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

sp. was challenged with high concentrations of toxic

selenite ions. The microbe was found to transform the

toxic, soluble, colorless selenite (SeO3

2-) oxyions to nontoxic,

insoluble, red elemental selenium (Se0). This process of

biotransformation was accompanied by cytoplasmic and

surface accumulation of electron dense selenium (Se0)

granules, as revealed in electron micrographs. The cells

grown in the presence of selenite oxyions secreted large

quantities of extracellular polymeric substances (EPS).

There were quantitative and qualitative differences in the

cell wall fatty acids of the culture grown in the presence of

selenite ions. The relative percentage of total saturated

fatty acid and cyclic fatty acid increased significantly,

whereas the amount of total unsaturated fatty acids

decreased when the cells were exposed to selenite stress.

All these physiological adaptive responses evidently

indicate a potentially important role of cell wall fatty acids

and extracellular polymeric substances in determining

bacterial adaptation towards selenite-induced toxicity,

which thereby explains the remarkable competitiveness

and ability of this microbe to survive the environmental

stress.

Keywords: Selenite, physiological response, Bacillus sp.,

exopolysaccharides, cellular fatty acids

Microbial interactions with metals are unavoidable in the

natural environment. Therefore, it is not surprising that

microbes have developed means to use a few metals

beneficially and develop defenses against those that are

toxic. Microbes significantly affect the distribution of metals

in the environment and a number of studies emphasize the

role played by microbe/microbial communities in the

process of metal biotransformation [1, 8, 9]. “Geosymbiosis”

is the term used to express the inseparable association of

the geological process and the microbial activity. During

this activity, both the geosymbiotic partners are affected as

in the case of microbe-metal interactions [11]. Microbial

interactions with metals alter their physical or chemical

state, with metals also affecting the activity, growth, and

survival of the microbe/microbial community in metalliferous

environments [14, 21, 26], and this aspect of the interaction

(i.e., how metals effect the microbial physiology) is

substantially less investigated [27]. Selenium (Se), an

essential trace mineral, is of fundamental importance to

human health, being a component of selenoproteins, which

have important enzymatic functions although its toxicity

cannot be ruled out at elevated levels [33]. Se pollution is a

worldwide phenomenon and is associated with a wide range

of natural and human activities, ranging from agricultural

practices to technologically advanced industrial processes.

Naturally, selenium exists in a number of oxidation states

[+6, selenate (SeO4

2-); +4, selenite (SeO3

2-) oxyions; 0,

elemental selenium (Se0); and selenide (Se

2-)] in inorganic

form. The toxicity of these states is related to their degrees

of solubility in water and hence their bioavailability for

biogeochemical cycling. Selenate (SeO4

2-) and selenite

(SeO3

2-) are highly water soluble, and thus toxic, having

tendency to bioaccumulate. Elemental selenium Se0 is an

insoluble red precipitate, being biologically unavailable

and therefore nontoxic. Selenide (Se2-

) is a toxic gas and

seldom a biological threat as it gets readily oxidized to

insoluble elemental form in the presence of air. Among the

various selenium species, selenite (SeO3

2-) reduction has

attracted a great deal of attention as a potential compound

for microbial reduction owing to its high toxicity. High

concentrations of Se in contaminated agricultural soils and

drainage waters have resulted in identification of adapted

microbes that efficiently reduce selenate/selenite to red

elemental Se or methylated forms of selenium [3, 7, 12,

24]. In this line of research, the present work was planned

*Corresponding authorPhone: +91-172-6665-224; Fax: +91-172-2690585;E-mail: [email protected]

# Supplementary data for this paper are available on-line only athttp://jmb.or.kr.

1185 Dhanjal and Cameotra

to study the adaptive response of Bacillus sp. (strain JS-2)

to survive the selenium toxicity and determine its

biotransformation potential, as the genus Bacillus represents

one of the dominant taxa of soil bacteria.

MATERIALS AND METHODS

Microorganism and Culture Conditions

The strain JS-2 was isolated by enrichment of seleniferous agricultural

soil samples collected from Jainpur located in the Nawahshahr

district (latitude 31o07' N and longitude 76

o08' E) of Punjab, India.

The pure isolate was routinely cultured on Tryptic Soya Agar (TSA)

plates supplemented with 2 mM sodium selenite at 37oC.

