Taxonomy and physiology of Pseudoxanthomonas arseniciresistens sp. nov., an arsenate and nitrate-reducing novel gammaproteobacterium from arsenic contaminated groundwater, India

Reductive transformation of toxic arsenic (As) species by As reducing bacteria (AsRB) is a key process in As-biogeochemical-cycling within the subsurface aquifer environment. In this study, we have characterized a Gram-stain-negative, non-spore-forming, rod-shaped As reducing bacterium designated KAs 5-3T, isolated from highly As-contaminated groundwater of India. Strain KAs 5-3T displayed high 16S rRNA gene sequence similarity to the members of the genus Pseudoxanthomonas, with P. mexicana AMX 26BT (99.25% similarity), P. japonensis 12-3T (98.9 0%), P. putridarboris WD-12T (98.02%), and P. indica P15T (97.27%) as closest phylogenetic neighbours. DNA-DNA hybridization study unambiguously indicated that strain KAs 5-3T represented a novel species that was separate from reference strains of P. mexicana AMX 26BT (35.7%), P. japonensis 12-3T (35.5%), P. suwonensis 4M1T (35.5%), P. wuyuanensis XC21-2T (35.0%), P. indica P15T (32.5%), P. daejeonensis TR6-08T (32.0%), and P. putridarboris WD12T (22.1%). The DNA G+C content of strain KAs 5-3T was 64.9 mol %. The predominant fatty acids were C15:0 (37.4%), C16:0 iso (12.6%), C17:1 iso ω9c (10.5%), C15:0 anteiso (9.5%), C11:0 iso 3-OH (8.5%), and C16:1 ω7c/ C16:1 ω6c (7.5%). The major polar lipids were diphosphatidylglycerol, phosphatidyldimethylethanolamine, phosphatidylcholine, and two unknown phospholipids (PL1, PL2). Ubiquinone 8 (Q8) was the predominant respiratory quinone and spermidine was the major polyamine of the strain KAs 5-3T. Cells of strain KAs 5-3T showed the ability to use O2, As5+, NO3-, NO2-, and Fe3+ as terminal electron acceptors as well as to reduce As5+ through the cytosolic process under aerobic incubations. Genes encoding arsenate reductase (arsC) for As-detoxification, nitrate- and nitrite reductase (narG and nirS) for denitrification were detected in the strain KAs 5-3T. Based on taxonomic and physiological data, strain KAs 5-3T is described as a new representative member of the genus Pseudoxanthomonas, for which the name Pseudoxanthomonas arseniciresistens sp. nov. is proposed. The type strain is KAs 5-3T (= LMG 29169T = MTCC 12116T = MCC 3121T).

Introduction Taxonomic hierarchy of the genus Pseudoxanthomonas denotes its affiliation to the class Gammaproteobacteria, family Xanthomonadaceae of phylum Proteobacteria. Members of the genera Xanthomonas, Xyllela, and Stenotrophomonas are found to be the nearest phylogenetic neighbours of Pseudoxanthomonas [1]. Finkmann et al. [2] reported the first validly described species of Pseudoxanthomonas, P. broegbernensis isolated from an experimental biofilter. The taxon has been subsequently emended by Thierry et al. [3] and Lee et al. [4]. Members of this genus were described as Gram-stain-negative, non-spore forming rods, with iso C 15:0 and anteiso C 15:0 as major fatty acids, ubiquinone (Q8) as major respiratory quinone and capable of performing strict respiratory metabolism with O 2 as preferred terminal electron acceptor [3]. The genus can be well differentiated from the two other related members Xanthomonas and Stenotrophomonas by the absence of fatty acid C 13:0 3-OH and from genus Xylella by the presence of branched-chain fatty acids (as described in the Bergey's Manual of Systematic Bacteriology, 2 nd edition, Volume II, The Proteobacteria [4]). At the time of writing this manuscript, 17 validly described and two non-validly described (but effectively published) type species of the genus Pseudoxanthomonas were reported from varied environments [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17]. The non-validly described members (but effectively published): P. kaohsiungensis and P. gei are isolated from an oil-polluted site and plant stem respectively [18,19]. The members of this genus are ecologically important due to their ability to reduce both nitrite and nitrate; degrade a variety of hydrocarbons (including benzene, toluene, ethyl-benzene and o-, m-, p-xylene) [20][21][22]. Recently, the presence of Pseudoxanthomonas and other members of Xanthomonadaceae have been reported for As-contaminated groundwater of alluvial aquifers in West Bengal and Bangladesh [23][24][25][26]. However, neither the taxonomic identity of these strains nor their eco-physiology towards As-transformation has been adequately studied. As a result, the role of such organisms in biogeochemical-cycling of As in contaminated groundwater remained highly unexplored.
The present study was therefore undertaken to investigate the taxonomic and eco-physiological properties of an As-resistant and -reducing Pseudoxanthomonas strain previously isolated from As rich groundwater of West Bengal [23]. A polyphasic taxonomic approach was undertaken to characterize and delineate the taxonomic position of the strain KAs 5-3 T . This strain was found to possess abilities for anaerobic As reduction and hydrocarbons utilization as well as several other traits potentially important for surviving in highly As-contaminated oligotrophic aquifer environment. To the best of our knowledge, till date no Pseudoxanthomonas type strain has been characterized from As-contaminated groundwater and capable of reducing toxic As 5+ while assimilating hydrocarbons.

