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Received: 28-Mar-2014; Revised: 05-Sep-2014; Accepted:14-Oct-2014
This article has been accepted for publication and undergone full peer review but has not been through the copyediting,
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Record. Please cite this article as doi: 10.1002/pmic.201400113.
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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
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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)
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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
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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
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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
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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.
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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
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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
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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.
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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.
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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.
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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
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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
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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
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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
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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.
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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
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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.
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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 + - + -
Control Tellurite + - + -
Control Tellurite + - + -
Metabolism Protein homeostasis
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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