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PubMed2006

A highly thermostable, homodimeric single-stranded DNA-binding protein from Deinococcus radiopugnans.

We report the identification and characterization of the single-stranded DNA-binding protein (SSB) from the mesophile and highly radiation-resistant Deinococcus radiopugnans (DrpSSB). PCR-derived DNA fragment containing the complete structural gene for DrpSSB protein was cloned and expressed in Escherichia coli. The gene consisting of an open reading frame of 900 nucleotides encodes a protein of 300 amino acids with a calculated molecular weight of 32.45 kDa and pI 5.34. The amino acids sequence exhibits 43, 44, 79 and 18% identity with Thermus aquaticus, Thermus thermophilus, Deinococcus radiodurans and E. coli SSBs, respectively. The DrpSSB includes two OB folds per monomer and functions as a homodimer. In fluorescence titrations with poly(dT), DrpSSB bound 24-31 nt depending on the salt concentration, and fluorescence was quenched by about 80%. In a complementation assay in E. coli, DrpSSB took over the in vivo function of EcoSSB. The half-lives of DrpSSB were 120 min at 90 degrees C, 60 min at 95 degrees C and 30 min at 100 degrees C. These results were surprising in the context of half-life of SSB from thermophilic T. aquaticus, which has only 30 s of half-life at 95 degrees C. DrpSSB is the most thermostable SSB-like protein identified to date, offering an attractive alternative for TaqSSB and TthSSB in their applications for molecular biology methods and analytical purposes.

Amino Acid Sequence↗
PubMed2005

Analysis of interaction between DNA and Deinococcus radiodurans PprA protein by atomic force microscopy.

A DNA repair-promoting protein, PprA, was isolated from a radiation resistant bacterium, Deinococcus radiodurans [I. Narumi, K. Sato, S. Cui, T. Funayama, S. Kitayama, H. Watanabe, PprA: a novel protein from Deinococcus radiodurans that stimulates DNA ligation, Mol. Microbiol. 54 (2004) 278-285]. Despite several studies, however, the function of PprA is not still clear. We used atomic force microscopy (AFM) to elucidate the role of this protein in the DNA repair pathway. In the present study, interaction between the linear DNA and PprA protein was imaged and analyzed by AFM without any fixation or staining. Though both end-bound and internally bound PprA was observed, the affinity of the end-bound protein was greater considering the proportion of features of binding analyzed by AFM. In some conditions, looping forms of the DNA-PprA complex were observed. Gel filtration high performance liquid chromatography (HPLC) was also conducted to estimate the molecular weight of this protein. The result of the HPLC analysis suggested that PprA formed multimers in buffer solution without DNA.

Bacterial Proteins↗
PubMed2003

Pyrroloquinoline-quinone synthesized in Escherichia coli by pyrroloquinoline-quinone synthase of Deinococcus radiodurans plays a role beyond mineral phosphate solubilization.

Deinococcus radiodurans, an extremely radioresistant bacterium, synthesizes coenzyme pyrroloquinoline-quinone (PQQ) but exhibits a negative phenotype for mineral phosphate solubilization. Gene for the putative PQQ synthesizing protein was PCR amplified and cloned from Deinococcus, sequenced, and expressed in Escherichia coli, under an inducible E. coli promoter. The transgenic E. coli expressed PQQ synthase protein of 42kDa and complemented the mineral phosphate solubilization phenotype of E. coli, suggesting the synthesis of an active protein. The cells expressing high levels of this protein showed increased protection against photodynamically produced reactive oxygen species. The effect could be attributed to the upregulation of antioxidant enzymes such as catalase and superoxide dismutase by PQQ in transgenic E. coli through an unknown mechanism. The study elucidates a hitherto unknown possible function of PQQ in bacteria.

Antioxidants↗
PubMedDate not supplied

Enantioselective synthesis of L-homophenylalanine by whole cells of recombinant Escherichia coli expressing L-aminoacylase and N-acylamino acid racemase genes from Deinococcus radiodurans BCRC12827.

