Metal removal by metallothionein and an acid phosphatase PhoN, surface-displayed on the cells of the extremophile, Deinococcus radiodurans.

The utility of surface layer proteins (Hpi and SlpA) of the radiation resistant bacterium, Deinococcus radiodurans, was investigated for surface display and bioremediation of cadmium and uranium. The smtA gene, from Synechococcus elongatus (encoding the metal binding metallothionein protein), was cloned and over-expressed in D. radiodurans, either as such or as a chimeric gene fused with hpi ORF (Hpi-SmtA), or fused to the nucleotide sequence encoding the SLH domain of the SlpA protein (SLH-SmtA). The expressed fusion proteins localized to the deinococcal cell surface, while the SmtA protein localized to the cytoplasm. Recombinant cells surface-displaying the SLH-SmtA or Hpi-SmtA fusion proteins respectively removed 1.5-3 times more cadmium than those expressing only cytosolic SmtA. The deinococcal Hpi protein layer per se also contributed to U binding, by conferring substantial negative charge to deinococcal cell surface. The ORF of an acid phosphatase, PhoN was fused with the hpi or SLH domain DNA sequence and purified. Isolated Hpi-PhoN and SLH-PhoN, immobilized on deinococcal peptidoglycan showed efficient uranium precipitation (446 and 160 mg U/g biomass used respectively). The study demonstrates effective exploitation of the deinococcal S layer protein components for (a) cell surface-based sequestration of cadmium, and (b) cell-free preparations for uranium remediation.

Effect of 60Co-Gamma Ionizing Radiation and Desiccation Stress on Protein Profile of Anabaena 7120.

Physiological stress can bring major molecular and cellular change to a living cell which further decide its survival or tolerance to the stress exposure. Cyanobacteria like Anabaena has been shown to tolerate high levels of different stresses like oxidative, desiccation, UV, and gamma radiation. They are able to withstand and recover remarkably without any lethal mutation when exposed to high doses of gamma radiation or prolonged duration of desiccation. In the present work, the modifications in protein profiles of Anabaena 7120 cells after exposure to 6 kGy of 60Co γ-rays and 6 days of desiccation, and the proteome dynamics during post stress recovery were investigated. Differentially expressed proteins during stress and recovery were identified by MALDI-ToF or LC-MS, which generated a partial proteome map of Anabaena 7120. Anabaena cells went through protein recycling-phase of protein degradation following by their resynthesis, which helped them to recover remarkably. The data suggests an overlap in proteome changes during recovery against radiation and desiccation stress.

Low concentrations of ethanol during irradiation drastically reduce DNA damage caused by very high doses of ionizing radiation.

Presence of low concentrations (1-2%) of ethanol during irradiation exhibited significant protection against DNA damage caused by very high doses (2-12 kGy) of 60 Co-gamma-rays in vitro. Radiation-induced DNA damage was substantially reduced in different types of DNA molecules (chromosomal DNA from Anabaena 7120 or Deinococcus radiodurans or bacteriophage Lambda, and plasmid pBluescript DNA) when irradiated in the presence of ethanol, thus indicating the generic nature of ethanol protection. The radioprotection appeared to be a consequence of the well known ability of ethanol to scavenge hydroxyl radicals. Addition of ethanol during 6 kGy irradiation also reduced DNA damage in vivo and improved post-irradiation growth recovery of Anabaena 7120 cultures. To our knowledge, this is the first instance of ability of very low ethanol concentrations to protect DNA from damage triggered by extremely high doses of 60 Co-gamma rays.

Regulation of potassium dependent ATPase (kdp) operon of Deinococcus radiodurans.

The genome of D. radiodurans harbors genes for structural and regulatory proteins of Kdp ATPase, in an operon pattern, on Mega plasmid 1. Organization of its two-component regulatory genes is unique. Here we demonstrate that both, the structural as well as regulatory components of the kdp operon of D. radiodurans are expressed quickly as the cells experience potassium limitation but are not expressed upon increase in osmolarity. The cognate DNA binding response regulator (RR) effects the expression of kdp operon during potassium deficiency through specific interaction with the kdp promoter. Deletion of the gene encoding RR protein renders the mutant D. radiodurans (ΔRR) unable to express kdp operon under potassium limitation. The ΔRR D. radiodurans displays no growth defect when grown on rich media or when exposed to oxidative or heat stress but shows reduced growth following gamma irradiation. The study elucidates the functional and regulatory aspects of the novel kdp operon of this extremophile, for the first time.

