Uranium biomineralization by immobilized Chryseobacterium sp. strain PMSZPI cells for efficient uranium removal.

Uranium (U) contamination is hazardous to human health and the environment owing to its radiotoxicity and chemical toxicity and needs immediate attention. In this study, the immobilized biomass of Chryseobacterium sp. strain PMSZPI isolated from U enriched site, was investigated for U(VI) biomineralization in batch and column set-up. Under batch mode, the fresh or lyophilized cells successfully entrapped in calcium alginate beads demonstrated effectual U precipitation under acid and alkaline conditions. The maximum removal was detected at pH 7 wherein ∼98-99% of uranium was precipitated from 1 mM uranyl carbonate solution loading ∼350 mg U/g of biomass within 24 h in the presence of organic phosphate substrate. The resulting uranyl phosphate precipitates within immobilized biomass loaded beads were observed by SEM-EDX and TEM while the formation of U biomineral was confirmed by FTIR and XRD. Retention of phosphatase activity without any loss of uranium precipitation ability was observed for alginate beads with lyophilized biomass stored for 90 d at 4 °C. Continuous flow through experiment with PMSZPI biomass immobilized in polyacrylamide gel exhibited U loading of 0.8 g U/g of biomass at pH 7 using 1 l of 1 mM uranyl solution. This investigation established the feasibility for the application of immobilized PMSZPI biomass for field studies. ENVIRONMENTAL IMPLICATION: Uranium contamination is currently a serious environmental concern owing to anthropogenic activities and needs immediate attention. We have developed here a biotechnological method for successful uranium removal using immobilized cells of a uranium tolerant environmental bacterium, Chryseobacterium sp. strain PMSZPI isolated from U ore deposit via phosphatase enzyme mediated uranium precipitation. The ability of immobilized PMSZPI cells to precipitate U(VI) as long-term stable U phosphates under environmental conditions relevant for contaminated waters containing high concentrations of U that exerts toxicity for biological systems is explored here. The long term stability of the immobilized biomass without compromising its U removal capacity shows the relevance of the bioremediation strategy for uranium contamination proposed in this work.

T9GPred: A Comprehensive Computational Tool for the Prediction of Type 9 Secretion System, Gliding Motility, and the Associated Secreted Proteins.

Type 9 secretion system (T9SS) is one of the least characterized secretion systems exclusively found in the Bacteroidetes phylum, which comprises various environmental and economically relevant bacteria. While T9SS plays a central role in bacterial movement termed gliding motility, survival, and pathogenicity, there is an unmet need for a comprehensive tool that predicts T9SS, gliding motility, and proteins secreted via T9SS. In this study, we develop such a computational tool, Type 9 secretion system and Gliding motility Prediction (T9GPred). To build this tool, we manually curated published experimental evidence and identified mandatory components for T9SS and gliding motility prediction. We also compiled experimentally characterized proteins secreted via T9SS and determined the presence of three unique types of C-terminal domain signals, and these insights were leveraged to predict proteins secreted via T9SS. Notably, using recently published experimental evidence, we show that T9GPred has high predictive power. Thus, we used T9GPred to predict the presence of T9SS, gliding motility, and associated secreted proteins across 693 completely sequenced Bacteroidetes strains. T9GPred predicted 402 strains to have T9SS, of which 327 strains are also predicted to exhibit gliding motility. Further, T9GPred also predicted putative secreted proteins for the 402 strains. In a nutshell, T9GPred is a novel computational tool for systems-level prediction of T9SS and streamlining future experimentation. The source code of the computational tool is available in our GitHub repository: https://github.com/asamallab/T9GPred. The tool and its predicted results are compiled in a web server available at: https://cb.imsc.res.in/t9gpred/.

Inorganic polyphosphate accumulation protects a marine, filamentous cyanobacterium, Anabaena torulosa against uranium toxicity.