16S rRNA gene phylogeny and multi locus sequence typing
Nearly complete stretch of 16S rRNA gene was PCR amplified using 27F/1492R primers (Table A in S1 File); individual sequences were edited and assembled by BioEdit version 7.1.11 [27], subjected to similarity search in NCBI BLAST [28], RDP II [29], and against validly described members in the EzBioCloud database (http://www.ezbiocloud.net/eztaxon; [30]). Multiple alignments with 16S rRNA gene sequences of Pseudoxanthomonas type strains were performed using the CLUSTAL W package of the MEGA software version 7.0 [31]. All ambiguous positions were removed for each sequence pair and a total of 1492 positions were taken in the final dataset for construction of phylogenetic trees. Phylogenetic reconstruction and validation were performed using neighbour-joining (NJ) method [32] (Fig 1) based on bootstrap analysis with 1000 replications using Jukes-Cantor [33] distance model. Both maximum-likelihood (ML) [34] and minimum-evolution (ME) [35] methods were employed to test the robustness of the trees (Figure A in S1 File). Multi locus sequence analysis (MLSA) was performed using single copy genes which include gyrB (1200 bp), dnaJ (1000 bp), atpG (400 bp), and rpoB (1200 bp). PCR primers and conditions are given (Table A in S1 File). All PCR products were gel purified, cloned into pTZ57R/T vector and sequenced using vector specific primer set (M13F/M13R). Sequences obtained were searched for similarity level using BLASTN, concatenated, and phylogeny was inferred by constructing NJ tree with 1000 bootstrap resampling ( Figure B in S1 File).

