Identification and characterization of siderophore producing arsenic tolerant Staphylococcus sp. TA6 and its possible involvement in arsenic geocycle

Increased concentration of arsenic in the groundwater of Brahmaputra valley has been a matter of serious concern for the region. Although the presence of Arsenic in sediments and carbonaceous rocks are geogenic, its entry into the groundwater is mediated by several processes including microorganisms. It is well known that microorganisms play a crucial role in the biogeochemical cycle of elements. However, the precise role of bacteria in regulating the concentration of arsenic in Brahmaputra valley has not been investigated in detail. In this paper, we report the isolation of bacterium Staphylococcus sp. (TA6) that can reduce arsenate to arsenite. The isolate was able to grow in arsenate concentration exceeding ≥ 250 mM and arsenite up to ≤ 30 mM. It also showed cross-tolerance to other heavy metals like Hg+2, Cd+2, Co+2, Ni+2, Cr+2. The bacterium also had a high siderophore activity (78.7 micromoles), which is positively correlated with increased arsenate reduction efficiency. The bacterium could significantly reduce ~88.2% of initial 2mM As(V) into As(III). The arsenate reductase enzyme showed the highest activity at pH 5.5 and temperature 50C. This bacterium can actively control the concentration of arsenate and arsenite through a reduction reaction.

.Arsenic is a metalloid widely distributed in the earth's crust and its concentration can exist from traces to up to hundreds of mg/kg or mg/l in both soil and in water. In groundwater, the element is predominantly found in two states viz., arsenate (AsV) and arsenite (AsIII). Arsenate is dominant in the oxic environment and gets strongly absorbed by chemicals like ferric-oxyhydroxide, ferrihydrite, apatite, and alumina. The arsenite form is dominant in an anoxic environment and is more mobile and toxic than arsenate [4].The geochemical cycling of arsenic is composite in nature; in fact, in addition to various physical and chemical factors, it also involves biological agents. Microorganisms play a critical role in mobilization and speciation of arsenical compounds in aquifer systems [5]. Microorganisms in the course of evolution have developed the necessary genetic makeup which confers them with resistance to high concentration of arsenic as well other toxic metalloids [6]. Microorganism arbitrated mechanisms of arsenic mobilization are still poorly understood and therefore, needs further studies to reveal their role in sediment-bound arsenic mobilization. They can either reduce, oxidize or can methylate the element to other organic compounds to generate energy [7]. Arsenate reducing bacteria are able to reduce As (V) to As (III) and use the same as an electron acceptor in a respiratory pathway or efflux the same as a mean of resistance mechanisms [8]. Arsenic resistant bacteria are frequently detected with siderophore activity. Siderophore are high-affinity iron chelating compounds produced and secreted by few microorganisms to forage the environmental iron from inorganic phase by formation of soluble Fe 3+ complex, which can be taken up by active transport mechanisms [9]. The Fe sequestering ability of bacteria through siderophore production confers them with added advantages over the non-producers in mobilizing arsenic. The previous study has shown that the rate of arsenic uptake and reduction efficiency of a bacteria significantly varies with varied siderophore concentration [10].
In this paper, we report the isolation of a gram-positive bacterium TA6, which is resistant to both arsenate and arsenite. Based on its morphological, molecular and chemotaxonomic characterization the isolate was identified as a species of the genus Staphylococcus that had facilitated arsenate reduction along with siderophore activity.  1), a village in Titabor subdivision of Jorhat district, Assam, India. The concentration of arsenic in the water was measured by atomic absorption spectrophotometer (AAS) following the standard protocol as described by Behari and Prakash [11].

Isolation of arsenic-tolerant bacteria
The collected sample was subjected to serial dilution and cultured in arsenate amended Lauria-Bertani (LB) agar plates (10mM Arsenate) and incubated at 30 0 C for 48 hrs. Individual colonies were picked up based on the morphological identities and sub-cultured to obtain the pure isolates.