The intricate dynamics of inorganic polyphosphate (polyP) in response to phosphorus (P) limitation and metal exposure typical of contaminated aquatic environments is poorly understood. Cyanobacteria are important primary producers in aquatic environments that are exposed to P stringency as well as metal contamination. There is a growing concern regarding migration of uranium, generated as a result of anthropogenic activities, into the aquatic environments owing to high mobility and solubility of stable aqueous complexes of uranyl ions. The polyP metabolism in cyanobacteria in context of uranium (U) exposure under P limitation has hardly been explored. In this study, we analyzed the polyP dynamics in a marine, filamentous cyanobacterium Anabaena torulosa under combination of variable phosphate concentrations (overplus and deficient) and uranyl exposure conditions typical of marine environments. Polyphosphate accumulation (polyP+) or deficient (polyP-) conditions were physiologically synthesized in the A. torulosa cultures and were ascertained by (a) toulidine blue staining followed by their visualization using bright field microscopy and (b) scanning electron microscopy in combination with energy dispersive X-ray spectroscopy (SEM/EDX). On exposure to 100 µM of uranyl carbonate at pH 7.8, it was observed that the growth of polyP+ cells under phosphate limitation was hardly affected and these cells exhibited larger amounts of uranium binding as compared to polyP- cells of A. torulosa. In contrast, the polyP- cells displayed extensive lysis when exposed to similar U exposure. Our findings suggest that polyP accumulation played an important role in conferring uranium tolerance in the marine cyanobacterium, A. torulosa. The polyP-mediated uranium tolerance and binding could serve as a suitable strategy for remediation of uranium contamination in aquatic environments.

Structural Analysis of Gliding Motility of a Bacteroidetes Bacterium by Correlative Light and Scanning Electron Microscopy (CLSEM).

The members of the Bacteroidetes phylum move on surfaces by gliding motility in the absence of external motility appendages, leading to the formation of spreading colonies. Here, the structural features of the spreading colony were assessed in a uranium-tolerant Bacteroidetes bacterium, Chryseobacterium sp. strain PMSZPI, by using correlative light and scanning electron microscopy (CLSEM). We developed a simple and convenient workflow for CLSEM using a shuttle and find software module and a correlative sample holding slide designed to transport samples between the light/fluorescence microscope (LM/FM) and the scanning electron microscope (SEM) to image spreading colony edges. The datasets from the CLSEM studies allowed convenient examination of the colonial organization by LM/FM followed by ultrastructural analysis by SEM. The regions of interest (ROIs) of the spreading colony edges that were observed in LM/FM in the absence and presence of uranium could be re-identified in the SEM quickly without prolonged searching. Perfect correlation between LM and SEM could be achieved with minimum preparation steps. Subsequently, imaging of the correlated regions was done at higher resolution in SEM to obtain more comprehensive information. We further showed the association of uranium with the gliding PMSZPI cells by energy-dispersive X-ray spectroscopy (EDS) attached to SEM.

Gliding motility of a uranium-tolerant Bacteroidetes bacterium Chryseobacterium sp. strain PMSZPI: insights into the architecture of spreading colonies.

Uranium-tolerant soil bacterium Chryseobacterium sp. strain PMSZPI moved over solid agar surfaces by gliding motility thereby forming spreading colonies which is a hallmark of members of Bacteroidetes phylum. PMSZPI genome harboured orthologs of all the gld and spr genes considered as core bacteroidetes gliding motility genes of which gldK, gldL, gldM and gldN were co-transcribed. Here, we present the intriguing interplay between gliding motility and cellular organization in PMSZPI spreading colonies. While nutrient deficiency enhanced colony spreading, high agar concentrations and presence of motility inhibitor like 5-hydroxyindole reduced the spreading. A detailed in situ structural analysis of spreading colonies revealed closely packed cells forming multiple layers at centre of colony while the edges showed clusters of cells periodically arranged in hexagonal lattices interconnected with each other. The cell migration within colony was visualized as branched structures wherein the cells were buried within extracellular matrix. PMSZPI colonies exhibited strong iridescence possibly as a result of periodicity within the cell population achieved through gliding motility. Presence of uranium reduced motility and iridescence and induced biofilm formation. The coordinated study of gliding motility and iridescence apparently influenced by uranium provides unique insights into the lifestyle of PMSZPI residing in uranium enriched environment.

Carbon nano-dot for cancer studies as dual nano-sensor for imaging intracellular temperature or pH variation.

