Proteomic analysis reveals contrasting stress response to uranium in two nitrogen-fixing Anabaena strains, differentially tolerant to uranium

Aquatic Toxicology 182 (2017) 205–213

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Aquatic Toxicology journal homepage: www.elsevier.com/locate/aquatox

Proteomic analysis reveals contrasting stress response to uranium in two nitrogen-fixing Anabaena strains, differentially tolerant to uranium Bandita Panda, Bhakti Basu, Celin Acharya, Hema Rajaram, Shree Kumar Apte ∗ Molecular Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400085, India

a r t i c l e

i n f o

Article history: Received 30 August 2016 Received in revised form 30 November 2016 Accepted 2 December 2016 Available online 5 December 2016 Keywords: Uranium exposure Diazotrophic cyanobacteria Anabaena 7120 Anabaena L-31 Proteomic response Photosynthesis Oxidative stress

a b s t r a c t Two strains of the nitrogen-fixing cyanobacterium Anabaena, native to Indian paddy fields, displayed differential sensitivity to exposure to uranyl carbonate at neutral pH. Anabaena sp. strain PCC 7120 and Anabaena sp. strain L-31 displayed 50% reduction in survival (LD50 dose), following 3 h exposure to 75 ␮M and 200 ␮M uranyl carbonate, respectively. Uranium responsive proteome alterations were visualized by 2D gel electrophoresis, followed by protein identification by MALDI-ToF mass spectrometry. The two strains displayed significant differences in levels of proteins associated with photosynthesis, carbon metabolism, and oxidative stress alleviation, commensurate with their uranium tolerance. Higher uranium tolerance of Anabaena sp. strain L-31 could be attributed to sustained photosynthesis and carbon metabolism and superior oxidative stress defense, as compared to the uranium sensitive Anabaena sp. strain PCC 7120. Significance: Uranium responsive proteome modulations in two nitrogen-fixing strains of Anabaena, native to Indian paddy fields, revealed that rapid adaptation to better oxidative stress management, and maintenance of metabolic and energy homeostasis underlies superior uranium tolerance of Anabaena sp. strain L-31 compared to Anabaena sp. strain PCC 7120. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Uranium is known to adversely influence life through its chemical toxicity, rather than its radiological toxicity (Qi et al., 2014). Anthropogenic activities like uranium mining, nuclear fuel production and reprocessing, and waste disposal can lead to migration of uranium to groundwater, leading to contamination of soil and water and metal bioaccumulation (Taylor and Taylor, 2011). Contemporary agriculture utilizes phosphate fertilizers (e.g. superphosphates) and biofertilizers (e.g. diazotrophic cyanobacteria) in paddy fields for enhancing crop productivity. Rock phosphates of sedimentary origin, generally used for the production of phosphate fertilizers, contain 1.0–5.7 Bq g−1 uranium (238 U) and serve as a potential source of natural radionuclide contamination (Barisic et al., 1992). Uranium concentrations ranging from 15.9 to 35.8 mg L−1 have been reported in phosphate fertilizers in India (Lal et al., 1985; Yamazaki and Geraldo, 2003), while in other countries, it ranges from 3.2 to 221 mg L−1 (Yamazaki and

∗ Corresponding author. E-mail addresses: [email protected], [email protected] (S.K. Apte). http://dx.doi.org/10.1016/j.aquatox.2016.12.002 0166-445X/© 2016 Elsevier B.V. All rights reserved.

Geraldo, 2003). Environmental contamination with radionuclides like uranium causes serious problems for safer use of agricultural land, including resident microbes therein, thereby posing a serious threat to ecosystem and human health (Taylor and Taylor, 2011). To improve soil and crop productivity, physio-chemical removal of uranium is necessary, but is difficult and rather expensive (Barisic et al., 1992). Photosynthetic, cyanobacteria naturally abound in tropical fields and some of them are common occurrence in metal contaminated environments, which often accumulate and detoxify metal contaminants from soil and water (El-Enany and Issa, 2000; Noraho and Gaur, 1996; Gale and Wixson, 1979; Li et al., 2004; Kanamaru et al., 1994). Two strains chosen for the present study, Anabaena sp. strain PCC 7120 is a sequenced strain (http:// genomekazusa.or.jp/Cyanobase/anabaena) and Anabaena strain L31 is native to Indian paddy fields. These cyanobacteria regularly experience environmental stresses like salinity, desiccation, heat, salinity and heavy metals (Apte, 2001). The genome of Anabaena sp. strain PCC 7120 is known to harbour a plethora of genes encoding reactive oxygen species (ROS) mitigating enzymes, to alleviate oxidative stress induced by different stresses, including heavy metals (Banerjee et al., 2013; Acharya and Apte, 2013; Bhargava et al., 2008; Panda et al., 2014; Zhao et al., 2007; Banerjee et al., 2012a,b;