Characterization of the Strain

Biochemical characterization of the strain JS-2 was performed following

standard methods as described in Bergy’s Manual of Systemic

Bacteriology, Vol. I and II, and Manual for the Identification of

Medical Bacteria by Cowan and Steel (2nd Ed., Cambridge

University Press) [5, 15]. For the 16S rRNA gene analysis, the

method described by Dhanjal and Cameotra [6] was followed.

Bacterial Growth Under Selenite Stress and Its Selenite Reducing

Ability

The effect of selenite on the growth of the bacterial isolate and

reduction of selenite by the bacterial isolate were studied as described

by Dhanjal and Cameotra [6]. Briefly, Se content in the supernatants

was determined by atomic absorption spectrophotometry (AA-6800,

Shimadzu) in hydride vapor generation mode, with a selenium

cathode lamp. An air-acetylene (oxidizing) flame was used and a

wavelength of 196 nm was chosen for the purpose of absorption of

incident light.

Effects of Temperature, pH, and Different Oxyions on Selenite

Reduction

The effect of temperature on growth and selenite reduction was

determined by estimating the selenium concentration by atomic

absorption spectrophotometry as described above, and bacterial

growth by quantifying the protein content of the biomass. For this, the

culture amended with required concentration of selenite ions was

incubated at different temperatures at 200 rpm for 48 h. The optimum

pH range was evaluated for selenite reduction by adding HCl/NaOH

(1N) to Tryptic Soya Broth to obtain the desirable pH. Percentage

(%) concentration of residual concentration of selenite in the culture

broth was measured as (Initial conc.- Final conc.)/Initial conc.×100.

The effect of different oxyions on the growth and selenite reduction

was determined by supplementing the culture media with various

oxyions like sulfate (Na2SO4), sulfite (Na2SO3), nitrate (NaNO3), nitrite

(NaNO2), thiosulfate (Na2S2O3·5H2O), molybdate (Na2MoO4·2H2O),

thiocyanate (NaSCN), tungsten (Na2WO4·2H2O), and chromate

(K2Cr2O7) along with selenite oxyions at 2 mM concentration. The

selenite concentration in the culture broth and growth of bacteria

were determined [6].

Electron Microscopy and Elemental Analysis

The effect of toxic selenite ions was examined using scanning electron

microscopy (SEM) and transmission electron microscopy (TEM) as

described earlier by Dhanjal and Cameotra [6].

Flow Cytometry

The relative changes in the cellular granularity and size of the cell

population under selenite stress were determined by Forward Scatter

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

centrifuged at 2,500 ×g for 10 min at 4oC. The cell pellet was gently

washed twice with phosphate-buffered saline (PBS), pH 7.2, and

resuspended in it for analysis by FACS. Culture without addition of

selenite oxyions served as the control.

Confocal Laser Scanning Microscopy (CLSM)

Selenite-stressed cells were washed with deionized water by

centrifugation at 2,500 ×g for 5 min at 4oC, and aliquots were stained

for 1 min with 20 µM SYTO9 (Molecular Probes, Invitrogen), a

nucleic acid dye that stains bacteria (green fluorescence). For

fluorescence detection of exopolysaccharides produced by bacteria,

the cell suspension was stained with lectin PHA-L conjugates (Alexa

Fluor 594-conjugated; 2 µg/ml; Molecular Probes, Invitrogen). Confocal

microscopy was performed by using a Nikon A1R inverted Confocal

Laser Scanning Microscope (CLSM) equipped with an Ar-ion laser

(488 nm) and a HeNe-laser (543 nm).

Whole-Cell Fatty Acid Profile

For cellular fatty acid analysis, strain JS-2 was grown with and

without addition of selenite oxyions for 24 h. Cells were harvested

and fatty acid methyl esters were prepared sequentially by

saponication, methylation, and extraction [36]. The extracted fatty

acid methyl esters were analyzed by gas chromatography (Hewlett

Packard Model 5890A, equipped with 5% phenylmethyl silicon

column [0.2 m × 25 m] and a flame ionization detector) followed by

the Microbial Identication System (MIDI, Inc., Newark, NJ, USA).

RESULTS AND DISCUSSION

Characterization of Selenite-Tolerant Bacterium

The strain JS-2 was a Gram-positive, spore-forming, rod-

shaped, motile, facultative anaerobic bacterium. The temperature

range for growth was 15oC-42oC (optimum 37oC) and pH

values 6-11 (optimum pH 7.5). Strain JS-2 showed high salt

tolerance and could grow at 12.5% NaCl concentration.