Cellular temperature and pH govern many cellular physiologies, especially of cancer cells. Besides, attaining higher cellular temperature plays key role in therapeutic efficacy of hyperthermia treatment of cancer. This requires bio-compatible, non-toxic and sensitive probe with dual sensing ability to detect temperature and pH variations. In this regard, fluorescence based nano-sensors for cancer studies play an important role. Therefore, a facile green synthesis of orange carbon nano-dots (CND) with high quantum yield of 90% was achieved and its application as dual nano-sensor for imaging intracellular temperature and pH was explored. CND was synthesized from readily available, bio-compatible citric acid and rhodamine 6G hydrazide using solvent-free and simple heating technique requiring purification by dialysis. Although the particle size of 19 nm (which is quite large for CND) was observed yet CND exhibits no surface defects leading to decrease in photoluminescence (PL). On the contrary, very high fluorescence was observed along with good photo-stability. Temperature and pH dependent fluorescence studies show linearity in fluorescence intensity which was replicated in breast cancer cells. In addition, molecular nature of PL of CND was established using pH dependent fluorescence study. Together, the current investigation showed synthesis of highly fluorescent orange CND, which acts as a sensitive bio-imaging probe: an optical nano-thermal or nano-pH sensor for cancer-related studies.

Transcriptome Response of the Tropical Marine Yeast Yarrowia lipolytica on Exposure to Uranium.

In our earlier investigation, we reported the consequences of uranium (U)-induced oxidative stress and cellular defense mechanisms alleviating uranium toxicity in the marine yeast Yarrowia lipolytica NCIM 3589. However, there is lack of information on stress response towards uranium toxicity at molecular level in this organism. To gain an insight on this, transcriptional response of Y. lipolytica after exposure to 50 µM uranium was investigated by RNA sequencing at the global level in this study. The de novo transcriptome analysis (in triplicates) revealed 56 differentially expressed genes with significant up-regulation and down-regulation of 33 and 23 transcripts, respectively, in U-exposed yeast cells as compared to the control, U-unexposed cells. Highly up-regulated genes under U-treated condition were identified to be primarily involved in transport, DNA damage repair and oxidative stress. The major reaction of Y. lipolytica to uranium exposure was the activation of oxidative stress response mechanisms to protect the important biomolecules of the cells. On the other hand, genes involved in cell wall and cell cycle regulation were significantly down-regulated. Overall, the transcriptional profiling by RNA sequencing to stress-inducing concentration of uranium sheds light on the various responses of Y. lipolytica for coping with uranium toxicity, providing a foundation for understanding the molecular interactions between uranium and this marine yeast.

AzuR From the SmtB/ArsR Family of Transcriptional Repressors Regulates Metallothionein in Anabaena sp. Strain PCC 7120.

Metallothioneins (MTs) are cysteine-rich, metal-sequestering cytosolic proteins that play a key role in maintaining metal homeostasis and detoxification. We had previously characterized NmtA, a MT from the heterocystous, nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120 and demonstrated its role in providing protection against cadmium toxicity. In this study, we illustrate the regulation of Anabaena NmtA by AzuR (Alr0831) belonging to the SmtB/ArsR family of transcriptional repressors. There is currently no experimental evidence for any functional role of AzuR. It is observed that azuR is located within the znuABC operon but in the opposite orientation and remotely away from the nmtA locus. Sequence analysis of AzuR revealed a high degree of sequence identity with Synechococcus SmtB and a distinct α5 metal binding site similar to that of SmtB. In order to characterize AzuR, we overexpressed it in Escherichia coli and purified it by chitin affinity chromatography. Far-UV circular dichroism spectroscopy indicated that the recombinant AzuR protein possessed a properly folded structure. Glutaraldehyde cross-linking and size-exclusion chromatography revealed that AzuR exists as a dimer of ∼28 kDa in solution. Analysis of its putative promoter region [100 bp upstream of nmtA open reading frame (ORF)] identified the presence of a 12-2-12 imperfect inverted repeat as the cis-acting element important for repressor binding. Electrophoretic mobility shift assays (EMSAs) showed concentration-dependent binding of recombinant dimeric AzuR with the promoter indicating that NmtA is indeed a regulatory target of AzuR. Binding of AzuR to DNA was disrupted in the presence of metal ions like Zn2+, Cd2+, Cu2+, Co2+, Ni2+, Pb2+, and Mn2+. The metal-dependent dissociation of protein-DNA complexes suggested the negative regulation of metal-inducible nmtA expression by AzuR. Overexpression of azuR in its native strain Anabaena 7120 enhanced the susceptibility to cadmium stress significantly. Overall, we propose a negative regulation of Anabaena MT by an α5 SmtB/ArsR metalloregulator AzuR.