Proximity of Radiation Desiccation Response Motif to the core promoter is essential for basal repression as well as gamma radiation-induced gyrB gene expression in Deinococcus radiodurans.

The radioresistant D. radiodurans regulates its DNA damage regulon (DDR) through interaction between a 17bp palindromic cis-regulatory element called the Radiation Desiccation Response Motif (RDRM), the DdrO repressor and a protease IrrE. The role of RDRM in regulation of DDR was dissected by constructing RDRM sequence-, position- or deletion-variants of Deinococcal gyrB gene (DR0906) promoter and by RDRM insertion in the non-RDRM groESL gene (DR0606) promoter, and monitoring the effect of such modifications on the basal as well as gamma radiation inducible promoter activity by quantifying fluorescence of a GFP reporter. RDRM sequence-variants revealed that the conservation of sequence at the 5th and 13th position and the ends of RDRM is essential for basal repression by interaction with DdrO. RDRM position-variants showed that the sequence acts as a negative regulatory element only when located around transcription start site (TSS) and within the span of RNA polymerase (RNAP) binding region. RDRM deletion-variants indicated that the 5' sequence of RDRM possibly possesses an enhancer-like element responsible for higher expression yields upon repressor clearance post-irradiation. The results suggest that RDRM plays both a negative as well as a positive role in the regulation of DDR in D. radiodurans.

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.

Interaction of Uranium with Bacterial Cell Surfaces: Inferences from Phosphatase-Mediated Uranium Precipitation.

UNLABELLED: Deinococcus radiodurans and Escherichia coli expressing either PhoN, a periplasmic acid phosphatase, or PhoK, an extracellular alkaline phosphatase, were evaluated for uranium (U) bioprecipitation under two specific geochemical conditions (GCs): (i) a carbonate-deficient condition at near-neutral pH (GC1), and (ii) a carbonate-abundant condition at alkaline pH (GC2). Transmission electron microscopy revealed that recombinant cells expressing PhoN/PhoK formed cell-associated uranyl phosphate precipitate under GC1, whereas the same cells displayed extracellular precipitation under GC2. These results implied that the cell-bound or extracellular location of the precipitate was governed by the uranyl species prevalent at that particular GC, rather than the location of phosphatase. MINTEQ modeling predicted the formation of predominantly positively charged uranium hydroxide ions under GC1 and negatively charged uranyl carbonate-hydroxide complexes under GC2. Both microbes adsorbed 6- to 10-fold more U under GC1 than under GC2, suggesting that higher biosorption of U to the bacterial cell surface under GC1 may lead to cell-associated U precipitation. In contrast, at alkaline pH and in the presence of excess carbonate under GC2, poor biosorption of negatively charged uranyl carbonate complexes on the cell surface might have resulted in extracellular precipitation. The toxicity of U observed under GC1 being higher than that under GC2 could also be attributed to the preferential adsorption of U on cell surfaces under GC1. This work provides a vivid description of the interaction of U complexes with bacterial cells. The findings have implications for the toxicity of various U species and for developing biological aqueous effluent waste treatment strategies. IMPORTANCE: The present study provides illustrative insights into the interaction of uranium (U) complexes with recombinant bacterial cells overexpressing phosphatases. This work demonstrates the effects of aqueous speciation of U on the biosorption of U and the localization pattern of uranyl phosphate precipitated as a result of phosphatase action. Transmission electron microscopy revealed that location of uranyl phosphate (cell associated or extracellular) was primarily influenced by aqueous uranyl species present under the given geochemical conditions. The data would be useful for understanding the toxicity of U under different geochemical conditions. Since cell-associated precipitation of metal facilitates easy downstream processing by simple gravity-based settling down of metal-loaded cells, compared to cumbersome separation techniques, the results from this study are of considerable relevance to effluent treatment using such cells.

