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Received: 28-Mar-2014; Revised: 05-Sep-2014; Accepted:14-Oct-2014

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Depletion of reduction potential and key energy generation

metabolic enzymes underlies tellurite toxicity in Deinococcus

radiodurans

Narasimha Anaganti1, Bhakti Basu1*, Alka Gupta1, Daisy Joseph2 and Shree Kumar

Apte1

1 Molecular Biology Division, Bhabha Atomic Research Centre, Mumbai - 400085,

India

2 Nuclear Physics Division, Bhabha Atomic Research Centre, Mumbai - 400 085,

India

* Author for correspondence: Dr. Bhakti Basu, Molecular Biology Division,

Bhabha Atomic Research Centre, Trombay, Mumbai – 400 085, India, Tel. No: +91-

22-25593887, Email: [email protected]

Running title: Mechanism of tellurite toxicity in D. radiodurans

Key words: Deinococcus radiodurans, tellurite reduction, ROS, reducing potential,

energy biogenesis

Abbreviations

cDNA, complementary DNA; DCFH-DA, 2'-7'-dichlorodihydrofluorescein diacetate; DCW,

dry cell weight; EDXRF, energy dispersive X-ray fluorescence; EF-TEM, energy filtered

transmission electron microscopy; ETF, electron transfer flavoprotein; GSH, glutathione;

KeV, kilo electron volt; LD50, lethal dose 50; MIC, minimum inhibitory concentration; qRT-

PCR, quantitative real time-PCR; ROS, reactive oxygen species; RSH, reduced thiols; TGY,

tryptone-glucose-yeast extract; Te, Tellurium; TerB, tellurium resistance protein B; TerD,

tellurium resistance protein D

mailto:[email protected]



Abstract

Oxidative stress resistant Deinococcus radiodurans surprisingly exhibited moderate

sensitivity to tellurite induced oxidative stress (LD50 = 40 μM tellurite, 40 min

exposure). The organism reduced 70% of 40 μM potassium tellurite within 5h.

Tellurite exposure significantly modulated cellular redox status. The level of ROS

and protein carbonyl contents increased while the cellular reduction potential

substantially decreased following tellurite exposure. Cellular thiols levels initially

increased (within 30 min) of tellurite exposure but decreased at later time points. At

proteome level, tellurite resistance proteins (TerB and TerD), tellurite reducing

enzymes (pyruvate dehydrogense subunits E1 and E3), ROS detoxification enzymes

(superoxide dismutase and thioredoxin reductase) and protein folding chaperones

(DnaK, EF-Ts and PPIase) displayed increased abundance in tellurite stressed cells.

However, remarkably decreased levels of key metabolic enzymes (aconitase,

transketolase, 3-hydroxy acyl-CoA dehydrogenase, acyl-CoA dehydrogenase,

electron transfer flavoprotein alpha and beta) involved in carbon and energy

metabolism were observed upon tellurite stress. The results demonstrate that

depletion of reduction potential in intensive tellurite reduction with impaired energy

metabolism lead to tellurite toxicity in D. radiodurans.

1. Introduction

Heavy metals, such as arsenic, chromium, cadmium, lead, mercury and tellurium,

are toxic to living organisms due to generation of intracellular ROS during reduction

of toxic metal oxides to non-toxic metals [1]. Tellurium is a rare element but its

readily soluble oxides are highly toxic to microflora even at < 1 µg/ml (4 µM)



concentration [2, 3]. Microorganisms employ several mechanisms such as intra/extra

cellular reduction, exclusion or volatilization to evade tellurite toxicity [2]. Metal

catalyzed oxidative stress has been envisaged as a leading cause of tellurite toxicity

when microorganisms attempt to reduce toxic tellurite (Te4+) to non-toxic metallic

tellurium (Te0). Metallic tellurium accumulates in intra/extra cellular black deposits as

an end product of tellurite reduction, rendering jet black color to tellurite challenged

cells [3-6].