Removal of uranium by immobilized biomass of a tropical marine yeast Yarrowia lipolytica.

A marine yeast, Yarrowia lipolytica isolated from an oil polluted sea water and shown earlier to sequester dissolved uranium (U) at pH 7.5, was utilized in the present study for developing an immobilized-cell process for U removal from aqueous solutions under batch and continuous flow through systems. In batch system, optimum biosorption conditions for U removal were assessed by investigating the effects of biomass dose, initial U concentration, contact time and pH of solution using Y. lipolytica cells immobilized in calcium alginate beads. Appreciable uranium-binding capabilities over a wide pH range (3-9) were observed with the alginate beads bearing yeast cells. Out of Langmuir and Freundlich models employed for describing the sorption equilibrium data under batch mode, uranyl adsorption followed Langmuir approach with satisfactory correlation coefficient higher than 0.9. Uranyl adsorption kinetics by Y. lipolytica entrapped in alginate beads was best described by the pseudo-second-order model. While the environmental scanning electron microscopy established the immobilization and the uniform distribution of Y. lipolytica cells in the alginate beads, the Energy Dispersive X-ray spectroscopy analysis confirmed the deposition of U in the beads following their exposure to uranyl solution. Fixed bed flow-through column comprising of Y. lipolytica biomass immobilized in polyacrylamide matrix displayed high efficacy for continuous removal of uranium at pH 7.5 up to five adsorption-desorption cycles. Adsorbed U by immobilized cells could be significantly desorbed using 0.1 N HCl. Overall, our results present the superior efficiency of immobilized Y. lipolytica biomass for U removal using batch and regenerative approaches.

Genomic and functional insights into the adaptation and survival of Chryseobacterium sp. strain PMSZPI in uranium enriched environment.

Metal enriched areas represent important and dynamic microbiological ecosystems. In this study, the draft genome of a uranium (U) tolerant bacterium, Chryseobacterium sp. strain PMSZPI, isolated from the subsurface soil of Domiasiat uranium ore deposit in Northeast India, was analyzed. The strain revealed a genome size of 3.8 Mb comprising of 3346 predicted protein-coding genes. The analysis indicated high abundance of genes associated with metal resistance and efflux, transporters, phosphatases, antibiotic resistance, polysaccharide synthesis, motility, protein secretion systems, oxidoreductases and DNA repair. Comparative genomics with other closely related Chryseobacterium strains led to the identification of unique inventory of genes which were of adaptive significance in PMSZPI. Consistent with the genome analysis, PMSZPI showed superior tolerance to uranium and other heavy metals. The metal exposed cells exhibited transcriptional induction of metal translocating PIB ATPases suggestive of their involvement in metal resistance. Efficient U binding (~90% of 100 µM U) and U bioprecipitation (~93-94% of 1 mM U at pH 5, 7 and 9) could be attributed as uranium tolerance strategies in PMSZPI. The strain demonstrated resistance to a large number of antibiotics which was in agreement with in silico prediction. Reduced gliding motility in the presence of cadmium and uranium, enhanced biofilm formation on uranium exposure and tolerance to 1.5 kGy of 60Co gamma radiation were perceived as adaptive responses in PMSZPI. Overall, the positive correlation observed between uranium/metal tolerance abilities predicted using genome analysis and the functional characterization reinforced the multifaceted adaptation strategies employed by PMSZPI for its survival in the soil of uranium ore deposit comprising of high concentrations of uranium and other heavy metals.

Impact of uranium exposure on marine yeast, Yarrowia lipolytica: Insights into the yeast strategies to withstand uranium stress.

