Selenite Stress Elicits Physiological Adaptations in Bacillus Sp

Home , Sodium selenite

J. Microbiol. Biotechnol. (2011), 21(11), 1184–1192 http://dx.doi.org/10.4014/jmb.1105.05038 First published online 6 September 2011

Selenite Stress Elicits Physiological Adaptations in Bacillus sp. (Strain JS-2)

Dhanjal, Soniya and Swaranjit Singh Cameotra*

Environmental Biotechnology and Microbial Biochemistry Laboratory, Institute of Microbial Technology (IMTECH) Sector-39A, Chandigarh-160 036, India Received: May 19, 2011 / Revised: July 21, 2011 / Accepted: July 22, 2011

A bacterial isolate (strain JS-2) characterized as Bacillus role played by microbe/microbial communities in the sp. was challenged with high concentrations of toxic process of metal biotransformation [1, 8, 9]. “Geosymbiosis” selenite ions. The microbe was found to transform the is the term used to express the inseparable association of 2- toxic, soluble, colorless selenite (SeO3 ) oxyions to nontoxic, the geological process and the microbial activity. During insoluble, red elemental selenium (Se0). This process of this activity, both the geosymbiotic partners are affected as biotransformation was accompanied by cytoplasmic and in the case of microbe-metal interactions [11]. Microbial 0 surface accumulation of electron dense selenium (Se ) interactions with metals alter their physical or chemical granules, as revealed in electron micrographs. The cells state, with metals also affecting the activity, growth, and grown in the presence of selenite oxyions secreted large survival of the microbe/microbial community in metalliferous quantities of extracellular polymeric substances (EPS). environments [14, 21, 26], and this aspect of the interaction There were quantitative and qualitative differences in the (i.e., how metals effect the microbial physiology) is cell wall fatty acids of the culture grown in the presence of substantially less investigated [27]. Selenium (Se), an selenite ions. The relative percentage of total saturated essential trace mineral, is of fundamental importance to fatty acid and cyclic fatty acid increased significantly, human health, being a component of selenoproteins, which whereas the amount of total unsaturated fatty acids have important enzymatic functions although its toxicity decreased when the cells were exposed to selenite stress. cannot be ruled out at elevated levels [33]. Se pollution is a All these physiological adaptive responses evidently worldwide phenomenon and is associated with a wide range indicate a potentially important role of cell wall fatty acids of natural and human activities, ranging from agricultural and extracellular polymeric substances in determining practices to technologically advanced industrial processes. bacterial adaptation towards selenite-induced toxicity, Naturally, selenium exists in a number of oxidation states 2- 2- which thereby explains the remarkable competitiveness [+6, selenate (SeO4 ); +4, selenite (SeO3 ) oxyions; 0, 0 2- and ability of this microbe to survive the environmental elemental selenium (Se ); and selenide (Se )] in inorganic stress. form. The toxicity of these states is related to their degrees of solubility in water and hence their bioavailability for Keywords: Selenite, physiological response, Bacillus sp., 2- biogeochemical cycling. Selenate (SeO4 ) and selenite exopolysaccharides, cellular fatty acids 2- (SeO3 ) are highly water soluble, and thus toxic, having 0 tendency to bioaccumulate. Elemental selenium Se is an insoluble red precipitate, being biologically unavailable 2- Microbial interactions with metals are unavoidable in the and therefore nontoxic. Selenide (Se ) is a toxic gas and natural environment. Therefore, it is not surprising that seldom a biological threat as it gets readily oxidized to microbes have developed means to use a few metals insoluble elemental form in the presence of air. Among the beneficially and develop defenses against those that are 2- various selenium species, selenite (SeO3 ) reduction has toxic. Microbes significantly affect the distribution of metals attracted a great deal of attention as a potential compound in the environment and a number of studies emphasize the for microbial reduction owing to its high toxicity. High concentrations of Se in contaminated agricultural soils and *Corresponding author drainage waters have resulted in identification of adapted Phone: +91-172-6665-224; Fax: +91-172-2690585; E-mail: [email protected] microbes that efficiently reduce selenate/selenite to red elemental Se or methylated forms of selenium [3, 7, 12, # Supplementary data for this paper are available on-line only at http://jmb.or.kr. 24]. In this line of research, the present work was planned 1185 Dhanjal and Cameotra to study the adaptive response of Bacillus sp. (strain JS-2) Flow Cytometry to survive the selenium toxicity and determine its The relative changes in the cellular granularity and size of the cell biotransformation potential, as the genus Bacillus represents population under selenite stress were determined by Forward Scatter one of the dominant taxa of soil bacteria. and Side Scatter using FACS Calibur (Becton-Dickinson). The bacterial strain was grown in the presence of 5 mM sodium selenite and an aliquot of 1 ml of bacterial culture was collected at regular time intervals of bacterial growth for analysis. The samples were MATERIALS AND METHODS o centrifuged at 2,500 ×g for 10 min at 4 C. The cell pellet was gently Microorganism and Culture Conditions washed twice with phosphate-buffered saline (PBS), pH 7.2, and The strain JS-2 was isolated by enrichment of seleniferous agricultural resuspended in it for analysis by FACS. Culture without addition of soil samples collected from Jainpur located in the Nawahshahr selenite oxyions served as the control. district (latitude 31o07' N and longitude 76o08' E) of Punjab, India. Confocal Laser Scanning Microscopy (CLSM) The pure isolate was routinely cultured on Tryptic Soya Agar (TSA) plates supplemented with 2 mM sodium selenite at 37oC. Selenite-stressed cells were washed with deionized water by centrifugation at 2,500 ×g for 5 min at 4oC, and aliquots were stained Characterization of the Strain for 1 min with 20 µM SYTO9 (Molecular Probes, Invitrogen), a Biochemical characterization of the strain JS-2 was performed following nucleic acid dye that stains bacteria (green fluorescence). For standard methods as described in Bergy’s Manual of Systemic fluorescence detection of exopolysaccharides produced by bacteria, Bacteriology, Vol. I and II, and Manual for the Identification of the cell suspension was stained with lectin PHA-L conjugates (Alexa Medical Bacteria by Cowan and Steel (2nd Ed., Cambridge Fluor 594-conjugated; 2 µg/ml; Molecular Probes, Invitrogen). Confocal University Press) [5, 15]. For the 16S rRNA gene analysis, the microscopy was performed by using a Nikon A1R inverted Confocal method described by Dhanjal and Cameotra [6] was followed. Laser Scanning Microscope (CLSM) equipped with an Ar-ion laser (488 nm) and a HeNe-laser (543 nm). Bacterial Growth Under Selenite Stress and Its Selenite Reducing Ability Whole-Cell Fatty Acid Profile The effect of selenite on the growth of the bacterial isolate and For cellular fatty acid analysis, strain JS-2 was grown with and reduction of selenite by the bacterial isolate were studied as described without addition of selenite oxyions for 24 h. Cells were harvested by Dhanjal and Cameotra [6]. Briefly, Se content in the supernatants and fatty acid methyl esters were prepared sequentially by was determined by atomic absorption spectrophotometry (AA-6800, saponication, methylation, and extraction [36]. The extracted fatty Shimadzu) in hydride vapor generation mode, with a selenium acid methyl esters were analyzed by gas chromatography (Hewlett cathode lamp. An air-acetylene (oxidizing) flame was used and a Packard Model 5890A, equipped with 5% phenylmethyl silicon wavelength of 196 nm was chosen for the purpose of absorption of column [0.2 m × 25 m] and a flame ionization detector) followed by incident light. the Microbial Identication System (MIDI, Inc., Newark, NJ, USA).