Minimum Inhibitory Concentration Test
The minimum inhibitory concentration (MIC) of arsenate [As (V)] and arsenite [As (III)] was evaluated to determine the resistance capacity of the isolated bacteria. The bacterial isolates were cultured in freshly prepared LB broth at 30 0 C for 48 hours and then 100 µl of the freshly cultured bacterial suspension was inoculated in minimal salt media (MSM) supplemented with different concentration of Arsenite (0.5-30 mM) added as sodium meta-arsenite (m-Na-AsO 2 ) and Arsenate (10-300mM) added as disodium hydrogen arsenate (Na 2 HAsO 4 .7H 2 O) and incubated for 72 hours at 30 0 C and 142 rpm. The microbial growth was recorded with a UV-Visible spectrophotometer at 600nm.

Growth of the bacterial isolate in the presence and the absence of arsenite/arsenate
Among all the isolates, TA6 showed the highest MIC and as such, was taken for studying the growth kinetics in presence and absence of arsenite and arsenate. The isolate was cultured in Luria-Bertani broth containing arsenate in a concentration of 1mM to 30mM and arsenite from 0.5 mM to 10 mM respectively. The growth of the isolate was monitored through measurement of the optical density (OD) with a spectrophotometer (Thermo-Scientific, India) at 600 nm (OD600) at a specified interval of time (4hr, 8hr, 12hr, 24hr, 48hr, and 72hr).

Biotransformation assay
The ability of the bacteria to reduce As (V) or to oxidize As (III) was evaluated using the silver nitrate (AgNO 3 ) method as described by Simeonova et al., [12]. Freshly cultured bacterium grown in minimal salt medium supplemented with 5mM glucose was cultured on two different LB agar plates supplemented with 2mM of Sodium Meta-Arsenite and Sodium Arsenate respectively and . CC-BY-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/231241 doi: bioRxiv preprint incubated for 48 hours at 30 0 C. The streaked plates were then flooded with 0.1M Silver Nitrate (AgNO 3 ) solution. Formation of light yellow color will indicate the precipitation of silver orthoarsenite (Ag3AsO3) due to As (III) and light brown-red color for precipitation of silver-ortho-arsenate (Ag3AsO4) due to As(V).
Quantitative assay of arsenate reduction was analyzed by growing the bacteria in arsenic amended LB broth (2mM of Arsenate). In a time interval of 6, 12, 24, 48, 72 hours the bacterial cells were collected at a time by centrifugation and arsenite content of the supernatant was determined by AAS following standard protocols as described by Aggett and Aspell [13].

Arsenate reductase enzyme assay
The enzyme assay was done using NADPH coupled assay as described by Gladysheva et al., [14].
Cell-free crude extracts of Escherichia sp. SD23 was used as positive control. Effect of pH and temperature on enzyme activity was also measured using this method.

Cross tolerance
The isolate was tested for its cross-tolerance efficiency with other heavy metals like Hg +2 added as HgCl 2 , Cd +2 added as CdCl 2 , Co +2 added as CoCl 2 , Ni +2 added as NiCl 2 and Al +3 added as AlCl 3 in a concentration ranging from 0.5 to 10mM in MSM broth culture and OD (OD600) was recorded after 48 hours to evaluate the bacterial growth.

Biochemical Tests
Biochemical tests for starch hydrolysis, catalase, oxidase, casein production, nitrate reduction, urease, malate, citrate, indole, and motility were done according to the standard protocol described by Krieg [15].

Identification based on 16S rRNA
Genomic DNA was extracted from approximately 100 mg of the cell as per standard phenolchloroform method. The 1500bp region of the 16S rRNA gene was amplified from the extracted genomic DNA using the universal primer 5' TACGGYTACCTTGTTACGACTT 3' (1492R), 5' The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/231241 doi: bioRxiv preprint the reverse primer, 2.5 µl (2.5 mM of each) dNTP mix, 2.5 µl of 10x PCR buffer, 1 µl (1U) of Taq DNA polymerase. A negative control (PCR mix without DNA) was included in all PCR experiments.
The PCR reaction conditions were set for 94 ºC for 3min, followed by 30 cycles of denaturation at 94 ºC for 30s, annealing at 58 ºC for 1min and extension at 72 ºC for 2 min, before a final extension at 72 ºC for 7min. The PCR products were sequenced and the forward and reverse sequences obtained were assembled using the Codon-Code Aligner software (version: 5.1). Nucleotide sequence identities were determined using the BLAST tool from the National Center for Biotechnology Information (NCBI) and Similarity index value from EzTaxon Server. Then the partial sequence data for the 16SrRNA genes have been submitted to GeneBank.