A marine yeast, Yarrowia lipolytica, was evaluated for morphological, physiological and biochemical responses towards uranium (U) exposure at pH 7.5. The yeast revealed biphasic U binding - a rapid biosorption resulting in ∼35% U binding within 15-30 min followed by a slow biomineralization process, binding up to ∼45.5% U by 24 h on exposure to 50 µM of uranyl carbonate. Scanning electron microscopy coupled with Energy Dispersive X-ray spectroscopy analysis of 24 h U challenged cells revealed the deposition of uranyl precipitates due to biomineralization. The loss of intracellular structures together with surface and subcellular localization of uranyl precipitates in 24 h U exposed cells were visualized by transmission electron microscopy. Cells treated with 50 µM U exhibited membrane permeabilization which was higher at 200 µM U. Enhanced reactive oxygen species (ROS) accumulation and lipid peroxidation, transient RNA degradation and protein oxidation were observed in U exposed cells. High superoxide dismutase levels coupled with uranium binding and bioprecipitation possibly helped in counteracting U stress in 50 µM U treated cells. Resistance to U toxicity apparently developed under prolonged uranyl (50 µM) incubations. However, cells could not cope up with toxicity at 200 µM U due to impairment of resistance mechanisms.

Responses exhibited by various microbial groups relevant to uranium exposure.

There is a strong interest in knowing how various microbial systems respond to the presence of uranium (U), largely in the context of bioremediation. There is no known biological role for uranium so far. Uranium is naturally present in rocks and minerals. The insoluble nature of the U(IV) minerals keeps uranium firmly bound in the earth's crust minimizing its bioavailability. However, anthropogenic nuclear reaction processes over the last few decades have resulted in introduction of uranium into the environment in soluble and toxic forms. Microbes adsorb, accumulate, reduce, oxidize, possibly respire, mineralize and precipitate uranium. This review focuses on the microbial responses to uranium exposure which allows the alteration of the forms and concentrations of uranium within the cell and in the local environment. Detailed information on the three major bioprocesses namely, biosorption, bioprecipitation and bioreduction exhibited by the microbes belonging to various groups and subgroups of bacteria, fungi and algae is provided in this review elucidating their intrinsic and engineered abilities for uranium removal. The survey also highlights the instances of the field trials undertaken for in situ uranium bioremediation. Advances in genomics and proteomics approaches providing the information on the regulatory and physiologically important determinants in the microbes in response to uranium challenge have been catalogued here. Recent developments in metagenomics and metaproteomics indicating the ecologically relevant traits required for the adaptation and survival of environmental microbes residing in uranium contaminated sites are also included. A comprehensive understanding of the microbial responses to uranium can facilitate the development of in situ U bioremediation strategies.

Uranium biomineralization induced by a metal tolerant Serratia strain under acid, alkaline and irradiated conditions.

It has become increasingly apparent that the environmental microorganisms residing in uranium (U) enriched sites offer the possibility of understanding the biological mechanisms catalyzing the processes important for uranium bioremediation. Here, we present the results of uranium biomineralization over a wide pH range by a metal tolerant Serratia sp. strain OT II 7 isolated from the subsurface soil of a U ore deposit at Domiasiat in India. The Serratia cells actively expressed acid and alkaline phosphatase enzymes which hydrolyzed differential amounts of phosphate from an organophosphate substrate in the presence of uranium between pH 5 to 9. These cells precipitated ∼91% uranium from aqueous solutions supplemented with 1 mM uranyl nitrate at pH 5 within 120 h. More rapid precipitation was observed at pH 7 and 9 wherein the cells removed ∼93-94% of uranium from solutions containing 1 mM uranyl carbonate within 24 h. The aqueous uranyl speciation prevalent under the studied pH conditions influenced the localization of crystalline uranyl phosphate precipitates, which in turn, impacted the cell viability to a great extent. Furthermore, the cells tolerated up to ∼1.6 kGy 60Co gamma radiation and their uranium precipitation abilities at pH 5, 7 and 9 were uncompromised even after exposure to a high dose of ionizing radiation. Overall, this study establishes the ecological adaptation of a natural strain like Serratia in a uranium enriched environment and corroborates its contribution towards uranium immobilization in contaminated subsurfaces through the formation of stable uranyl phosphate minerals over a wide pH range.