Effects of Temperature, pH, and Different Oxyions on Selenite Reduction RESULTS AND DISCUSSION The effect of temperature on growth and selenite reduction was determined by estimating the selenium concentration by atomic Characterization of Selenite-Tolerant Bacterium absorption spectrophotometry as described above, and bacterial The strain JS-2 was a Gram-positive, spore-forming, rod- growth by quantifying the protein content of the biomass. For this, the shaped, motile, facultative anaerobic bacterium. The temperature culture amended with required concentration of selenite ions was o - o o incubated at different temperatures at 200 rpm for 48 h. The optimum range for growth was 15 C 42 C (optimum 37 C) and pH - pH range was evaluated for selenite reduction by adding HCl/NaOH values 6 11 (optimum pH 7.5). Strain JS-2 showed high salt (1N) to Tryptic Soya Broth to obtain the desirable pH. Percentage tolerance and could grow at 12.5% NaCl concentration. (%) concentration of residual concentration of selenite in the culture The strain was positive for gelatin hydrolysis, starch broth was measured as (Initial conc.- Final conc.)/Initial conc.×100. hydrolysis, and fermentation of glucose, catalase, and The effect of different oxyions on the growth and selenite reduction oxidase. Acid was produced from carbohydrates, namely, was determined by supplementing the culture media with various dextrose, fructose, maltose, mannitol, mannose, salicin, oxyions like sulfate (Na2SO4), sulfite (Na2SO3), nitrate (NaNO3), nitrite sucrose, trehalose, and xylose. Phylogenetically, the strain (NaNO2), thiosulfate (Na2S2O3·5H2O), molybdate (Na2MoO4·2H2O), JS-2 showed the highest sequence similarity of 99.8% with thiocyanate (NaSCN), tungsten (Na2WO4·2H2O), and chromate Bacillus sp. (GenBank: HQ330528). (K2Cr2O7) along with selenite oxyions at 2 mM concentration. The selenite concentration in the culture broth and growth of bacteria 2- were determined [6]. Evaluation of Selenite (SeO3 ) Reducing Ability of Bacillus sp. (Strain JS-2) 2- Electron Microscopy and Elemental Analysis To determine the toxicity of selenite (SeO3 ), the growth The effect of toxic selenite ions was examined using scanning electron profile was studied by addition of different concentrations microscopy (SEM) and transmission electron microscopy (TEM) as of sodium selenite (0.5 mM-10 mM) in the growth medium described earlier by Dhanjal and Cameotra [6]. under aerobic conditions. The total protein content was SELENITE STRESS ELICITS PHYSIOLOGICAL ADAPTATIONS IN BACILLUS SP. (STRAIN JS-2) 1186