In situ real-time evaluation of radiation-responsive promoters in the extremely radioresistant microbe Deinococcus radiodurans.

A third generation promoter probe shuttle vector pKG was constructed, using the green fluorescent protein as a reporter, for in situ evaluation of Deinococcal promoter activity in Escherichia coli or Deinococcus radiodurans. The construct yielded zero background fluorescence in both the organisms, in the absence of promoter sequences. Fifteen Deinococcal promoters, either harbouring Radiation and Desiccation Response Motif (RDRM) or not, were cloned in vector pKG. Only the RDRM-promoter constructs displayed (i) gamma radiation inducible GFP expression in D. radiodurans, following gamma irradiation, (ii) DdrO-mediated repression of GFP expression in heterologous E. coli, or (iii) abolition in GFP induction following gamma irradiation, in pprI mutant of D. radiodurans. Utility of pKG vector for real-time in situ assessment of Deinococcal promoter function was, thus, successfully demonstrated.

Involvement of phosphoesterases in tributyl phosphate degradation in Sphingobium sp. strain RSMS.

A tri- and dibutyl phosphate (TBP/DBP) non-degrading spontaneous mutant, Sphingobium SS22, was derived from the Sphingobium sp. strain RSMS (wild type). Unlike the wild type strain, Sphingobium SS22 could not grow in a minimal medium supplemented with TBP or DBP as the sole source of carbon or phosphorous. Sphingobium SS22 also did not form any of the intermediates or end products of TBP or DBP degradation, namely DBP, butanol or inorganic phosphate. Proteomic analysis revealed the absence of three prominent proteins in Sphingobium SS22 as compared to wild type. These proteins were identified by MALDI mass spectrometry, and they showed similarities to phosphohydrolase- and exopolyphosphatase-like proteins from other bacteria, which belong to the class of phosphoesterases. Cellular proteins of Sphingobium SS22 showed none or negligible phosphodiesterase (PDE) and phosphomonoesterase (PME) activities at pH 7 and displayed approximately five- and approximately twofold less DBP and monobutyl phosphate (MBP) degradation activity, respectively, in comparison to the wild type strain. In-gel zymographic analysis revealed two PDE and PME activity bands in the wild type strain, one of which was absent in the Sphingobium SS22 mutant. The corresponding proteins from the wild type strain could degrade DBP and MBP. The results demonstrate the involvement of phosphoesterase enzymes in the TBP degradation pathway elucidated earlier.

Surface (S)-layer proteins of Deinococcus radiodurans and their utility as vehicles for surface localization of functional proteins.

The radiation resistant bacterium, Deinococcus radiodurans contains two major surface (S)-layer proteins, Hpi and SlpA. The Hpi protein was shown to (a) undergo specific in vivo cleavage, and (b) closely associate with the SlpA protein. Using a non-specific acid phosphatase from Salmonella enterica serovar Typhi, PhoN as a reporter, the Surface Layer Homology (SLH) domain of SlpA was shown to bind deinococcal peptidoglycan-containing cell wall sacculi. The association of SlpA with Hpi on one side and peptidoglycan on the other, localizes this protein in the 'interstitial' layer of the deinoccocal cell wall. Gene chimeras of hpi-phoN and slh-phoN were constructed to test efficacy of S-layer proteins, as vehicles for cell surface localization in D. radiodurans. The Hpi-PhoN protein localized exclusively in the membrane fraction, and displayed cell-based phosphatase activity in vivo. The SLH-PhoN, which localized to both cytosolic and membrane fractions, displayed in vitro activity but no cell-based in vivo activity. Hpi, therefore, emerged as an efficient surface localizing protein and can be exploited for suitable applications of this superbug.

Membrane targeting of MnSOD is essential for oxidative stress tolerance of nitrogen-fixing cultures of Anabaena sp. strain PCC7120.