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Raghavan et al., 2011). Unravelling of the molecular mechanisms involved in heavy metal detoxification in these paddy-field inhabitants will help in establishing cost effective in situ bioremediation strategies for contaminated sites. Proteomic analyses are extensively useful in revealing the molecular mechanisms employed by the microbes to combat various stresses (Bhargava et al., 2008; Panda et al., 2014). The present study reports proteome modifications in two filamentous, photosynthetic, nitrogen-fixing cyanobacteria, Anabaena sp. strain PCC 7120 (hereafter referred to as Anabaena 7120) and Anabaena sp. strain L-31 (hereafter referred to as Anabaena L-31), exposed to sub-lethal (50% growth inhibitory or LD50 ) concentrations of uranium. A total of 79 proteins from Anabaena 7120 and 64 proteins from Anabaena L-31 were identified by MALDI mass spectrometry in response to uranium exposure, of which levels of 45 and 27 proteins, respectively were found to be differentially modulated in the two strains. The results provide an insight into the adaptive response of nitrogen-fixing Anabaena strains, involved in alleviation of uranium stress. 2. Materials and methods 2.1. Cyanobacterial strains, growth conditions and uranium treatment

untreated control cells were harvested by centrifugation at 5000 rpm for 5 min and resuspended in lysis buffer [1 mM Tris-HCl, pH 8 containing 1 mM phenylmethylsulfonyl fluoride (PMSF)]. The cells were lysed by freeze and thaw method (Panda et al., 2014) and proteins extracted from each strain by centrifugation at 14,000 rpm for 30 min at 4 ◦ C. The protein content was estimated using a Lowry protein estimation kit (Sigma, India). The protein extracts were subjected to simultaneous DNase I and RNase I (10 ␮g ml−1 each) for 1 h on ice in each case. For protein separation, proteins (∼1 mg) from each strain were concentrated under vacuum to 10 ␮L and then solubilized in 80 ␮L of rehydration buffer {8 M urea, 1 M thiourea, 4% 3-[(3-cholamidopropyl) dimethylammonio]1-1-propanesulfonate (CHAPS), 150 mM dithiothreitol (DTT), 2% immobilized pH gradient (IPG) buffer, traces of Bromophenol Blue} for 1 h at room temperature and loaded on the IPG strips, nonlinear pH 3–10, 17 cm (Bio-Rad, India) by cup-loading method following the protocol as described earlier (Panda et al., 2014). The iso-electric focusing (IEF) was performed with the Protean Isoelectric Focusing Cell (Bio-Rad, India) at 20 ◦ C and the 2nd dimensional resolution was performed by 14% SDS-PAGE. Each experiment was repeated three times in each strain resulting in 3 biological replicates each. 2.4. Gel imaging and spot analysis

Two cyanobacterial strains, namely Anabaena PCC 7120 and Anabaena L-31 were grown in combined nitrogen free BG-11 medium, pH 7.2 under continuous illumination (30 ␮E m−2 s−1 ) and aeration (3 L min−1 ) at 27 ± 2 ◦ C (Mackinney, 1941). Growth was measured in terms of chlorophyll a estimated in 90% methanolic extracts at 665.4 nm, as described earlier (Castenholz, 1998). For determination of LD50 for uranium, exponential phase cultures of the two strains were exposed to different concentrations of uranyl carbonate (50–300 ␮M) prepared as described earlier (Acharya et al., 2009), at a density of 10 ␮g chlorophyll a mL−1 , and incubated under shaking (120 rpm) and illumination (30 ␮E m−2 s−1 ) for specified periods of time (1–6 h). Survival following uranium exposure was evaluated by plating 100 ␮L culture aliquots on combined nitrogen free BG-11 agar plates and counting the number of colony forming units (CFU) after 10 days of incubation, under continuous illumination at 27 ± 2 ◦ C. Each experiment comprised of three replicates and the observed variation between the experiments was found to be less than 10%.

Gels were imaged by Dyversity-6 gel imager (Syngene, UK) using Gene Snap software (Syngene, UK). PD-Quest (version 8.1.0, Bio-Rad) was used to generate a first level match set from three biological replicates of 2D gels with a minimum correlation coefficient value of 0.6. Spot detection and matching between replicate gels were done in automatic detection mode, followed by manual editing to exclude those spots that were not present on all replicate gels. 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 altered between control and treated sample of three strains. Eighty three protein spots from Anabaena 7120 and 64 protein spots from Anabaena L-31 were excised from the gel, followed by repeated washing with 50 mM NH4 HCO3 /ACN, reduction with DTT (Sigma, India), alkylation with 55 mM iodoacetamide (Sigma, India). A standard protocol for in-gel trypsin digestion and elution of oligopeptides was used (Panda et al., 2014). Eluted peptides were vacuum concentrated to a final volume of 5 ␮L, if necessary.