Tellurite reduction is accompanied by generation of ROS such as hydrogen

peroxide, superoxide anions and hydroxyl radicals leading to increased protein

carbonyl content and thiobarbituric acid-reactive substances in E. coli [4, 7]. Several

specific knockout mutagenesis or individual protein studies have stressed upon the

involvement of tellurite resistance proteins, oxidative stress alleviation proteins and

NADH/NADPH producing metabolic enzymes in tellurite reduction in bacteria [5, 6,

8-11]. A proteomic comparison between tellurite sensitive (LD50 = 0.5 µM tellurite, 10

min exposure) and resistant (LD50 = 3.9 µM tellurite, 10 min exposure) strains of E.

coli revealed upregulation of Ter determinants in tellurite resistant strain or of

oxidative defense in tellurite sensitive strain, following exposure to 3.9 µM tellurite

[12]. Thus, available literature suggests oxidative stress as major determinant of

tellurite toxicity in tellurite sensitive organisms.

Deinococcus radiodurans, well known for its extreme gamma radiation

resistance, also exhibits extraordinary oxidative stress resistance owing to the

presence of highly efficient enzymatic and non-enzymatic small molecule

antioxidants [13]. Response to tellurite, however, remains unexplored in this

microbe. In the present study, Deinococcus radiodurans was found to be moderately

sensitive to potassium tellurite [minimum inhibitory concentration (MIC) = 40 µM



tellurite and LD50 = 40 μM tellurite, 40 min exposure] and quickly reduced it to

elemental tellurium intracellularly. We examined 40 µM tellurite mediated changes in

cellular redox status and proteome constitution. Tellurite exposure enhanced cellular

ROS level and protein carbonyl content by 3 fold but decreased reduction potential in

tellurite exposed cells as compared to control cells. Cellular thiols levels displayed an

initial increase till 30min and then decreased in response to tellurite reduction by

tellurite treated cells. At proteome level, tellurite resistance proteins, metabolic

enzymes involved in tellurite reduction, ROS detoxification proteins, transcriptional

regulators and protein renaturation components were found in higher abundance in

tellurite treated cells. However, level of key metabolic enzymes involved in pathways

of energy production and protein homeostasis components decreased in response to

tellurite exposure. In contrast, exposure to 4 µM tellurite produced only 1.5 fold

increase in ROS, no major proteomic changes, and complete reduction of tellurite in

3h resulting in growth recovery. Thus, metabolic impairments and perpetual

diversion of reduction potential for tellurite reduction contribute to sensitivity of D.

radiodurans to high concentrations of tellurite.

2. Materials and methods

2.1 Bacterial strain, growth and determination of MIC

Deinococcus radiodurans R1 strain ATCC BAA-816 was grown at 32°C in either

TGY or in minimal medium with 150 rpm agitation. The minimal medium [14] was

used wherein individual amino acids were replaced with 0.05% casamino acids.

Bacterial growth was monitored either by measuring turbidity (A600) or by determining

colony forming units (CFU) on TGY agar plates following 48 h of incubation at 32°C.

Minimum inhibitory concentration (MIC) of tellurite was determined as described



previously [15], using increasing concentrations of potassium tellurite (1 - 50 μg/ml,

hereafter referred as tellurite).

2.2 Tellurite exposure and uptake measurement

D. radiodurans cells were grown to late log phase in TGY, washed twice, transferred

to fresh TGY or minimal medium (A600 = 0.5 ± 0.05) containing 4 or 40 μM (1 or 10

µg/ml) tellurite and incubated under optimal growth conditions. Cells grown similarly

but without tellurite served as control for testing physiological parameters. Medium

containing tellurite but devoid of cells was used as control for tellurite uptake

measurements. At different time intervals, the tellurite concentration in the culture

supernatant was measured using diethyldithiocarbomate (DDTC) (Himedia, India)

reagent [16].

2.3 Energy dispersive X-ray fluorescence (EDXRF)

The control or 40 μM tellurite treated cells were harvested, washed and processed

further to obtain pellets which were used to acquire EDXRF spectra exactly as

detailed earlier [17].