NmtA, a novel metallothionein of Anabaena sp. strain PCC 7120 imparts protection against cadmium stress but not oxidative stress.

Metallothioneins (MTs) are low molecular weight, sulfhydryl-containing, cysteine-rich, metal-binding proteins. Eukaryotes have multiple metallothionein genes; however, there is dearth of reports on prokaryotic metallothioneins. Bacterial MTs with SmtA from Synechococcus PCC 7942 as prototype have been studied in the context of cadmium detoxification. In this study, a smtA related ORF, namely nmtA, was identified in the heterocystous, nitrogen-fixing cyanobacterium, Anabaena PCC 7120. A recombinant N-terminal histidine-tagged Anabaena NmtA protein was overexpressed in Escherichia coli and purified. The protein was identified by peptide mass fingerprinting using MALDI-TOF Mass Spectrometry as putative metallothionein of Anabaena PCC 7120 with a calculated mass of ∼6.1 kDa. While the native metallated NmtA exhibited resistance against proteolysis, metal free apo-NmtA resulting from acid and dithiothreitol (DTT) treatment could be digested by proteinase K revealing a metal dependent proteolytic protection of NmtA. Expression of nmtA in Anabaena PCC 7120 was induced evidently by cadmium, zinc and copper but not by uranium or hydrogen peroxide. Recombinant Anabaena PCC 7120 overexpressing NmtA protein revealed superior cadmium tolerance but showed limited influence against oxidative stress tolerance as compared with the strain carrying vector alone. In contrast, a mutant of Synechococcus PCC 7942 deficient in MT locus was found to be highly susceptible to H2O2 indicating a likely involvement of cyanobacterial MT in protection against oxidative damage. Overall, the study improved our understanding of metal tolerance mechanisms in Anabaena PCC 7120 by demonstrating a key role of NmtA in cadmium tolerance.

Unusual Versatility of the Filamentous, Diazotrophic Cyanobacterium Anabaena torulosa Revealed for Its Survival during Prolonged Uranium Exposure.

Reports on interactions between cyanobacteria and uranyl carbonate are rare. Here, we present an interesting succession of the metabolic responses employed by a marine, filamentous, diazotrophic cyanobacterium, Anabaena torulosa for its survival following prolonged exposure to uranyl carbonate extending up to 384 h at pH 7.8 under phosphate-limited conditions. The cells sequestered uranium (U) within polyphosphates on initial exposure to 100 µM uranyl carbonate for 24 to 28 h. Further incubation until 120 h resulted in (i) significant degradation of cellular polyphosphates causing extensive chlorosis and cell lysis, (ii) akinete differentiation followed by (iii) extracellular uranyl precipitation. X-ray diffraction (XRD) analysis, fluorescence spectroscopy, X-ray absorption near edge structure (XANES), and extended X-ray absorption fine structure (EXAFS) spectroscopy established the identity of the bioprecipitated uranium as a U(VI) autunite-type mineral, which settled at the bottom of the vessel. Surprisingly, A. torulosa cells resurfaced as small green flakes typical of actively growing colonies on top of the test solutions within 192 to 240 h of U exposure. A consolidated investigation using kinetics, microscopy, and physiological and biochemical analyses suggested a role of inducible alkaline phosphatase activity of cell aggregates/akinetes in facilitating the germination of akinetes leading to substantial regeneration of A. torulosa by 384 h of uranyl incubation. The biomineralized uranium appeared to be stable following cell regeneration. Altogether, our results reveal novel insights into the survival mechanism adopted by A. torulosa to resist sustained uranium toxicity under phosphate-limited oxic conditions.IMPORTANCE Long-term effects of uranyl exposure in cyanobacteria under oxic phosphate-limited conditions have been inadequately explored. We conducted a comprehensive examination of the metabolic responses displayed by a marine cyanobacterium, Anabaena torulosa, to cope with prolonged exposure to uranyl carbonate at pH 7.8 under phosphate limitation. Our results highlight distinct adaptive mechanisms harbored by this cyanobacterium that enabled its natural regeneration following extensive cell lysis and uranium biomineralization under sustained uranium exposure. Such complex interactions between environmental microbes such as Anabaena torulosa and uranium over a broader time range advance our understanding on the impact of microbial processes on uranium biogeochemistry.