2.2. Estimation of uranyl binding

2.5. Mass spectrometry and protein identification

Experimental media (BG-11 medium) were allowed to equilibrate for 30 min after addition of LD50 concentration (75 ␮M and 200 ␮M for Anabaena 7120 and Anabaena L31, respectively) of uranyl carbonate [UO2 (CO3 )2 ]2− . Experiments were initiated by inoculating an equivalent density (10 ␮g of chlorophyll a mL−1 ) of both the cyanobacterial strains, separately in the test solutions and incubating them under continuous shaking and illumination for 3 h. Aliquots (100 ␮L) were withdrawn at timed intervals and centrifuged at 13,000 rpm for 3 min. The supernatants (residual uranium) were acidified with 0.01 N HCl to prevent precipitation. The uranium loaded cell pellets (washed with distilled water to remove loosely bound uranium) were digested with 0.2% HCl at room temperature. Both the mineralized fractions were assayed for uranium using arsenazo III method (Savvin, 1961).

The eluted polypeptides were co-crystallized with ␣-cyano4-hydroxycinnamic acid (CHCA) matrix [5 mg ml−1 in 0.1% trifluoroacetic acid (TFA) and 30% acetonitrile (ACN)] on a 384well ground steel target plate (Bruker Daltonics, Germany). The Matrix-assisted laser desorption/ionization-Time of Flight (MALDI ToF/ToF) UltraFlexIII mass spectrometer was externally calibrated using Peptide calibration mix I (Bruker Daltonics, Germany) as per the manufacturer’s protocol. The analysis was carried out in positive ion reflector mode and the mass spectra were acquired with standard ToF-MS protocol in the mass range of 600–4500 Da. Spectra were acquired using FlexAnalysis software 3.0 (Bruker Daltonics) as detailed earlier (Panda et al., 2014). The mass spectra were imported into the database search engine (BioTools v3.1 connected to Mascot, Version 2.2.04, Matrix Science). Settings of Mascot search from NCBI nonredundant database (released Jan 2012 or later, at least 17910093 entries actually searched) or SwissProt database (released Jan 2012 or later, at least 539616 entries actually searched) chosen for identification were: number of missed cleavages permitted 1or 2; fixed modifications such as carbamidomethyl on cysteine; variable modification of oxidation

2.3. Extraction and separation of proteins by 2-D electrophoresis Uranium treated (75 ␮M and 200 ␮M Uranyl carbonate for Anabaena 7120 and Anabaena L-31 respectively, for 3 h) cyanobacterial cells (equivalent of ∼150 ␮g chl a) along with respective

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Survival (% control)

90 80

7120

70

L-31

60 50 40 30 20 10 0

5050 μM

100 150 200 250 300 75 μM 100 μM 150 μM 200 μM 300 μM

Uranyl carbonate (μM) Fig. 1. Survival of Anabaena 7120 (䊉) and Anabaena L-31 (䊏) in response to uranyl carbonate exposure. Three day old cultures of each Anabaena strain were concentrated to a chlorophyll a density of 10 ␮g ml−1 and exposed to 0–300 ␮M concentrations of uranyl carbonate, for 3 h. Survival was estimated by plating 100 ␮L culture aliquot on to BG-11 N− agar plates followed by incubation under constant illumination for 10 days and determination of the colony forming units (CFUs). The CFUs have been expressed as percent of unstressed control culture. The dotted line shows the LD50 dose (75 ␮M for Anabaena 7120 and 200 ␮M for Anabaena L-31), chosen for further proteomic analyses.

on methionine residue; peptide tolerance 50/100 ppm; enzyme used as trypsin and peptide charge setting as +1. A match with Anabaena 7120 protein with the best score in each Mascot search was accepted as successful identification. Protein identification was considered to be significant if minimum 2 of the following 3 criteria were fulfilled: a Mascot score of >60 (p < 0.05), a minimum match of 5 peptides, and sequence coverage ≥20%.

3. Results and discussions Heavy metals induce generation of reactive oxygen species (ROS) in cyanobacteria (Castielli et al., 2009). Actinide like uranium plays no biological role but exerts cellular toxicity through ROS mediated oxidative stress (Pourahmad et al., 2006; Shaki et al., 2012). Although no uranium transporters have as yet been discovered in microbes (Shaki et al., 2012), its transportation into the microbial cells has occasionally been demonstrated and is thought to be due to increased membrane permeability, resulting from uranium toxicity (Alan Di Spirito et al., 1983).