Genotypic characterization
Molar G+C content was (mol %) determined following the thermal denaturation method [36]. DNA-DNA hybridization was carried out between strain KAs 5-3 T and reference type members (P. mexicana AMX 26B T , P. japonensis 12-3 T , P. indica P15 T , P. suwonensis 4M1 T , P. wuyuanensis XC21-2 T , P. putridarboris WD12 T , and P. daejeonensis TR6-08 T ) using a thermal denaturation procedure involving SYBR green dye-DNA binding method [37]. Optimum renaturation temperature (T OR ) was calculated and hybridization was performed as described by Mohapatra et al. [38]. DNA-DNA hybridization value < 70% or difference in T m values of 5˚C or higher was considered as the cut-off for distinct microbial species [39].
increments of 1.0 pH unit) was investigated using appropriate buffer system [pH 3-5 (0.1 M citric acid/0.1 M sodium citrate), pH 6-8 (0.1 M KH 2 PO 4 /0.1 M NaOH, pH 9-10 (0.1 M NaHCO 3 /0.1 M Na 2 CO 3 )] in LB broth, where no significant pH change of the medium was noticed after autoclaving. NaCl tolerance [0-10% (w/v) with increments of 0.5%] was examined in LB broth, where appropriate volume of NaCl was added (from 0-5%) to the autoclaved medium from a sterile stock solution (20%, w/v). For > 5% of NaCl concentrations, the culture medium was prepared in the double strength (2 X) to avoid the dilution done with the addition of higher NaCl stock solution. For sensitivity towards temperature, pH, and NaCl concentrations, cellular growth was assessed by measuring absorbance (growth optical density, OD 600 nm) at 0, 12, 24, and 48 h. Tests for catalase, oxidase, nitrate reduction to N 2 , utilization of gelatin, esculin, citrate, and urea were performed following the standard procedures [41][42][43]. Other biochemical properties were studied using API 20NE kit (Bio-Merieux) at 30˚C for 24-48 h and GEN-III microplate (Biolog) following the manufacturer's instructions and are presented in Table 1. Gram-staining was performed using Gram staining kit (HiMedia). Susceptibility towards various antibiotics was tested following disc diffusion susceptibility method [44] involving commercially prepared paper antibiotic disks (HiMedia, India): cefixime (5 μg), ceftriaxone (30 μg), amikacin (30 μg), cefotaxime (30 μg), chloramphenicol (30 μg), ofloxacin (5 μg), polymyxin-B (300 units), tetracycline (30 μg), ciprofloxacin (5 μg), and erythromycin (15 μg). Freshly grown bacterial cultures (approximately 2×10 7 CFU/mL) were spreaded onto the surface of Mueller-Hinton (MH) agar plates and are incubated for 18-24 h at 30˚C. The zones of growth inhibition around each antibiotic disks were correlated to the susceptibility of the isolate using the criteria published by the clinical and laboratory standards institute (CLSI, formerly the National Committee for Clinical Laboratory Standards or NCCLS) [45]. Minimum inhibitory concentration (MIC) of As and various heavy metals was evaluated by growing the cells in LB supplemented agar medium under aerobic condition by following the plate dilution protocol of Zhu et al. [46]. Increasing concentrations of As [0.1-200 mM] (As 3+ as  as NiCl 2 , Zn 2+ as ZnCl 2 ) were amended into the medium and medium without any heavy metal was treated as control. The lowest concentration of metals, which inhibited cellular growth completely, was considered for MIC evaluation ( Table 2). Strains of Escherichia coli NCIM 2931 T and Cupriavidus metallidurans DSM 2839 T were used as negative and positive control respectively, as the strains are found to have the lowest and highest resistance respectively to the heavy metals tested. The analysis of cellular fatty acid methyl esters (FAMEs) was performed after growth of bacterial strains (KAs 5-3 T , P. mexicana AMX 26B T , P. japonensis 12-3 T , P. indica P15 T , P. suwonensis 4M1 T , P. wuyuanensis XC21-2 T , P. putridarboris WD12 T , and P. daejeonensis TR6-08 T ) on Tryptic Soy agar (TSA) for 24 h at 30˚C. One loopful of bacterial colony was harvested at exponential phase, subjected to saponification, methylation, and extraction. Fatty acids were determined by Microbial ID using the fully automated GC Sherlock Microbial Identification System (MIDI) using MIDI standard procedures [47]. Isoprenoid quinones were extracted from overnight grown culture following the procedure of Komagata & Suzuki [48] and analysed using high performance liquid chromatography (HPLC, Agilent 1100; column: Sorbax C18 reverse phase, Agilent), where methanol: isopropanol (2:1, v/v), was used as mobile phase with peak detection at 275 nm. The ubiquinone fractions were separated and identified by liquid chromatography-mass spectrometry (LC-MS, WATERS 2695) in a positive-mode electrospray analysis. Polar lipids were extracted and analyzed by two-dimensional TLC following protocol of Komagata & Suzuki [48] (Figure D in S1 File). Polyamines were extracted as described by Kumari et al., [13] and analysed by TLC (Silica gel 60 F254, 20×20 cm, Merck, Germany).

Utilization of carbon substrates, electron acceptors, and As-reductive growth
To test the utilization of different carbon substrates by strain KAs 5-3 T , a range of hydrocarbon compounds (benzene, toluene, xylene, catechol, benzoic acid, naphthalene, phenanthrene, anthracene, pyrene, dodecane, pentadecane, hexadecane, nonadecane, docosane) were  amended into MSM medium at a concentration of 500 μM. Freshly grown cell suspension (MSM culture medium) was centrifuged at 10,000 rpm for 5 min, washed twice with 0.85% saline, resuspended in the MSM (without any amendment), inoculated (1%, v/v) into the medium (OD 600 0.03-0.05 at t 0 ), and incubated for 72 h at 30˚C. Growth was monitored at regular intervals by measuring colony forming unit (CFU)/mL, by plating 0.1 mL of the culture onto MSM plates supplemented with respective hydrocarbon sources. Utilization of various terminal electron acceptors (TEAs) was tested following anaerobic growth (OD at 600 nm) with As 5+ (5 mM), Fe 3+ (5 mM), NO 3 -(5 mM), NO 2 -(5 mM) or SO 4 2-(5 mM) in MSM [37] as alternate electron acceptors following addition of either sugar substrates (glucose or lactate, 20 mM each) and hydrocarbons (pentadecane or naphthalene, 750 μM each) as the sole carbon/energy source (Fig 2). Medium with added TEAs and without any inoculum was used as abiotic control. The concentration of TEAs in growth medium was measured in duplicate at regular intervals using standard procedures [43,[49][50][51]. Cytosolic As 5+ reduction was also checked by growing strain KAs 5-3 T in MSM supplemented with carbon sources (as described above) and incubated at 30˚C for 24 h. The growth parameters and rate of reduction of As 5+ were calculated by checking growth OD (at 600 nm) and residual As 5+ concentration in the medium by spectrophotometric method [52] and validated by atomic absorption spectrophotometer (AAS; PinAAcle900H, Perkin Elmer).

