Bioremediation of heavy metals by an unexplored bacterium, Pseudoxanthomonas mexicana strain GTZY, isolated from aerobic-biofilm wastewater system

Bioremediation of heavy metals by an unexplored bacterium, Pseudoxanthomonas mexicana strain GTZY, isolated from aerobic-biofilm wastewater system | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Bioremediation of heavy metals by an unexplored bacterium, Pseudoxanthomonas mexicana strain GTZY, isolated from aerobic-biofilm wastewater system Abdul Raheem Nelofer, Ganesh-Kumar Selvaraj, Kalimuthu Karuppanan, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4104815/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Aug, 2024 Read the published version in Environmental Science and Pollution Research → Version 1 posted 5 You are reading this latest preprint version Abstract We prompted first to characterize a wastewater bacterium, Pseudoxanthomonas mexicana GTZY, that efficiently transforms toxic mercury and arsenic and to explore its bioremediation capability and to reveal their relevant gene resistance operons. The Isolated strain was characterized for its phylogenetic, biochemical and phenotypic properties. The strain GTZY potentially removed 84.3% of mercury and their mercury volatilization (Hg(II) to Hg(0)) was confirmed using X-ray film method, and its respective merA gene was PCR amplified. In addition, strain GTZY was efficiently removed arsenate (68.5%) and arsenite (63.2%), and showed resistance up to >175 and >55 mM, respectively. Their genomic annotations were disclosed the linkage of Tn2 -transposon and int1 in both ends of mer operon ( merAPTR) . The co-existence of arsP and arsH proteins in its intrinsic ars operon ( arsCPRH) was extremely diverse from its ancestral species. We believe that mercury resistance-conferring mer operon of P. mexicana GTZY presumably derived horizontally from other species in the reactor, while arsenic resistance-conferring intrinsic ars operon was highly diversified and evolved from its ancestral species. By considering the potential of the strain GTZY to transform heavy metals, this can be used to recover contaminated sites. Pseudoxanthomonas mexicana Mercury Arsenic Resistance gene operon Bioremediation wastewater Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Highlights First detailed heavy metals bioremediation study in the genus, Pseudoxanthomonas Isolate has great potential to resist, reduce and remove the toxic Hg(II) and As(V) An acquired mobile elements linked mer gene operon was observed in its chromosome. An intrinsic ars gene operon was extremely diverse from its ancestral species 1. Introduction Heavy metals are inorganic ill-defined chemical compounds that ubiquitous in nature. In which, some heavy metals such as cadmium (Cd), arsenic (As), nickel (Ni), lead (Pb), chromium (Cr), mercury (Hg), copper (Cu) and zinc (Zn) are mainly persisting in the environments due to the core release of anthropical sources (Loganathan et al. 2015 ; Govarthanan et al. 2016b , a , 2018 , 2019 ; Jobby et al. 2018 ; Briffa et al. 2020 ; Khalid et al. 2020 ). This persistence is considered as an emerging situation due to their potential toxicity in the entire ecosystem, including humans, animals and plants (Olmedo et al. 2013 ; Rebelo and Caldas 2016 ; Luo et al. 2020 ; Qin et al. 2021 ). Some heavy metals are considered highly toxic as they could cause human illness even at their relatively low levels. Among those, arsenic and mercury are the prime consideration for their chemical toxicity, persistant, frequency and potential to human exposure(Hou et al. 2020 ). According to the national priorities list released by the Agency for Toxic Substances and Disease Registry (ATSDR), among the 275 toxic substances, arsenic and mercury were ranked as first and third, respectively(Watters and Rayman 2014 , 2018). These heavy metals are non-biodegradable and can not catabolized by microorganisms. Alternatively, these heavy metals can be transformed from toxic to non-toxic forms by the nuclear structural changes via microbial activities(Wuana and Okieimen 2011 ; Jebeli et al. 2017 ; Wang et al. 2020 ). In general, microbial mercury bioremediation mainly occurs by the mechanism of reduction or volatilization, which is governed by mer operon in microorganisms(Boyd and Barkay 2012 ; Raj and Maiti 2019 ). In mer operon, merA protein is one of the main constituents along with Hg binding ( merP ), regulatory ( merR and D ), and mercury transporting proteins ( merT and E ), and are mainly located in the bacterial chromosome or mobile genetic elements (e.g. plasmids, integrons or transposons)(Oregaard and Sørensen 2007 ; Boyd and Barkay 2012 ; Møller et al. 2014 ; Naguib et al. 2018 ). Relying on the availability of toxic mercury (Hg 2 ), merR protein binds specifically into the promoter-operator region and it is followed by regulating the transcription of structural genes. As a result, it produces a key enzyme in the detoxifying system known as mercury reductase (encoded by merA gene), an NAD(P)H-dependent flavin oxidoreductase, reducing toxic mercury (Hg 2 ) into a volatile natured less toxic mercury (Hg 0 )(Freedman et al. 2012 ; Dash et al. 2017 ). Likewise, microbial arsenic bioremediation mainly involves arsenic reduction ( arsC ), oxidation ( AoxAB, arsH ), methylation and intra-cellular bioaccumulation(Pepi et al. 2007 ; Rahman and Hassler 2014 ; Govarthanan et al. 2015 , 2016a , 2019 ; Satyapal et al. 2018 ; Ben Fekih et al. 2018 ). The ars operon is an energy-gaining detoxifying respiratory system that facilitates the reduction of pentavalent arsenate (V) into trivalent-arsenite (III)(Suhadolnik et al. 2017 ). The interaction of transcriptional repressor protein (arsR) with promoter region of ars operon, followed by the binding of arsR protein with arsenate, dissociates the repressor protein from a specific DNA site permitting the ars operon’s transcription(Páez-Espino et al. 2015 ; Li et al. 2016 ). This organoarsenic reduction process is governed by the arsC encoding arsenic reductase enzyme. The ars operons, such as arsRB, arsRBC, arsRDABC and arsHRBC are widely demonstrated in the gram-negative bacteria(Dey et al. 2016 ; Ben Fekih et al. 2018 ; Taran et al. 2019 ). The existence and sustainability of these metal resistance operons among the various bacterial communities are usually maintained and transmitted with the help of mobile genetic elements via horizontal gene transfer(Møller et al. 2014 ; Suhadolnik et al. 2017 ). In some bacteria, this metal resistance sustainability is preserved intrinsically in the bacterial chromosome, the consequence of vertical gene transfer from its ancestral species. Many bacterial species reportedly have arsenic and mercury resistance operons in their chromosome or mobile genetics elements(Møller et al. 2011 ; Ghaly et al. 2017 ; Agarwal et al. 2019 ). However, so far, none among the species in the genus Pseudoxanthomonas have been demonstrated for any heavy metal resistance and their respective operons. The 25 members of this genus are gram-negative, rod-shaped, non-pathogenic that are long been known for their prevalence in diverse ecological sources, including soil, water, plants, animal and human tissues(Thierry et al. 2004 ; Rani et al. 2010 ; Kittiwongwattana and Thawai 2016 ; Selvaraj et al. 2018 ). Although the genus Pseudoxanthomonas is well characterized for its biodegradation capability and surfactant production(Choi et al. 2013 ; Talwar and Ninnekar 2015 ; Biswas et al. 2017 ; Astuti et al. 2019 ; Lu et al. 2019 ; Purwasena et al. 2020 ), it is unremarked in many environmental processes, particularly in terms of its bioremediation capability. This study thereby deals (i) isolation and characterization of Hg and As resistant strain GTZY from an bioreactor treating antibiotic-containing wastewater, (ii) evaluation of Hg and As removal efficiency of strain GTZY in batch-culture experiments, (iii) disclosure of the mer and ars resistance operon from strain GTZY and (iv) phylogenetic distribution and relationship of identified mer and ars operon of strain GTZY with other bacterial communities. 2. Materials and Methods 2.1. Bacterial strain The bacterial samples were collected from the aerobic-biofilm wastewater reactor treating aminoglycoside antibiotic, streptomycin (50 mg L − 1 ) (State key laboratory of RCEES, CAS Institute, Beijing, China). The ten-fold serial dilution method was used to isolate and enrich bacterial isolate from the collected sludge samples. Inoculated (100 µL aliquots) plates were incubated at 30 ºC for 12–18 h. Tryptone soy agar (TSA) and Reasoner’s 2A agar (R2A) media were used for the purpose of bacterial isolation. Gram’s staining was routinely performed to check the microbial purity. Initially, there were 23 bacterial isolates harvested from the wastewater reactor. To obtain the potential heavy metal transforming bacteria, the purified isolates were further screened on the growth media amended with As (V); 1 µM and HgCl 2 ; 10 µM. Among the 23 isolates, strain GTZY was choosen for further studies due to its fast growing ability and surviving capability in the presence of increased concentration of As (V); 100 µM and HgCl 2 ; 50 µM. These screened isolates were stored as 40% glycerol stock for further studies. 2.2. General characterization of bacterial isolate The morphological, biochemical and physiological characterization of isolated bacterial strain was performed based on the standard microbiological methods. The optimum temperature and pH were identified by culturing the strain on the nutrient agar media in the various temperatures (25−40 ºC) and pH (5−8). After 12−18 h incubation, bacterial colony morphology was determined using a single colony which grown on the nutrient agar, TSA, LB and R2A media. The hanging drop method was performed to check the motility of isolates under the light microscopy. Gram’s staining solutions were manually prepared and isolates were viewed under the light microscopy magnifications (100×). A salt tolerance test was carried out by preparing Brain-heart infusion agar plates with the addition of various NaCl concentrations 0−7% (w/v). Activitiy of various enzymes (catalase, oxidase, urease and indole), hydrogen sulfide production, utilization of various carbon sources, nitrate reductability and hydrolysis of biochemicals (gelatin, starch and casein) tests were also carried out by the standard microbial methods. 2.3. 16S rRNA gene amplification and phylogenetic tree analysis The genomic DNA isolation and 16rRNA gene PCR amplifications were carried out as described in the previous study(Selvaraj et al. 2018 ). In brief, the genomic DNA was harvested from the bacterial samples using the TIANamp bacteria DNA kit (Tiangen, China), and the isolated DNA was quantified using Nanodrop 1000 spectrophotometer (Nanodrop, USA). The 16SrRNA fragments were amplified using the universal primers (27F-5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R-5'-AAGGAGGTGATCCAGCCGCA-3'), sanger sequenced (Biobase, China) and interpreted by the Ribosomal Database Project (RDP) and GenBank-National Center for Biotechnology Information (NCBI) database. The MEGA6.06 and Clustal W softwares were used to constructing a phylogenetic tree for isolated strain against its closest 19 bacterial strains. The Neighbour-joining method and bootstrap analysis were executed for the bacterial phylogeny and statistical sequence analysis, respectively. In bootstrap analysis, 1000 replicates were applied for determining the confidence values of bacterial phylogenetic tree nodes. Finally, the partial 16S rRNA gene sequence was submitted in the GenBank. 2.4. Determination of mercury MIC After bacterial isolation, the bacterial efficiency on mercury resistance was maintained in LB media by the supplement of mercury chloride solution (10−30 µM; HgCl 2 ). The bacterial isolates were grown overnight on the LB media added with 10 µM of HgCl 2 . Subsequently, those cultures were resuspended with 2−5 mL of Tris-buffered saline (TBS) solution, and absorbance (OD 600 nm ) was adjusted to 0.100 OD. Further, 125 µL of appropriate bacterial dilutions were inoculated on the 96-well plate containing 125 µL of LB broth (in triplicate) amended with various concentrations (0, 1, 5, 10, 20, 30, 40, 50, 60, 70 and 80 µM) of HgCl 2 and incubated at 30 ºC for 72 h in the dark. After the incubation, bacterial growth was observed by measuring the absorbance (OD 600 nm ) using a microplate reader. The inhibition effect of mercury on the microbial growth, values of MIC and MIC 50 were calculated. 2.5. Mercury removal assay Overnight grown bacterial cultures (in triplicate) added with 10 µM of HgCl 2 were subjected for the mercury removal assay. Every 12 h of incubation, 4 mL of culture was centrifuged for 10 min at 7500 rpm,thereby recovered bacterial pellets were resuspended in 2.8 mL of Tris-buffered saline solution. In the resuspended solution, 0.4 mL of 5-diphenyl-3-thiocarbazone (Dithizone) reagent solution (0.50 mg of dithizone mixed with 20 mL of acetone) was added, followed by the addition of 0.4 mL of sulphuric acid (2 N) to maintain the acid pH (pH 2−3) and finally added by 0.4 mL of Dioxane (Naguib et al. 2019 ). The acidic pH of the solution enhanced the formation of orange-red colour by coupling of dithizone and mercury (II). These final solutions were diluted to 5 mL using distilled water and checked mercury removal values at 488 nm in a UV/Visible spectrophotometer. Meanwhile, a similar experimental set up was carried out using dead bacterial cells (autoclaved cells) for treating as control. The standard curve was also prepared for HgCl 2 solution without bacterial suspension. The detection limit for this method was estimated as < 1 ppb (Nordlander). The actual Hg concentrations during the incubation period was measured and thereby the mercury removal percentage was calculated as follows, 2.6. Mercury volatilization assay A simplified X-ray film method was used to confirm the mercury volatilization from Hg (II) state to Hg (0) state by the strain GTZY. Overnight grown culture added with 10 µM; HgCl 2 was centrifuged at 6000 rpm for 5 min. Harvested bacterial pellets were resuspended into a 200 µL assay solution containing 0.07 M phosphate buffer (pH 7), 0.2 mM magnesium acetate, 5 mM sodium thioglycolate and 50 µM HgCl 2 . The microtiter plate containing the above-mentioned solution were fully covered using an X-ray film (Kodak scientific imaging, Hyderabad, India)(Møller et al. 2011 , 2014 ) and incubated at 35 ºC in the dark. A similar solution mixture that without bacterial cells and bacterial suspension without HgCl 2 were utilized as control samples. 