L-Homophenylalanine (l-HPA) is a chiral unnatural amino acid used in the synthesis of angiotensin converting enzyme inhibitors and many pharmaceuticals. To develop a bioconversion process with dynamic resolution of N-acylamino acids for the l-HPA production, N-acylamino acid racemase (NAAAR) and l-aminoacylase (LAA) genes were cloned from Deinococcus radiodurans BCRC12827 and expressed in Escherichia coli XLIBlue. The recombinant enzymes were purified by nickel-chelate chromatography, and their biochemical properties were determined. The NAAAR had high racemization activity toward chiral N-acetyl-homophenylalanine (NAc-HPA). The LAA exhibited strict l-enantioselection to hydrolyze the NAc-l-HPA. A stirred glass vessel containing transformed E. coli cells expressing D. radiodurans NAAAR and LAA was used for the conversion of NAc-d-HPA to l-HPA. Unbalance activities of LAA and NAAAR were found in E. coli cell coexpressing laa and naaar genes, which resulted in the accumulation of an intermediate, NAc-l-HPA, in the early stage of conversion and a low productivity of 0.83 mmol l-HPA/L h. The results indicated that low activity of LAA present in the biomass is the rate-limiting factor in l-HPA production. In the case of two whole cells with separately expressed enzyme, the enzymatic activities of LAA and NAAAR could be balanced by changing the loading of individual cells. When the activities of two enzymes were fixed at 3600 U/L, 99.9% yield of l-HPA could be reached in 1 h, with a productivity of 10 mmol l-HPA/L h. The cells can be reused at least six cycles at a conversion yield of more than 96%. This is the first NAAAR/LAA process using NAc-HPA as substrate and recombinant whole cells containing Deinococcus enzymes as catalysts for the production of l-HPA to be reported.

Amidohydrolases↗
PubMed2004

Accumulation of Mn(II) in Deinococcus radiodurans facilitates gamma-radiation resistance.

Deinococcus radiodurans is extremely resistant to ionizing radiation. How this bacterium can grow under chronic gamma radiation [50 grays (Gy) per hour] or recover from acute doses greater than 10 kGy is unknown. We show that D. radiodurans accumulates very high intracellular manganese and low iron levels compared with radiation-sensitive bacteria and that resistance exhibits a concentration-dependent response to manganous chloride [Mn(II)]. Among the most radiation-resistant bacterial groups reported, Deinococcus, Enterococcus, Lactobacillus, and cyanobacteria accumulate Mn(II). In contrast, Shewanella oneidensis and Pseudomonas putida have high iron but low intracellular manganese concentrations and are very sensitive. We propose that Mn(II) accumulation facilitates recovery from radiation injury.

Culture Media↗
PubMed2003

Engineering Deinococcus geothermalis for bioremediation of high-temperature radioactive waste environments.

Deinococcus geothermalis is an extremely radiation-resistant thermophilic bacterium closely related to the mesophile Deinococcus radiodurans, which is being engineered for in situ bioremediation of radioactive wastes. We report that D. geothermalis is transformable with plasmids designed for D. radiodurans and have generated a Hg(II)-resistant D. geothermalis strain capable of reducing Hg(II) at elevated temperatures and in the presence of 50 Gy/h. Additionally, D. geothermalis is capable of reducing Fe(III)-nitrilotriacetic acid, U(VI), and Cr(VI). These characteristics support the prospective development of this thermophilic radiophile for bioremediation of radioactive mixed waste environments with temperatures as high as 55 degrees C.

Biodegradation, Environmental↗
PubMed2005

Extensive diversity of ionizing-radiation-resistant bacteria recovered from Sonoran Desert soil and description of nine new species of the genus Deinococcus obtained from a single soil sample.