oxyions in the culture broth within a period of 24 h. Therefore, the ability of strain JS-2 to rapidly reduce 2- soluble and toxic selenite (SeO3 ) to insoluble and 0 unavailable Se highlights it as a promising exploitable microbe for bioremediating selenium-laden effluents/soil.

Effects of Abiotic Factors on Selenite Reduction: Temperature, pH, and Presence of Various Oxyions Abiotic factors like temperature, pH, or similar oxyions influence the microbial growth and their activities. The temperature range of 30-42oC was observed to be most favorable for the growth and selenite reduction. Maximum growth as well as selenite reduction was observed at 37oC. Temperatures ≥45oC and ≤15oC severely affected the bacterial growth, thereby resulting in a decrease in selenite reduction activity (Fig. 2A). Fig. 2B clearly shows a direct positive correlation between selenite reduction and pH. Below pH 6, the microbial growth was severely affected and selenite reduction was suppressed as well. In Bacillus sp., selenite reduction was appreciable between pH 6 and 10, whereas maximum selenite reduction occurred between pH 6 and 8. In a previous study on Bacillus sp., the biological transformation of selenite to volatile selenium was observed to be dependent on several factors like incubation temperature, pH, incubation period, and substrate concentration [34]. Fig. 1. Growth and selenite reducing ability of Bacillus sp. (strain Another study demonstrated the effect of temperature and JS-2). dissolved oxygen on selenite reduction by Shewanella (A) Growth profile of Bacillus sp. (strain JS-2) under selenite stress. (B) sp. HN-41 [20]. There are several reports on various The relationship between cell growth expressed as the concentration of Bacillus parameters that affect selenite reduction in both aerobic cellular protein and selenite reduction in sp. (strain JS-2). and anaerobic conditions by microorganisms [10, 16, 39]. The reduction of selenium oxyions (selenate and selenite) estimated at regular time intervals and correlated with the by Pseudomonas stutzeri was observed under aerobic growth of the microbe, as the optical interference of red conditions between pH 7.0 and 9.0 and at temperatures of 0 Se particles made it difficult to measure the growth by 25 to 35oC [22]. However, it can be stated that selenite spectroscopy. The growth profile in the presence of reduction is an enzyme-mediated process, and extreme selenite (0.5-10 mM) was comparable to that of control temperature and pH changes may affect the enzyme lacking selenite oxyions (Fig. 1A). Graphically, representation ionization rate, and change in protein conformation, and of growth is shown in the presence of 5 mM and 10 mM consequently affect the enzymatic activity of the reductases. selenite for clarity; however, growth studies were done in In the natural environment, metal-contaminated sites are the presence of selenite concentrations ranging from 0.5- often found to be co-contaminated with various other 10 mM, as mentioned above. There was rapid decrease of inorganic ions. Therefore, the influence of different oxyions selenite oxyions in the culture broth within a period of (sulfate, sulfite, nitrate, nitrite, thiosulfate, molybdate, 24 h, as shown in Fig. 1B. Cultures challenged with toxic thiocyanate, tungsten, and chromate) on selenite reduction selenite oxyions begin to turn red within 8-10 h of was studied. In the case of Bacillus sp. (strain JS-2), incubation with corresponding decrease in selenite content chromate and tungsten ions significantly affected selenite of the culture broth. The significance of the growth profile reduction by the microbe (Fig. 2C). In most of industrial studies was to understand the response of the test organism wastewaters containing selenium compounds, nitrate and to high concentrations of selenite that are several orders other similar anions are also present at high concentration, of magnitude higher than usual levels present in the which may interfere with the reduction of selenium environment [7]. Apparently, high concentrations of oxyions. At relatively high concentrations, nitrate and selenite oxyions did not affect the growth of the bacterial nitrite have been shown to inhibit selenate and selenite isolate as indicated by the increase in protein level, and reduction [28], although the inhibition was isolate there was rapid decrease in the concentration of selenite dependent [1]. In addition, because nitrate is a common 1187 Dhanjal and Cameotra