FAME analysis
The fatty acid methyl ester (FAME) profile was analyzed using Sherlock-Midi system and compared with few reference strains of Staphylococcus genus for taxonomical validation.

Siderophore production
Production of siderophore was studied using Chrome Azurol S (CAS) agar media as described by Schwyn and Neilands [16]. CAS agar was prepared from four solutions which were sterilized separately before mixing. The solution I: Blue dye was prepared by mixing 10 ml of 1 mM FeCl 3 ,

Siderophore quantification
The method of Alexander et al. [18] was used to measure siderophore production in vitro. The bacterial cells were grown at 30 0 C for 24 h in 50 ml of Chrome Azurol S (CAS) medium with 5 mM MES (2-(N-morpholino ethanesulfonic acid)-KOH buffer at pH 6.8. After the culture growth attains exponential phase at OD-600, the cells were pelleted by centrifugation at 10,000g for 10 min and the supernatant was filtered through 0.25 µm filter. Siderophore concentration in the filtrate was measured by mixing 500 µl of modified CAS assay solution with 500 µl filtrates. The standard solution of deferoxamine mesylate was used for siderophore quantification. The sterile CAS-MES-KOH solution was used as a reference solution, which did not contain siderophores. A standard curve was prepared by analyzing the absorbance (630 nm) of the reference solution (A/Aref) as a function of the siderophore concentration.

Resistance to Arsenic in comparison to siderophore mutant
The role of siderophore in arsenic tolerance was determined following the protocol described by Ghosh et al., [10] using one siderophore mutant (non-producer) Pseudomonas putida (Lp10L02M) and one control Acinetobacter guillourie (S02Ar2) with low siderophore production ability (10.8µmol). Arsenic tolerance of the isolate was measured as a percentage of growth rate and As(V) reduction at 5 and 10 mM of As(V) modified LB medium incubated at 30 0 C for 24 h shaking at 142 rpm and compared with the TA6. Growth was measured as OD at 600nm on UV-Vis spectrophotometer. All the data were taken in triplicates.

Groundwater Sample
The contaminated groundwater sample collected from Titabor subdivision had pH 6.2 and arsenic concentration of 356µg/l.

Isolation of arsenic-resistant bacteria and MIC
. CC-BY-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/231241 doi: bioRxiv preprint The collected groundwater sample was inoculated in arsenate amended LB medium and morphologically different bacterial colonies were picked up and tested for minimum inhibitory concentration of arsenate and arsenite. Among the isolates, TA6 showed highest MIC and was able to grow in medium with ≥ 250mM of arsenate and ≤ 30mM of arsenite.

Bacterial growth in presence of arsenate and arsenite
Growth curve analysis showed the effect of arsenate and arsenite in the bacterial growth pattern. The isolate TA6 was cultured in fresh LB broth with a concentration of arsenate varying from 1mM -30mM and arsenite from 0.5mM -10mM respectively. Bacterial growth was not much affected in the presence of arsenate as compared with control. However, the presence of arsenite in the medium greatly affected the rate of growth. In the presence of arsenate, TA6 started doubling at the lowest time of 4hrs but in the presence of arsenite, it took approximately 24hrs to start multiplying. At the highest concentration of arsenate (~30mM) taken for the test and at 72 hours of incubation time, OD was measured as 1.474 ± 0.067, which is statistically at par with the OD 1.962 ± 0.058 for control, at the same time of incubation. But in comparison, at 72hrs of incubation in the presence of 10mM of arsenite growth was rigidly reduced when compared to the control. For control, OD was recorded as 1.962 ± 0.058, whereas in the 10mM of arsenite, the growth was recorded as OD 0.1036 ± 0.043. At lowest concentration of arsenite 0.5mM, the bacterial cell (TA6) approximately took 8 ± 2 hrs of incubation to multiply. Arsenite can strongly cease the bacterial growth even at a minimal concentration or as low as 0.5mM when compared to arsenate (Fig. 2). Both arsenate and arsenite are toxic to bacteria but arsenite is more toxic as evident from the present growth kinetics studies.