2.4 Microscopic investigations and Elemental mapping

Tellurite exposed or unexposed (control) D. radiodurans cells were harvested,

washed and observed under light microscope (Carl Zeiss Axioskop 40) with 100X oil

immersion objective under hydrous conditions. Alternatively, the cells were fixed with

2.5% glutaraldehyde - 0.5% paraformaldehyde mixture (1:1), dehydrated with

increasing concentrations of ethanol and embedded in epoxy resin. Then 50 nm

sections were cut and examined under Carl Zeiss Libra 120 keV transmission



electron microscope without staining. Elemental mapping was performed on the

electron dense areas using Energy Filtered Transmission Electron Microscopy (EF-

TEM) as described earlier [18]. The slit width of the energy filter was set for Tellurium

specific edge of 572 eV and 631 eV peak maxima in the electron energy loss

spectrum. Three images were taken using iTEM software, first at 475 eV as high

contrast image, second at 553 eV (before tellurium specific electron energy loss) and

third at 631 eV (tellurium specific electron energy loss maxima). The final elemental

map was produced after background subtraction of 631 eV image from 553 eV

image.

2.5 Estimation of cellular redox status

Protein oxidation was determined using Oxy-Blot protein oxidation detection kit

(Millipore, India). The cellular proteins (100µg) from control and tellurite treated cells

were derivatized by dinitro-phenylhydrazine (DNPH) and resolved by 12% SDS-

PAGE. The oxidized proteins were immuno-detected using an anti-DNP antibody

followed by anti-IgG antibody tagged with alkaline phosphatase (Sigma, India). At

appropriate time intervals, the tellurite exposed or control cells were washed and

resuspended in PBS for ROS, redox potential and RSH estimation. ROS estimation

was carried out as described earlier [19]. The D. radiodurans cells were treated with

2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA) (5µM/ml) for 30 min at 32°C

and then exposed to tellurite (4 or 40 μM). At appropriate time intervals, DCFH-DA

fluorescence was monitored fluorometrically (Jasco, FP-6500, Japan; λex 488nm/ λem

520nm). Redox potential was determined as described previously [20]. 3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, 0.5 mg/ml) was added

to each cell suspension (100µl) and the incubation was continued for 1 hr at 37°C.



Formazan crystals were dissolved in DMSO (100 µl), at 37°C, for 30 min.

Absorbance was measured at 550 nm using multi-channel plate reader (Bio-Tek

instruments, India). The GSH content of control or tellurite treated cells was

estimated as detailed earlier [21]. Monobromobimane was added to the cell

suspension (40 µM) and the cells were incubated for 30 min at 37°C. The cells were

washed, resuspended in 100µl of PBS and the fluorescence was measured using

Infinite-M200 plate reader (TECAN, Switzerland, λex 398nm/ λem 490nm). The relative

levels of ROS, reduction potential or GSH were expressed per mg dry cell weight

(DCW). DCW measurements were carried out as described previously [22].

2.6 Resolution of cellular proteins and image analysis

Protein samples were prepared and resolved by two dimensional electrophoresis as

per the protocol detailed earlier [23]. The cellular protein extracts were prepared from

tellurite treated or control cells following 3h of tellurite (4 or 40 μM) exposure. Cellular

proteins (700 μg) were resolved by IEF (11 or 17cm IPG strip, pI 4-7, Bio-Rad, India)

by cup loading method followed by 14% SDS-PAGE. The gels were stained by CBB

G250 and imaged by Dyversity-6 gel imager (Syngene, UK) using GeneSnap

software (Syngene, UK). The experiment was repeated at least three times to

generate three biological replicates and one 2D gel set was analyzed in each

experiment. First level match set from three biological replicates of 2D gels was

generated using PDQuest 2D analysis software (version 8.1.0, Bio-Rad). Minimum

correlation coefficient value between the replicate sets was 0.7. Spot detection and

matching between replicate gels were done in automatic detection mode, followed by

manual editing to exclude spuriously detected spots. The spot densities were

normalized using local regression method. Statistical analysis was performed by



independent Student’s t-test and the protein spots with p values less than 0.05 were

considered as significantly modulated between control and tellurite treated cells. The

protein spots that displayed greater than ± 2.0 fold change were considered as

differentially expressed. The protein spots of interest were manually excised from the

gel and processed for identification.