Fig. 2. Abiotic factors affecting selenite reduction. (A) Growth profile of Bacillus sp. (strain JS-2) and selenite reduction at different temperatures. (B) Growth profile of Bacillus sp. (strain JS-2) and selenite reduction at different pH values. (C) Effect of various anions on selenite reduction by Bacillus sp. (strain JS-2). pollutant in selenium-contaminated wastewater, studying X-ray (EDX) analysis of the spherical particles produced the effect of nitrate and the denitrification intermediate, specific selenium absorption peaks at 1.37 keV (peak nitrite or selenate and selenite reduction is of interest. In SeLα), 11.22 keV (peak SeKα), and 12.49 keV (peak SeKβ) our study, as selenite reduction was not appreciably (Fig. 3B). affected by the co-existence of nitrate and sulfur oxyions. Transmission electron microscopic (TEM) analysis These findings suggest that selenium, nitrate, and sulfur demonstrated that there is an accumulation of intracellular have different reductive pathways in this bacterium. The deposits inside the cells that were grown in the presence of 0 reductive pathway of selenium in this bacterium may toxic selenite ions. Such accumulation of Se crystals is in function as a system for the detoxification of selenium accordance with earlier reports on bacterial reduction of oxyanions, which may be independent of other reduction selenite [6, 32, 35]. However, interestingly, these electron- pathways related to nitrate and sulfur. dense deposits were seen in the cytoplasm, periplasmic space, and on the cell wall of bacterial cells (Fig. 3C). The Determining the Location of Transformed Selenium by chemical microanalysis (TEM-EDX) of reddish colonies Electron Microscopy of the bacterial isolates grown in the presence of selenite Scanning electron microscopic (SEM) studies of the revealed cytoplasmic and surface attached electron-dense 0 bacterial isolate grown in the presence of selenite ions Se granules. The EDX spectrum showed specific selenium 0 were done to study the effect of toxic selenite ions on the absorption peaks (Fig. 3D). Adherence of Se crystals to bacterial cells. In Bacillus sp. (strain JS-2), several irregular the bacterial cell membrane during anaerobic reduction and spherical deposits were observed, which adhered to was recently reported in the case of selenite reduction by the bacterial cell surface (Fig. 3A). The Energy Dispersive Shewanella oneidensis [19]. Similarly, another report suggested SELENITE STRESS ELICITS PHYSIOLOGICAL ADAPTATIONS IN BACILLUS SP. (STRAIN JS-2) 1188