3.1. Physiological response of Anabaena strains to uranium exposure Physiological effect of uranium (uranyl carbonate) on Anabaena strains was studied by exposing cells to a range of uranium concentrations (50–300 ␮M) for different time periods. LD50 concentration of uranium was determined to be 75 ␮M for Anabaena 7120 and 200 ␮M for Anabaena L-31, for 3 h (Fig. 1 and Supplementary Fig. 1). After 3 h exposure, Anabaena 7120 cells bound 60% of the initially added 75 ␮M uranium (17.85 mg U g−1 of dry wt.). The more tolerant Anabaena L-31 displayed a binding of 66% of 200 ␮M uranium initially added (52.42 mg U g−1 of dry wt.) at 3 h. Similar values for uranium binding have been earlier reported in the unicellular (Synechococcus) and filamentous (Anabaena torulosa) cyanobacteria (Kanamaru et al., 1994; Acharya and Apte, 2013).

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3.2. Proteomic response of Anabaena strains to LD50 dose of uranium Comparative proteomic response of the two Anabaena strains to uranium was studied at their respective LD50 concentrations (Supplementary Figs. 2 and 3). On an average, 334 and 285 protein spots were respectively detected in biological triplicate gels of control or uranium exposed cultures of Anabaena 7120, (Supplementary Table 1). Corresponding numbers for Anabaena L-31 were 337 and 334 protein spots (Supplementary Table 1). Anabaena 7120 and Anabaena L-31 showed differential expression of 55 and 41 protein spots respectively, during exposure to uranium (Supplementary Figs. 2 and 3). A total of 79 proteins from Anabaena 7120 and 64 proteins from Anabaena L-31 were identified by MALDI mass spectrometry (Supplementary Table 2 and Supplementary mass spectrometry data). All the identified proteins were grouped into functional categories such as photosynthesis, carbon fixation and carbon metabolism, protein translation and folding, oxidative stress alleviation, DNA repair, and others. 3.3. Effect of uranium on photosynthesis Light harvesting phycobillisome complexes on the thylakoid membrane of cyanobacteria comprise of phycobiliproteins [allophycocyanin (Apc), phycocyanin (Cpc) and phycoerythrin (PE)] and associated linker proteins. Phycobiliproteins serve as the antenna proteins and play a major role in light harvesting and oxidation of water in cyanobacterial photosynthesis (Zhang et al., 2009). In the present study phycobilisome proteins were detected in 31 and 22 protein spots, respectively in Anabaena 7120 and Anabaena L-31 (Supplementary Figs. 2 and 3, and Supplementary Table 2). Phycobiliproteins were observed in multiple spots in both the strains, as has also been reported earlier (Panda et al., 2015, 2014; Zhang et al., 2009) and were represented by numbers (Table 1 and Supplementary Table 2). For interpretation of differential modulations in their abundance, the logic used was as described previously (Panda et al., 2014). In brief, (i) when molecular mass of the protein was close to the theoretical mass, higher/lower abundance was indicative of either increase in its synthesis/decrease in its degradation or decrease in its synthesis/increase in its degradation, respectively and (ii) when molecular mass of the protein was lower than its theoretical mass, increased/decreased level of processed protein was indicative of enhanced/reduced degradation of such proteins. At the LD50 uranium concentration of 75 ␮M uranium, the levels of phycobilisome core component ApcF (ApcF-1), phycobilisome rod core linker protein (CpcG1 & CpcG4) and phycocyanobilin lyase CpcS2 (CpcS2-1) were significantly increased in Anabaena 7120 while those of phycobilisome core- membrane linker (ApcE-1 and ApcE-2), phycoerythrocyanin alpha chain (PecA1) and phycoerythrocyanin associated rod linker protein (PecC-1) decreased (Fig. 2, Table 1, Supplementary Fig. 2 and Supplementary Table 2). Degradation of ApcF (ApcF-2) was unaffected but that of ApcE (ApcE-2), PecC (PecC-2) and phycocyanobilin lyase (CpcS2-2) was enhanced (Table 1, Supplementary Fig. 3 and Supplementary Table 2). Uranium exposure did not affect levels of allophycocyanin subunit alpha (ApcA), phycococyanin alpha chain (CpcA-1), phycococyanin beta chain (CpcB-1), phycococyanin associated rod linker protein (CpcC-1), CpcG2 (CpcG2-1), phycoerythrocyanin beta chain (PecB) and ATP synthase A (AtpA). However, it enhanced degradation of CpcA (CpcA-2), CpcB (CpcB-2), CpcC (CpcC-2 and CpcC-3) and CpcG2 (CpcG2-2 and CpcG2-3) (Fig. 2, Table 1, Supplementary Fig. 2 and Supplementary Table 2). Thus, the overall photosynthetic machinery was found to be strongly impaired by uranium exposure in Anabaena 7120. In Anabaena L-31 exposed to 200 ␮M uranium, abundance of ATP synthase F0F1 subunit alpha (AtpA) as well as subunit beta