The nitrogen-fixing cyanobacterium, Anabaena PCC7120 encodes for a membrane-targeted 30 kDa Mn-superoxide dismutase (MnSOD) and a cytosolic FeSOD. The MnSOD is post-translationally processed to 27 and 24 kDa forms in the cytosol and periplasm/thylakoid lumen. The extent of cleavage of signal and linker peptides at the N-terminus is dependent on the availability of combined nitrogen during growth. While the 24 and 27 kDa forms are present in near equal proportions under nitrogen-fixing conditions, the 24 kDa form is predominant under nitrogen-supplemented conditions. Individual contribution of these forms of MnSOD to total oxidative stress tolerance was analysed using recombinant Anabaena strains overexpressing either different molecular forms of MnSOD or MnSOD defective in the cleavage of signal/linker peptide. Targeting of MnSOD to the membrane and subsequent cleavage to release both the 24 and 27 kDa forms was essential for oxidative stress tolerance under nitrogen-fixing conditions. On the other hand, the cleavage of linker peptide was absolutely essential and the release of cytosolic 24 kDa form of MnSOD was obligatory for developing oxidative stress tolerance under nitrogen-supplemented conditions. Thus, a single MnSOD caters to the reduction of superoxide radical in both cytosol and thylakoid lumen/periplasm irrespective of the N-status of growth by regulating its cleavage. This is the first report on the physiological advantage of membrane-targeting and processing of MnSOD in either bacteria or plants. The higher oxidative stress tolerance offered by the cytosolic form of MnSOD has possibly resulted in retention of only the cytosolic form in bacterial non-nitrogen-fixers during evolution.

Engineered Deinococcus radiodurans R1 with NiCoT genes for bioremoval of trace cobalt from spent decontamination solutions of nuclear power reactors.

The aim of the present work was to engineer bacteria for the removal of Co in contaminated effluents. Radioactive cobalt ((60)Co) is known as a major contributor for person-sievert budgetary because of its long half-life and high γ-energy values. Some bacterial Ni/Co transporter (NiCoT) genes were described to have preferential uptake for cobalt. In this study, the NiCoT genes nxiA and nvoA from Rhodopseudomonas palustris CGA009 (RP) and Novosphingobium aromaticivorans F-199 (NA), respectively, were cloned under the control of the groESL promoter. These genes were expressed in Deinococcus radiodurans in reason of its high resistance to radiation as compared to other bacterial strains. Using qualitative real time-PCR, we showed that the expression of NiCoT-RP and NiCoT-NA is induced by cobalt and nickel. The functional expression of these genes in bioengineered D. radiodurans R1 strains resulted in >60 % removal of (60)Co (≥5.1 nM) within 90 min from simulated spent decontamination solution containing 8.5 nM of Co, even in the presence of >10 mM of Fe, Cr, and Ni. D. radiodurans R1 (DR-RP and DR-NA) showed superior survival to recombinant E. coli (ARY023) expressing NiCoT-RP and NA and efficiency in Co remediation up to 6.4 kGy. Thus, the present study reports a remarkable reduction in biomass requirements (2 kg) compared to previous studies using wild-type bacteria (50 kg) or ion-exchanger resins (8000 kg) for treatment of ~10(5)-l spent decontamination solutions (SDS).

Comparative proteomics of oxidative stress response in three cyanobacterial strains native to Indian paddy fields.

Three strains of photoautotrophic, heterocystous, nitrogen-fixing cyanobacterium Anabaena, native to Indian paddy fields, were examined for their tolerance and proteomic response to the frequently used weedicide paraquat (methyl viologen). Anabaena 7120 (LD50 dose: 2µM for 6h) and Anabaena L-31 (LD50 dose: 2µM for 5h) showed distinctly better tolerance than Anabaena doliolum (LD50 dose: 2µM for 3h), to methyl viologen induced oxidative stress. The proteomic response, at respective LD50 dose, was mapped by 2D gel protein electrophoresis followed by protein identification by MALDI-ToF mass spectrometry. About 92 and 41 oxidative stress-responsive proteins were identified from Anabaena L-31 and A. doliolum, respectively, and compared with methyl viologen responsive proteins reported from Anabaena 7120 earlier. Upregulation of proteins involved in oxidative stress alleviation and protein homeostasis and downregulation of photosynthesis and carbon metabolism related enzymes appeared to underlie the oxidative stress response in all three Anabaena strains. Reduced photosynthesis and cellular reserves of molecular energy [ATP+NAD(P)H] seemed to overwhelm the cellular machinery to combat oxidative stress and protein denaturation, in preference to other adaptations, while the strain specific differences observed in proteome response appeared to determine the methyl viologen tolerance of individual cyanobacterial strains. This article is part of a Special Issue entitled: Proteomics in India.