2.7 Mass spectrometry and protein identification

Treatment of protein gel plugs for destaining, reduction, alkylation, in-gel trypsin

digestion and elution of oligopeptides was performed exactly as described earlier

[24]. Protein samples were subjected to mass spectrometry (UltraFlex III MALDI-

TOF/TOF mass spectrometer, Bruker Daltonics, Germany). The oligopeptides were

co-crystallized with CHCA (5 mg/ml in 0.1% TFA and 30% ACN) on target plate

(384-well stainless steel plate, Bruker Daltonics, Germany). The machine was

externally calibrated using Peptide calibration mix I (Bruker Daltonics, Germany) or

with the trypsin autodigest peptides. The mass spectra were generated in the mass

range of 600-4500 Da using standard ToF-MS protocol in positive ion reflection

mode. Laser was set to fire 150 times per spot. Peak list was generated using

FlexAnalysis software 3.0 (Bruker Daltonics, Germany) and mass spectra were

imported into the database search engine (BioTools v3.1 connected to Mascot,

Version 2.2.04, Matrix Science). Mascot searches were conducted using the NCBI

nonredundant database (released in March 2012 or later with minimum of 17612906

entries actually searched) with the following settings: Number of miscleavages

permitted was 1 (or 2 for spots Gap-2, Mdh-1, Rpl15, Ctc and Rps4); fixed

modifications such as Carbamidomethyl on cysteine, variable modification of

oxidation on methionine residue; peptide tolerance as 100 ppm; enzyme used as



trypsin and a peptide charge setting as +1. A match with D. radiodurans protein with

the best score in each Mascot search was accepted as successful identification. A

Mascot score of >65 with a minimum of 6 peptide matches was considered to be a

significant identification (p < 0.05) when sequence coverage was at least 25%.

2.8 RNA isolation and quantitative real-time PCR (qRT-PCR)

RNA was isolated from control or 40 µM tellurite treated cells at 30 min or 3h of

exposure using RNeasy kit (Qiagen) and its quality and quantity were determined

spectrophotometrically. RNA was treated with DNase (Fermantas) to remove

chromosomal DNA contamination. The cDNA was synthesized from RNA (3µg)

using Revertaid H- First strand cDNA synthesis kit (Fermantas). qRT-PCR was

performed as described earlier [25]. In brief, about 150 bp sequence from the genes

of interest was amplified using specific primer sets (Supporting information Table 3).

The PCR reaction (20 µl) containing cDNA template (1µl) in qARTA evagreen qPCR

mix (QARTA Bio) with gene specific primers (3 pM) was performed using Realplex 4

qRT-PCR (Eppendorf, India). The program details were 95°C, 2 min, followed by

94°C, 15 sec 55°C, 15 sec 72°C, 20 sec (40 cycles). Melting curve analysis

was performed at the end of amplification. Comparative CT method [26] was

employed to calculate relative expression levels of individual genes. CT values of

specific genes were normalized using CT value of gap gene whose intensity did not

change in tellurite treated cells as compared to control cells.



3. Results and discussion

3.1 Tellurite sensitive D. radiodurans reduces tellurite and sequesters metallic

tellurium in intracellular granules

D. radiodurans exhibited moderate tellurite sensitivity and exposure of the organism

to 40 μM tellurite completely inhibited growth on TGY agar plates (Data not shown).

Forty minutes exposure to 4 or 40 μM tellurite in liquid medium reduced the surviving

population of D. radiodurans by 50% (LD50). The cells exposed to 4 μM tellurite

displayed growth recovery after 3h as against 40 μM tellurite exposed cells which

showed exponential loss in viability with time (Fig. 1A). D. radiodurans cells removed

nearly 70% of 40 μM tellurite from the culture supernatant in 5 h and no further

increase in tellurite removal was observed up till 20h (Fig. 1A). In contrast, cells

exposed to 4 μM tellurite removed > 95% of tellurite in 3h (Fig. 1A) resulting in

growth recovery. In the absence of cells, the concentration of tellurite in the medium

did not change over a period of 20 h, indicating that the cells removed tellurite (Fig.