The ionizing-radiation-resistant fractions of two soil bacterial communities were investigated by exposing an arid soil from the Sonoran Desert and a nonarid soil from a Louisiana forest to various doses of ionizing radiation using a (60)Co source. The numbers of surviving bacteria decreased as the dose of gamma radiation to which the soils were exposed increased. Bacterial isolates surviving doses of 30 kGy were recovered from the Sonoran Desert soil, while no isolates were recovered from the nonarid forest soil after exposure to doses greater than 13 kGy. The phylogenetic diversities of the surviving culturable bacteria were compared for the two soils using 16S rRNA gene sequence analysis. In addition to a bacterial population that was more resistant to higher doses of ionizing radiation, the diversity of the isolates was greater in the arid soil. The taxonomic diversity of the isolates recovered was found to decrease as the level of ionizing-radiation exposure increased. Bacterial isolates of the genera Deinococcus, Geodermatophilus, and Hymenobacter were still recovered from the arid soil after exposure to doses of 17 to 30 kGy. The recovery of large numbers of extremely ionizing-radiation-resistant bacteria from an arid soil and not from a nonarid soil provides further ecological support for the hypothesis that the ionizing-radiation resistance phenotype is a consequence of the evolution of other DNA repair systems that protect cells against commonly encountered environmental stressors, such as desiccation. The diverse group of bacterial strains isolated from the arid soil sample included 60 Deinococcus strains, the characterization of which revealed nine novel species of this genus.

DNA, Bacterial↗
PubMed2006

Characterization of adhesion threads of Deinococcus geothermalis as type IV pili.

Deinococcus geothermalis E50051 forms tenuous biofilms on paper machine surfaces. Field emission electron microscopy analysis revealed peritrichous appendages which mediated cell-to-surface and cell-to-cell interactions but were absent in planktonically grown cells. The major protein component of the extracellular extract of D. geothermalis had an N-terminal sequence similar to the fimbrial protein pilin annotated in the D. geothermalis DSM 11300 draft sequence. It also showed similarity to the type IV pilin sequence of D. radiodurans and several gram-negative pathogenic bacteria. Other proteins in the extract had N-terminal sequences identical to D. geothermalis proteins with conservative motifs for serine proteases, metallophosphoesterases, and proteins whose function is unknown. Periodic acid-Schiff staining for carbohydrates indicated that these extracellular proteins may be glycosylated. A further confirmation for the presence of glycoconjugates on the cell surface was obtained by confocal laser scanning imaging of living D. geothermalis cells stained with Amaranthus caudatus lectin, which specifically binds to galactose residues. The results indicate that the thread-like appendages of D. geothermalis E50051 are glycosylated type IV pili, bacterial attachment organelles which have thus far not been described for the genus Deinococcus.

Bacterial Adhesion↗
PubMed2006

Identification and characterization of a novel bacterial sulfite oxidase with no heme binding domain from Deinococcus radiodurans.

An open reading frame (draSO) encoding a putative sulfite oxidase (SO) was identified in the sequence of chromosome II of Deinococcus radiodurans; the predicted gene product showed significant amino acid sequence homology to several bacterial and eukaryotic SOs, such as the biochemically and structurally characterized enzyme from Arabidopsis thaliana. Cloning of the Deinococcus SO gene was performed by PCR amplification from the bacterial genomic DNA, and heterologous gene expression of a histidine-tagged polypeptide was obtained in a molybdopterin-overproducing strain of Escherichia coli. The recombinant protein was purified to homogeneity by nickel chelating affinity chromatography, and its main kinetic and chemical physical parameters were determined. Northern blot and enzyme activity analyses indicated that draSO gene expression is constitutive in D. radiodurans and that there is no increase upon exposure to thiosulfate and/or molybdenum(II).

Amino Acid Sequence↗
PubMed2005

[Cloning and expression of superoxide dismutase gene from Deinococcus radiodurans in E. coli].

OBJECTIVE: To construct expressing recombinant of Mn-SOD of Deinococcus radiodurans and express the target protein in E. coli BL21(DE3). METHODS: SOD gene was amplified by PCR from genomic DNA of Deinococcus radiodurans and inserted into expression plasmid pET-30a(+) to create the recombinant pET-SOD. After being analyzed by the restriction endonuclease, the plasmid was transformed into E. coli BL21(DE3), and the recombinant protein was expressed after induction by the isopropyl-beta-D-thiogalactopyranoside (IPTG) and was analyzed with SDS-PAGE. RESULTS: The recombinant plasmid pET-SOD was obtained, and the recombinant protein was highly expressed in E. coli BL21(DE3). The activity of recombinant superoxide dismutase was 51,800 U per gram of wet bacteria. CONCLUSION: This study has provided a foundation for further studies and applications of the recombinant Mn-SOD.

Cloning, Molecular↗
PubMed2006

Occurrence of thioredoxin reductase in Deinococcus species, the UV resistant bacteria.