The strain was positive for gelatin hydrolysis, starch

hydrolysis, and fermentation of glucose, catalase, and

oxidase. Acid was produced from carbohydrates, namely,

dextrose, fructose, maltose, mannitol, mannose, salicin,

sucrose, trehalose, and xylose. Phylogenetically, the strain

JS-2 showed the highest sequence similarity of 99.8% with

Bacillus sp. (GenBank: HQ330528).

Evaluation of Selenite (SeO3

2-) Reducing Ability of

Bacillus sp. (Strain JS-2)

To determine the toxicity of selenite (SeO3

2-), the growth

profile was studied by addition of different concentrations

of sodium selenite (0.5 mM-10 mM) in the growth medium

under aerobic conditions. The total protein content was

SELENITE STRESS ELICITS PHYSIOLOGICAL ADAPTATIONS IN BACILLUS SP. (STRAIN JS-2) 1186

estimated at regular time intervals and correlated with the

growth of the microbe, as the optical interference of red

Se0 particles made it difficult to measure the growth by

spectroscopy. The growth profile in the presence of

selenite (0.5-10 mM) was comparable to that of control

lacking selenite oxyions (Fig. 1A). Graphically, representation

of growth is shown in the presence of 5 mM and 10 mM

selenite for clarity; however, growth studies were done in

the presence of selenite concentrations ranging from 0.5-

10 mM, as mentioned above. There was rapid decrease of

selenite oxyions in the culture broth within a period of

24 h, as shown in Fig. 1B. Cultures challenged with toxic

selenite oxyions begin to turn red within 8-10 h of

incubation with corresponding decrease in selenite content

of the culture broth. The significance of the growth profile

studies was to understand the response of the test organism

to high concentrations of selenite that are several orders

of magnitude higher than usual levels present in the

environment [7]. Apparently, high concentrations of

selenite oxyions did not affect the growth of the bacterial

isolate as indicated by the increase in protein level, and

there was rapid decrease in the concentration of selenite

oxyions in the culture broth within a period of 24 h.

Therefore, the ability of strain JS-2 to rapidly reduce

soluble and toxic selenite (SeO3

2-) to insoluble and

unavailable Se0 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].

Another study demonstrated the effect of temperature and

dissolved oxygen on selenite reduction by Shewanella

sp. HN-41 [20]. There are several reports on various

parameters that affect selenite reduction in both aerobic

and anaerobic conditions by microorganisms [10, 16, 39].

The reduction of selenium oxyions (selenate and selenite)

by Pseudomonas stutzeri was observed under aerobic

conditions between pH 7.0 and 9.0 and at temperatures of

25 to 35oC [22]. However, it can be stated that selenite

reduction is an enzyme-mediated process, and extreme

temperature and pH changes may affect the enzyme

ionization rate, and change in protein conformation, and

consequently affect the enzymatic activity of the reductases.

In the natural environment, metal-contaminated sites are

often found to be co-contaminated with various other

inorganic ions. Therefore, the influence of different oxyions

(sulfate, sulfite, nitrate, nitrite, thiosulfate, molybdate,

thiocyanate, tungsten, and chromate) on selenite reduction

was studied. In the case of Bacillus sp. (strain JS-2),

chromate and tungsten ions significantly affected selenite

reduction by the microbe (Fig. 2C). In most of industrial

wastewaters containing selenium compounds, nitrate and

other similar anions are also present at high concentration,

which may interfere with the reduction of selenium

oxyions. At relatively high concentrations, nitrate and

nitrite have been shown to inhibit selenate and selenite

reduction [28], although the inhibition was isolate

dependent [1]. In addition, because nitrate is a common

Fig. 1. Growth and selenite reducing ability of Bacillus sp. (strainJS-2).(A) Growth profile of Bacillus sp. (strain JS-2) under selenite stress. (B)

The relationship between cell growth expressed as the concentration of

cellular protein and selenite reduction in Bacillus sp. (strain JS-2).


pollutant in selenium-contaminated wastewater, studying

the effect of nitrate and the denitrification intermediate,

nitrite or selenate and selenite reduction is of interest. In

our study, as selenite reduction was not appreciably

affected by the co-existence of nitrate and sulfur oxyions.

These findings suggest that selenium, nitrate, and sulfur

have different reductive pathways in this bacterium. The

reductive pathway of selenium in this bacterium may

function as a system for the detoxification of selenium

oxyanions, which may be independent of other reduction

pathways related to nitrate and sulfur.