1A). Tellurium specific Kα and Kβ peaks with X-ray energies of 27.4 keV and 30.99

keV, respectively, were exclusively observed in the 40 µM tellurite challenged cells

(Fig. 1B). Tellurite (40 µM) exposed D. radiodurans cells turned black within 3 h and

displayed presence of distinct, large, black, intracellular granules that were visible by

light microscopy. The black granules comprised of crystalline nanorod like structures

as revealed by transmission electron microscopy (Fig 1C). Energy loss images

obtained by EF-TEM taken at tellurium specific edge (572 eV) clearly showed that

the electron dense deposits observed in zero loss image contained tellurium (Fig.

1D). Thus, D. radiodurans employed tellurite reduction to overcome tellurite toxicity

and accumulated metallic tellurium intracellularly.



3.2 Exposure to tellurite modulates cellular redox status in D. radiodurans

Tellurite reduction is coupled with generation of ROS that causes severe oxidative

stress in many tellurite sensitive organisms and the process is envisaged to utilize

NAD(P)H as reductant [3-6, 19]. Forty micromolar tellurite exposed cells had 3 and

7.5 fold more ROS at 3h and 20h, respectively as against marginal increase in ROS

(~ 1.5 fold) in 4 μM tellurite exposed cells (Fig 2A). Since exposure to 4 μM tellurite

did not elicit significant changes in ROS levels and resulted in its rapid reduction, all

further experiments were carried out at 40 μM tellurite concentration. Exposure to 40

μM tellurite for 3h resulted in 2.93 fold increase in protein carbonylation (Fig 2B).

However, cellular reduction potential was substantially decreased upon 40 μM

tellurite exposure (Fig 2C). Cellular reduced thiols (RSH) increased in the first 30 min

of 40 μM tellurite exposure, dropped to control levels by 2-3h and then decreased

further by 20h (Fig. 2D). Thus, tellurite consumed both, NAD(P)H and RSH, for its

reduction as observed in other organisms [8, 10]. The initial increase in RSH content

in D. radiodurans contrasts sharply with the reported immediate decrease or no

change in RSH content respectively in tellurite sensitive or tellurite resistant E. coli

[7, 8, 10]. Cellular thiols react with tellurite contribute to tellurite resistance [7]. High

level of thiols in the first 1h of tellurite exposure in D. radiodurans would promote

tellurite reduction. Thus, tellurite exposure elicited oxidative stress in D. radiodurans

and consumed reduction potential and thiols for its reduction.



3.3 Tellurite upregulated proteome contributes to functions that promote

tellurite reduction and improve defense against oxidative stress

D. radiodurans cells exposed to 4 µM tellurite did not display significant modulations

in proteome profile (data not shown), however, exposure to 40 µM tellurite

modulated the abundance of several proteins as revealed by 2D gel-based

comparative proteomics (Supporting Information Fig. 1). On an average, PDQuest

2D analysis software detected 553 or 535 spots from biological triplicate 2D gels of

control or tellurite treated samples, respectively. Among these, the levels of 25 or 17

spots respectively displayed increased (fold change ≥ 2.0, p = <0.05) or decreased

(fold change ≤ -2.0, p = <0.05) abundance in tellurite treated cells as compared to

control cells (Supporting Information Table 1). Sixty seven proteins were identified by

mass spectrometry (Supporting Information Fig. 1 and Table 2, Supporting MS data).

Proteome level modulation of 10 genes was validated at transcript levels by

quantitative RT-PCR analysis (Supporting Information Table 3).