The occurrence of thioredoxin reductase (NAD(P)H: oxidized-thioredoxin reductase, EC 1.6.4.5, TrxR) in five mesophilic species of Deinococcus was investigated by PAGE. Each species possessed a unique TrxR pattern, for example, a single TrxR characterized D. radiopugnans while multiple forms of TrxR occurred in other Deinococcal spp. Most of TrxRs occurring in Deinococcus showed dual cofactor specificity, active with either NADH or NADPH, although the NADPH specific-TrxR was observed in D. radiophilus and D.proteolyticus.

Deinococcus↗
PubMed1997

The sequences of heat shock protein 40 (DnaJ) homologs provide evidence for a close evolutionary relationship between the Deinococcus-thermus group and cyanobacteria.

The genes encoding for heat shock protein 40 (Hsp40 or DnaJ) homologs were cloned and sequenced from the archaebacterium Halobacterium cutirubrum and the eubacterium Deinococcus proteolyticus to add to sequences from the gene banks. These genes were identified downstream of the Hsp70 (or DnaK) genes in genomic fragments spanning this region and, as in other prokaryotic species, Hsp70-Hsp40 genes are likely part of the same operon. The Hsp40 homolog from D. proteolyticus was found to be lacking a central 204 base pair region present in H. cutirubrum that encodes for the four cysteine-rich domains of the repeat consensus sequence CxxCxGxG (where x is any amino acid), present in most Hsp40 homologs. The available sequences from various archaebacteria, eubacteria, and eukaryotes show that the same deletion is also present in the homologs from Thermus aquaticus and two cyanobacteria, but in no other species tested. This unique deletion and the clustering of homologs from the Deinococcus-Thermus group and cyanobacterial species in the Hsp40 phylogenetic trees suggest a close evolutionary relationship between these groups as was also shown recently for Hsp70 sequences (R.S. Gupta et al., J Bacteriol 179:345-357, 1997). Sequence comparisons indicate that the Hsp40 homologs are not as conserved as the Hsp70 sequences. Phylogenetic analysis provides no reliable information concerning evolutionary relationship between prokaryotes and eukaryotes and their usefulness in this regard is limited. However, in phylogenetic trees based on Hsp40 sequences, the two archaebacterial homologs showed a polyphyletic branching within Gram-positive bacteria, similar to that seen with Hsp70 sequences.

Amino Acid Sequence↗
PubMed1998

Signature sequences in diverse proteins provide evidence of a close evolutionary relationship between the Deinococcus-thermus group and cyanobacteria.

A number of proteins have been identified that contain prominent sequence signatures that are uniquely shared by the members of the Deinococcus-Thermus genera and the cyanobacterial species but which are not found in any of the other eubacterial or archaebacterial homologs. The proteins containing such sequence signatures include (1) the DnaJ/Hsp40 family of proteins, (2) DNA polymerase I, (3) the protein synthesis elongation factor EF-Tu, and (4) the elongation factor EF-Ts. A strong affinity of the Deinococcus-Thermus species to cyanobacteria is also seen in the phylogenetic trees based on Hsp70 and DnaJ sequences. These results provide strong evidence of a close and specific evolutionary relationship between species belonging to these two eubacterial divisions.

Amino Acid Sequence↗
PubMed1996

Sequence analysis of the L-lactate dehydrogenase-encoding gene of Deinococcus radiodurans, a suitable mesophilic counterpart for Thermus.

A gene encoding L-lactate dehydrogenase (LDH; EC 1.1.1.27) from Deinococcus radiodurans (Dr) was cloned and sequenced. The deduced amino acid (aa) sequence was compared to those of the Thermus aquaticus (Ta) and T. caldophilus (Tc) LDH. The similarity of the G + C contents between the Dr and Thermus genes permits the comparison of the aa sequences of LDH without considering the difference in the G + C contents. Compared to the mesophilic Dr LDH, increased numbers of hydrophobic aa, together with hydrophilic Arg, were found in the LDH from Ta and Tc, suggesting that these features would contribute to protein thermostability in part via hydrophobic and electrostatic interactions in the protein globule. Deinococcus would be a suitable mesophilic counterpart for Thermus to investigate the thermostability of proteins.