Depletion of reduction potential and key energy generation metabolic enzymes underlies tellurite toxicity in Deinococcus radiodurans.

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 5 h. 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.

Harnessing a radiation inducible promoter of Deinococcus radiodurans for enhanced precipitation of uranium.

Bioremediation is an attractive option for the treatment of radioactive waste. We provide a proof of principle for augmentation of uranium bioprecipitation using the radiation inducible promoter, Pssb from Deinococcus radiodurans. Recombinant cells of D. radiodurans carrying acid phosphatase gene, phoN under the regulation of Pssb when exposed to 7 kGy gamma radiation at two different dose rates of 56.8 Gy/min and 4 Gy/min, showed 8-9 fold increase in acid phosphatase activity. Highest whole cell PhoN activity was obtained after 2h in post irradiation recovery following 8 kGy of high dose rate radiation. Such cells showed faster removal of high concentrations of uranium than recombinant cells expressing PhoN under a radiation non-inducible deinococcal promoter, PgroESL and could precipitate uranium even after continuous exposure to 0.6 Gy/min gamma radiation for 10 days. Radiation induced recombinant D. radiodurans cells when lyophilized retained high levels of PhoN activity and precipitated uranium efficiently. These results highlight the importance of using a suitable promoter for removal of radionuclides from solution.

The hypothetical protein 'All4779', and not the annotated 'Alr0088' and 'Alr7579' proteins, is the major typical single-stranded DNA binding protein of the cyanobacterium, Anabaena sp. PCC7120.

Single-stranded DNA binding (SSB) proteins are essential for all DNA-dependent cellular processes. Typical SSB proteins have an N-terminal Oligonucleotide-Binding (OB) fold, a Proline/Glycine rich region, followed by a C-terminal acidic tail. In the genome of the heterocystous nitrogen-fixing cyanobacterium, Anabaena sp. strain PCC7120, alr0088 and alr7579 are annotated as coding for SSB, but are truncated and have only the OB-fold. In silico analysis of whole genome of Anabaena sp. strain PCC7120 revealed the presence of another ORF 'all4779', annotated as a hypothetical protein, but having an N-terminal OB-fold, a P/G-rich region and a C-terminal acidic tail. Biochemical characterisation of all three purified recombinant proteins revealed that they exist either as monomer or dimer and bind ssDNA, but differently. The All4779 bound ssDNA in two binding modes i.e. (All4779)35 and (All4779)66 depending on salt concentration and with a binding affinity similar to that of Escherichia coli SSB. On the other hand, Alr0088 bound in a single binding mode of 50-mer and Alr7579 only to large stretches of ssDNA, suggesting that All4779, in all likelihood, is the major typical bacterial SSB in Anabaena. Overexpression of All4779 in Anabaena sp. strain PCC7120 led to enhancement of tolerance to DNA-damaging stresses, such as γ-rays, UV-irradiation, desiccation and mitomycinC exposure. The tolerance appears to be a consequence of reduced DNA damage or efficient DNA repair due to increased availability of All4779. The ORF all4779 is proposed to be re-annotated as Anabaena ssb gene.

Cyanobacterial heat-shock response: role and regulation of molecular chaperones.