2.7. Determination of arsenic MIC The bacterial arsenic resistance was evaluated by adding the different concentrations of sodium arsenate (10−400 mM) and sodium arsenite (10−200 mM) into the overnight grown bacterial culture, while the control samples have no metals. The cultures were resuspended with 2−5 mL of TBS solution with an absorbance (OD 600 nm ) of 0.100. Further, 125 µL of appropriate bacterial dilutions and 125 µL of LB broth (in triplicate) were added into the 96-well plate containing suspended concentrations of arsenic and incubated at 30 ºC for 48 h in the dark. After the incubation, tested bacterial samples were estimated for their optical density at 600 nm using a microplate reader. The inhibition effect of arsenic on the microbial growth, values of MIC and MIC 50 were calculated. . 2.8. Arsenic removal assay After the bacterial isolation, the efficiency of arsenic resistance was maintained by adding sodium arsenate (100 mM) and sodium arsenite (100 mM) into the bacteria growth media (NA). Arsenic utilization by bacterial isolate was determined by the Silver Diethyldithiocarbamate (SDDC) method(Tahir et al. 2012 ). The detection limit for this method was evaluated as 1–5 ppb. Initially, the isolate (1%) was inoculated (in triplicate) in LB broth added with 5 µM sodium arsenate and sodium arsenite solution (as sources for As (V) and As (III), respectively) and incubated at 30 ºC for various time intervals (viz. 8, 12, 18, 24, 48 and 72 h). After the definite time points, the actual arsenic concentrations during the incubation period was estimated using the SDDC method. Briefly, the production of arsine carrying hydrogen passed through a reduction column impregnated with lead acetate and then into the absorbing solution SDDC dissolved pyridine. The end red-coloured product (arsine with SDDC solution) was measured spectrophotometrically at 535 nm. 2.9. Arsenic reduction and oxidation assay The ability of the bacterial isolate to oxidize Arsenic (As III) and reduce Arsenic (As V) was checked by the silver nitrate solution(Dey et al. 2016 ). After 24 and 48 h of incubation, 100 µL of silver nitrate solution (0.1 M) was added into the grown bacterial culture (100 µL) amended with 1 µM sodium arsenate and sodium arsenite. If the culture broth turns brown, it represents the presence of silver arsenate, while if the culture broth turns yellow, represents the presence of silver arsenite. 2.10. merA and arsC genes sequencing The extracted and purified genomic DNA was used as a template DNA to screen mercury ( merA ) and arsenical ( arsC ) resistance genes. The designed merA ( merA- FP- 5′-CTACCCCGCGCAACAGGACA-3′ and merA- RP- 5′-TGATGGCGCCTTGCGCATTG-3′) and arsC ( arsC- FP-5′-TCAGGACGAGGCGCCGATCT-3′ and arsC- RP-3′- ATGGACCGCCCCTACAACCT-5′) primers were used to amplify the partial regions merA and arsC . The purified genomic DNA (50 ng) was used as a template for preparing the PCR reaction. The triplicate PCR mixture (50 µL) comprised of 1× PCR buffer (with MgCl 2+ ), 2.5 mM of the dNTPs mix, 0.5 µM of each primer, and 1 U of Taq DNA polymerase (Takara, Bio Inc., Shiga, Japan). The PCR conditions were performed as follows: initial denaturation stage at 95°C for 5 min, followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 2 min and a final extension at 72°C for 10 min. The amplified specific PCR products were resolved using 1.2% agarose (Biowest, Hong Kong) gel and sequenced (Sanger sequencing). The gene sequences were analyzed using BLASTN. 2.11. Annotations of mer and ars gene operons The extracted total genome of isolated strain GTZY was sequenced using the third-generation single-molecule real-time (SMRT) technology (Pacific Biosciences Inc., USA), and the sequenced genome was submitted to the GenBank. The whole-genome sequence of Pseudoxanthomonas mexicana strain GTZY (Accession No.: CP060028) was used for the resistance gene cluster’s interpretation. The mer and ars resistance gene clusters related data were initially annotated using the online ORF prediction server, Rapid Annotations using Subsystems Technology (RAST), and their manual annotations were performed using BLAST. A Python application software, Easyfig (ver. 2.1) was used for the linear comparison and visualization of mer and ars resistance gene clusters. In addition, the BLAST comparison between the gene annotated files was manually uploaded, output files (blastn) generated, interactively coloured and enabled a rapid transition using the software, Easyfig. The SnapGene Viewer (ver. 4.1.3) was used for the gene annotation and visualization. 3. Results 3.1. Characterization of bacterial isolate The isolated bacterial strain was aerobic, gram-negative, rod-shaped and highly motile. The colonies were pale yellow, circular, convex and mucoid-natured on the solid nutrient media (Fig. S1). The isolate can grow in the media up to 40 ºC and pH 8, while its optimum temperature and pH were 34−37 ºC and 7−7.5, respectively. Isolate can tolerate the presence of sodium chloride up to 8% (w/v); however, its optimum concentration was 3.5–5.5%. Their biochemical and phenotypic characterizations were given in Table S1. Biochemically, the isolate turned positive for catalase while remained negative for oxidase and urease, which in turn ferments glucose and mannitol but not sucrose and lactose. The hydrolysis test turned positive for the gelatin and casein, but remained negative for starch. Moreover, it showed negative results for indole production, nitrate reductase, Voges-Proskauer (VP) test, and positive for the Tween-20 and Methyl Red (MR) test. The length of the 16S rDNA gene sequence was 1424 bp, and it shared the closest relationship (99.39%) with P. mexicana CP4 (Accession No.: MT549102) in the phylogenetic tree by the neighbour-joining method (Fig. 1 ). Hence, the isolated bacterium was named as Pseudoxanthomonas mexicana strain GTZY and submitted to the GenBank (Accession No.: MZ242608). 3.2. MIC, volatilization and bioremediation of mercury The effect of mercury on microbial growth was studied. Further, MIC and MIC 50 were determined by supplementing different mercury concentrations into LB broth inoculated with strain GTZY. After 48 h of incubation, mean values of OD 600nm were plotted against 0−80 µM of HgCl 2 . The MIC value of mercury against strain GTZY was 33.4 µM of HgCl 2, and its MIC 50 value was at 40.8 µM of HgCl 2 . Microbial growth was notably inhibited by 35.5 µM of HgCl 2, and it was completely inhibited (0.21 OD 600nm ) at 49.7 µM of HgCl 2 (Fig. 2 A). After 60 h of incubation, the residual concentration of Hg was 1.7 ± 0.2 µM and it was sustained for the remaining time periods (Fig. S3A). The mercury reducing ability of the strain GTZY was confirmed by a mercury volatilization assay conducted at 48 h of incubation. The strain formed a foggy area (dark circle) due to the reaction between Hg (0) and Ag emulsion on the X-ray film (Fig. S2). However, the isolate started to produce foggy formation from 4 h onwards. Hence, the strain GTZY reduced inorganic Hg (II) and formed volatile Hg (0), which reacted with the film. There was no interference of foggy circles observed in the control samples. Mercury removal by the strain GTZY was determined using the spectrophotometric dithizone method. Initial and residual Hg concentrations were evaluated against the standard calibration curve of various concentrations of Hg. The strain GTZY removed 16.3% of Hg from 4 h onwards, and their removal process was stopped at 48 h of incubation (Fig. 2 B). The strain GTZY removed a maximum of 84.3% mercury at 48 h and their removal rate was maintained until 96 h of incubation. At the same time, no mercury removal was found in the control samples. MIC, reduction and bioremediation of arsenic metals The isolate GTZY was resistance to the high concentrations of arsenate and arsenite solutions. Also, it removed the arsenic compound at various time periods. Isolate GTZY was efficiently removed arsenate (68.5%) and arsenite (63.2%) at 72 h of incubation (Fig. 3 ). At 12 h of incubation, the removal rate (%) of arsenate and arsenite were accounted for 34.2% and 28.3%. Meanwhile, no notable removal of arsenical compounds was observed in the control samples. The MIC values of isolate towards arsenate and arsenite were > 175 and > 55 mM, and their corresponding MIC 50 were 240 and 60.5 mM, respectively. The bacterial growth was completely arrested with 330 mM; arsenate and 69.4 mM; arsenite (Fig. S4). After 60 h of incubation, the residual concentration of As (III) and As (V) were 22 ± 2 µM and 27.5 ± 2.5 µM (Fig. S3B), thereafter it was sustained for the remaining time periods. After 24 and 48 h of incubation, 100 µL of silver nitrate solution was added to both bacterial cultures (As III and V), and the suspensions were turned into the light yellow colour, which indicated the presence of As (III). Meanwhile, there was no brownish colour observed which indicating the absence of silver arsenate. The result confirmed that isolate GTZY could reduce As (V) to As (III), while the isolate could not oxidize As (III) to the As (V). 3.3. Resistance genes sequencing From the specific gene PCR, arsC (495 bp) and partial merA (550 bp) genes were amplified successfully and this was confirmed by the agarose gel electrophoresis (Fig. S5). The amplified DNA fragments were sequenced and annotated in GenBank. The partial sequence of merA from isolate GTZY was closely similar (coverage: 96%, similarity: 83.37%) to the merA sequence of Lysobacter oculi strain 83 − 4 (Accession No.: CP029556), which encodes for mercury (II) reductase enzyme. arsC gene of isolate GTZY was closely similar (coverage: 100%, similarity: 99.88%) with the arsC encoding arsenate reductase enzyme of Stenotrophomonas acidaminiphila strain ZAC14D2_NAIMI4 (Accession No.: CP012900). 3.4. Mercury gene cluster and its interpretation From the genome analysis of strain GTZY, an entire set of mercury resistance gene cluster, named merAPTR (2829 bp) was identified (Fig. 4 A). The gene cluster was possessed 1695 bp of merA (mercury (II) reductase), 279 bp of merP (mercury (II) binding protein), 351 bp of metT (mercuric transport protein) and 408 bp of merR (mercuric (II) resistance transcription regulator) genes. This gene cluster was flanked by a linkage of Tn2 -family transposase, ISII (579 bp) and a site-specific integrase, int1 (1080 bp) on both sides. IS-II family transposase was located adjacent to the gene encoding lead, cadmium, zinc and mercury-transporting ATPase ( zntA ). The entire mer gene cluster consists of 34 restriction enzyme sites in its respective gene sites. Seven different bacterial mercury gene clusters ( Thermomonas sp., Stenotrophomonas acidaminiphila, Pseudomonas aeruginosa, Cupriavidus metallidurans, Achromobacter xylosoxidans, Delftia lacustris and Burkholderia cenocepacia ) along with the strain GTZY were chosen for the gene cluster interpretation, and their comparative visualization was given in Fig. 5 . The strain GTZY mer gene cluster’s downstream and upstream were highly conserved against three of seven bacterial species. Besides, it had the closest relationship (coverage: 100%, similarity: 99.89%) with the mercury gene cluster of Thermomonas sp. XSG (Accession No.: CP061497) and were followed by the relationship (coverage: 100%, similarity: 99.82%) with Stenotrophomonas acidaminiphila strain ZAC14D2 (Accession No.: AM743169) and Pseudomonas aeruginosa H47921 (Accession No.: CP008861) (coverage: 99%, similarity: 82.17%). From these interpretations, it was clearly evidenced that merA encoding mercury (II) reductase and merP encoding mercury (II) binding protein were invariably conserved in mercury gene clusters of all the bacterial species except Cupriavidus metallidurans CH34 (Accession No.: CP000352). Meanwhile, merR encoding mercury (II) transcription regulator was invariably conserved in all the bacterial species except Achromobacter xylosoxidans FDAARGO 984 (Accession No.: CP066291). 3.5. Arsenic gene cluster and its interpretation From the genome analysis of the strain GTZY, an entire set of arsenic resistance gene clusters, arsCPRH (2791 bp), was identified (Fig. 4 B). This gene cluster possessed 495 bp of arsC (arsenate reductase), 1026 bp of arsP (arsenate permease protein), 336 bp of arsR (arsenate repressor protein) and 819 bp arsH (arsenate resistance protein). The identified ars gene cluster comprised of 41 restriction enzyme sites in its respective gene sites. Three different bacterial mercury gene cluster ( Dokdonella koreensis, Luteimonas sp. and Lysobacter oculi ) and the strain GTZY were chosen for the gene cluster interpretation and visualization (Fig. 6 ). However, strain GTZY arsCPRH gene cluster’s downstream and upstream were more conserved against only to Dokdonella koreensis DS-123 (Accession No.: CP015249) (coverage: 94%, similarity: 79.14%) and least conserved with Lysobacter oculi 83 − 4 (Accession No.: CP029556) (coverage: 64%, similarity: 77.51%). The arsenic resistance genes arsP, arsR and arsH were conserved among all four bacterial genera. In the meantime, the arsC gene was observed only in the isolate GTZY and Dokdonella koreensi DS-123. The arsenic gene cluster’s arrangement in Luteimonas sp. ( arsPRH) and Lysobacter oculi (arsRPH ) were extremely different from the strains, GTZY and Dokdonella koreensis (arsCPRH). 