and helix α5 (equivalent to α4 in ArsR, PDB: 1GXP) interact with DNA in Helix-turn-helix (HTH)–DNA complex structures (Fig. 5A). The DNA-binding sequence of ArsR^M in the P_arsRM region included a region that contained a palindromic sequence “CTTTATATAAAG” located upstream of the initiation codon of arsR^M (Fig. 5A). This region is similar to the A/T-rich binding sites of ArsR. The interaction residues include R20 and E42, and, in helix α5, N96, A97, A98, D99, D100, L103, and E104 (Fig. 5B).

We tested biotin-labeled probes of the arsR^M promoter region (215-bp long) in EMSAs: P_arsRM-1 (containing the predicted target sequence), P_arsRM-2, and P_arsRM-3 (Fig. 5C). Sufficient quantities of recombinant full-length ArsR^M–His₆ protein were produced in E. coli BL21 (DE3). The soluble protein was eluted from a Ni-NTA column in buffer containing 150 mM imidazole (Fig. 5D). Probe P_arsRM-1, containing the predicted binding sequence of ArsR^M to P_arsRM, was shifted in EMSAs by the addition of purified ArsR^M (Fig. 5E). This result confirmed the binding of ArsR^M to its own promoter region, which might regulate the transcription of the methionine biosynthesis gene cluster in Brevundimonas sp. M20.

ArsR^M contributes to the methylation of As(III)

The process of arsenic methylation was originally proposed to be a detoxification mechanism, by which trivalent inorganic arsenic is biotransformed to a less toxic pentavalent methylated form. The MTD of ArsR^M was predicted to interact with SAM and trivalent arsenic (Fig. 6A). Amino acid residues C134, H223, Q224, H227, F254, R263, H268, and P296 were identified to be the SAM-binding residues (Fig. 6A). Unlike in the arsenite methyltransferases shown in Figure S1 where Cys interacts with As(III), in ArsR^M, Q224 and Y228 were the As(III) interacting residues, at a distance of 2.3–2.4 Å (Fig. 6B). These distances are similar to those for residues (C174 and C224) in C. merolae ArsM (2.2–2.3 Å) [30].

The methyltransferase activity of ArsR^M was measured using a Methyltransferase Activity Assay Kit. Activity was detected in the presence of a mixture of SAM, arsenious acid, and ArsR^M (Fig. 6C). This confirmed that arsenious acid can be methylated to monomethylarsonic acid by ArsR^M with 0.87U/mg, although the transformation rate was low.

Arsenic is a metalloid found worldwide [4]. Natural and anthropogenic activities both led to arsenic accumulation and transfer into biosphere [32]. The higher affinity and ability to form metallic compounds make As(III) more toxic than As(V) [2]. In the environment, inorganic arsenic, such as arsenate [As(V)], arsenite [As(III)] [2], are thought to be more toxic than organic arsenic, such as pentavalent roxarsone [Rox(V)] and trivalent roxarsone [Rox(III)] [7]. Exposure to arsenic leads to diseases, including diabetes, peripheral vascular disease, and cancer [33, 34]. The toxicity, mobility, and fate of arsenic in the environment are influenced by lots of factors, including its speciation, arsenic speciation changes and biogeochemical cycle [2]. In this study, we isolated an arsenic-resistant bacterial strain, Brevundimonas sp. M20, which was tolerant of organic and inorganic arsenicals. Genomic annotation indicated the presence of various arsenic-tolerance genes, including for valence transition of inorganic arsenic, transformation of organic and inorganic arsenicals, and arsenic efflux pump.

ars cluster is a genetic system to cope with arsenic toxicity in microorganisms. Genes of ars cluster occur in most prokaryotic genomes and are widely distributed in bacterial and archaeal species [35]. arsRDABC and arsRBC variants are the usual operons found in bacteria [15]. In the present study, we identified a novel ars cluster variant in the chromosome of Brevundimonas sp. M20; this ars cluster contained five genes with the arrangement arsH–arsR–arsN–arsB–arsC. Homologous genes of these in arsHRNBC cluster were proved to contribute to organoarsenical oxidation [19, 36, 37], cluster transcriptional repression [38], organoarsenical acetyl transfer [9], formation of arsenite efflux pump[3] and arsenate reduction[15]. Thus, novel arsHRNBC cluster is probably a contributor to As(III) resistance from gene functional prediction.