Determining the Location of Transformed Selenium by

Electron Microscopy

Scanning electron microscopic (SEM) studies of the

bacterial isolate grown in the presence of selenite ions

were done to study the effect of toxic selenite ions on the

bacterial cells. In Bacillus sp. (strain JS-2), several irregular

and spherical deposits were observed, which adhered to

the bacterial cell surface (Fig. 3A). The Energy Dispersive

X-ray (EDX) analysis of the spherical particles produced

specific selenium absorption peaks at 1.37 keV (peak

SeLα), 11.22 keV (peak SeKα), and 12.49 keV (peak SeKβ)

(Fig. 3B).

Transmission electron microscopic (TEM) analysis

demonstrated that there is an accumulation of intracellular

deposits inside the cells that were grown in the presence of

toxic selenite ions. Such accumulation of Se0 crystals is in

accordance with earlier reports on bacterial reduction of

selenite [6, 32, 35]. However, interestingly, these electron-

dense deposits were seen in the cytoplasm, periplasmic

space, and on the cell wall of bacterial cells (Fig. 3C). The

chemical microanalysis (TEM-EDX) of reddish colonies

of the bacterial isolates grown in the presence of selenite

revealed cytoplasmic and surface attached electron-dense

Se0 granules. The EDX spectrum showed specific selenium

absorption peaks (Fig. 3D). Adherence of Se0 crystals to

the bacterial cell membrane during anaerobic reduction

was recently reported in the case of selenite reduction by

Shewanella oneidensis [19]. Similarly, another report suggested

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).


that the reduction of selenite occurs close to the membrane,

possibly due to a membrane- associated reductase, and that

the particles are rapidly expelled by a membrane efflux

pump [23].

Flow Cytometric Studies

The intracellular deposits of elemental selenium in the

bacterial cells may lead to change in the granularity of the

cells, which was further studied by side scattering using

flow cytometry. The bacterial cells exposed to toxic selenite

ions were analyzed for the difference in granularity at

different time intervals. There was a gradual increase in the

granularity of the cell population exposed to selenite ions.

At 12 h of growth, a marginal shift of the test population

was observed as compared with the control cell population.

At 24 h and 36 h of growth, the shift of the test population

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)

Transmission electron micrograph showing the intracellular and extracellular depositions (indicated by arrows) of elemental Se0 in the cells. (D) EDX

spectrum of electron-dense particles, showing characteristic selenium peaks.


was quite prominent. After 48 h of cell growth in the presence

of toxic selenite ions, there was a significant shift in the

test population of the cells, indicating increased granularity

of the cells (Fig. S1). However, there was no change in

the size of the cells as observed by forward scatter plots

(Fig. S2). The increase in granularity of the cells of the test

population accompanied by reduction of selenite ions to

red elemental selenium Se0 signifies the accumulation of

Se0 inside the cells, which was observed in transmission

electron micrographs. In the literature, there are no reports

on the study of granularity of metal-transforming bacteria.

The interesting results obtained in our study suggest that

flow cytometry would be a useful technique to study the

kinetics of selenium transformation by bacteria. The

monitoring of the change in granularity of the cells along

with selenium reduction can be related to better understanding

of the mechanism of selenium transformation by the

microorganisms.

Exopolysaccharide Secretion Under Selenite Stress

Exopolysaccharides or extracellular polysaccharides are

known to protect bacterial cells from desiccation, aid in

cellular aggregation and adhesion, provide resistance to

harmful exogenous materials or other environmental stresses,

including host-immune responses, and produce biofilms,

thus enabling the cells to colonize special ecological

niches [18]. In addition, exopolymers serve as biosorbing

agents by accumulating nutrients from the surrounding

environment and also play a crucial role in biosorption of

metals. Therefore, with an aim to directly visualize the

exopolysaccharides secreted by the bacterial cells under

selenite stress, the cell suspension was stained with

lectin PHA-L conjugates (Alexa Fluor 594-conjugated;

Molecular Probes, Invitrogen) for fluorescence detection

of exopolysaccharides. In Bacillus sp. (strain JS-2), cells

grown in the presence of toxic selenite ions were found to

secrete exopolysaccharides as compared with the control

cells, which were not exposed to selenite ions during their

growth period (Fig. 4).