4. Discussion Only a few gene clusters encoding toxic chemical degradationhave been reported so far in Pseudoxanthomonas species(Choi et al. 2013; Wang et al. 2013). Thereby, in this study, we looked into the bioremediation capability of two different heavy metals (Hg and As) and their related resistance gene operons in the environmental non-pathogenic bacterium, P. mexicana strain GTZY, for the first time. Initially, PCR amplification was confirmed the presence of metal resistance genes ( merA and arsC ). Interestingly, further genome annotation studies revealed the occurrence of corresponding resistance operons set ( merAPTR and arsCPRH) in the genome of strain GTZY. In particular, 2829 bp size mercury operon was located initially with mercury ion reducing reductase enzyme ( merA ), which has been proven to volatilize or reduce the toxic Hg (II) to non-toxic Hg (0), then it might extrude passively from the microbial cellular environment (Fig. 8)(Sotero-Martins et al. 2008; Agarwal et al. 2019). This cytoplasmic enzyme helps to minimize the environmental re-pollution of mercury (II) caused by the mercury efflux-containing bacteria, which usually pumped out mercury rather than mercury reduction (Boyd and Barkay, 2012). In merAPTR , periplasmic Hg (II) binding protein-encoding merP and Hg (II) transporting inner membrane protein-encoding merT were exactly located at 10 and 12 bp upstream of merA gene, respectively. It is followed by the cytoplasmic mercury regulatory protein-encoding merR , located before the transporter gene and 729 bp downstream of the merA gene. The gene cluster orientation and specifications of the strain GTZY’s mer operon were found to be in a close relationship (99.72 %) only with mer operons of other gram-negative bacteria, Thermomonas sp. (Accession No.: CP059266) and Stenotrophomonas sp. (Accession No.: AM743169). Despite the orientation ( IS2-merA-merP-mer-T-merR-int1 ) of mer operon of Pseudomonas sp. and Delftia sp. was similar to the mer operon of strain GTZY; they have only 82.17 % and 79.19 % of gene similarities, respectively. More than 80 % of bacterial communities were demonstrated for the presence of their mer operon embedded in their chromosomal DNA(Boyd and Barkay 2012) and the strain GTZY does the same, and no plasmids were detected in the strain GTZY. The ancestor species of Pseudoxanthomonas was not detected for any mer operon, excluding the process of strain GTZY does mercury bioremediation. Besides, another strain Pseudoxanthomonas mexicana GTZY2 (Accession No.: CP060731) isolated from a wastewater reactor (control) not exposed to any antibiotics was not detected for any heavy metals related operons in its genome. However, mer operon of the strain GTZY has distinctly flanked both ends by incorporating Tn2 family transposase ( IS-II ) and a site-specific integrase ( int1 ). Previously, members of Bacillus, Stenotrophomonas, Pseudomonas, Paenibacillus, Enterobacter and Exiguobacterium were reported for the mer operon linked with elements of transposons ( TnMERI1, Tn5083, Tn6294 and Tn5085 )(Ramírez-Díaz et al. 2011; Wang et al. 2013; Matsui et al. 2016; Agarwal et al. 2019) and integron gene ( int1 )(Ghaly et al. 2017). These two mobile genetic fragments were proven for the transmission of functionally specific genes among the various bacterial communities(Wang et al. 2013; Ghaly et al. 2017; Ben Fekih et al. 2018). Hence, we speculated that mer operon of strain GTZY is not to be inherited through vertical gene transfer from its ancestral species of Pseudoxanthomonas; instead, it might be received from other related bacterial communities by the horizontal gene transfer mechanisms such as conjugation, transposition or phage attack with mer operon. Besides, it seems that the strain GTZY can be a perfect fit for the heavy metal selected genome plasticity and interaction with other bacterial communities, which often have consequences with evolved bacterial species in the toxic heavy metals contaminated sites. Since the strain GTZY was isolated from the high-dose streptomycin treating reactor, it could have a high fitness cost that leads to the out-competence by wild type sensitive strains existing in the heavy metal contaminated sites. On the other side, the strain GTZY possessed an arsenic gene operon ( arsCPRH ) in its chromosome with the size of 2791 bp. The identified arsCPRH of strain GTZY was diverse from commonly known prokaryotic ars operons, arsRBC and arsRDABC identified in the Escherichia coli, Staphylococcus xylosus, Pseudomonas aeruginosa, Pseudomonas fluorescens, Acidiphilium multivorum and Staphylococcus aureus (Páez-Espino et al. 2015; Chen et al. 2016; Ben Fekih et al. 2018) rientation of strain GTZY and their gene components had only shown matching with a soil bacterium, Dokdonella koreensis DS-123 (Accession No.: CP015249). The multiple sequence alignment between these two ars operons revealed their maximum gene cluster coverage and basepairs similarities accounted for 94 % and 79.14 %. It is thereby likely that identified ars operon was distinct from other bacterial genera containing ars operons. Also, the identified ars operon from the strain GTZY has not flanked with any mobile genetic elements in both ends and was located intrinsically in a chromosome, which might be received from its ancestral strains by the process of vertical gene transfer. From the genome interpretation study, we confirm the existence of ars operon in its ancestral bacterial species, Pseudoxanthomonas suwonensis 11-1 (Accession No.: CP002446), isolated from cotton waste compost(Weon et al. 2006) and Pseudoxanthomonas spadix BD-a59 (Accession No.: CP003093), known for BETX biodegradation(Choi et al. 2013) (Fig. 7). This further reveals that the arsenic gene cluster orientation (arsC-arsP-arsR-arsH) of strain GTZY as extremely divergent (< 38% similarity) from their inherited BD-a59 (arsH-arsB-arsC-arsR) and 11-1 (arsR-arsH-arsB-arsC) strains. In particular, arsR and arsH of the strain GTZY were similar with the nucleotides of BD-a59 and 11-1 strains, while arsC was diverse in their ancestral strains, respectively. The genome of strain GTZY is comprised of the membrane permease transporter encoding arsP genewhile,BD-a59 and 11-1 strains are located with the most frequent acr3 family efflux transporterencoding arsB gene, which extrudes arsenate (III). Apart from arsCPRH , strain GTZY has also been distributed with additional arsB (963 bp), arsR (219 bp) and arsC (420 bp) genes in its chromosome. The presence of arsP and its co-existence with the arsH gene in the ars operon of strain GTZY makes them versatile among other known bacterial ars operons. The arsP encoding efflux related permease system is known for the detoxification or extrudes the toxic organic arsenicals (III) such as roxarsone and monomethylarsonous acid (MMA)(Shen et al. 2014; Chen et al. 2015b). The arsP gene encoding ArsP permease was initially identified in the ars operon of Campylobacter jejuni (Wang et al. 2009) , and later it was believed to be a prokaryotic cell membrane pump that could extrude arsenical roxarsone (III)(Shen et al. 2014). The recent genomic interpretation study suggests that the ancestral origin of arsP and their homologs, existence in the bacteria, archaea and a few other eukaryotes (Yang et al. 2015). The existence of a gene encoding flavoprotein, arsH, utilizing NADP+ to resist and oxidize the toxic methylarsenite (MAs (III)) into MAs (V) was reported in Campylobacter jejuni strain(Li et al. 2016). These arsH enzymes are widely distributed in gram-negative, mostly Gammaproteobacteri a including Pseudomonas putida, Sinorhizobium meliloti, Acidithiobacillus ferrooxidans, Rhodopseudomonas palustris, Ochrobactrum tritici, Thiobacillus ferrooxidans, Herminiimonas arsenicoxydans and Pseudomonas aeruginosa , but not in gram-positive bacteria (Yang et al. 2005; Chen et al. 2015a; Yang and Rosen 2016; Falgenhauer et al. 2017). The ArsP protein from both P. putida and S. meliloti was evaluated as an organoarsenical oxidase enzyme conferring resistance to trivalent methyl As derivatives. We assumed that arsCPRH of strain GTZY has highly evolved from its ancestral species, in response to the environmental mutations, genetic reshuffling and recombination with other bacterial communities. Initially, P. mexicana GTZY isolate was characterized for its phylogeny, morphology and biochemical properties, and was found specifically relevant to the properties of various reported members of Pseudoxanthomonas. Like other heavy metal transforming bacteria, strain GTZY did not utilize the mercury and arsenic as main carbon sources in the basal media (data not shown). Since these heavy metals are not catabolized by the heavy metal transforming bacteria, their bioremediations were usually evaluated by the removal, reduction, oxidation and volatilization assays(Wuana and Okieimen 2011; Jebeli et al. 2017; Wang et al. 2020). TZY was successfully fulfilled the above chemical strategies against the mercury and arsenic metals in the growth media. The strain GTZY can reduce inorganic Hg (II) and into foggy forming volatile Hg (0) in 4-h incubation. Previously, Mahbub et al. (2017) have proved the ability of Sphingopyxis sp. SE2 to volatilize or remove 44 % of mercury (II) in 6-h incubation. This study found 49.6 % of mercury removal achieved by the strain GTZY in 6 h. However, Sphingobium sp. SA2 was recorded for the maximum Hg (II) volatilization and reduction in 6 h account for 79 % (Mahbub et al. 2016, 2017). The strain GTZY’s mercury volatilization and reduction ability were further confirmed by the occurrence of the merA gene encoding for mercury (II) reductase. Also, the isolate was proven to resist (> 33.4 µM) and remove (84.3 %) the toxic form of mercury (II) in 48-h incubation. The Minimum inhibitory concentrations (MIC and MIC 50 ) of strain GTZY against Hg (II) was observed to be 33.4 µM and 40.8 µM;, respectively, whereas in other bacterial classes, Alphaproteobacteria , Betaproteobacteria, Flavobacteria, Firmcutes and Sphingobacteria, MICs were observed in the range of 5-30 µM; Hg (II)(Møller et al. 2011, 2014). After 60 h incubation, the concentration of Hg (II) was remained stable at 1.9 ± 0.2 µM along with strain GTZY. In the meantime, thermophilic species Aquificae was proven to be sustainable at 3.0 µM; Hg (II) in the basel media(Freedman et al. 2012). These results represent that the strain GTZY could be useful for preventing the formation of a more toxic form of methyl mercury (MeHg). In contaminated environments, high concentration and precipitation of Hg (II) always end up with more accumulation of toxic MeHg(Eckley et al. 2020). Hence, this is arguably a better solution to reduce the formation of toxic MeHg, in which a high concentration of Hg (II) should be cleaned up first so that the strain GTZY would be more suitable for the detoxification. On the other side, strain GTZY can remove (68.5 %) and resist (> 175 mM) As (V) in the culture media at 72-h incubation. The Minimum inhibitory concentrations of strain GTZY towards arsenate and arsenite were > 175 and > 55 mM. Previous studies revealed the arsenate and arsenite MICs of Gammaproteobacteria, Firmcutes and Kocuria, in which the ranges of 75 – 125 mM and 10 – 50 mM, respectively, whereas in the Bacillus fusiformis ORAs1 , B. thuringiensis ORAs2, , Pseudomonas sp. ORAs5, Aeromonas molluscorum ORAs6, MICs of arsenate and arsenite were > 16.68 mM and > 133.47 mM, respectively(Pepi et al. 2007). In addition, strain GTZY was reduced organic As (V) into As (III) form in the liquid solution, and it was further confirmed by the amplification of the arsC gene (495 bp) encoding arsenate reductase. Meanwhile, the strain GTZY was also able to remove (63.2 %) and resist (> 55 mM) As (III); however, it could not oxidize organic As (III) into As (V) form in the liquid solution. Similarly, Dey et al. (2016) isolated Bacillus sp. and Aneurinibacillus aneurinilyticus and found that both strains could remove As (V) (51.45–51.99 %) and As (III) (50.37–53.29 %). Besides, the strains also resisted the arsenate (4500 ppm) and arsenite (550 ppm). However, both strains have oxidized the arsenite, but none of them reduced arsenate into arsenite. Many previous reports suggest different detoxification mechanisms of arsenic resistance bacteria, including arsenic reduction and oxidation, heavy metal efflux pumping, metal adsorption to the cell surface, bacterial cell membrane binding and complex formation with exopolysaccharides (Zhang et al. 2016; Govarthanan et al. 2016a). Despite the absence of arsenite oxidase enzyme, we presume that the strain GTZY might be removed the arsenite (III) by following any of the above-proposed detoxification mechanisms. Conclusion From this study, it is therefore concluded that Pesudoxanthomoas mexicana strain GTZY has the potential for the bioremediation of highly toxic heavy metals such as mercury and arsenic. In short, this is the first investigation that opens up the resistance mechanism of mer (2.8 kb) and ars (2.7 kb) operons in the chromosome of P. mexicana . The existence of mer operon might be derived via horizontal gene transfer from other bacterial communities, because of the linkage of mobile genetic elements ( IS2 and int1 ) in its both terminuses. The occurrence of ars operon could be rendered vertically from its ancestral Pseudoxanthomonas species. This intrinsic ars operon was highly diversified and evolved from its ancestral species, which might be due to their mutations, genetic reshuffling and recombination with other bacterial communities. Initially, strain GTZY was isolated from an aerobic wastewater reactor and characterized for its biochemical, physiological and phylogeny, besides the isolated strain showed its potential resistance towards heavy metals (Hg and As) and their increased removal and reduction in short periods. The evaluations from this study was further confirmed by the existence of resistance genes ( merA and arsC ). It is thereby claimable that the strain GTZY could be an exceedingly valuable addition to the mercury and arsenic-contaminated sites for time- Declarations Author Contributions: Conceptualization, Methodology, Software using, Data analysis, Writing—original draft preparation - Abdul Raheem Nelofer; Project administration, Conceptualization - Selvaraj Ganesh-Kumar (corresponding author) and S. Balachandran. Software using - Soorangkattan Saravanan, Software using, Data analysis- Karuppanan Kalimuthu; Software using, Data analysis - Ganesan Govindarajan; Data analysis - Rajah Dilip Kumar; Shivani Ramamurthy Baluraj - Data analysis; Software using, Data analysis - Imran Hasan; All authors have read and agreed to the published version of the manuscript. Funding The authors express their gratitude to King Saud University, Researchers Supporting Project Number (RSPD2024R670), King Saud University, Riyadh, Saudi Arabia. We thank Prof. Yang Min and Prof. Yu Zhang (RCEES, CAS Institute, Beijing, China) for kindly providing us P. mexicana strain GTZY. The work was supported by the Ministry of Education and State Administration of Foreign Experts Affairs (Grant No.: G20190011010), People’s Republic of China. Declarations Ethics approval: Not applicable. Consent to participate: All persons who meet authorship criteria are listed as authors. Consent for publication: All authors certify that they have participated sufciently in the work to take public responsibility for the content. Furthermore, each author certifes that this material or similar material has not been and will not be submitted to or published in any other publication. Competing interests: The authors declare no competing interests. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4104815","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":292650099,"identity":"40861448-8fbf-47c6-a61a-e6513a984b31","order_by":0,"name":"Abdul Raheem Nelofer","email":"","orcid":"","institution":"St Peter's Institute of Higher Education and Research","correspondingAuthor":false,"prefix":"","firstName":"Abdul","middleName":"Raheem","lastName":"Nelofer","suffix":""},{"id":292650100,"identity":"35e72768-47f9-4105-bd76-6c9848731841","order_by":1,"name":"Ganesh-Kumar Selvaraj","email":"","orcid":"","institution":"St Peter's Institute of Higher Education and Research","correspondingAuthor":false,"prefix":"","firstName":"Ganesh-Kumar","middleName":"","lastName":"Selvaraj","suffix":""},{"id":292650101,"identity":"f60ed3da-c7ca-414e-9bfd-3e2c6136e86d","order_by":2,"name":"Kalimuthu Karuppanan","email":"","orcid":"","institution":"SRM Institute of Science and Technology (Deemed to be University) Research Kattankulathur","correspondingAuthor":false,"prefix":"","firstName":"Kalimuthu","middleName":"","lastName":"Karuppanan","suffix":""},{"id":292650102,"identity":"884e27f7-ce25-4766-b6fd-1b526a78929e","order_by":3,"name":"Govindarajan Ganesan","email":"","orcid":"","institution":"Sri Shanmuga college of Engineering and Technology","correspondingAuthor":false,"prefix":"","firstName":"Govindarajan","middleName":"","lastName":"Ganesan","suffix":""},{"id":292650103,"identity":"749afaaa-075a-40fc-83df-1ff1d2956901","order_by":4,"name":"Saravanan Soorangkattan","email":"","orcid":"","institution":"Madura College","correspondingAuthor":false,"prefix":"","firstName":"Saravanan","middleName":"","lastName":"Soorangkattan","suffix":""},{"id":292650104,"identity":"5d7075db-ca35-490c-b1b2-49e8caef3c86","order_by":5,"name":"Balachandran Subramanian","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIiWNgGAWjYBACAwbmBgbGBgYGNv7mA0C+hAwRWhghWvgkjiWAtPAQr0WOIccAJEBYizn7wcYPP3fYRLMxnPn86kaNBQ8D++GjG/BpsexJbJbsPZOW28bcu8065xjQYTxpaTfwOuxAYoM0Y9vh3DaGs9uMc9iAWiR4zPBrOf+w+Tdj23+glpxnxjn/iNFyI7ENaMsBkBbmx7ltRGixnPGwzbK3LRmo+JgZc26fBA8bIb+Y8ycfvvGzzS53fn/z48853+rk+NkPH8OrBRmwSYBJYpWDAPMHUlSPglEwCkbByAEAgjtLfTVRQTIAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-4173-2032","institution":"SIMATS Deemed University Saveetha Dental College","correspondingAuthor":true,"prefix":"","firstName":"Balachandran","middleName":"","lastName":"Subramanian","suffix":""},{"id":292650105,"identity":"780d144f-16f4-41f6-90c2-375cf579fee1","order_by":6,"name":"Shivani Ramamurthy Baluraj","email":"","orcid":"","institution":"SIMATS Deemed University Saveetha Dental College","correspondingAuthor":false,"prefix":"","firstName":"Shivani","middleName":"Ramamurthy","lastName":"Baluraj","suffix":""},{"id":292650106,"identity":"4f299046-cb72-4a8e-9774-f7b0e2791e85","order_by":7,"name":"Dhilip Kumar Rajaiah","email":"","orcid":"","institution":"University of Ulsan","correspondingAuthor":false,"prefix":"","firstName":"Dhilip","middleName":"Kumar","lastName":"Rajaiah","suffix":""},{"id":292650107,"identity":"7e2aae7f-21cf-415d-9e6f-108e13f73b21","order_by":8,"name":"Imran Hasan","email":"","orcid":"","institution":"King Saud University","correspondingAuthor":false,"prefix":"","firstName":"Imran","middleName":"","lastName":"Hasan","suffix":""}],"badges":[],"createdAt":"2024-03-15 04:51:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4104815/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4104815/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11356-024-34602-1","type":"published","date":"2024-08-08T15:57:01+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":55329887,"identity":"65ad7f55-3680-470d-a86d-d6b6913b8c75","added_by":"auto","created_at":"2024-04-25 19:04:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":98181,"visible":true,"origin":"","legend":"\u003cp\u003eA phylogenetic tree showing the clustering of twenty 16S rRNA gene sequences along with an isolated bacterial strain GTZY. The neighbour-joining constructed tree was evaluated by the bootstrap method. The scale bar 0.0002 represents the nucleotide substitution level, and numbers indicate bootstrap values.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4104815/v1/9563eca7117953fb2fa5f007.png"},{"id":55329891,"identity":"0b6646e3-f73e-46e9-b9da-50d6b2eb587d","added_by":"auto","created_at":"2024-04-25 19:04:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":130948,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of mercury on the growth of strain GTZY with different concentrations of mercury amended LB media. The microbial growth was estimated by the absorbance at 600 nm (A). Percent removal of mercury by the strain GTZY was determined at various periodic intervals (B).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4104815/v1/0463f8f14f410f3b4c364727.png"},{"id":55329889,"identity":"67f9061e-7ad2-4a51-8ada-8b4b9485ebfb","added_by":"auto","created_at":"2024-04-25 19:04:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":44278,"visible":true,"origin":"","legend":"\u003cp\u003ePercent removal of\u003cstrong\u003e \u003c/strong\u003earsenite (III) and arsenate (V) by the isolate GTZY was estimated using Silver Diethyldithiocarbamate (SDDC) method. The experimental strain (1 %) was inoculated in LB broth amended with 5 µM sodium arsenate and sodium arsenite solution (as sources for As (V) and As (III), respectively) and incubated at 30 ºC.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4104815/v1/40c2dc4d68eed6691b198754.png"},{"id":55330279,"identity":"e29ab3da-0ea7-4b4a-bcac-f8befde08ae0","added_by":"auto","created_at":"2024-04-25 19:12:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":99659,"visible":true,"origin":"","legend":"\u003cp\u003eThe genetic organization of heavy metal resistance operons from \u003cem\u003ePsudoxanthomonas mexicana\u003c/em\u003e GTZY. Arrows indicate the orientation of transcription and its open reading frame. Arrow length indicates the basepairs length of particular genes. The particular gene descriptions are given in detail in the text. (A) Mercury resistance operon and (B) Arsenic resistance operon.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4104815/v1/f83e807f9085045355f9acb4.png"},{"id":55329896,"identity":"1b2f0701-b06c-4559-8938-6de433507229","added_by":"auto","created_at":"2024-04-25 19:04:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":544881,"visible":true,"origin":"","legend":"\u003cp\u003eA phylogenetic distribution and genetic organization of mercury resistance operon of \u003cem\u003ePsudoxanthomonas mexicana\u003c/em\u003e GTZY. \u003cem\u003emer\u003c/em\u003e gene clusters of different bacterial strains were annotated by BLASTn and compared with the EasyFig tool. The \u003cem\u003emer\u003c/em\u003e operon of strain GTZY has the closest relationship (coverage: 100 %, similarity: 99.89 %) with the \u003cem\u003emer\u003c/em\u003e operon of \u003cem\u003eThermomonas \u003c/em\u003esp. XSG (Accession No.: CP061497). The degree of sequence homology (%) between the operons was shown by the grey shades, and shade intensity indicates the relationship level between the \u003cem\u003emer \u003c/em\u003eoperons. Arrows indicate the orientation of transcription and its open reading frames. The bar indicates 100 bp lengths of nucleotides.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4104815/v1/bcebe3693501efbd86d4707d.png"},{"id":55329894,"identity":"8f079884-185e-4e4e-b696-1cf004a76045","added_by":"auto","created_at":"2024-04-25 19:04:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":284138,"visible":true,"origin":"","legend":"\u003cp\u003eA graphical representation of BLASTn comparison of arsenic resistance operon of \u003cem\u003ePsudoxanthomonas mexicana\u003c/em\u003e GTZY along with various bacterial species. \u003cem\u003ears\u003c/em\u003e operon of \u003cem\u003eDokdonella koreensis\u003c/em\u003e DS-123 (Accession No.: CP015249) is the only bacterial strain shown the maximum relationship (coverage: 94 %, similarity: 79.14 %) with \u003cem\u003ears\u003c/em\u003eoperon of isolate GTZY and least relationship (coverage: 64 %, similarity: 77.51 %) with \u003cem\u003eLysobacter oculi \u003c/em\u003e83-4 (Accession No.: CP029556).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4104815/v1/6fa1591ee005fb363ec2cfbc.png"},{"id":55329893,"identity":"70543b33-3a3b-49b8-ad63-01aea216299c","added_by":"auto","created_at":"2024-04-25 19:04:05","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":227869,"visible":true,"origin":"","legend":"\u003cp\u003eThe genomic comparison of intrinsic ars operon of \u003cem\u003ePsudoxanthomonas mexicana\u003c/em\u003e GTZY along with its bacterial ancestral species (EasyFig visualization). An extremely diverse relationship (\u0026lt; 38 %) was detected against ancestral strains, \u003cem\u003ePseudoxanthomonas spadix\u003c/em\u003e BD-a59 (Accession No.: CP003093), reported for BETX biodegradation (Choi et al. 2013a) and \u003cem\u003ePseudoxanthomonas suwonensis\u003c/em\u003e 11-1 (Accession No.: CP002446) isolated from cotton waste compost.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4104815/v1/8ea192fb5110f8c4445d3d93.png"},{"id":55329892,"identity":"b0e84785-c1c2-41cc-a6bf-f6d62639c390","added_by":"auto","created_at":"2024-04-25 19:04:05","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":157180,"visible":true,"origin":"","legend":"\u003cp\u003eThe role and cellular mechanisms of \u003cem\u003emer\u003c/em\u003e and \u003cem\u003ears\u003c/em\u003e systems occurred in the \u003cem\u003ePseudoxanthomonas mexicana\u003c/em\u003e strain GTZY. P – Periplasmic permease facilitate the cellular uptake of heavy metals (Hg and As), R – Regulator initiates the transcription of corresponding genes, T – Inner membrane protein transport the Hg (II) to reducing enzyme, A – \u003cem\u003emerA\u003c/em\u003e encoding mercury reductase reducing toxic Hg (II) to non-toxic Hg (0), C – \u003cem\u003earsC\u003c/em\u003e encoding arsenic reductase reducing As (V) to As (III), H – \u003cem\u003earsH\u003c/em\u003e encoding arsenical (III) oxidase enzyme transferring toxic MAs (III) to less toxic MAs (V). Reduced or oxidized metals extrude via cell membrane efflux system. The numbers represent the steps of heavy metal detoxification. The reduced As (III) and Hg (0) are further extruded via cell membrane efflux system, \u003cem\u003earsB\u003c/em\u003e encoding Ars3-family transporter and Hg/Cd/Pb transporting protein, respectively.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-4104815/v1/026c89bae34e697eccee398b.png"},{"id":62298202,"identity":"c82d0148-bfbd-4075-95df-9788d3ea0b0f","added_by":"auto","created_at":"2024-08-12 16:10:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2116786,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4104815/v1/3984769f-2dde-43b3-933d-98b0d0c8131c.pdf"},{"id":55330280,"identity":"915d157f-3986-468f-99ba-3f657a249b19","added_by":"auto","created_at":"2024-04-25 19:12:10","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":176962,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformations.docx","url":"https://assets-eu.researchsquare.com/files/rs-4104815/v1/bb296f581f9569b6a4edb4fc.docx"},{"id":55329890,"identity":"24df3f51-e808-4d63-9a08-b0d87200d325","added_by":"auto","created_at":"2024-04-25 19:04:05","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":143011,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical Abstract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"GraphicalAbstract.png","url":"https://assets-eu.researchsquare.com/files/rs-4104815/v1/54e76f3fc674f62f0c2d7165.png"}],"financialInterests":"","formattedTitle":"Bioremediation of heavy metals by an unexplored bacterium, Pseudoxanthomonas mexicana strain GTZY, isolated from aerobic-biofilm wastewater system","fulltext":[{"header":"Highlights","content":"\u003cul\u003e\n \u003cli\u003eFirst detailed heavy metals bioremediation study in the genus, \u003cem\u003ePseudoxanthomonas\u003c/em\u003e\u003c/li\u003e\n \u003cli\u003eIsolate has great potential to resist, reduce and remove the toxic Hg(II) and As(V)\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eAn acquired mobile elements linked\u003cem\u003e\u0026nbsp;mer\u003c/em\u003e gene operon was observed in its chromosome.\u003c/li\u003e\n \u003cli\u003eAn intrinsic \u003cem\u003ears\u003c/em\u003e gene operon was extremely diverse from its ancestral species\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"1. Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eHeavy metals are inorganic ill-defined chemical compounds that ubiquitous in nature. In which, some heavy metals such as cadmium (Cd), arsenic (As), nickel (Ni), lead (Pb), chromium (Cr), mercury (Hg), copper (Cu) and zinc (Zn) are mainly persisting in the environments due to the core release of anthropical sources (Loganathan et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Govarthanan et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016b\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003ea\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Jobby et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Briffa et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Khalid et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This persistence is considered as an emerging situation due to their potential toxicity in the entire ecosystem, including humans, animals and plants (Olmedo et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Rebelo and Caldas \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Luo et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Qin et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Some heavy metals are considered highly toxic as they could cause human illness even at their relatively low levels. Among those, arsenic and mercury are the prime consideration for their chemical toxicity, persistant, frequency and potential to human exposure(Hou et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). According to the national priorities list released by the Agency for Toxic Substances and Disease Registry (ATSDR), among the 275 toxic substances, arsenic and mercury were ranked as first and third, respectively(Watters and Rayman \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, 2018). These heavy metals are non-biodegradable and can not catabolized by microorganisms. Alternatively, these heavy metals can be transformed from toxic to non-toxic forms by the nuclear structural changes via microbial activities(Wuana and Okieimen \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Jebeli et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn general, microbial mercury bioremediation mainly occurs by the mechanism of reduction or volatilization, which is governed by \u003cem\u003emer\u003c/em\u003e operon in microorganisms(Boyd and Barkay \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Raj and Maiti \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In \u003cem\u003emer\u003c/em\u003e operon, merA protein is one of the main constituents along with Hg binding (\u003cem\u003emerP\u003c/em\u003e), regulatory (\u003cem\u003emerR\u003c/em\u003e and \u003cem\u003eD\u003c/em\u003e), and mercury transporting proteins (\u003cem\u003emerT\u003c/em\u003e and \u003cem\u003eE\u003c/em\u003e), and are mainly located in the bacterial chromosome or mobile genetic elements (e.g. plasmids, integrons or transposons)(Oregaard and S\u0026oslash;rensen \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Boyd and Barkay \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; M\u0026oslash;ller et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Naguib et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Relying on the availability of toxic mercury (Hg\u003csup\u003e2\u003c/sup\u003e), merR protein binds specifically into the promoter-operator region and it is followed by regulating the transcription of structural genes. As a result, it produces a key enzyme in the detoxifying system known as mercury reductase (encoded by \u003cem\u003emerA\u003c/em\u003e gene), an NAD(P)H-dependent flavin oxidoreductase, reducing toxic mercury (Hg\u003csup\u003e2\u003c/sup\u003e) into a volatile natured less toxic mercury (Hg\u003csup\u003e0\u003c/sup\u003e)(Freedman et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Dash et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Likewise, microbial arsenic bioremediation mainly involves arsenic reduction (\u003cem\u003earsC\u003c/em\u003e), oxidation (\u003cem\u003eAoxAB, arsH\u003c/em\u003e), methylation and intra-cellular bioaccumulation(Pepi et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Rahman and Hassler \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Govarthanan et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016a\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Satyapal et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ben Fekih et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The \u003cem\u003ears\u003c/em\u003e operon is an energy-gaining detoxifying respiratory system that facilitates the reduction of pentavalent arsenate (V) into trivalent-arsenite (III)(Suhadolnik et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The interaction of transcriptional repressor protein (arsR) with promoter region of \u003cem\u003ears\u003c/em\u003e operon, followed by the binding of arsR protein with arsenate, dissociates the repressor protein from a specific DNA site permitting the \u003cem\u003ears\u003c/em\u003e operon\u0026rsquo;s transcription(P\u0026aacute;ez-Espino et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). This organoarsenic reduction process is governed by the \u003cem\u003earsC\u003c/em\u003e encoding arsenic reductase enzyme. The \u003cem\u003ears\u003c/em\u003e operons, such as \u003cem\u003earsRB, arsRBC, arsRDABC\u003c/em\u003e and \u003cem\u003earsHRBC\u003c/em\u003e are widely demonstrated in the gram-negative bacteria(Dey et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Ben Fekih et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Taran et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe existence and sustainability of these metal resistance operons among the various bacterial communities are usually maintained and transmitted with the help of mobile genetic elements via horizontal gene transfer(M\u0026oslash;ller et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Suhadolnik et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In some bacteria, this metal resistance sustainability is preserved intrinsically in the bacterial chromosome, the consequence of vertical gene transfer from its ancestral species. Many bacterial species reportedly have arsenic and mercury resistance operons in their chromosome or mobile genetics elements(M\u0026oslash;ller et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Ghaly et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Agarwal et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, so far, none among the species in the genus \u003cem\u003ePseudoxanthomonas\u003c/em\u003e have been demonstrated for any heavy metal resistance and their respective operons. The 25 members of this genus are gram-negative, rod-shaped, non-pathogenic that are long been known for their prevalence in diverse ecological sources, including soil, water, plants, animal and human tissues(Thierry et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Rani et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Kittiwongwattana and Thawai \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Selvaraj et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Although the genus \u003cem\u003ePseudoxanthomonas\u003c/em\u003e is well characterized for its biodegradation capability and surfactant production(Choi et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Talwar and Ninnekar \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Biswas et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Astuti et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Lu et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Purwasena et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), it is unremarked in many environmental processes, particularly in terms of its bioremediation capability. This study thereby deals (i) isolation and characterization of Hg and As resistant strain GTZY from an bioreactor treating antibiotic-containing wastewater, (ii) evaluation of Hg and As removal efficiency of strain GTZY in batch-culture experiments, (iii) disclosure of the \u003cem\u003emer\u003c/em\u003e and \u003cem\u003ears\u003c/em\u003e resistance operon from strain GTZY and (iv) phylogenetic distribution and relationship of identified \u003cem\u003emer\u003c/em\u003e and \u003cem\u003ears\u003c/em\u003e operon of strain GTZY with other bacterial communities.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1. Bacterial strain\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThe bacterial samples were collected from the aerobic-biofilm wastewater reactor treating aminoglycoside antibiotic, streptomycin (50 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (State key laboratory of RCEES, CAS Institute, Beijing, China). The ten-fold serial dilution method was used to isolate and enrich bacterial isolate from the collected sludge samples. Inoculated (100 \u0026micro;L aliquots) plates were incubated at 30 \u0026ordm;C for 12\u0026ndash;18 h. Tryptone soy agar (TSA) and Reasoner\u0026rsquo;s 2A agar (R2A) media were used for the purpose of bacterial isolation. Gram\u0026rsquo;s staining was routinely performed to check the microbial purity. Initially, there were 23 bacterial isolates harvested from the wastewater reactor. To obtain the potential heavy metal transforming bacteria, the purified isolates were further screened on the growth media amended with As (V); 1 \u0026micro;M and HgCl\u003csub\u003e2\u003c/sub\u003e; 10 \u0026micro;M. Among the 23 isolates, strain GTZY was choosen for further studies due to its fast growing ability and surviving capability in the presence of increased concentration of As (V); 100 \u0026micro;M and HgCl\u003csub\u003e2\u003c/sub\u003e; 50 \u0026micro;M. These screened isolates were stored as 40% glycerol stock for further studies.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2. General characterization of bacterial isolate\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThe morphological, biochemical and physiological characterization of isolated bacterial strain was performed based on the standard microbiological methods. The optimum temperature and pH were identified by culturing the strain on the nutrient agar media in the various temperatures (25\u0026minus;40 \u0026ordm;C) and pH (5\u0026minus;8). After 12\u0026minus;18 h incubation, bacterial colony morphology was determined using a single colony which grown on the nutrient agar, TSA, LB and R2A media. The hanging drop method was performed to check the motility of isolates under the light microscopy. Gram\u0026rsquo;s staining solutions were manually prepared and isolates were viewed under the light microscopy magnifications (100\u0026times;). A salt tolerance test was carried out by preparing Brain-heart infusion agar plates with the addition of various NaCl concentrations 0\u0026minus;7% (w/v). Activitiy of various enzymes (catalase, oxidase, urease and indole), hydrogen sulfide production, utilization of various carbon sources, nitrate reductability and hydrolysis of biochemicals (gelatin, starch and casein) tests were also carried out by the standard microbial methods.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3. 16S rRNA gene amplification and phylogenetic tree analysis\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThe genomic DNA isolation and \u003cem\u003e16rRNA\u003c/em\u003e gene PCR amplifications were carried out as described in the previous study(Selvaraj et al. \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). In brief, the genomic DNA was harvested from the bacterial samples using the TIANamp bacteria DNA kit (Tiangen, China), and the isolated DNA was quantified using Nanodrop 1000 spectrophotometer (Nanodrop, USA). The 16SrRNA fragments were amplified using the universal primers (27F-5\u0026apos;-AGAGTTTGATCCTGGCTCAG-3\u0026apos; and 1492R-5\u0026apos;-AAGGAGGTGATCCAGCCGCA-3\u0026apos;), sanger sequenced (Biobase, China) and interpreted by the Ribosomal Database Project (RDP) and GenBank-National Center for Biotechnology Information (NCBI) database. The MEGA6.06 and Clustal W softwares were used to constructing a phylogenetic tree for isolated strain against its closest 19 bacterial strains. The Neighbour-joining method and bootstrap analysis were executed for the bacterial phylogeny and statistical sequence analysis, respectively. In bootstrap analysis, 1000 replicates were applied for determining the confidence values of bacterial phylogenetic tree nodes. Finally, the partial \u003cem\u003e16S rRNA\u003c/em\u003e gene sequence was submitted in the GenBank.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4. Determination of mercury MIC\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eAfter bacterial isolation, the bacterial efficiency on mercury resistance was maintained in LB media by the supplement of mercury chloride solution (10\u0026minus;30 \u0026micro;M; HgCl\u003csub\u003e2\u003c/sub\u003e). The bacterial isolates were grown overnight on the LB media added with 10 \u0026micro;M of HgCl\u003csub\u003e2\u003c/sub\u003e. Subsequently, those cultures were resuspended with 2\u0026minus;5 mL of Tris-buffered saline (TBS) solution, and absorbance (OD\u003csub\u003e600 nm\u003c/sub\u003e) was adjusted to 0.100 OD. Further, 125 \u0026micro;L of appropriate bacterial dilutions were inoculated on the 96-well plate containing 125 \u0026micro;L of LB broth (in triplicate) amended with various concentrations (0, 1, 5, 10, 20, 30, 40, 50, 60, 70 and 80 \u0026micro;M) of HgCl\u003csub\u003e2\u003c/sub\u003e and incubated at 30 \u0026ordm;C for 72 h in the dark. After the incubation, bacterial growth was observed by measuring the absorbance (OD\u003csub\u003e600 nm\u003c/sub\u003e) using a microplate reader. The inhibition effect of mercury on the microbial growth, values of MIC and MIC\u003csub\u003e50\u003c/sub\u003e were calculated.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5. Mercury removal assay\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eOvernight grown bacterial cultures (in triplicate) added with 10 \u0026micro;M of HgCl\u003csub\u003e2\u003c/sub\u003e were subjected for the mercury removal assay. Every 12 h of incubation, 4 mL of culture was centrifuged for 10 min at 7500 rpm,thereby recovered bacterial pellets were resuspended in 2.8 mL of Tris-buffered saline solution. In the resuspended solution, 0.4 mL of 5-diphenyl-3-thiocarbazone (Dithizone) reagent solution (0.50 mg of dithizone mixed with 20 mL of acetone) was added, followed by the addition of 0.4 mL of sulphuric acid (2 N) to maintain the acid pH (pH 2\u0026minus;3) and finally added by 0.4 mL of Dioxane (Naguib et al. \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). The acidic pH of the solution enhanced the formation of orange-red colour by coupling of dithizone and mercury (II). These final solutions were diluted to 5 mL using distilled water and checked mercury removal values at 488 nm in a UV/Visible spectrophotometer. Meanwhile, a similar experimental set up was carried out using dead bacterial cells (autoclaved cells) for treating as control. The standard curve was also prepared for HgCl\u003csub\u003e2\u003c/sub\u003e solution without bacterial suspension. The detection limit for this method was estimated as \u0026lt;\u0026thinsp;1 ppb (Nordlander). The actual Hg concentrations during the incubation period was measured and thereby the mercury removal percentage was calculated as follows,\u003c/p\u003e\n \u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6. Mercury volatilization assay\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eA simplified X-ray film method was used to confirm the mercury volatilization from Hg (II) state to Hg (0) state by the strain GTZY. Overnight grown culture added with 10 \u0026micro;M; HgCl\u003csub\u003e2\u003c/sub\u003e was centrifuged at 6000 rpm for 5 min. Harvested bacterial pellets were resuspended into a 200 \u0026micro;L assay solution containing 0.07 M phosphate buffer (pH 7), 0.2 mM magnesium acetate, 5 mM sodium thioglycolate and 50 \u0026micro;M HgCl\u003csub\u003e2\u003c/sub\u003e. The microtiter plate containing the above-mentioned solution were fully covered using an X-ray film (Kodak scientific imaging, Hyderabad, India)(M\u0026oslash;ller et al. \u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e) and incubated at 35 \u0026ordm;C in the dark. A similar solution mixture that without bacterial cells and bacterial suspension without HgCl\u003csub\u003e2\u003c/sub\u003e were utilized as control samples.