Fig. 3. Electron Microscopic Studies. (A) Scanning electron micrograph of the Bacillus sp. (strain JS-2) grown in the presence of toxic selenite oxyions. The selenite stressed cells show the presence of granular deposits of elemental Se. (B) Characteristic Se peaks in EDX spectrum confirm the round spherical deposits as element selenium. (C) 0 Transmission electron micrograph showing the intracellular and extracellular depositions (indicated by arrows) of elemental Se in the cells. (D) EDX spectrum of electron-dense particles, showing characteristic selenium peaks. that the reduction of selenite occurs close to the membrane, cells, which was further studied by side scattering using possibly due to a membrane- associated reductase, and that flow cytometry. The bacterial cells exposed to toxic selenite the particles are rapidly expelled by a membrane efflux ions were analyzed for the difference in granularity at pump [23]. different time intervals. There was a gradual increase in the granularity of the cell population exposed to selenite ions. Flow Cytometric Studies At 12 h of growth, a marginal shift of the test population The intracellular deposits of elemental selenium in the was observed as compared with the control cell population. bacterial cells may lead to change in the granularity of the At 24 h and 36 h of growth, the shift of the test population 1189 Dhanjal and Cameotra was quite prominent. After 48 h of cell growth in the presence niches [18]. In addition, exopolymers serve as biosorbing of toxic selenite ions, there was a significant shift in the agents by accumulating nutrients from the surrounding test population of the cells, indicating increased granularity environment and also play a crucial role in biosorption of of the cells (Fig. S1). However, there was no change in metals. Therefore, with an aim to directly visualize the the size of the cells as observed by forward scatter plots exopolysaccharides secreted by the bacterial cells under (Fig. S2). The increase in granularity of the cells of the test selenite stress, the cell suspension was stained with population accompanied by reduction of selenite ions to lectin PHA-L conjugates (Alexa Fluor 594-conjugated; 0 red elemental selenium Se signifies the accumulation of Molecular Probes, Invitrogen) for fluorescence detection 0 Se inside the cells, which was observed in transmission of exopolysaccharides. In Bacillus sp. (strain JS-2), cells electron micrographs. In the literature, there are no reports grown in the presence of toxic selenite ions were found to on the study of granularity of metal-transforming bacteria. secrete exopolysaccharides as compared with the control The interesting results obtained in our study suggest that cells, which were not exposed to selenite ions during their flow cytometry would be a useful technique to study the growth period (Fig. 4). kinetics of selenium transformation by bacteria. The Exopolysaccharides are a complex mixture of biopolymers monitoring of the change in granularity of the cells along comprising polysaccharides, proteins, nucleic acids, uronic with selenium reduction can be related to better understanding acids, humic substances, lipids, etc. These are polyionic in of the mechanism of selenium transformation by the nature and form complexes with metal ions, resulting in microorganisms. metal immobilization with the exopolymeric matrix [2, 25, 6+ 30]. In a previous study, it was observed that Cr -resistant Exopolysaccharide Secretion Under Selenite Stress isolates produced high amounts of EPS, and sensitive Exopolysaccharides or extracellular polysaccharides are isolates produced low amounts of EPS. It was shown that 6+ known to protect bacterial cells from desiccation, aid in Cr is an important stress factor that increases EPS cellular aggregation and adhesion, provide resistance to production in cyanobacteria [29]. Therefore, toxic substances harmful exogenous materials or other environmental stresses, like metal ions may stimulate the production of EPS. In including host-immune responses, and produce biofilms, context to selenium, various studies have been reported thus enabling the cells to colonize special ecological involving the interaction of extracellular polymeric