Exopolysaccharides are a complex mixture of biopolymers

comprising polysaccharides, proteins, nucleic acids, uronic

acids, humic substances, lipids, etc. These are polyionic in

nature and form complexes with metal ions, resulting in

metal immobilization with the exopolymeric matrix [2, 25,

30]. In a previous study, it was observed that Cr6+

-resistant

isolates produced high amounts of EPS, and sensitive

isolates produced low amounts of EPS. It was shown that

Cr6+

is an important stress factor that increases EPS

production in cyanobacteria [29]. Therefore, toxic substances

like metal ions may stimulate the production of EPS. In

context to selenium, various studies have been reported

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.


substances (EPS) and selenium ions. The selenium-tolerant

exopolysaccharide-producing bacterial strain Enterobacter

cloacae, when grown in the presence of selenite ions,

transformed the inorganic selenite into organic forms and

produced selenium-enriched exopolysaccharide (Se-ECZ-

EPS-1), which acted as a potent immunomodulatory agent

[40]. It has also been reported that algae Nostoc linckia is

able to accumulate Cu, Zn, and Se from the environment,

and exopolysaccharides are reported to play a dominating

role in the accumulation of these metals [37]. Another

study showed that optimal concentrations of Se are required

in the culture medium for yield of immunostimulatory-

active selenated exopolysaccharides in Shiitake mushroom

[38]. Therefore, the presence of selenite ions in the growth

medium may stimulate the secretion of exopolysaccharides

by the Bacillus sp., as observed in our study.

Effect of Toxic Selenite Ions on Cellular Fatty Acids

Earlier studies have demonstrated the bacterial adaptations

to be articulated via alteration in cell morphology and

changes in the total cellular fatty acid composition [17,

36]. The cellular fatty acid analysis showed significant

alterations in the fatty acid composition with the presence

of additional saturated fatty acids C9:0, C10:0, C12:0,

C16:0iso, C17:0 anteiso, and C20:0, and the absence of

unsaturated fatty acids C18:1ω9c, under selenite stress

(Table 1). The relative percentage concentration of total

saturated fatty acid and cyclic fatty acid increased

significantly, whereas the amount of total unsaturated fatty

acids decreased when the strains were exposed to selenite

stress. The adaptation towards the toxic selenite ions is

marked by synthesis of branched fatty acids such as C12:0

iso, C16:0 iso, and C17:0 anteiso. These observations

clearly indicate a potentially important role of saturated,

unsaturated, and branched fatty acids in determining the

bacterial adaptation towards selenite-induced toxicity. These

fatty acids may regulate the membrane fluidity under metal

stress conditions during adaptation [13, 31]. An earlier

report has shown a nearly exclusive role of branched fatty

acids for adaptations towards environmental toxicity, where

the ratio of anteiso/iso fatty acid was used as one of the

most important determinants for bacterial cell membrane

fluidity and consequently their adaptation towards

environmental stress [4]. The observation for the putative

role of saturated fatty acid and unsaturated fatty acid is that

they can regulate adaptive response. The increased ratios

of saturated fatty acids to unsaturated fatty acids probably

suggest the increase in membrane fluidity resulting in easy

expulsion of precipitated granular elemental selenium

(Se0) from the cells.

The present study reveals that microorganisms employ

different mechanisms to achieve tolerance towards toxic

selenite oxyions. In particular, the strains that are tolerant

to high concentrations of metals exhibit coordinated

activities of several mechanisms of metal resistance, which

include intracellular or extracellular sequestration, secretion

of substances like exopolysaccharides, metal ion chelators,

etc., to alter their microenvironment.

The biotransformation of toxic soluble selenite ions to

insoluble nontoxic red precipitate by the microbe Bacillus

sp. (strain JS-2) seems to be the most obvious microbial activity

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

studies. Apart from this, some of the physiological adaptations

are secretion of exopolysaccharides and change in cellular

fatty acids, which probably aid in the biotransformation of

selenite oxyions. This study helps to gain insights into some

of the physiological mechanisms adapted by microorganisms

to sustain in toxic environments, which could be exploited

to understand the vast metabolic potential and unique

growth characteristics of these microbes in the presence of

toxic metals/metalloids.

Acknowledgment

We are thankful to the Council of Scientific and Industrial

Research (CSIR), India for the financial support.

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Fatty acid type Control (without selenite)

Test (with 2mM selenite)

Saturated fatty acids

C9:0

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C12:0

C14:0

C15:0 iso

C15:0 anteiso

C16:0 iso

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5.05

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21.81

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