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e2.7. Determination of arsenic MIC\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThe bacterial arsenic resistance was evaluated by adding the different concentrations of sodium arsenate (10\u0026minus;400 mM) and sodium arsenite (10\u0026minus;200 mM) into the overnight grown bacterial culture, while the control samples have no metals. The cultures were resuspended with 2\u0026minus;5 mL of TBS solution with an absorbance (OD\u003csub\u003e600 nm\u003c/sub\u003e) of 0.100. Further, 125 \u0026micro;L of appropriate bacterial dilutions and 125 \u0026micro;L of LB broth (in triplicate) were added into the 96-well plate containing suspended concentrations of arsenic and incubated at 30 \u0026ordm;C for 48 h in the dark. After the incubation, tested bacterial samples were estimated for their optical density at 600 nm using a microplate reader. The inhibition effect of arsenic on the microbial growth, values of MIC and MIC\u003csub\u003e50\u003c/sub\u003e were calculated. .\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e2.8. Arsenic removal assay\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eAfter the bacterial isolation, the efficiency of arsenic resistance was maintained by adding sodium arsenate (100 mM) and sodium arsenite (100 mM) into the bacteria growth media (NA). Arsenic utilization by bacterial isolate was determined by the Silver Diethyldithiocarbamate (SDDC) method(Tahir et al. \u003cspan class=\"CitationRef\"\u003e2012\u003c/span\u003e). The detection limit for this method was evaluated as 1\u0026ndash;5 ppb. Initially, the isolate (1%) was inoculated (in triplicate) in LB broth added with 5 \u0026micro;M sodium arsenate and sodium arsenite solution (as sources for As (V) and As (III), respectively) and incubated at 30 \u0026ordm;C for various time intervals (viz. 8, 12, 18, 24, 48 and 72 h). After the definite time points, the actual arsenic concentrations during the incubation period was estimated using the SDDC method. Briefly, the production of arsine carrying hydrogen passed through a reduction column impregnated with lead acetate and then into the absorbing solution SDDC dissolved pyridine. The end red-coloured product (arsine with SDDC solution) was measured spectrophotometrically at 535 nm.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e2.9. Arsenic reduction and oxidation assay\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThe ability of the bacterial isolate to oxidize Arsenic (As III) and reduce Arsenic (As V) was checked by the silver nitrate solution(Dey et al. \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). After 24 and 48 h of incubation, 100 \u0026micro;L of silver nitrate solution (0.1 M) was added into the grown bacterial culture (100 \u0026micro;L) amended with 1 \u0026micro;M sodium arsenate and sodium arsenite. If the culture broth turns brown, it represents the presence of silver arsenate, while if the culture broth turns yellow, represents the presence of silver arsenite.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e2.10. merA and arsC genes sequencing\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThe extracted and purified genomic DNA was used as a template DNA to screen mercury (\u003cem\u003emerA\u003c/em\u003e) and arsenical (\u003cem\u003earsC\u003c/em\u003e) resistance genes. The designed \u003cem\u003emerA\u003c/em\u003e (\u003cem\u003emerA-\u003c/em\u003eFP- 5\u0026prime;-CTACCCCGCGCAACAGGACA-3\u0026prime; and \u003cem\u003emerA-\u003c/em\u003eRP- 5\u0026prime;-TGATGGCGCCTTGCGCATTG-3\u0026prime;) and \u003cem\u003earsC\u003c/em\u003e (\u003cem\u003earsC-\u003c/em\u003eFP-5\u0026prime;-TCAGGACGAGGCGCCGATCT-3\u0026prime; and \u003cem\u003earsC-\u003c/em\u003eRP-3\u0026prime;- ATGGACCGCCCCTACAACCT-5\u0026prime;) primers were used to amplify the partial regions \u003cem\u003emerA\u003c/em\u003e and \u003cem\u003earsC\u003c/em\u003e. The purified genomic DNA (50 ng) was used as a template for preparing the PCR reaction. The triplicate PCR mixture (50 \u0026micro;L) comprised of 1\u0026times; PCR buffer (with MgCl\u003csup\u003e2+\u003c/sup\u003e), 2.5 mM of the dNTPs mix, 0.5 \u0026micro;M of each primer, and 1 U of Taq DNA polymerase (Takara, Bio Inc., Shiga, Japan). The PCR conditions were performed as follows: initial denaturation stage at 95\u0026deg;C for 5 min, followed by 30 cycles of denaturation at 95\u0026deg;C for 30 s, annealing at 56\u0026deg;C for 30 s, extension at 72\u0026deg;C for 2 min and a final extension at 72\u0026deg;C for 10 min. The amplified specific PCR products were resolved using 1.2% agarose (Biowest, Hong Kong) gel and sequenced (Sanger sequencing). The gene sequences were analyzed using BLASTN.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e2.11. Annotations of mer and ars gene operons\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThe extracted total genome of isolated strain GTZY was sequenced using the third-generation single-molecule real-time (SMRT) technology (Pacific Biosciences Inc., USA), and the sequenced genome was submitted to the GenBank. The whole-genome sequence of \u003cem\u003ePseudoxanthomonas mexicana\u003c/em\u003e strain GTZY (Accession No.: CP060028) was used for the resistance gene cluster\u0026rsquo;s interpretation. The \u003cem\u003emer\u003c/em\u003e and \u003cem\u003ears\u003c/em\u003e resistance gene clusters related data were initially annotated using the online ORF prediction server, Rapid Annotations using Subsystems Technology (RAST), and their manual annotations were performed using BLAST. A Python application software, Easyfig (ver. 2.1) was used for the linear comparison and visualization of \u003cem\u003emer\u003c/em\u003e and \u003cem\u003ears\u003c/em\u003e resistance gene clusters. In addition, the BLAST comparison between the gene annotated files was manually uploaded, output files (blastn) generated, interactively coloured and enabled a rapid transition using the software, Easyfig. The SnapGene Viewer (ver. 4.1.3) was used for the gene annotation and visualization.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Characterization of bacterial isolate\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe isolated bacterial strain was aerobic, gram-negative, rod-shaped and highly motile. The colonies were pale yellow, circular, convex and mucoid-natured on the solid nutrient media (Fig. S1). The isolate can grow in the media up to 40 \u0026ordm;C and pH 8, while its optimum temperature and pH were 34\u0026minus;37 \u0026ordm;C and 7\u0026minus;7.5, respectively. Isolate can tolerate the presence of sodium chloride up to 8% (w/v); however, its optimum concentration was 3.5\u0026ndash;5.5%. Their biochemical and phenotypic characterizations were given in Table S1. Biochemically, the isolate turned positive for catalase while remained negative for oxidase and urease, which in turn ferments glucose and mannitol but not sucrose and lactose. The hydrolysis test turned positive for the gelatin and casein, but remained negative for starch. Moreover, it showed negative results for indole production, nitrate reductase, Voges-Proskauer (VP) test, and positive for the Tween-20 and Methyl Red (MR) test. The length of the 16S rDNA gene sequence was 1424 bp, and it shared the closest relationship (99.39%) with \u003cem\u003eP. mexicana\u003c/em\u003e CP4 (Accession No.: MT549102) in the phylogenetic tree by the neighbour-joining method (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Hence, the isolated bacterium was named as \u003cem\u003ePseudoxanthomonas mexicana\u003c/em\u003e strain GTZY and submitted to the GenBank (Accession No.: MZ242608).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2. MIC, volatilization and bioremediation of mercury\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe effect of mercury on microbial growth was studied. Further, MIC and MIC\u003csub\u003e50\u003c/sub\u003e were determined by supplementing different mercury concentrations into LB broth inoculated with strain GTZY. After 48 h of incubation, mean values of OD\u003csub\u003e600nm\u003c/sub\u003e were plotted against 0\u0026minus;80 \u0026micro;M of HgCl\u003csub\u003e2\u003c/sub\u003e. The MIC value of mercury against strain GTZY was 33.4 \u0026micro;M of HgCl\u003csub\u003e2,\u003c/sub\u003e and its MIC\u003csub\u003e50\u003c/sub\u003e value was at 40.8 \u0026micro;M of HgCl\u003csub\u003e2\u003c/sub\u003e. Microbial growth was notably inhibited by 35.5 \u0026micro;M of HgCl\u003csub\u003e2,\u003c/sub\u003e and it was completely inhibited (0.21 OD\u003csub\u003e600nm\u003c/sub\u003e) at 49.7 \u0026micro;M of HgCl\u003csub\u003e2\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). After 60 h of incubation, the residual concentration of Hg was 1.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 \u0026micro;M and it was sustained for the remaining time periods (Fig. S3A). The mercury reducing ability of the strain GTZY was confirmed by a mercury volatilization assay conducted at 48 h of incubation. The strain formed a foggy area (dark circle) due to the reaction between Hg (0) and Ag emulsion on the X-ray film (Fig. S2). However, the isolate started to produce foggy formation from 4 h onwards. Hence, the strain GTZY reduced inorganic Hg (II) and formed volatile Hg (0), which reacted with the film. There was no interference of foggy circles observed in the control samples.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMercury removal by the strain GTZY was determined using the spectrophotometric dithizone method. Initial and residual Hg concentrations were evaluated against the standard calibration curve of various concentrations of Hg. The strain GTZY removed 16.3% of Hg from 4 h onwards, and their removal process was stopped at 48 h of incubation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The strain GTZY removed a maximum of 84.3% mercury at 48 h and their removal rate was maintained until 96 h of incubation. At the same time, no mercury removal was found in the control samples.\u003c/p\u003e \u003cp\u003e \u003cem\u003eMIC, reduction and bioremediation of arsenic metals\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe isolate GTZY was resistance to the high concentrations of arsenate and arsenite solutions. Also, it removed the arsenic compound at various time periods. Isolate GTZY was efficiently removed arsenate (68.5%) and arsenite (63.2%) at 72 h of incubation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003e). At 12 h of incubation, the removal rate (%) of arsenate and arsenite were accounted for 34.2% and 28.3%. Meanwhile, no notable removal of arsenical compounds was observed in the control samples. The MIC values of isolate towards arsenate and arsenite were \u0026gt;\u0026thinsp;175 and \u0026gt;\u0026thinsp;55 mM, and their corresponding MIC\u003csub\u003e50\u003c/sub\u003e were 240 and 60.5 mM, respectively. The bacterial growth was completely arrested with 330 mM; arsenate and 69.4 mM; arsenite (Fig. S4). After 60 h of incubation, the residual concentration of As (III) and As (V) were 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2 \u0026micro;M and 27.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5 \u0026micro;M (Fig. S3B), thereafter it was sustained for the remaining time periods. After 24 and 48 h of incubation, 100 \u0026micro;L of silver nitrate solution was added to both bacterial cultures (As III and V), and the suspensions were turned into the light yellow colour, which indicated the presence of As (III). Meanwhile, there was no brownish colour observed which indicating the absence of silver arsenate. The result confirmed that isolate GTZY could reduce As (V) to As (III), while the isolate could not oxidize As (III) to the As (V).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Resistance genes sequencing\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFrom the specific gene PCR, \u003cem\u003earsC\u003c/em\u003e (495 bp) and partial \u003cem\u003emerA\u003c/em\u003e (550 bp) genes were amplified successfully and this was confirmed by the agarose gel electrophoresis (Fig. S5). The amplified DNA fragments were sequenced and annotated in GenBank. The partial sequence of \u003cem\u003emerA\u003c/em\u003e from isolate GTZY was closely similar (coverage: 96%, similarity: 83.37%) to the \u003cem\u003emerA\u003c/em\u003e sequence of \u003cem\u003eLysobacter oculi\u003c/em\u003e strain 83\u0026thinsp;\u0026minus;\u0026thinsp;4 (Accession No.: CP029556), which encodes for mercury (II) reductase enzyme. \u003cem\u003earsC\u003c/em\u003e gene of isolate GTZY was closely similar (coverage: 100%, similarity: 99.88%) with the \u003cem\u003earsC\u003c/em\u003e encoding arsenate reductase enzyme of \u003cem\u003eStenotrophomonas acidaminiphila\u003c/em\u003e strain ZAC14D2_NAIMI4 (Accession No.: CP012900).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Mercury gene cluster and its interpretation\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFrom the genome analysis of strain GTZY, an entire set of mercury resistance gene cluster, named \u003cem\u003emerAPTR\u003c/em\u003e (2829 bp) was identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The gene cluster was possessed 1695 bp of \u003cem\u003emerA\u003c/em\u003e (mercury (II) reductase), 279 bp of \u003cem\u003emerP\u003c/em\u003e (mercury (II) binding protein), 351 bp of \u003cem\u003emetT\u003c/em\u003e (mercuric transport protein) and 408 bp of \u003cem\u003emerR\u003c/em\u003e (mercuric (II) resistance transcription regulator) genes. This gene cluster was flanked by a linkage of \u003cem\u003eTn2\u003c/em\u003e-family transposase, \u003cem\u003eISII\u003c/em\u003e (579 bp) and a site-specific integrase, \u003cem\u003eint1\u003c/em\u003e (1080 bp) on both sides. IS-II family transposase was located adjacent to the gene encoding lead, cadmium, zinc and mercury-transporting ATPase (\u003cem\u003ezntA\u003c/em\u003e). The entire \u003cem\u003emer\u003c/em\u003e gene cluster consists of 34 restriction enzyme sites in its respective gene sites.