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Table 1 Differentially expressed proteins in Anabaena PCC 7120 and Anabaena L-31, following exposure to 75 ␮M and 200 ␮M uranium, respectively. Gene ID Photosynthesis (Light Reaction) alr0021 alr0022 alr0020

all2327 alr0529 alr0528 alr0530 alr0534 alr0535 alr0537 alr0524 alr0525

Protein name

Spot name

Anabaena PCC7120 (Fold Change)

Anabaena L-31 (Fold change)

Allophycocyanin subunit alpha Allophycocyanin subunit beta Phycobilisome core-membrane linker protein

ApcA ApcB ApcE

ApcA (−3.33 ± 0.29) ↓ ApcB (−1.45 ± 0.24) ↓ ApcE-1 (−4.0 ± 0.14) ↓

Phycobilisome core component Phycococyanin alpha chain Phycococyanin beta chain Phycococyanin associated rod linker protein Phycobilisome rod core linker protein Phycobilisome rod core linker protein Phycobilisome rod core linker protein Phycoerythrocyanin alpha chain Phycoerythrocyanin associated rod linker protein

ApcF CpcA CpcB CpcC

No change No change ApcE-1 (−2.2 ± 0.09) ↓ ApcE-2 (−1.56 ± 0.4)↓ ApcE-4 (1.9 ± 0.32) ↑ ApcF-1 (2.29 ± 0.04) ↑ CpcA-2 (2.85 ± 0.29) ↑ CpcB-2 (1.50 ± 0.47) ↑ CpcC-2 (2.15 ± 1.25) ↑ CpcC-3 (1.19 ± 0.15) ↑ CpcG1 (1.45 ± 0.9) ↑

CpcG1

– No change CpcB-2 (−1.47 ± 0.56) ↓ CpcC-1 (−2.3 ± 0.28) ↓ CpcC-2 (3.2 ± 1.9) ↑ No change –

CpcG4

CpcG2-2 (1.31 ± 0.3) ↑ CpcG2-3 (2.8 ± 0.8) ↑ CpcG4 (1.51 ± 0.5) ↑

PecA

PecA-1 (−1.36 ± 0.5) ↓

No change

PecC

PecC-1 (−1.72 ± 0.2) ↓ PecC-2 (1.63 ± 1.1) ↑

PecC-2 (−1.51 ± 0.41) ↓ PecC-3 (−2.71 ±0.18) ↓ PecC-4 (−1.53 ± 0.34) ↓ NI

CpcG2

No change

all5292

Putative phycocyanobilin lyase

CpcS2

all0005

ATP synthase F0F1 subunit alpha ATP synthase F0F1 subunit beta

AtpA

CpcS2-1(2.19 ± 0.9) ↑ CpcS2-2 (1.99 ± 1.4) ↑ No change

AtpB

NI

AtpB-1 (1.63 ± 0.41) ↑ AtpB-2 (2.49 ± 0.3) ↑

RbcL

RbcL-a (2.05 ± 0.38) ↑ RbcL-b (2.08 ± 0.93) ↑ RbcL-c (1.8 ± 0.78) ↑ RbcL-1 (1.41 ± 0.28) ↑ RbcL-2 (−1.61 ± 0.4) ↓ RbcS (1.40 ± 0.62) ↑

RbcL-a (3.35 ± 0.76) ↑ RbcL-b (2.46 ± 0.5) ↑ RbcL-c (2.38 ± 0.75) ↑

NI No change

AhcY

Eno (−1.14 ± 0.46) ↓ Fda-1 (−1.88 ± 0.77) ↓ Fda-3 (1.63 ± 0.69) ↑ Prk (1.64 ± 0.05) ↑ Tkt-a (1.90 ± 0.86) ↑ Tkt-2 (2.2 ± 1.12) ↑ No change Pde1-2 (1.84 ± 1.32) ↑ AhcY (1.47 ± 0.68) ↑

all5039

Photosynthesis (Calvin Cycle)/CO2 fixation alr1524 Ribulose-1,5- bisphosphate carboxylase/oxygenase

AtpA (2.72 ± 1.0) ↑

Ribulose-1,5- bisphosphate carboxylase small subunit

RbcS

Enolase Fructose-1,6-Bisphosphate aldolase Phosphoribulokinase Transketolase

Eno Fda

Pde1

Pgdh

No change

Pgdh (1.8 ± 0.49) ↑

alr4416

Pyruvate Dehydrogenase E1 subunit S-adenosyl-l-homocysteine hydrolase D-3-phosphoglycerate dehydrogenase Cysteine synthase A

CysA

CysA-1 (1.75 ± 1.12) ↑ CysA-2 (1.59 ± 0.61) ↑

NI

Protein folding and translation alr1594

DNA directed RNA Polymerase

RpoB

ND

PNPase GroEL DnaK EF-Tu EF-G PPIase

PNPase (−1.53± 0.22) ↓ GroEL (−2.24 ± 0.44) ↓ DnaK (−1.23 ± 0.81) ↓ EF-Tu-2 (1.75 ± 1.64) ↑ ND PPIase (−1.19 ± 0.27) ↓

all4203 all4802

Polynucleotide phosphorylase Chaperon GroEL Heat shock protein DnaK Elongation Factor Elongation Factor G FKBP-type peptidylprolyl isomerase 50S ribosomal protein L5 Probable 30S ribosomal protein