Following tellurite exposure, the levels of tellurite resistance/reduction

proteins, ROS detoxification enzymes, transcriptional regulators and protein

renaturation components prominently increased (≥ 2.0 fold change). Proteins

involved in tellurite resistance/reduction were tellurite resistance proteins TerB and

TerD, pyruvate dehydrogenase subunit E1 and dihydrolipoamide dehydrogenase E3

(Fig. 3A and Supporting Information Table 2). Among these, expression of terD,

aceE and lpdA genes was analyzed at transcript levels and was found be increased

(Supporting Information Table 3). Upregulation of Ter proteins has been reported

following exposure to tellurite or oxidative stress [12, 27] . Microbes harboring ter

genes are found to be tellurite resistant [12, 27] and in E. coli, TerB maintains the



intracellular reducing environment [6]. Pyruvate dehydrogenase complex possesses

tellurite reduction activity and confers tellurite resistance [8].

The abundance of ROS detoxification proteins thioredoxin reductase and superoxide

dismutase increased following tellurite exposure (Fig. 3B and Supporting Information

Table 2) while levels of catalase, thiosulfate sulfurtransferase, cysteine desulfurase

activator ATPase, stress response protein DR1199, WrbA and LuxA displayed minor

modifications following tellurite exposure (Supporting Information Fig. 1 and Table 2).

Thioredoxin reductase (DR1982) of D. radiodurans reduces oxidized thioredoxin

using NADPH [28] and its deletion mutant is hypersensitive to tellurite stress [15].

Reduced thioredoxin contributes to the pool of reduced cellular thiols and in

presence of ter determinants, contributes to tellurite resistance [11]. Tellurite

exposure generates superoxide radicals, upregulates superoxide dismutase

expression and activity whereas sodAsodB mutation imparts hypersensitivity to

tellurite in E. coli [4, 9]. Catalase of Staphylococcus epidermidis has been shown to

possess NADPH dependent tellurite reductase activity [10]. Hypothetical protein

DR1199 has been implicated in protection of D. radiodurans against oxidative stress

[29]. Proteins WrbA and LuxA possess redox activities. Two oxidative stress

responsive transcriptional regulators, PspA and CspA, were found to be upregulated

in tellurite exposed cells (Fig. 3B). The phage shock protein responds to

extracytoplasmic stress that reduces the energy status of the cell [30]. Two fold

induction of cspA gene has been reported at transcript level in response to tellurite

[4].

Proteins such as DnaK, EF-Ts and peptidyl-prolyl cis-trans isomerase, involved in

renaturation or folding of denatured proteins were observed at higher levels in

tellurite exposed cells (Fig. 3C and Supporting Information Table 2). Increased



transcript abundance was also observed for dnaK and DR1314 genes (Supporting

Information Table 3). These proteins might assist in restoration of functional

competency of oxidatively damaged proteins. Three spots differing in molecular

mass and pI, and highly enhanced in tellurite treated cells, were identified as

hypothetical protein DR1314 (Fig 3D and Supporting Information Table 2). DR1314

apparently protects D. radiodurans from heat shock [31]. Cell division protein FtsZ

and uncharacterized protein DR2377 were present in higher abundance in tellurite

treated cells (Fig 3D and Supporting Information Table 2).

3.4 Key metabolic enzymes and protein homeostasis components display

lower abundance following tellurite exposure

The levels of key metabolic enzymes such as aconitase (Acn), transketolase (Tkt), 3-

hydroxy acyl-CoA dehydrogenase (DR2477), acyl-CoA dehydrogenase (DR2361),

and electron transfer flavoproteins alpha and beta (EtfA and EtfB), decreased

following tellurite exposure (≤ -2.0 fold change, p = < 0.05) (Fig 4A and Supporting

Information Table 2). Concomitantly, acn, DR2477 and etfB transcripts decreased at

30 min or at 3h, except acn which showed slight increase at 3h in tellurite treated

cells (Supporting Information Table 3). Other identified metabolic enzymes -

fructose-bisphosphate aldolase, enolase, flavoprotein subunit of succinate

dehydrogenase, fumerate hydratase, malate dehydrogenase, glyceraldehyde-3-

phosphate dehydrogenase, citrate synthase and isocitrate dehydrogenase, acetyl-

CoA acetyltransferase, acetyl-CoA acyl transferase, V-type ATP synthase (A, B and

C subunits) and nucleoside diphosphate kinase, displayed minor alterations in their

levels following tellurite exposure (Supporting Information Fig. 1 and Table 2).