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSeven different bacterial mercury gene clusters (\u003cem\u003eThermomonas\u003c/em\u003e sp., \u003cem\u003eStenotrophomonas acidaminiphila, Pseudomonas aeruginosa, Cupriavidus metallidurans, Achromobacter xylosoxidans, Delftia lacustris\u003c/em\u003e and \u003cem\u003eBurkholderia cenocepacia\u003c/em\u003e) along with the strain GTZY were chosen for the gene cluster interpretation, and their comparative visualization was given in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003e. The strain GTZY \u003cem\u003emer\u003c/em\u003e gene cluster\u0026rsquo;s downstream and upstream were highly conserved against three of seven bacterial species. Besides, it had the closest relationship (coverage: 100%, similarity: 99.89%) with the mercury gene cluster of \u003cem\u003eThermomonas\u003c/em\u003e sp. XSG (Accession No.: CP061497) and were followed by the relationship (coverage: 100%, similarity: 99.82%) with \u003cem\u003eStenotrophomonas acidaminiphila\u003c/em\u003e strain ZAC14D2 (Accession No.: AM743169) and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e H47921 (Accession No.: CP008861) (coverage: 99%, similarity: 82.17%). From these interpretations, it was clearly evidenced that \u003cem\u003emerA\u003c/em\u003e encoding mercury (II) reductase and \u003cem\u003emerP\u003c/em\u003e encoding mercury (II) binding protein were invariably conserved in mercury gene clusters of all the bacterial species except \u003cem\u003eCupriavidus metallidurans\u003c/em\u003e CH34 (Accession No.: CP000352). Meanwhile, \u003cem\u003emerR\u003c/em\u003e encoding mercury (II) transcription regulator was invariably conserved in all the bacterial species except \u003cem\u003eAchromobacter xylosoxidans\u003c/em\u003e FDAARGO 984 (Accession No.: CP066291).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Arsenic gene cluster and its interpretation\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFrom the genome analysis of the strain GTZY, an entire set of arsenic resistance gene clusters, \u003cem\u003earsCPRH\u003c/em\u003e (2791 bp), was identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). This gene cluster possessed 495 bp of \u003cem\u003earsC\u003c/em\u003e (arsenate reductase), 1026 bp of \u003cem\u003earsP\u003c/em\u003e (arsenate permease protein), 336 bp of \u003cem\u003earsR\u003c/em\u003e (arsenate repressor protein) and 819 bp \u003cem\u003earsH\u003c/em\u003e (arsenate resistance protein). The identified \u003cem\u003ears\u003c/em\u003e gene cluster comprised of 41 restriction enzyme sites in its respective gene sites. Three different bacterial mercury gene cluster (\u003cem\u003eDokdonella koreensis, Luteimonas\u003c/em\u003e sp. and \u003cem\u003eLysobacter oculi\u003c/em\u003e) and the strain GTZY were chosen for the gene cluster interpretation and visualization (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003e). However, strain GTZY \u003cem\u003earsCPRH\u003c/em\u003e gene cluster\u0026rsquo;s downstream and upstream were more conserved against only to \u003cem\u003eDokdonella koreensis\u003c/em\u003e DS-123 (Accession No.: CP015249) (coverage: 94%, similarity: 79.14%) and least conserved with \u003cem\u003eLysobacter oculi\u003c/em\u003e 83\u0026thinsp;\u0026minus;\u0026thinsp;4 (Accession No.: CP029556) (coverage: 64%, similarity: 77.51%). The arsenic resistance genes \u003cem\u003earsP, arsR\u003c/em\u003e and \u003cem\u003earsH\u003c/em\u003e were conserved among all four bacterial genera. In the meantime, the \u003cem\u003earsC\u003c/em\u003e gene was observed only in the isolate GTZY and \u003cem\u003eDokdonella koreensi\u003c/em\u003e DS-123. The arsenic gene cluster\u0026rsquo;s arrangement in \u003cem\u003eLuteimonas\u003c/em\u003e sp. (\u003cem\u003earsPRH)\u003c/em\u003e and \u003cem\u003eLysobacter oculi (arsRPH\u003c/em\u003e) were extremely different from the strains, GTZY and \u003cem\u003eDokdonella koreensis (arsCPRH).\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eOnly a few gene clusters encoding toxic chemical degradationhave been reported so far in \u003cem\u003ePseudoxanthomonas\u003c/em\u003e species(Choi et al. 2013; Wang et al. 2013). Thereby, in this study, we looked into the bioremediation capability of two different heavy metals (Hg and As) and their related resistance gene operons in the environmental non-pathogenic bacterium, \u003cem\u003eP. mexicana\u003c/em\u003e strain GTZY, for the first time. Initially, PCR amplification was confirmed the presence of metal resistance genes (\u003cem\u003emerA\u003c/em\u003e and \u003cem\u003earsC\u003c/em\u003e). Interestingly, further genome annotation studies revealed the occurrence of corresponding resistance operons set (\u003cem\u003emerAPTR\u0026nbsp;\u003c/em\u003eand \u003cem\u003earsCPRH)\u003c/em\u003e in the genome of strain GTZY. In particular, 2829 bp size mercury operon was located initially with mercury ion reducing reductase enzyme (\u003cem\u003emerA\u003c/em\u003e), which has been proven to volatilize or reduce the toxic Hg (II) to non-toxic Hg (0), then it might extrude passively from the microbial cellular environment (Fig. 8)(Sotero-Martins et al. 2008; Agarwal et al. 2019). This cytoplasmic enzyme helps to minimize the environmental re-pollution of mercury (II) caused by the mercury efflux-containing bacteria, which usually pumped out mercury rather than mercury reduction\u0026nbsp;(Boyd and Barkay, 2012). In \u003cem\u003emerAPTR\u003c/em\u003e, periplasmic Hg (II) binding protein-encoding \u003cem\u003emerP\u003c/em\u003e and Hg (II) transporting inner membrane protein-encoding \u003cem\u003emerT\u003c/em\u003e were exactly located at 10 and 12 bp upstream of \u003cem\u003emerA\u003c/em\u003e gene, respectively. It is followed by the cytoplasmic mercury regulatory protein-encoding \u003cem\u003emerR\u003c/em\u003e, located before the transporter gene and 729 bp downstream of the \u003cem\u003emerA\u003c/em\u003e gene. The gene cluster orientation and specifications of the strain GTZY’s\u003cem\u003e\u0026nbsp;mer\u0026nbsp;\u003c/em\u003eoperon were found to be in a close relationship (99.72 %) only with \u003cem\u003emer\u003c/em\u003e operons of other gram-negative bacteria, \u003cem\u003eThermomonas\u0026nbsp;\u003c/em\u003esp. (Accession No.: CP059266) and \u003cem\u003eStenotrophomonas\u003c/em\u003e sp. (Accession No.: AM743169). Despite the orientation (\u003cem\u003eIS2-merA-merP-mer-T-merR-int1\u003c/em\u003e) of \u003cem\u003emer\u0026nbsp;\u003c/em\u003eoperon of \u003cem\u003ePseudomonas\u0026nbsp;\u003c/em\u003esp. and \u003cem\u003eDelftia\u003c/em\u003e sp. was similar to the \u003cem\u003emer\u0026nbsp;\u003c/em\u003eoperon of strain GTZY; they have only 82.17 % and 79.19 % of gene similarities, respectively. More than 80 % of bacterial communities were demonstrated for the presence of their \u003cem\u003emer\u003c/em\u003e operon embedded in their chromosomal DNA(Boyd and Barkay 2012)\u0026nbsp;and the strain GTZY does the same, and no plasmids were detected in the strain GTZY. The ancestor species of \u003cem\u003ePseudoxanthomonas\u0026nbsp;\u003c/em\u003ewas not detected for any \u003cem\u003emer\u003c/em\u003e operon, excluding the process of strain GTZY does mercury bioremediation. Besides, another strain \u003cem\u003ePseudoxanthomonas\u003c/em\u003e \u003cem\u003emexicana\u003c/em\u003e GTZY2 (Accession No.: CP060731) isolated from a wastewater reactor (control) not exposed to any antibiotics was not detected for any heavy metals related operons in its genome. However, \u0026nbsp;\u003cem\u003emer\u003c/em\u003e operon of the strain GTZY has distinctly flanked both ends by incorporating \u003cem\u003eTn2\u0026nbsp;\u003c/em\u003efamily transposase (\u003cem\u003eIS-II\u003c/em\u003e) and a site-specific integrase (\u003cem\u003eint1\u003c/em\u003e). Previously, members of \u003cem\u003eBacillus, Stenotrophomonas, Pseudomonas, Paenibacillus, Enterobacter\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Exiguobacterium\u003c/em\u003e were reported for the \u003cem\u003emer\u0026nbsp;\u003c/em\u003eoperon linked with elements of transposons (\u003cem\u003eTnMERI1, Tn5083, Tn6294\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Tn5085\u003c/em\u003e)(Ramírez-Díaz et al. 2011; Wang et al. 2013; Matsui et al. 2016; Agarwal et al. 2019)\u0026nbsp;and integron gene (\u003cem\u003eint1\u003c/em\u003e)(Ghaly et al. 2017). These two mobile genetic fragments were proven for the transmission of functionally specific genes among the various bacterial communities(Wang et al. 2013; Ghaly et al. 2017; Ben Fekih et al. 2018). Hence, we speculated that \u003cem\u003emer\u003c/em\u003e operon of strain GTZY is not to be inherited through vertical gene transfer from its ancestral species of \u003cem\u003ePseudoxanthomonas;\u0026nbsp;\u003c/em\u003einstead, it might be received from other related bacterial communities by the horizontal gene transfer mechanisms such as conjugation, transposition or phage attack with \u003cem\u003emer\u003c/em\u003e operon. Besides, it seems that the strain GTZY can be a perfect fit for the heavy metal selected genome plasticity and interaction with other bacterial communities, which often have consequences with evolved bacterial species in the toxic heavy metals contaminated sites. Since the strain GTZY was isolated from the high-dose streptomycin treating reactor, it could have a high fitness cost that leads to the out-competence by wild type sensitive strains existing in the heavy metal contaminated sites. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;On the other side, the strain GTZY possessed an arsenic gene operon (\u003cem\u003earsCPRH\u003c/em\u003e) in its chromosome with the size of 2791 bp. The identified \u003cem\u003earsCPRH\u0026nbsp;\u003c/em\u003eof strain GTZY was diverse from commonly known prokaryotic \u003cem\u003ears\u003c/em\u003e operons, \u003cem\u003earsRBC\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;arsRDABC\u003c/em\u003e identified in the \u003cem\u003eEscherichia coli, Staphylococcus xylosus, Pseudomonas aeruginosa, Pseudomonas fluorescens, Acidiphilium multivorum\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Staphylococcus aureus\u003c/em\u003e(Páez-Espino et al. 2015; Chen et al. 2016; Ben Fekih et al. 2018)\u0026nbsp;rientation of strain GTZY and their gene components had only shown matching with a soil bacterium, \u003cem\u003eDokdonella koreensis\u003c/em\u003e DS-123 (Accession No.: CP015249). The multiple sequence alignment between these two \u003cem\u003ears\u003c/em\u003e operons revealed their maximum gene cluster coverage and basepairs similarities accounted for 94 % and 79.14 %. It is thereby likely that identified \u003cem\u003ears\u003c/em\u003e operon was distinct from other bacterial genera containing \u003cem\u003ears\u003c/em\u003e operons. Also, the identified \u003cem\u003ears\u003c/em\u003e operon from the strain GTZY has not flanked with any mobile genetic elements in both ends and was located intrinsically in a chromosome, which might be received from its ancestral strains by the process of vertical gene transfer. From the genome interpretation study, we confirm the existence of \u003cem\u003ears\u003c/em\u003e operon in its ancestral bacterial species, \u003cem\u003ePseudoxanthomonas suwonensis\u003c/em\u003e 11-1 (Accession No.: CP002446), isolated from cotton waste compost(Weon et al. 2006)\u0026nbsp;and \u003cem\u003ePseudoxanthomonas spadix\u003c/em\u003e BD-a59 (Accession No.: CP003093), known for BETX biodegradation(Choi et al. 2013)\u0026nbsp;(Fig. 7). This further reveals that the arsenic gene cluster orientation \u003cem\u003e(arsC-arsP-arsR-arsH)\u003c/em\u003e of strain GTZY as extremely divergent (\u0026lt; 38% similarity) from their inherited BD-a59 \u003cem\u003e(arsH-arsB-arsC-arsR)\u003c/em\u003e and 11-1 \u003cem\u003e(arsR-arsH-arsB-arsC)\u003c/em\u003e strains. In particular, \u003cem\u003earsR\u003c/em\u003e and \u003cem\u003earsH\u003c/em\u003e of the strain GTZY were similar with the nucleotides of BD-a59 and 11-1 strains, while \u003cem\u003earsC\u003c/em\u003e was diverse in their ancestral strains, respectively. The genome of strain GTZY is comprised of the membrane permease transporter encoding\u003cem\u003e\u0026nbsp;arsP\u003c/em\u003e genewhile,BD-a59 and 11-1 strains are located with the most frequent \u003cem\u003eacr3\u003c/em\u003e family efflux transporterencoding\u003cem\u003e\u0026nbsp;arsB\u003c/em\u003e gene, which extrudes arsenate (III). Apart from \u003cem\u003earsCPRH\u003c/em\u003e, strain GTZY has also been distributed with additional \u003cem\u003earsB\u003c/em\u003e (963 bp), \u003cem\u003earsR\u003c/em\u003e (219 bp) and \u003cem\u003earsC\u003c/em\u003e (420 bp) genes in its chromosome. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe presence of \u003cem\u003earsP\u003c/em\u003e and its co-existence with the \u003cem\u003earsH\u003c/em\u003e gene in the \u003cem\u003ears\u003c/em\u003e operon of strain GTZY makes them versatile among other known bacterial \u003cem\u003ears\u003c/em\u003e operons. The \u003cem\u003earsP\u0026nbsp;\u003c/em\u003eencoding efflux related permease system is known for the detoxification or extrudes the toxic organic arsenicals (III) such as roxarsone and monomethylarsonous acid (MMA)(Shen et al. 2014; Chen et al. 2015b). The \u003cem\u003earsP\u003c/em\u003e gene encoding ArsP permease was initially identified in the \u003cem\u003ears\u003c/em\u003e operon of \u003cem\u003eCampylobacter jejuni\u0026nbsp;\u003c/em\u003e(Wang et al. 2009)\u003cem\u003e,\u0026nbsp;\u003c/em\u003eand later it was believed to be a prokaryotic cell membrane pump that could extrude arsenical roxarsone (III)(Shen et al. 2014). The recent genomic interpretation study suggests that the ancestral origin of arsP and their homologs, existence in the bacteria, archaea and a few other eukaryotes\u0026nbsp;(Yang et al. 2015). The existence of a gene encoding flavoprotein, arsH, utilizing NADP+ to resist and oxidize the toxic methylarsenite (MAs (III)) into MAs (V) was reported in \u003cem\u003eCampylobacter jejuni\u003c/em\u003e strain(Li et al. 2016). These arsH enzymes are widely distributed in gram-negative, mostly \u003cem\u003eGammaproteobacteri\u003c/em\u003ea \u0026nbsp;including \u003cem\u003ePseudomonas putida, Sinorhizobium meliloti, Acidithiobacillus ferrooxidans, Rhodopseudomonas palustris, Ochrobactrum tritici, Thiobacillus ferrooxidans, Herminiimonas arsenicoxydans\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Pseudomonas aeruginosa\u003c/em\u003e, but not in gram-positive bacteria\u0026nbsp;(Yang et al. 2005; Chen et al. 2015a; Yang and Rosen 2016; Falgenhauer et al. 2017). The ArsP protein from both \u003cem\u003eP. putida\u0026nbsp;\u003c/em\u003eand \u003cem\u003eS. meliloti\u003c/em\u003e was evaluated as an organoarsenical oxidase enzyme conferring resistance to trivalent methyl As derivatives. We assumed that \u003cem\u003earsCPRH\u003c/em\u003e of strain GTZY has highly evolved from its ancestral species, in response to the environmental mutations, genetic reshuffling and recombination with other bacterial communities.