Functional gene-based analysis
Genes responsible for cytosolic As 5+ reduction (arsC), dissimilatory nitrate-(narG) and nitrite reduction (nirS) were also amplified through PCR based approach (Table A in S1 File). All PCR products were gel purified, cloned and sequenced (as described above for MLSA). Nucleotide sequences obtained were searched for similarity level using BLASTN. The corresponding nucleotides were translated to amino acids in ExPasy tool [53] using appropriate open reading frames (ORFs) and searched in BLASTP, (nr database) excluding options for uncultured/environmental sequences and including option for type material. Conserved domain was predicted through CDD database and phylogeny was inferred through neighbour-joining method (Figs 3 and 4) considering the translated amino acid sequence of strain KAs 5-3 T and similar sequences (>90% similarity value). The nucleotide sequences were analyzed for GC content (mol %), GC % deviation from their respective genomes (Table B in S1 File) as well as p-distance calculations through MEGA 7.0. Phylogenetic network analysis was performed using SplitsTree software [54] (Figures E and F in S1 File).

16S rRNA gene phylogeny and multi locus sequence typing
Comparison of nearly complete (1,495 nucleotides) 16S rRNA gene sequence indicated taxonomic affiliation of strain KAs 5-3 T to the genus Pseudoxanthomonas, with highest sequence similarity to the type strains of P. mexicana AMX 26B T (99.25%), P. japonensis 12-3 T (98.9%), followed by P. putridarboris WD-12 T (98.02%), P. indica P15 T (97.27%), P. wuyuanensis XC21-2 T (97.12%), P. suwonensis 4M1 T (97.0%), and P. daejeonensis TR6-08 T (96.99%). The NJ phylogenetic analysis showed that strain KAs 5-3 T formed a coherent cluster of monophyletic pattern with the type strains of P. mexicana AMX 26B T and P. japonensis 12-3 T (bootstrap support of 100.0%) and claded to the type members of Pseudoxanthomonas (Fig 1). Both ML and ME phylogenetic reconstruction methods indicated a consistent tree topology cladding strain KAs 5-3 T to the AMX 26B T , 12-3 T , P15 T , and WD12 T as the nearest phylogenetic neighbours, while the clade comprising the near distant members of the strain KAs 5-3 T was only supported by either of the methods (Figure A in S1 File). On the basis of high percentage of 16S rRNA gene sequence homology and coherent monophyletic cladding of strain KAs 5-3 T , the type strains P. mexicana AMX 26B T ,P. japonensis 12-3 T , P. indica P15 T , and P. putridarboris WD12 T are inferred to be the closest phylogenetic neighbours. Multi locus sequence typing (MLST) involving various hose-keeping genes have been employed as a taxonomic marker for species level comparisons and clonal relationship [55,56]. Sequence analysis of gyrB, dnaJ, rpoB, and atpG genes of strain KAs 5-3 T showed > 92.0% sequence similarity to the type strains P. mexicana AMX 26B T and P. japonensis12-3 T but formed a separate clade in the NJ phylogenetic reconstruction ( Figure B in S1 File) indicating its non-clonal nature and species distinction from both the closest phylogenetic neighbours.