Fig. 4. Exopolysaccharide secretion by Bacillus sp. under selenite stress. The control panel shows the absence of exopolysaccharides in the cells that were grown without the addition of selenite in the medium, as compared with the test population. The rod-shaped structures are the bacterial cells. SELENITE STRESS ELICITS PHYSIOLOGICAL ADAPTATIONS IN BACILLUS SP. (STRAIN JS-2) 1190 substances (EPS) and selenium ions. The selenium-tolerant marked by synthesis of branched fatty acids such as C12:0 exopolysaccharide-producing bacterial strain Enterobacter iso, C16:0 iso, and C17:0 anteiso. These observations cloacae, when grown in the presence of selenite ions, clearly indicate a potentially important role of saturated, transformed the inorganic selenite into organic forms and unsaturated, and branched fatty acids in determining the produced selenium-enriched exopolysaccharide (Se-ECZ- bacterial adaptation towards selenite-induced toxicity. These EPS-1), which acted as a potent immunomodulatory agent fatty acids may regulate the membrane fluidity under metal [40]. It has also been reported that algae Nostoc linckia is stress conditions during adaptation [13, 31]. An earlier able to accumulate Cu, Zn, and Se from the environment, report has shown a nearly exclusive role of branched fatty and exopolysaccharides are reported to play a dominating acids for adaptations towards environmental toxicity, where role in the accumulation of these metals [37]. Another the ratio of anteiso/iso fatty acid was used as one of the study showed that optimal concentrations of Se are required most important determinants for bacterial cell membrane in the culture medium for yield of immunostimulatory- fluidity and consequently their adaptation towards active selenated exopolysaccharides in Shiitake mushroom environmental stress [4]. The observation for the putative [38]. Therefore, the presence of selenite ions in the growth role of saturated fatty acid and unsaturated fatty acid is that medium may stimulate the secretion of exopolysaccharides they can regulate adaptive response. The increased ratios by the Bacillus sp., as observed in our study. of saturated fatty acids to unsaturated fatty acids probably suggest the increase in membrane fluidity resulting in easy Effect of Toxic Selenite Ions on Cellular Fatty Acids expulsion of precipitated granular elemental selenium 0 Earlier studies have demonstrated the bacterial adaptations (Se ) from the cells. to be articulated via alteration in cell morphology and The present study reveals that microorganisms employ changes in the total cellular fatty acid composition [17, different mechanisms to achieve tolerance towards toxic 36]. The cellular fatty acid analysis showed significant selenite oxyions. In particular, the strains that are tolerant alterations in the fatty acid composition with the presence to high concentrations of metals exhibit coordinated of additional saturated fatty acids C9:0, C10:0, C12:0, activities of several mechanisms of metal resistance, which C16:0iso, C17:0 anteiso, and C20:0, and the absence of include intracellular or extracellular sequestration, secretion unsaturated fatty acids C18:1ω9c, under selenite stress of substances like exopolysaccharides, metal ion chelators, (Table 1). The relative percentage concentration of total etc., to alter their microenvironment. saturated fatty acid and cyclic fatty acid increased The biotransformation of toxic soluble selenite ions to significantly, whereas the amount of total unsaturated fatty insoluble nontoxic red precipitate by the microbe Bacillus acids decreased when the strains were exposed to selenite sp. (strain JS-2) seems to be the most obvious microbial activity stress. The adaptation towards the toxic selenite ions is observed to combat selenium toxicity. This bioaccumulation of reduced selenium inside the cells leads to increased granularity of the cells, as determined by flow cytometry

Table 1. Fatty acid profile of Bacillus sp. (strain JS-2). studies. Apart from this, some of the physiological adaptations are secretion of exopolysaccharides and change in cellular Control Test (with Fatty acid type (without selenite) 2mM selenite) fatty acids, which probably aid in the biotransformation of selenite oxyions. This study helps to gain insights into some Saturated fatty acids of the physiological mechanisms adapted by microorganisms C9:0 ND 1.02 to sustain in toxic environments, which could be exploited C10:0 ND 1.94 to understand the vast metabolic potential and unique C12:0 ND 2.18 C14:0 3.13 4.02 growth characteristics of these microbes in the presence of C15:0 iso 22.43 16.39 toxic metals/metalloids. C15:0 anteiso 11.22 12.12 C16:0 iso ND 2.31 C16:0 27.94 25.58 Acknowledgment C17:0 iso 5.05 6.83 C17:0 anteiso ND 2.69 We are thankful to the Council of Scientific and Industrial C18:0 21.81 20.82 Research (CSIR), India for the financial support. C20:0 ND 4.11 Unsaturated fatty acids C18:1ω9c 8.41 ND REFERENCES Values represent percentages; ND, not detected. Bold fonts show the distinct differences in fatty acid composition between cells grown with and 1. Adams, D. J. and T. M. Pickett. 1998. Microbial and cell-free without the selenite oxyions. selenium bioreduction in mining waters, pp. 479-499. In W. T. 1191 Dhanjal and Cameotra

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