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Initially, \u003cem\u003eP. mexicana\u0026nbsp;\u003c/em\u003eGTZY isolate was characterized for its phylogeny, morphology and biochemical properties, and was found specifically relevant to the properties of various reported members of \u003cem\u003ePseudoxanthomonas.\u0026nbsp;\u003c/em\u003eLike other heavy metal transforming bacteria, \u0026nbsp;strain GTZY did not utilize the mercury and arsenic as main carbon sources in the basal media (data not shown). Since these heavy metals are not catabolized by the heavy metal transforming bacteria, their bioremediations were usually evaluated by the removal, reduction, oxidation and volatilization assays(Wuana and Okieimen 2011; Jebeli et al. 2017; Wang et al. 2020). TZY was successfully fulfilled the above chemical strategies against the mercury and arsenic metals in the growth media.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe strain GTZY can reduce inorganic Hg (II) and into foggy forming volatile Hg (0) in 4-h incubation. Previously,\u0026nbsp;Mahbub et al. (2017)\u0026nbsp;have proved the ability of \u003cem\u003eSphingopyxis\u003c/em\u003e sp. SE2 to volatilize or remove 44 % of mercury (II) in 6-h incubation. This study found 49.6 % of mercury removal achieved by the strain GTZY in 6 h. However, \u003cem\u003eSphingobium\u003c/em\u003e sp. SA2 was recorded for the maximum Hg (II) volatilization and reduction in 6 h account for 79 %\u0026nbsp;(Mahbub et al. 2016, 2017). The strain GTZY’s mercury volatilization and reduction ability were further confirmed by the occurrence of the \u003cem\u003emerA\u0026nbsp;\u003c/em\u003egene encoding for mercury (II) reductase. Also, the isolate was proven to resist (\u0026gt; 33.4 µM) and remove (84.3 %) the toxic form of mercury (II) in 48-h incubation. The Minimum inhibitory concentrations (MIC and MIC\u003csub\u003e50\u003c/sub\u003e) of strain GTZY against Hg (II) was observed to be 33.4 µM \u0026nbsp;and 40.8 µM;, respectively, whereas in other bacterial classes, \u003cem\u003eAlphaproteobacteria\u003c/em\u003e, \u003cem\u003eBetaproteobacteria, Flavobacteria, Firmcutes\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Sphingobacteria,\u003c/em\u003e MICs were observed in the range of 5-30 µM; Hg (II)(Møller et al. 2011, 2014). After 60 h incubation, the concentration of Hg (II) was remained stable at 1.9 ± 0.2 µM along with strain GTZY. In the meantime, thermophilic species\u003cem\u003e\u0026nbsp;Aquificae\u003c/em\u003e was proven to be sustainable at 3.0 µM; Hg (II) in the basel media(Freedman et al. 2012). These results represent that the strain GTZY could be useful for preventing the formation of a more toxic form of methyl mercury (MeHg). In contaminated environments, high concentration and precipitation of Hg (II) always end up with more accumulation of toxic MeHg(Eckley et al. 2020). Hence, this is arguably a better solution to reduce the formation of toxic MeHg, in which a high concentration of Hg (II) should be cleaned up first so that the strain GTZY would be more suitable for the detoxification. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOn the other side, strain GTZY can remove (68.5 %) and resist (\u0026gt; 175 mM) As (V) in the culture media at 72-h incubation. The Minimum inhibitory concentrations of strain GTZY towards arsenate and arsenite were \u0026gt; 175 and \u0026gt; 55 mM. Previous studies revealed the arsenate and arsenite MICs of \u003cem\u003eGammaproteobacteria, Firmcutes\u0026nbsp;\u003c/em\u003eand \u003cem\u003eKocuria,\u003c/em\u003e in which the ranges of 75 – 125 mM and 10 – 50 mM, respectively, whereas in the \u003cem\u003eBacillus fusiformis\u0026nbsp;\u003c/em\u003eORAs1\u003cem\u003e, B. thuringiensis\u0026nbsp;\u003c/em\u003eORAs2,\u003cem\u003e, Pseudomonas\u0026nbsp;\u003c/em\u003esp. ORAs5, \u003cem\u003eAeromonas molluscorum\u0026nbsp;\u003c/em\u003eORAs6, MICs of arsenate and arsenite were \u0026gt; 16.68 mM and \u0026gt; 133.47 mM, respectively(Pepi et al. 2007). In addition, strain GTZY was reduced organic As (V) into As (III) form in the liquid solution, and it was further confirmed by the amplification of the \u003cem\u003earsC\u003c/em\u003e gene (495 bp) encoding arsenate reductase. Meanwhile, the strain GTZY was also able to remove (63.2 %) and resist (\u0026gt; 55 mM) As (III); however, it could not oxidize organic As (III) into As (V) form in the liquid solution. Similarly,\u0026nbsp;Dey et al. (2016)\u0026nbsp;isolated \u003cem\u003eBacillus\u003c/em\u003e sp. and \u003cem\u003eAneurinibacillus aneurinilyticus\u003c/em\u003e and found that both strains could remove As (V) (51.45–51.99 %) and As (III) (50.37–53.29 %). Besides, the strains also resisted the arsenate (4500 ppm) and arsenite (550 ppm). However, both strains have oxidized the arsenite, but none of them reduced arsenate into arsenite. Many previous reports suggest different detoxification mechanisms of arsenic resistance bacteria, including arsenic reduction and oxidation, heavy metal efflux pumping, metal adsorption to the cell surface, bacterial cell membrane binding and complex formation with exopolysaccharides (Zhang et al. 2016; Govarthanan et al. 2016a). Despite the absence of arsenite oxidase enzyme, we presume that the strain GTZY might be removed the arsenite (III) by following any of the above-proposed detoxification mechanisms.\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusion ","content":"\u003cp\u003eFrom this study, it is therefore concluded that \u003cem\u003ePesudoxanthomoas mexicana\u003c/em\u003e strain GTZY has the potential for the bioremediation of highly toxic heavy metals such as mercury and arsenic. In short, this is the first investigation that opens up the resistance mechanism of \u003cem\u003emer\u003c/em\u003e (2.8 kb) and \u003cem\u003ears\u003c/em\u003e (2.7 kb) operons in the chromosome of \u003cem\u003eP. mexicana\u003c/em\u003e. The existence of \u003cem\u003emer\u003c/em\u003e operon might be derived via horizontal gene transfer from other bacterial communities, because of the linkage of mobile genetic elements (\u003cem\u003eIS2\u003c/em\u003e and \u003cem\u003eint1\u003c/em\u003e) in its both terminuses. The occurrence of \u003cem\u003ears\u003c/em\u003e operon could be rendered vertically from its ancestral \u003cem\u003ePseudoxanthomonas\u0026nbsp;\u003c/em\u003especies. This intrinsic \u003cem\u003ears\u003c/em\u003e operon was highly diversified and evolved from its ancestral species, which might be due to their mutations, genetic reshuffling and recombination with other bacterial communities. Initially, strain GTZY was isolated from an aerobic wastewater reactor and characterized for its biochemical, physiological and phylogeny, besides the isolated strain showed its potential resistance towards heavy metals (Hg and As) and their increased removal and reduction in short periods. The evaluations from this study was further confirmed by the existence of resistance genes (\u003cem\u003emerA\u003c/em\u003e and \u003cem\u003earsC\u003c/em\u003e). It is thereby claimable that the strain GTZY could be an exceedingly valuable addition to the mercury and arsenic-contaminated sites for time-\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e Conceptualization, Methodology, Software using, Data analysis, Writing—original draft preparation - Abdul Raheem Nelofer; Project administration, Conceptualization - Selvaraj Ganesh-Kumar (corresponding author) and S. Balachandran. Software using - Soorangkattan Saravanan, Software using, Data analysis- Karuppanan Kalimuthu; Software using, Data analysis - Ganesan Govindarajan; Data analysis - Rajah Dilip Kumar; Shivani Ramamurthy Baluraj - Data analysis; Software using, Data analysis - Imran Hasan; All authors have read and agreed to the published version of the manuscript. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors express their gratitude to King Saud University, Researchers Supporting Project Number (RSPD2024R670), King Saud University, Riyadh, Saudi Arabia. We thank Prof. Yang Min and Prof. Yu Zhang (RCEES, CAS Institute, Beijing, China) for kindly providing us \u003cem\u003eP. mexicana\u003c/em\u003e strain GTZY. The work was supported by the\u0026nbsp;Ministry of Education and State Administration of Foreign Experts Affairs\u0026nbsp;(Grant No.: G20190011010), People’s Republic of China.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval:\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate:\u003c/strong\u003e All persons who meet authorship criteria are listed as authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e All authors certify that they have participated sufciently in the work to take public responsibility for the content. Furthermore, each author certifes that this material or similar material has not been and will not be submitted to or published in any other publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e The authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAgarwal M, Rathore RS, Jagoe C, Chauhan A (2019) Multiple lines of evidences reveal mechanisms underpinning mercury resistance and volatilization by Stenotrophomonas sp. MA5 isolated from the Savannah River Site (SRS), USA. Cells 8:309\u003c/li\u003e\n \u003cli\u003eAstuti DI, Purwasena IA, Putri RE, et al (2019) Screening and characterization of biosurfactant produced by Pseudoxanthomonas sp. G3 and its applicability for enhanced oil recovery. J Pet Explor Prod Technol 9:2279\u0026ndash;2289\u003c/li\u003e\n \u003cli\u003eBen Fekih I, Zhang C, Li YP, et al (2018) Distribution of arsenic resistance genes in prokaryotes. 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Accessed 14 Feb 2024\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"environmental-science-and-pollution-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"espr","sideBox":"Learn more about [Environmental Science and Pollution Research](https://www.springer.com/journal/11356)","snPcode":"11356","submissionUrl":"https://submission.nature.com/new-submission/11356/3","title":"Environmental Science and Pollution Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Pseudoxanthomonas mexicana, Mercury, Arsenic, Resistance gene operon, Bioremediation, wastewater ","lastPublishedDoi":"10.21203/rs.3.rs-4104815/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4104815/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWe prompted first to characterize a wastewater bacterium, \u003cem\u003ePseudoxanthomonas mexicana \u003c/em\u003eGTZY, that efficiently transforms toxic mercury and arsenic and to explore its bioremediation capability and to reveal their relevant gene resistance operons. The Isolated strain was characterized for its phylogenetic, biochemical and phenotypic properties. The strain GTZY potentially removed 84.3% of mercury and their mercury volatilization (Hg(II) to Hg(0)) was confirmed using X-ray film method, and its respective \u003cem\u003emerA\u003c/em\u003e gene was PCR amplified. In addition, strain GTZY was efficiently removed arsenate (68.5%) and arsenite (63.2%), and showed resistance up to \u0026gt;175 and \u0026gt;55 mM, respectively. Their genomic annotations were disclosed the linkage of \u003cem\u003eTn2\u003c/em\u003e-transposon and \u003cem\u003eint1\u003c/em\u003e in both ends of \u003cem\u003emer\u003c/em\u003eoperon (\u003cem\u003emerAPTR)\u003c/em\u003e. The co-existence of arsP and arsH proteins in its intrinsic \u003cem\u003ears\u003c/em\u003e operon (\u003cem\u003earsCPRH)\u003c/em\u003e was extremely diverse from its ancestral species. We believe that mercury resistance-conferring \u003cem\u003emer\u003c/em\u003eoperon of \u003cem\u003eP. mexicana \u003c/em\u003eGTZY presumably derived horizontally from other species in the reactor, while arsenic resistance-conferring intrinsic \u003cem\u003ears \u003c/em\u003eoperon was highly diversified and evolved from its ancestral species. By considering the potential of the strain GTZY to transform heavy metals, this can be used to recover contaminated sites.\u003c/p\u003e","manuscriptTitle":"Bioremediation of heavy metals by an unexplored bacterium, Pseudoxanthomonas mexicana strain GTZY, isolated from aerobic-biofilm wastewater system","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-25 19:04:00","doi":"10.21203/rs.3.rs-4104815/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-04-22T14:44:35+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-18T07:20:35+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Environmental Science and Pollution Research","date":"2024-04-16T12:57:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-01T05:13:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Environmental Science and Pollution Research","date":"2024-03-29T01:19:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"environmental-science-and-pollution-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"espr","sideBox":"Learn more about [Environmental Science and Pollution Research](https://www.springer.com/journal/11356)","snPcode":"11356","submissionUrl":"https://submission.nature.com/new-submission/11356/3","title":"Environmental Science and Pollution Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0f283007-3e3b-49da-b8f0-f25816ff4bf2","owner":[],"postedDate":"April 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-12T15:59:38+00:00","versionOfRecord":{"articleIdentity":"rs-4104815","link":"https://doi.org/10.1007/s11356-024-34602-1","journal":{"identity":"environmental-science-and-pollution-research","isVorOnly":false,"title":"Environmental Science and Pollution Research"},"publishedOn":"2024-08-08 15:57:01","publishedOnDateReadable":"August 8th, 2024"},"versionCreatedAt":"2024-04-25 19:04:00","video":"","vorDoi":"10.1007/s11356-024-34602-1","vorDoiUrl":"https://doi.org/10.1007/s11356-024-34602-1","workflowStages":[]},"version":"v1","identity":"rs-4104815","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4104815","identity":"rs-4104815","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}