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

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 3h exposure to 75µM and 200µM 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.

Cesium and strontium tolerant Arthrobacter sp. strain KMSZP6 isolated from a pristine uranium ore deposit.

Arthrobacter sp. KMSZP6 isolated from a pristine uranium ore deposit at Domiasiat located in North-East India exhibited noteworthy tolerance for cesium (Cs) and strontium (Sr). The strain displayed a high minimum inhibitory concentration (MIC) of 400 mM for CsCl and for SrCl2. Flow cytometric analysis employing membrane integrity indicators like propidium iodide (PI) and thiazole orange (TO) indicated a greater sensitivity of Arthrobacter cells to cesium than to strontium. On being challenged with 75 mM of Cs, the cells sequestered 9612 mg Cs g(-1) dry weight of cells in 12 h. On being challenged with 75 mM of Sr, the cells sequestered 9989 mg Sr g(-1) dry weight of cells in 18 h. Heat killed cells exhibited limited Cs and Sr binding as compared to live cells highlighting the importance of cell viability for optimal binding. The association of the metals with Arthrobacter sp. KMSZP6 was further substantiated by Field Emission-Scanning Electron Microscopy (FE-SEM) coupled with Energy dispersive X-ray (EDX) spectroscopy. This organism tolerated up to 1 kGy (60)Co-gamma rays without loss of survival. The present report highlights the superior tolerance and binding capacity of the KMSZP6 strain for cesium and strontium over other earlier reported strains and reveals its potential for bioremediation of nuclear waste.

Unexpected Interactions of the Cyanobacterial Metallothionein SmtA with Uranium.

Molecules for remediating or recovering uranium from contaminated environmental resources are of high current interest, with protein-based ligands coming into focus recently. Metallothioneins either bind or redox-silence a range of heavy metals, conferring protection against metal stress in many organisms. Here, we report that the cyanobacterial metallothionein SmtA competes with carbonate for uranyl binding, leading to formation of heterometallic (UO2)(n)Zn4SmtA species, without thiol oxidation, zinc loss, or compromising secondary or tertiary structure of SmtA. In turn, only metalated and folded SmtA species were found to be capable of uranyl binding. (1)H NMR studies and molecular modeling identified Glu34/Asp38 and Glu12/C-terminus as likely adventitious, but surprisingly strong, bidentate binding sites. While it is unlikely that these interactions correspond to an evolved biological function of this metallothionein, their occurrence may offer new possibilities for designing novel multipurpose bacterial metallothioneins with dual ability to sequester both soft metal ions including Cu(+), Zn(2+), Cd(2+), Hg(2+), and Pb(2+) and hard, high-oxidation state heavy metals such as U(VI). The concomitant protection from the chemical toxicity of uranium may be valuable for the development of bacterial strains for bio-remediation.

Insights into the interactions of cyanobacteria with uranium.

Due to various activities associated with nuclear industry, uranium is migrated to aquatic environments like groundwater, ponds or oceans. Uranium forms stable carbonate complexes in the oxic waters of pH 7-10 which results in a high degree of uranium mobility. Microorganisms employ various mechanisms which significantly influence the mobility and the speciation of uranium in aquatic environments. Uranyl bioremediation studies, this far, have generally focussed on low pH conditions and related to adsorption of positively charged UO2 (2+) onto negatively charged microbial surfaces. Sequestration of anionic uranium species, i.e. [UO2(CO3) 2 (2-) ] and [UO2(CO3) 3 (4-) ] onto microbial surfaces has received only scant attention. Marine cyanobacteria are effective metal adsorbents and represent an important sink for metals in aquatic environment. This article addresses the cyanobacterial interactions with toxic metals in general while stressing on uranium. It focusses on the possible mechanisms employed by cyanobacteria to sequester uranium from aqueous solutions above circumneutral pH where negatively charged uranyl carbonate complexes dominate aqueous uranium speciation. The mechanisms demonstrated by cyanobacteria are important components of biogeochemical cycle of uranium and are useful for the development of appropriate strategies, either to recover or remediate uranium from the aquatic environments.