Cross tolerance
Other heavy metal tolerance test also showed the resistive capacity of the isolate to various heavy metals like Hg +2 , Cd +2 , Co +2 , Ni +2 , Cr +2 . MIC was found as 0.8mM, 1.0mM, 4mM, and 6mM respectively.

Biotransformation assay
TA6 was found to be an arsenate reducer. Reduction of arsenate in the petri dish formed a yellow precipitation of silver ortho-arsenite (Ag 3 AsO 3 ) which indicates the presence of arsenite. In the quantitative assay, it was also found that with a gradual increase in time and with the increased . CC-BY-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/231241 doi: bioRxiv preprint bacterial cell count, the concentration of As(V) gradually decreased with increased concentration of AS(III). In the duration of 72 hours, nearly 88.2% of the initial 2mM As(V) is reduced to As(III) (Fig.   3).

Arsenate reductase enzyme activity
Arsenate reductase activity was measured using NADPH coupled oxidation method. A Km of 0.44 mM arsenate and Vmax of 6395 U/mg were measured (Fig. 4). There was no change in activity for 500µM and 1mM of arsenate. Temperature and pH are some critical factors for enzyme activity. Temperaturedependent activity assay revealed that 50 0 C was the optimal temperature for highest enzymatic activity and in pH-dependent activity assay, pH 5.5 was measured as optimal for highest enzymatic activity ( Fig. 5). Graphical representation of both the data formed a characteristic bell-shaped curved, where initial increased pH and temperature raised the activity till it reaches the optimal point of maximum activity and then the activity was found to gradually cease after the respective optimal value of pH 5.5 and temperature 50 0 C.

Biochemical test
The bacterium (TA6) was a gram-positive, nonmotile, coccus shaped bacterium. It was able to hydrolyze starch, casein and utilize citrate, reduce catalase and showed high siderophore activity (78.7 ± 0.004 µmol) but tested negative for oxidase, nitrate, urease and indole.

Chemotaxonomic and Molecular Identification
16S rRNA sequence similarity search identified the isolate as one of the species of the genus Staphylococcus having 97.6% pairwise similarity with Staphylococcus saprophyticus subsp. bovis (AB233327) and 97.5% with Staphylococcus saprophyticus subsp. Saprophyticus ( Table 1) saprophyticus showed considerable differences of C17:0, iso C17:0, iso C18:0 (Table 2). Therefore, based on both molecular and chemotaxonomic data the bacterium was identified as Staphylococcus sp. and the sequence was submitted under the GeneBank accession: KF134542.1 for further references.

Siderophore associated arsenate reduction
. CC-BY-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/231241 doi: bioRxiv preprint Microorganisms are the primary chelator of iron which dissociates Fe +3 ions with their siderophore activity. Siderophore associated arsenic resistance assay revealed that bacteria with high siderophore TA6 (78.7 ± 0.004 µmol) was significantly a strong As(V) reducer than the mutant strain Lp10L02M (non-producer). The growth of TA6 was also found reflective in comparison to the control and the mutant implying the added resistance ability of the strain to arsenate. In 5mM and 10mM arsenate broth, the TA6 showed higher growth as compared to the control strain S02Ar2. However, the mutant strain (Lp10L02M) had slower growth rate as compared to control and showed lesser reduction efficiency (Fig. 6).