Phenotypic and chemotaxonomic characterization
Culture characteristics revealed that on LB agar plates, colonies of strain KAs 5-3 T were creamy to pale yellow, circular, with entire margin and a diameter range of 1-2 mm after 24-48 h. Cells were Gram-stain-negative, rod-shaped, aerobic to facultative anaerobic, non-motile, catalase and oxidase positive, with a cell size of 1.2-1.5 μm length × 0.3-0.5 μm width ( Figure C in S1 File). The strain was found to grow well at temperature range of 10-38˚C (optimum at 28-32˚C), pH range of 6.0-8.0 (optimum at 7.0) and over a broad spectrum of NaCl concentrations (0.5-5%; optimum of 1%) and growth did not occur without NaCl in the medium. The other details of phenotypic characteristics of the strain KAs 5-3 T are presented in the species description and Table 1. Compared with other type members of the same genus (Pseudoxanthomonas), strain KAs 5-3 T exhibited phenotypic differences ( Table 1). The strain KAs 5-3 T showed ability to reduce nitrate to N 2 , assimilate esculin, casein, gelatin, urea, adipate, malate, citrate, and Nacetyl glucosamine (NAG) and showed negative response for tween 80, arabinose, mannose, gluconate, and caprate. The catalase-, oxidase-positive, mesophilic, slightly alkalophilic and heterotrophic growth pattern confirmed relatedness of KAs 5-3 T to the same genus [13]. The differential phenotypic properties viz., motility, assimilation of tween 80, urea, maltose, adipate, and production β-glucosidase confirmed the species level distinction of KAs 5-3 T from the compared Pseudoxanthomonas members. In comparison with the phylogenetic neighbours, strain KAs 5-3 T showed considerably higher resistance towards several metals Co 2+ , Cu 2+ , Se 6+ , Fe 3+ , As 3+ , and As 5+ ( Table 2). The strain's ability to withstand Fe 3+ was comparable to multi-metal resistant C. metallidurans and for As species, it was highest amongst all the strains tested.
The predominant quinone of the strain KAs 5-3 T was found to be Q8. This seems to be a familiar character as prevalence of Q8 was previously reported as the major quinone in members of the genus Pseudoxanthomonas [3,5,13]. The overall FAME profile of the strain KAs 5-3 T was found to be consistent to that of other type strains compared with some observed quantitative differences ( Table 3). The major cellular fatty acids (> 5% of the total fatty acids) of strain KAs 5-3 T consisted of C 15:0 (37.4%), C 16:0 iso (12.6%), C 17:1 iso ω9c (10.5%), C 15:0 anteiso (9.5%), C 11:0 iso 3-OH (8.5%), and C 16:1 ω7c/ C 16:1 ω6c (7.5%). The overall FAME profile was similar with the type strains compared, but the differential presence of C 11:0 anteiso, C 16:0, as well as absence of C 15:1 iso F and C 16:1 iso H distinguished the strain KAs 5-3 T from the reference type strains.
The polar lipid profile of the strain KAs 5-3 T was found to be consisting of diphosphatidylglycerol (DPG), phosphatidyldimethylethanolamine (PDE), phosphatidylcholine (PC), and unknown phospholipids (PL1, PL2, PL3). The presence of DPG, PDE and PL1 was found to be consistent in all the compared members (except P. indica P15 T ), indicating the affiliation of strain KAs 5-3 T to the members of the genus Pseudoxanthomonas. The appearance of spot corresponding to PC and absence of PE, unknown lipids (UL1, UL2) uniquely distinguished the strain KAs 5-3 T from all the compared members ( Figure D in S1 File).

Utilization of carbon substrates, electron acceptors, and As-reductive growth
Cells of the strain KAs 5-3 T were found to utilize catechol, naphthalene, dodecane, and pentadecane as sole carbon sources. Among various tested electron acceptors, strain KAs 5-3 T showed growth on As 5+ , NO 3 -, NO 2 -, and Fe 3+ , while no growth was observed in SO 4 2-. But, the preferential pattern [net reduction of each added TEA (mM) vs time] was found to be NO 3 -> NO 2 -> As 5+ > Fe 3+ . The growth of the strain while growing under these preferred electron acceptors showed that after 48 h, it reduced NO 3 preferably (5 mM to avg. of 1 mM) followed by NO 2 -(5 mM to avg. of 2.0 mM), As 5+ (5 mM to avg. of 2.5 mM), and Fe 3+ (5 mM to avg. of 2.8 mM) (Fig 2). Substantial growth [with a maximum growth OD of 1.2-1.3, μ = 0.11 h -1 ] along with the formation of As 3+ in the aqueous medium, confirmed its reductive transformation ability. Cells of strain KAs 5-3 T were also found to reduce As 5+ (from 1 mM to 0.2 mM) within 30 h of aerobic growth with the concomitant release of As 3+ in the supernatant, indicating its potential of cytosolic reduction of As 5+ . The ability of Pseudoxanthomonas members to metabolize alkyl and aromatic hydrocarbons (BTEX, chrysene, and phenanthrene) and degrade pollutants has been recently studied [20,21,[58][59][60]. The As-rich groundwater of Bengal basin harbours low amount of petroleum-derived hydrocarbons (that naturally seeps into the groundwater from deeper mature sediments), presence and hydrocarbon metabolizing activity of Pseudoxanthomonas strains is highly justified [26,61,62]. Except for P. kausinghensis and P. dokdonensis, Pseudoxanthomonas type members have been known to reduce nitrite. Thus, the ability of strain KAs 5-3 T to preferentially utilize NO 3 over NO 2 is considered to be a unique metabolic character, distinguishing the strain from its closest relatives. Strain's ability in utilizing diverse electron acceptor sources, thus corroborates its potential to dwell at the interface of aerobic-anaerobic zones of groundwater [26,[62][63][64][65].