Microbial arsenic methylation and demethylation play a crucial role in the arsenic biogeochemical cycle [9]. Because of the arsenic methylation, organoarsenic compounds are found naturally [39]. For methylation, methionine is a universal biological cofactor that transfers its methyl group to substrates such as lipids, proteins, DNA, and other small molecules [9] in the presents of SAM, which is a conjugate of the nucleotide adenosine and amino acid methionine and an essential substrate/cofactor in numerous enzyme-catalyzed reactions, including transmethylation [40]. In Brevundimonas sp. M20, we found a methionine biosynthesis gene cluster, the met operon, downstream of the ars cluster. The first gene, arsR^M, encodes a transcriptional regulator fused with a methyltransferase. Molecular simulation and laboratory experiments confirmed the mixed function of ArsR^M. The TRD of ArsR^M binds to the palindromic sequence “CTTTATATAAAG” in its own promoter region, P_arsRM, to regulate expression of the methionine biosynthesis gene cluster, while the MTD of ArsR^M presented a SAM-dependent arsenic methylation function. ArsR^M thus connects methionine biosynthesis and arsenic methylation to provide a precise and efficient pathway for arsenic resistance related to metabolism of methionine. Moreover, the organification of As(III), mediated by ArsH along with ArsR^M, broadens the microbial resistance spectrum of ars operons from inorganic to organic arsenicals.

The data presented here indicate a contribution to arsenic resistance mediated by the methyltransferase–transcriptional regulator fusion protein ArsR^M in Brevundimonas sp. M20 (Fig. 7). In this pathway, inorganic arsenic, organic arsenic, As(V), and As(III) are mutually transformed. As(III) can be extruded by the pump ArsB or methylated by ArsR^M, which also controls the biosynthesis of methionine, a component of SAM. During the arsenic methylation, the methyl group of SAM is transferred to As(III) by ArsR^M to form methylarsenite [MAs(III)]. MAs(III) is oxidized to Mas(V) by the organoarsenical oxidase ArsH. Elucidation of this novel arsenic-resistance pathway adds important data for our understanding of microbial arsenic resistance.

Supplementary Information

The online version contains supplementary material available at https:// xxxxxxxxxxx

Additional file 1. Table S1. Bacterial strains and plasmids used in this study. Table S2. Primers used in this study. Table S3. MICs of strains for organic and inorganic arsenics. Table S4. General features of the Brevundimonas sp. M20 genome. Figure S1. Multisequence comparison of the MTD domain of ArsR^M from Brevundimonas sp. M20 with methyltransferase proteins from other species.

Nucleotide sequence accession number

The complete sequence of the Brevundimonas sp. M20 genome was deposited in GenBank under accession no. CP041243.

Acknowledgments

We thank Liwen Bianji (Edanz) (www.liwenbianji.cn) for editing the language of a draft of this manuscript.

Author’s contributions

CCL prepared the aquaculture sewage, co-led the data analysis and wrote the manuscript. GLZ co-led the data analysis and edited the manuscript. WHG co-led the data analysis and co-led the performance of bioinformatics analysis. XC co-led the performance of bioinformatics analysis. MXT helped to analyze data. JFF helped revised the manuscript. PPZ helped to analyze data and revised the manuscript. BW co-led the project and revised the manuscript. GXC co-led led the project and revised the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by the Key Research and Development Project of Shandong Province (No. 2021ZDSYS27), the Academic Promotion Program of Shandong First Medical University [grant number LJ001, 2019], the Innovation Project of the Shandong Academy of Medical Sciences [grant number 2022], and the National College Students’ Innovation and Entrepreneurship Training Program [grant number 2029, 2022].

Availability of data and materials

The datasets supporting the conclusions of this article are available from the lead authors upon a reasonable request.

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests.

Author details

¹Shandong Quancheng Test & Technology Limited Company, Ji’nan 250101, China

²Biomedical Sciences College & Shandong Medicinal Biotechnology Centre, Shandong First Medical University & Shandong Academy of Medical Sciences, Ji’nan 250062, China

³NHC Key Laboratory of Biotechnology Drugs (Shandong Academy of Medical Sciences), Ji’nan 250117, China

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You are reading this latest preprint version

Bifunctional protein ArsRM contributes to arsenite methylation and resistance in Brevundimonas sp. M20

Status:

Journal Publication

Version 1

Abstract

Background

Methods

Results

Conclusions

Figures

Introduction

Materials And Methods

Whole-genome Sequencing And Genomic Analysis

Bioinformatic Analysis Of Arsenic Resistance Genes And Proteins

Expression And Purification Of Fusion Protein Arsr

Determination Of Minimum Inhibitory Concentrations (Mics)

Molecular Simulation And Electromobility Shift Assays (Emsas)

Methyltransferase Activity Analysis Of Arsr

Results And Discussion

Isolation and identification of arsenic-resistant strain M20

ArsR^M binds its own promoter region

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

Bifunctional protein ArsRM contributes to arsenite methylation and resistance in Brevundimonas sp. M20

Status:

Journal Publication

Version 1

Abstract

Background

Methods

Results

Conclusions

Figures

Introduction

Materials And Methods

Whole-genome Sequencing And Genomic Analysis

Bioinformatic Analysis Of Arsenic Resistance Genes And Proteins

Expression And Purification Of Fusion Protein Arsr

Determination Of Minimum Inhibitory Concentrations (Mics)

Molecular Simulation And Electromobility Shift Assays (Emsas)

Methyltransferase Activity Analysis Of Arsr

Results And Discussion

Isolation and identification of arsenic-resistant strain M20

ArsRM binds its own promoter region

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

ArsR^M binds its own promoter region