RpoB-1 (2.56 ± 0.95) ↑ RpoB-2 (1.69 ± 0.9) ↑ No change GroEL (−1.92 ± 0.46) ↓ DnaK (−1.75 ± 0.39) ↓ No change No change NI

Rpl5 PSRP-3

Rpl5 (−2.32 ± 0.25) ↓ PSRP-3 (1.52 ± 0.28) ↑

NI NI

Oxidative stress alleviation alr2938

Super oxide Dismutase

SodB

No change

all0070 alr4641 all4121

Super oxide Dismutase Peroxiredoxin Ferredoxin—NADP+ reductase

SodA Prx FNR

FeSOD-1 (−1.29 ± 0.17) ↓ FeSOD-2 (−1.56 ± 0.43) ↓ NI Prx (1.95 ± 1.89) ↑ FNR (1.38 ± 0.80) ↑

MnSOD (2.5 ± 0.47) ↑ Prx (−3.33 ± 0.24) ↓ FNR (−1.40 ± 0.54) ↓

Others synpcc7942 0563 alr4995

UvrABC system protein Alr4995 protein

UvrB Alr4995

NI NI

UvrB (1.4 ± 0.92) ↑ Alr4995 (−2.08 ± 0.4) ↓

alr1526 Carbon metabolism all3538 all4563 alr4132 alr3344 all0122 alr1414 alr1890

all4396 alr3662 alr1742 all4337 all4338 alr0577

NI: Not identified. ND: Not determined. Upward and downward arrows indicate up- and down-regulation, respectively.

Prk Tkt

NI

NI No change Tkt-c (2.27 ± 0.67) ↑ Pde1(−1.56 ± 0.37) ↓ No change

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Fig. 2. Uranium stress responsive differentially expressed proteins involved in photosynthesis. (A) Anabaena 7120 and (B) Anabaena L-31. Differentially expressed proteins with significant increased or decreased abundance are shown by red or green arrowheads in the uranyl carbonate treated or control gel, respectively. Proteins with no significant change in abundance are shown with blue arrowheads. Hyphenated numbers represent main protein (1) or its degradation products (2–4). Protein abbreviations are as mentioned in Supplementary Table 2. ND: Not determined, NI: Not identified. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 3. Uranium stress responsive differentially expressed proteins involved in carbon metabolism. (A) Anabaena 7120 and (B) Anabaena L-31. Hyphenated alphabets (a–d) represent likely post-translational modified forms of the protein. Other details were as described in the legend to Fig. 2.

(AtpB-1 and AtpB-2) was increased (Fig. 2, Table 1, Supplementary Fig. 3 and Supplementary Table 2), whereas levels of ApcA, ApcB, ApcE and CpcC (CpcC-1) were decreased, and CpcC (CpcC-2) degradation was enhanced (Fig. 2, Table 1, Supplementary Fig. 3 and Supplementary Table 2). No significant changes were observed in the resident levels of CpcA, CpcB (CpcB-1), CpcG1, CpcG4, PecA (PecA-1), PecB and PecC (PecC-1) and further degradation of CpcB (CpcB-2) and PecC (PecC-2, PecC-3 and PecC-4) was reduced (Fig. 2, Table 1, Supplementary Fig. 3 and Supplementary Table 2). Thus, photosynthetic machinery of Anabaena L-31 tolerated uranium exposure rather well, compared to Anabaena 7120.

3.4. Effect of uranium on carbon fixation and carbon metabolism Calvin cycle utilizes ATP and NADPH produced during light reaction of photosynthesis to assimilate carbon. Abundance of ribulose-1,5-bisphosphate carboxylase/oxygenase, large subunit and its post-translational modification products (RbcL-a-c) were increased in both Anabaena strains upon uranium exposure. Uranium exposed Anabaena 7120 also displayed increased levels of ribulose-1,5-bisphosphate carboxylase/oxygenase, small subunit