Functional gene-based analysis
The presence of cytosolic As 5+ reductase (arsC; 118 AA), nitrate reductase (narG; 214 AA) and nitrite reductase (nirS; 146 AA) were noted for the strains KAs 5-3 T but not for the other closest related strains. BLASTP search showed highest identity (100%) of arsC and narG genes to the same genes from Escherichia coli followed by several Pseudoxanthomonas and other Xanthomonas, while the sequence of nirS showed highest similarity with Pseudoxanthomonas helianthi roo 10. Elaborate phylogenetic analysis was conducted for the arsC and narG genes. Phylogenetic analysis (Figs 3 and 4), p-distance matrix based net amino acid substitution (Figures E and F in S1 File), and phylogenetic neighbour network (Figures E and F in S1 File) showed a close phylogenetic proximity among KAs 5-3 T and E. coli with respect to both of these genes. The data further indicated presence of similar mutational (insertion/deletion) events in these genes from the organisms, thus suggesting their possible transfer through horizontal gene transfer events. So, the observed phylogenetic incongruence between these functional genes and 16S rRNA gene was further studied with respect to GC mol %. Measure of unrelated GC mol % of the functional genes in the genome of organisms is considered to be the possible site of gene transfer events [24,66,67]. Hence, GC content (mol % and mol % deviation) of both the genes was compared with the genomic GC mol % for Pseudoxanthomonas reference genomes (Table B in S1 File). The GC mol % of both arsC and narG of strain KAs 5-3 T were close to the genomic GC content of E.coli genomes, but not to the genomes of any of the nearest Pseudoxanthomonas members, further supporting the possibility of horizontal gene transfer events [68,69]. Unlike, nitrite reduction, a universal property for the genus Pseudoxanthomonas; nitrate reduction by strain KAs 5-3 T , is a unique trait. The abilities to utilize multiple hydrocarbons, different electron acceptors with As 5+ reduction abilities and genetic validation of this potential clearly demonstrated the metabolic flexibility of the strain. Alluvial aquifer of West Bengal is oligotrophic in nature with low dissolved carbon, low oxygen tension, fluctuating availability of electron donors and acceptors, with a low concentration of naturally derived hydrocarbons [26,38,61,62]. Considering the overall hydrogeochemistry of West Bengal groundwater, the metabolic versatility of the strain KAs 5-3 T seems highly justified for its competitive niche adaptation.

Emended description of the genus Pseudoxanthomonas Finkmann et al. 2000 emend. Lee et al. 2008
As per the descriptions of Pseudoxanthomonas by Finkmann et al., emended by Lee et al. (2008) and properties tested in this study, an emended description of the genus Pseudoxanthomonas is provided. Type strains of all Pseudoxanthomonas species except P. kaohsiungensis, P. dokdonensis, and P. arseniciresistens have no nitrate reduction (to N 2 ) ability.

Conclusion
The phylogenetic, chemotaxonomic and phenotypic analysis supported the affiliation of strain KAs 5-3 T to the genus Pseudoxanthomonas. The strain KAs 5-3 T showed distinguishing physiological, phenotypic as well as molecular characteristic. Multi locus sequence analysis involving four house-keeping genes and DNA-DNA relatedness unambiguously demarcated the species novelty. Dissimilatory reduction of nitrate and nitrite as well as ability to metabolize hydrocarbons and reduce As 5+ through cytosolic processes highlighted the unique properties of the strain KAs 5-3 T , which are of ecological significance. On the basis of phenotypic and physiological characteristics, chemotaxonomic analysis, multi locus sequence analysis, and DNA-DNA relatedness data, the isolate represents a novel species of the genus Pseudoxanthomonas, therefore, the name Pseudoxanthomonas arseniciresistens sp. nov. is proposed.
Supporting information S1 File. Table A