Aconitase is sensitive to oxidative or tellurite stress [5] and its abundance was found



to be significantly reduced, in this study. A decrease in the levels of these important

enzymes involved in glycolysis, TCA cycle, pentose phosphate pathway, fatty acid

metabolism and energy metabolism may severely affect regeneration of molecular

energy [NAD(P)H and ATP] in tellurite challenged D. radiodurans.

Protein homeostasis components such as the GTPase HflX, general stress protein

Ctc and methionine aminopeptidase were substantially downregulated (Fig 4B and

Supporting Information Table 2). At transcript level, hflX was upregulated while ctc

was upregulated at 30 min but downregulated by 5 fold at 3h, in tellurite treated cells

(Supporting Information Table 3). Other important proteins involved in protein

homeostasis – GroESL, DnaJ, EF-Tu, EF-G, ribosomal proteins, serine protease and

aminopeptidase showed no significant change in their levels (Supporting Information

Fig. 1 and Table 2). The down regulation of these mechanisms could partially impair

the cellular protein homeostasis.

4. Conclusions

Although oxidative stress resistant, D. radiodurans displayed unexpected sensitivity

towards tellurite and employed tellurite reduction to evade toxicity. The battery of

inherent enzymatic and non-enzymatic oxidative stress tolerance present in D.

radiodurans promoted rapid reduction of tellurite without significantly elevating ROS

level or eliciting metabolic response at low tellurite concentrations (4 μM). However,

40 μM tellurite elevated ROS levels and protein carbonyls and consumed reduction

potential and cellular thiols towards its own reduction. At proteomic level, the

organism responded by elevating the levels of proteins that are associated with

tellurite resistance. For example Ter proteins, pyruvate dehydrogenase complex and

ROS detoxification enzymes were upregulated following tellurite exposure, reflective



of the organism’s quick response to tellurite mediated oxidative stress and its

alleviation by tellurite reduction. However, diminished levels of key metabolic

enzymes involved in carbon and energy metabolism (Acn, Tkt, DR2477, DR2361,

Etf) decreased cellular reduction potential. Thus, consumption of reducing potential

for tellurite reduction far exceeded its generation, thereby, reducing the overall

energy status of the cells. Major findings of this study are summarized in a model

(Fig.5). Although, induction of oxidative stress response is associated with tellurite

resistance, reduced levels of GSH and reduction potential may impair tellurite

reduction and impart tellurite sensitivity [32]. In conclusion, perpetual diversion of

reduction potential for tellurite reduction and inability of the cells to keep up with this

extra demand due to suboptimal functioning of major metabolic pathways of energy

production, appear to be major factors integral to tellurite toxicity in D. radiodurans.

Acknowledgments

Deinococcus radiodurans strain R1 ATCC BAA-816 was kindly provided by K. W.

Minton and M. J. Daly, Uniformed Services University of the Health Sciences,

Bethesda, USA.



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Figure 1. Tellurite uptake by D. radiodurans. (A) Tellurite concentration in culture

medium in the presence or absence of D. radiodurans when exposed to 4 or 40 μM

tellurite. Survival of D. radiodurans exposed to 4 or 40 μM tellurite. (B) Detection of

two tellurium specific peaks (Kα at 27.4 KeV and Kβ at 30.9keV) exclusively in

tellurite exposed cells (continuous line) as compared to control cells (dotted line), by

EDXRF analysis. (C) The light or transmission electron microscopic pictures of D.

radiodurans following 3h exposure to 40 μM tellurite compared to unexposed cells

(control). Tellurium deposits are indicated by arrows. (D) EF-TEM micrographs

confirming presence of tellurium in black deposits in the cell.