Cyanobacteria constitute a morphologically diverse group of oxygenic photoautotrophic microbes which range from unicellular to multicellular, and non-nitrogen-fixing to nitrogen-fixing types. Sustained long-term exposure to changing environmental conditions, during their three billion years of evolution, has presumably led to their adaptation to diverse ecological niches. The ability to maintain protein conformational homeostasis (folding-misfolding-refolding or aggregation-degradation) by molecular chaperones holds the key to the stress adaptability of cyanobacteria. Although cyanobacteria possess several genes encoding DnaK and DnaJ family proteins, these are not the most abundant heat-shock proteins (Hsps), as is the case in other bacteria. Instead, the Hsp60 family of proteins, comprising two phylogenetically conserved proteins, and small Hsps are more abundant during heat stress. The contribution of the Hsp100 (ClpB) family of proteins and of small Hsps in the unicellular cyanobacteria (Synechocystis and Synechococcus) as well as that of Hsp60 proteins in the filamentous cyanobacteria (Anabaena) to thermotolerance has been elucidated. The regulation of chaperone genes by several cis-elements and trans-acting factors has also been well documented. Recent studies have demonstrated novel transcriptional and translational (mRNA secondary structure) regulatory mechanisms in unicellular cyanobacteria. This article provides an insight into the heat-shock response: its organization, and ecophysiological regulation and role of molecular chaperones, in unicellular and filamentous nitrogen-fixing cyanobacterial strains.

Tributyl phosphate biodegradation to butanol and phosphate and utilization by a novel bacterial isolate, Sphingobium sp. strain RSMS.

A Sphingobium sp. strain isolated from radioactive solid waste management site (RSMS) completely degraded 7.98 g/L of tributyl phosphate (TBP) from TBP containing suspensions in 3 days. It also completely degraded 20 mM dibutyl phosphate (DBP) within 2 days. The strain tolerated high levels of TBP and showed excellent stability with respect to TBP degradation over several repeated subcultures. On solid minimal media or Luria Bertani media supplemented with TBP, the RSMS strain showed a clear zone of TBP degradation around the colony. Gas chromatography and spectrophotometry analyses identified DBP as the intermediate and butanol and phosphate as the products of TBP biodegradation. The RSMS strain utilized both TBP and DBP as the sole source of carbon and phosphorous for its growth. The butanol released was completely utilized by the strain as a carbon source thereby leaving no toxic residue in the medium. Degradation of TBP or DBP was found to be suppressed by high concentration of glucose which also inhibited TBP or DBP dependent growth. The results highlight the potential of Sphingobium sp. strain RSMS for bioremediation of TBP and for further molecular investigation.

High radiation and desiccation tolerance of nitrogen-fixing cultures of the cyanobacterium Anabaena sp. strain PCC 7120 emanates from genome/proteome repair capabilities.

The filamentous nitrogen-fixing cyanobacterium, Anabaena sp. strain PCC 7120 was found to tolerate very high doses of 60Co-gamma radiation or prolonged desiccation. Post-stress, cells remained intact and revived all the vital functions. A remarkable capacity to repair highly disintegrated genome and recycle the damaged proteome appeared to underlie such high radioresistance and desiccation tolerance. The close similarity observed between the cellular response to irradiation or desiccation stress lends strong support to the notion that tolerance to these stresses may involve similar mechanisms.

Bioprecipitation of uranium from alkaline waste solutions using recombinant Deinococcus radiodurans.

Bioremediation of uranium (U) from alkaline waste solutions remains inadequately explored. We engineered the phoK gene (encoding a novel alkaline phosphatase, PhoK) from Sphingomonas sp. for overexpression in the radioresistant bacterium Deinococcus radiodurans. The recombinant strain thus obtained (Deino-PhoK) exhibited remarkably high alkaline phosphatase activity as evidenced by zymographic and enzyme activity assays. Deino-PhoK cells could efficiently precipitate uranium over a wide range of input U concentrations. At low uranyl concentrations (1 mM), the strain precipitated >90% of uranium within 2h while a high loading capacity of around 10.7 g U/g of dry weight of cells was achieved at 10 mM U concentration. Uranium bioprecipitation by Deino-PhoK cells was not affected in the presence of Cs and Sr, commonly present in intermediate and low level liquid radioactive waste, or after exposure to very high doses of ionizing radiation. Transmission electron micrographs revealed the extracellular nature of bioprecipitated U, while X-ray diffraction and fluorescence analysis identified the precipitated uranyl phosphate species as chernikovite. When immobilized into calcium alginate beads, Deino-PhoK cells efficiently removed uranium, which remained trapped in beads, thus accomplishing physical separation of precipitated uranyl phosphate from solutions. The data demonstrate superior ability of Deino-PhoK, over earlier reported strains, in removal of uranium from alkaline solutions and its potential use in bioremediation of nuclear and other waste.