(RbcS). Increased levels of RubisCo indicate an attempt to harness adequate molecular energy [ATP and NAD(P)H] from uraniumdamaged photosynthetic complex. In Anabaena 7120, uranium exposure increased the levels of phosphoribulokinase (Prk), transketolase (and its post-translational modification product Tkt-a-c), S-adenosyl-lhomocysteine hydrolase (AhcY) and cysteine synthase A (CysA-1) (Fig. 3, Table 1, Supplementary Fig. 2 and Supplementary Table 2). The treatment also enhanced degradation of transketolase (Tkt-2) and cysteine synthase A (CysA-2) (Table 1, Supplementary Fig. 2 and Supplementary Table 2). Levels of enolase (Eno) and Fructose 1,6 bisphosphate aldolase (Fda-1) were decreased and degradation of Fda (Fda-3) and Pde1 (Pde1-2) was enhanced (Fig. 3, Table 1, Supplementary Fig. 2 and Supplementary Table 2). Abundance of all other identified metabolic enzymes remained unaffected by uranium exposure in Anabaena 7120 (Fig. 3, Supplementary Fig. 2 and Supplementary Table 2). Levels of majority of metabolic enzymes remained unaffected in Anabaena L-31, with the exception of (a) Pyruvate Dehydrogenase E1 subunit (Pde1) which decreased, and (b) D-3-phosphoglycerate dehydrogenase (Pgdh) and transketolase (Tkt-b) whose levels increased, following

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Fig. 4. Uranium stress responsive differentially expressed proteins involved in transcription and translation. (A) Anabaena 7120 and (B) Anabaena L-31. Other details were as described in the legend to Fig. 2.

Fig. 5. Uranium stress responsive differentially expressed proteins involved in oxidative stress alleviation. (A) Anabaena 7120 and (B) Anabaena L-31. Other details were as described in the legend to Fig. 2.

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uranium exposure (Fig. 3, Table 1, Supplementary Fig. 3 and Supplementary Table 2). Up-regulation of cysteine synthase has also been reported during arsenic stress in Anabaena PCC 7120 and helps to increase the synthesis of cysteine rich metallothionein (Pandey et al., 2012). Compared to Anabaena 7120, Anabaena L-31 maintained its metabolic functions well, even in the presence of uranium. Maintenance of metabolic homeostasis is essential for generation of molecular energy and precursor pool for synthesis of necessary biomolecules, and is crucial for general fitness of a cell experiencing stress. Thus, at proteome level, Anabaena −31 appeared to be better equipped for combating uranium stress. 3.5. Effect of uranium on transcription, translation and protein folding Uranium exposure, reduced the abundance of polynucleotide phosphorylase (PNPase), molecular chaperones GroEL (chaperonin) and DnaK (heat shock protein), FK506 binding protein-type peptidylprolyl isomerase (FKBP-type PPIase) and 50S ribosomal protein L5 (Rpl5), and enhanced degradation of EF-Tu (EF-Tu-2) (Fig. 4, Table 1, Supplementary Fig. 2 and Supplementary Table 2) in Anabaena 7120, but level of ribosomal protein PSRP-3 increased. In comparison, in Anabaena L-31 the levels of DNA directed RNA polymerase (RpoB-1) as well as its degradation (RpoB-2) were increased, while that of both the chaperones GroEL and DnaK were reduced (Fig. 4, Table 1, Supplementary Fig. 3 and Supplementary Table 2). Expression of other identified proteins in this category remained unaffected by uranium exposure. The results demonstrate lesser sensitivity of Anabaena L-31 to uranium than that of Anabaena 7120. 3.6. Effect of uranium on oxidative stress alleviation and DNA repair The two Anabaena strains exhibited somewhat different response of oxidative stress alleviating proteins to uranium stress. The levels of peroxiredoxins (Prx) and ferredoxin-NADP(+) reductase (FNR) increased, while that of Fe-superoxide dismutase (FeSOD) decreased in uranium exposed Anabaena 7120 cells (Fig. 5, Table 1, Supplementary Fig. 2 and Supplementary Table 2). In uranium exposed Anabaena L-31, on the other hand, the abundance of Mn-superoxide dismutase (MnSOD) increased while that of Prx and FNR decreased (Fig. 5, Table 1, Supplementary Fig. 3 and Supplementary Table 2). Increased abundance of MnSOD in response to uranium exposure has also been shown for Caulobacter crescentus (Yung et al., 2013; Hu et al., 2005). Anabaena L-31 also maintained normal levels of glutathione reductase (Gor), peroxiredoxin (Alr7524) and thioredoxin reductase (NTR) during uranium exposure (Supplementary Fig. 3 and Supplementary Table 2). Thus, higher sensitivity of Anabaena 7120 to uranium appeared to stem from its somewhat compromised oxidative stress defence capability. Superoxide dismutases constitute primary defense against ROS. Elevated levels of oxidative stress defence proteins appear to be responsible for mitigation of uranium induced oxidative stress, by expeditious ROS scavenging (Rai et al., 2013), in Anabaena L31, as compared to Anabaena 7120. Among other proteins, DNA repair protein UvrB was elevated in Anabaena L-31 following uranium exposure (Table 1, Supplementary Fig. 3 and Supplementary Table 2). Oxidative stress is known to result in DNA damage and Anabaena L-31 appears to be able to handle it well. It is worthwhile to explore the proteomic responses of diazotrophic cyanobacteria following uranium exposure since they are likely candidates to experience direct or indirect uranium contamination in paddy fields, where they play an important role as naturally abundant N-biofertilizers (Rai et al., 2013). Among the