Amino Acid Sequence↗
PubMed2000

Specific expansion of protein families in the radioresistant bacterium Deinococcus radiodurans.

Computer analysis of the complete genome of Deinococcus radiodurans R1 reveals a number of protein families, which are over-represented in this organism, compared to most other bacteria with known genome sequences. These families include both previously characterized and uncharacterized proteins. Most of the families whose functions are known or could be predicted seem to be related to stress-response and elimination of damage products (cell-cleaning). The two most prominent family expansions are the Nudix (MutT) family of pyrophosphohydrolases and a previously unnoticed family of proteins related to Bacillus subtilis DinB that could possess a metal-dependent enzymatic activity whose exact nature remains to be determined. Several proteins of the expanded families, particularly the Nudix family, are fused to other domains and form multidomain proteins that are so far unique for Deinococcus. The domain composition of some of these proteins indicates that they could be involved in novel DNA-repair pathways. Such unique proteins are good targets for knock-out and gene expression studies, which are aimed to shed light on the unusual features of this interesting bacterium.

Bacteria↗
PubMed2001

Predicted highly expressed and putative alien genes of Deinococcus radiodurans and implications for resistance to ionizing radiation damage.

Predicted highly expressed (PHX) and putative alien genes determined by codon usages are characterized in the genome of Deinococcus radiodurans (strain R1). Deinococcus radiodurans (DEIRA) can survive very high doses of ionizing radiation that are lethal to virtually all other organisms. It has been argued that DEIRA is endowed with enhanced repair systems that provide protection and stability. However, predicted expression levels of DNA repair proteins with the exception of RecA tend to be low and do not distinguish DEIRA from other prokaryotes. In this paper, the capability of DEIRA to resist extreme doses of ionizing and UV radiation is attributed to an unusually high number of PHX chaperone/degradation, protease, and detoxification genes. Explicitly, compared with all current complete prokaryotic genomes, DEIRA contains the greatest number of PHX detoxification and protease proteins. Other sources of environmental protection against severe conditions of UV radiation, desiccation, and thermal effects for DEIRA are the several S-layer (surface structure) PHX proteins. The top PHX gene of DEIRA is the multifunctional tricarboxylic acid (TCA) gene aconitase, which, apart from its role in respiration, also alerts the cell to oxidative damage.

Chromosomes, Bacterial↗
PubMed1999

Genome sequence of the radioresistant bacterium Deinococcus radiodurans R1.

The complete genome sequence of the radiation-resistant bacterium Deinococcus radiodurans R1 is composed of two chromosomes (2,648,638 and 412,348 base pairs), a megaplasmid (177,466 base pairs), and a small plasmid (45,704 base pairs), yielding a total genome of 3,284, 156 base pairs. Multiple components distributed on the chromosomes and megaplasmid that contribute to the ability of D. radiodurans to survive under conditions of starvation, oxidative stress, and high amounts of DNA damage were identified. Deinococcus radiodurans represents an organism in which all systems for DNA repair, DNA damage export, desiccation and starvation recovery, and genetic redundancy are present in one cell.

Bacterial Proteins↗
PubMed1986

Molecular cloning, expression, and characterization of the gene for the surface (HPI)-layer protein of Deinococcus radiodurans in Escherichia coli.

The HPI protein of Deinococcus radiodurans belongs to the class of surface layer proteins which form crystalline two-dimensional arrays on bacterial cell envelopes. We have cloned and expressed the gene for this protein of Mr about 100,000 by using plasmid pUC8 in Escherichia coli. As judged by immunoreaction with monospecific antibodies, apparent Mr, and limited proteolysis, a single clone contained the gene encoding the complete polypeptide on an 8.9-kilobase (kb) insert. The insert was reduced to a 5.7-kb HindIII fragment, cloned in pUC18 in both orientations, and subjected to unilateral processive deletion with exonuclease III. The library of deletion derivatives was mapped and, in conjunction with protein immunoblotting of expressed polypeptides, was used to locate the positions of the structural gene and the Deinococcus promoter region that was responsible for expression of the HPI polypeptide. The HPI gene was confined to a stretch 2.95 kb in length.

Bacterial Proteins↗