Discussion
Increased arsenic concentration in groundwater is a matter of serious concern due to its carcinogenic effects on human health. The Brahmaputra river basin is considered as one of the severely affected basins [19]. Flood-line areas of the river have been detected with higher arsenic concentration than the standard permissible limit. which has become a major health risk for the people residing within these vicinities as they solely depend on the natural streams and groundwater for potable water.
Titabor subdivision of Jorhat district, Assam has been severely affected by a alarming concentration of arsenic (194-657 µg/l) [3]. Although, several studies on arsenic poisoning and geogenic distribution in this region has been documented, the role of microbes in the geocycle is unknown. The study of contaminated aquifers is still a long way to go. Bacteria play an important role in the biogeochemical cycle of arsenic and they are contemplated as one of the major factors that have a pivotal role in the mobilization of sediment-bound arsenic [20]. They can change the bioavailability, solubility and mobility status of arsenical species by inter-conversion through redox reactions and thus, control toxicity level. Bacteria can resist the toxic arsenic using an array of cellular and metabolic mechanisms which basically includes active cellular transport of the toxic material out of the cellular environment, entrapment by cellular capsules or by precipitation, oxidation-reduction reaction [5,21]. We isolated a bacterium TA6 from groundwater sample containing 356µg/l of arsenic. The bacterium was identified as Staphylococcus sp. based on phylogenetic analysis of 16S rDNA sequence and FAME analysis.
Both 16S rDNA and FAME profile showed significant differences with the reference strains of The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. ≤ 30 mM). Resistance to arsenite concentration greater than 10 mM and arsenate greater than 100 mM has been considered as very high, whereas resistance to 200 mM As 5+ and 30 mM As3 + is a hyper-tolerance property [22]. The bacterium was also showed resistance to other heavy metals like Hg +2 , Cd +2 , Co +2 , Ni +2 , Cr +2 that ranged from ≥ 0.5 -10.0 mM and produced siderophore (78.7 ± 0.004 µmol). Microorganisms are known for their ability to produce different biogenic chelating agents like siderophore in iron-limiting environment. Siderophore solubilizes the ferric iron in the iron-starved environment and transports the Fe +3 into the cell [23]. It determines the growth of microorganisms in an environment where iron is the limiting factor [24]. Above and beyond being a limiting factor for growth in iron-starved environment, siderophore confers an added advantage of higher arsenic resistance as compared to the non-producers. In this study, screening of comparative resistance efficiency of TA6 (78.7 ± 0.004 µmol) revealed a higher siderophore activity against the control strain Acinetobacter guillourie S02Ar2 (10.8 ± 0.003 µmol) and a mutant strain Pseudomonas putida (Lp10L02M). The isolate TA6 not only had a higher siderophore activity in comparison to the mutant strain but also displayed a significantly higher resistance to arsenate and higher growth rate in an incubation period of 24h at 30 0 C. Siderophore assisted resistance to arsenical compounds has been reported earlier [10]. High siderophore concentration confers higher resistance to arsenite and that the reduction efficiency of bacteria is significantly influenced by varied siderophore concentration [10].
Biotransformation assay indicated the isolate as one of the members of the arsenate-reducing genera that can actively catalyze the reduction of As(V) to As(III) using an enzyme arsenate reductase encoded by arsC gene of the ars operon. Aerobic arsenate reduction is the most distributed detoxification mechanism among the microorganisms and the ars operon has been detected in more than 50 organisms within the domains of bacteria, yeast, and protist. The first recognized arsenate reductase gene was identified on a gram-positive Staphylococcus plasmid [21]. Since  conversion of arsenate to more mobile arsenite in the shallow aquifers that leads to its accumulation over a time period and could be one of the major reason for the increasing carcinogenic development in the northeastern region. In a transitory, the production of siderophore by bacteria helps in the mobilization of the sedimentary arsenate by displacing iron from the iron-arseno compounds forming a soluble Fe +3 ion. Increase concentration of arsenate in surrounding milieu competes with the phosphate ion and structural homology of the arsenate with phosphate gives an added advantage to enter the cellular system through pit/pst phosphate transporter channel. Cellular arsenate is then converted to arsenite by arsenate reductase enzyme and soon effluxes out of the system through arsenite transporter channel to maintain the cellular homeostasis. This essentially increases the concentration of both arsenite and arsenate in the aquifer system and eventually increases the arsenic contamination in the aquifers.