two Anabaena strains, native to rice fields in India, Anabaena L31 displayed superior tolerance to uranium (LD50 = 200 ␮M uranyl carbonate for 3 h), while Anabaena 7120 was found to be more sensitive (LD50 = 75 ␮M uranyl carbonate for 3 h). At proteome level, response of Anabaena L-31 to uranium exposure combined ROS alleviation with maintenance of molecular energy generation, through sustained photosynthesis and central carbon metabolism, thereby justifying better uranium tolerance. In contrast, the sensitivity of Anabaena 7120 is explained by major disruptions in photosynthetic complex and carbon metabolism, and failure to sustain/elevate ROS detoxification system. This is the first study to describe proteomic modulations associated with differential uranium tolerance in two cyanobacterial strains of Anabaena. Conflict of interest Authors declare no conflict of interest. Acknowledgements Authors are thankful to the Department of Science and Technology (DST), India for a research Grant (No.: SR/SO/PS-44/09). SKA gratefully acknowledges DST, India for award of the J. C. Bose National Fellowship (No. SERB/F/2569/2013-14 dated 29.07.2013) and the Department of Atomic Energy (DAE), India for award of the Raja Ramanna Fellowship (D.O. No. 10/1(20)/2014/RRF-R&DII/3208 dated March 11, 2015). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aquatox.2016.12. 002. References Acharya, C., Apte, S.K., 2013. Novel surface associated polyphosphate bodies sequester uranium in the filamentous, marine cyanobacterium, Anabaena torulosa. Metallomics 5, 1595–1598. Acharya, C., Joseph, D., Apte, S.K., 2009. Uranium sequestration by a marine cyanobacterium, Synechococcus elongatus strain BDU/75042. Bioresour. Technol. 100, 2176–2181. Alan Di Spirito, A., Joseph, W., Talnagi, J.H., Tuovinen, O., 1983. Accumulation and cellular distribution of uranium in Thiobacillus ferrooxidans. Arch. Microbiol. 153, 250–253. Apte, S.K., 2001. Coping with salinity/water stress: cyanobacteria show the way. Proc. Ind. Natl. Sci. Acad. B 67, 285–310. Banerjee, M., Ballal, A., Apte, S.K., 2012a. A novel glutaredoxin domain-containing peroxiredoxin All1541 protects the nitrogen-fixing cyanobacterium Anabaena PCC 7120 from oxidative stress. Biochem. J. 442, 671–680. Banerjee, M., Ballal, A., Apte, S.K., 2012b. Mn-catalase (Alr0998) protects the photosynthetic, nitrogen fixing cyanobacterium Anabaena PCC 7120 from oxidative stress. Environ. Microbiol. 14, 2891–2900. Banerjee, M., Raghavan, P.S., Ballal, A., Rajaram, H., Apte, S.K., 2013. Oxidative stress management in the filamentous, heterocystous, diazotrophic cyanobacterium Anabaena PCC 7120. Photosynth. Res. 118, 59–70. Barisic, D., Lulic, S., Miletic, P., 1992. Radium and uranium in phosphate fertilizers and their impact on the radioactivity of waters. Water Res. 26, 607–611. Bhargava, P., Mishra, Y., Srivastva, A.K., Narayan, O.P., Rai, L.C., 2008. Excess copper induces anoxygenic photosynthesis in Anabaena doliolum: a homology based proteomic assessment of its survival strategy. Photosynth. Res. 96, 61–74. Castenholz, R.W., 1998. Culturing of cyanobacteria. Methods Enzymol. 167, 68–93. Castielli, O., Cerda, B.D., Navarro, J., Hervas, A.M., De la Rosa, M.A., 2009. Proteomics analyses of the response of cyanobacteria to different stress conditions. FEBS Lett. 583, 1753–1758. El-Enany, A.E., Issa, A.A., 2000. Cyanobacteria as a biosorbent of heavy metals in sewage water. Environ. Toxicol. Pharmacol. 8, 95–101. Gale, N.L., Wixson, B.G., 1979. Removal of heavy metals from industrial effluents by algae. Dev. Ind. Microbiol. 20, 259–273. Hu, P., Brodie, E.L., Suzuki, Y., Mc-Adams, H.H., Andersen, G.L., 2005. Whole-genome transcriptional analysis of heavy metal stresses in Caulobacter crescentus. J. Bacteriol. 187, 8437–8449. Kanamaru, K., Kashiwagi, S., Mizuno, S., 1994. A copper-transporting P-type ATPase found in the thylakoid membrane of the cyanobacterium Synechococcus species PCC 7942. Mol. Microbiol. 3, 369–377.

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