Figure 2. Effect of tellurite exposure on cellular redox status (A) ROS levels were

monitored following addition of tellurite (4 or 40 µM, 0-20h) by DCFH-DA method. (B)

Protein carbonyls were detected by immuno-staining at 3h of tellurite exposure. In

brief, DNPH derivatized cellular proteins were resolved by 12% SDS-PAGE and

protein carbonyls were detected using anti-DNP antibody. (C) Cellular redox

potential was estimated by employing MTT assay and (D) RSH contents were

estimated using monobromobimane reagent. The data were derived from three

independent replicates and technical duplicates in each replicated experiment and

presented as means ± standard deviations.3



Figure 3. Tellurite responsive increase in abundance (≥ 2.0 fold, p < 0.05) of proteins

involved in (A) tellurite resistance/reduction, (B) response to oxidative stress, (C)

protein homeostasis and (D) proteins with unknown function. 2D protein profiles of

control and tellurite treated (40 µM) cells at 3h of tellurite exposure are shown in

Supporting Information Fig. 1. Identified proteins are marked with arrows in control or

tellurite panels. ‘+’ and ‘-’ signs at the top of the gel indicate acidic and basic sides of

the gel. Fold change values derived by PDQuest analysis are given below each spot.

TerB TerD AceE LpdA

1 2.69

1 2.45

1 2.41

1 2.45

1 2.83 1 2.2

1 3.91

1 4.82

1 2.1

DR1314 DR2377 FtsZ

1 20.87 1 6.35 1 2.18

1 2

3

1 2 3

DnaK PPIase EF-Ts

1 2.94

1 2.52

1 2.35

1 2.05

Control

Tellurite + - +

-

Control

Tellurite

Control

Tellurite + - +

- + - + -

Control

Tellurite + - +

-

Response to oxidative stress

Protein homeostasis

Unknown function

Tellurite resistance/reductio

n

TrxR SodA-1 PspA CspA

A.

B.

C.

D.



Figure 4. Tellurite responsive decrease in abundance (≤ -2.0 fold, p<0.05) of proteins

involved in (A) metabolism and (B) protein homeostasis. Other details were as

described in the legend to Figure 3.

Acn Tkt DR2477

1 -3.41

1 -2.78

1 -2.28

1 -3.34

1 -2.96

1 -2.16

DR2361 EtfA EtfB

1 -3.53

1 -5.93

1 -5.86

HflX Ctc Map

Control Tellurite + - + -



Metabolism Protein homeostasis



Figure 5. A proteome based model for response of D. radiodurans to tellurite

exposure. The tellurite (TeO3-2) and reduced metallic tellurium (Te°) are represented

by open and filled circles, respectively. The proteins or bio-molecules with enhanced,

reduced or constitutive abundance are shown in red, green or yellow, respectively.

For color information, please refer to web version. The protein abbreviations are as

detailed in Supporting information Table 2. The repressed (metabolism, translation

and folding, ETC or ATP synthesis) or induced (tellurite reduction, ROS production,

oxidation of RSH and redox potential or transcription) pathways are indicated by

dotted or solid bold lines, respectively. Other abbreviations used are: ETC – electron

transport chain, PPP – pentose phosphate pathway, TCA – tricarboxylic acid.

TeO3

-2

Te0

deposits

EtfB ETC

H+

EtfA As-B

As-C

H+

H+

ADP + Pi

ATP

As-A

ETC

TCA cycle PPP

Fatty acid

metab.

Glyco- lysis

KatA

e-

H2O

2

H2O

ROS

SodA

TetR

Ctc

GroEL DnaJ

DnaK HflX

Translation and folding DNA

mRNA

Proteins

GroES

EF-Ts

PPIase

Rpl Rps

EF-G EF-Tu

Transcription

CspA PspA

Acetyl CoA

ROS detoxification

LuxA WrbA

SufC

CysA-1 DR1199

TrxR TerB TerD

AceE LpdA

Te0

Metabolism

Tkt

Eno Gap FbaA

LpdA

AceE

AcdA-3 DR2477

AtoB-4 AtoB-3

SdhA FumC

Mdh Icd

CitA

Acn

NAD(P)H

NAD(P)+

RSH

R-SOH R-SS-R

depletion of reduction potential and key energy generation metabolic enzymes underlies tellurite...

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