Deinococcus Radiopugnans Research Articles

Hydrogen peroxide treatment induces the transposition of an insertion sequence in Deinococcus radiopugnans DY59

Deinococcus radiopugnans DY59 (formerly Deinococcus swuensis DY59) is a radiation-resistant bacterium isolated from soil. From the 3.5 Mb genomic DNA sequence of strain DY59 (December 2014), 31 insertion sequence (IS) elements of six IS families including IS1, IS4, IS5, IS66, IS630, and IS701 and five unclassified IS elements were detected. Upon induction of oxidative stress with 80 and 100 mM H2O2, the unique ISs of the IS4 family member were actively translocated into a carotenoid biosynthesis gene phytoene desaturase (QR90_10400), resulting in non-pigment phenotypic selection. Therefore, these active transpositions of a specific IS family member were induced by oxidative stress at 80 and 100 mM H2O2. Furthermore, D. radiopugnans DY59 exhibited extremely higher MIC values against H2O2 treatment. To explain this phenomenon, qRT-PCR was conducted to assess the expression levels of catalase and three LysR family regulators. Our findings indicated that the ISDrpg2 and ISDrpg3 elements of the IS4 family were actively transposed into the phytoene desaturase gene by H2O2 treatment via replicative transposition. However, high H2O2 resistance did not originate from H2O2-induced expression of catalase and LysR family regulators.

Transposition of insertion sequences by dielectric barrier discharge plasma and gamma irradiation in the radiation-resistant bacterium Deinococcus geothermalis

Insertion sequences (ISs) of the radiation-resistant bacterium Deinococcus geothermalis are transposed into other loci by oxidative stress through hydrogen peroxide treatment. Gamma irradiation and dielectric barrier discharge (DBD) plasma radiation are known to produce a variety of oxidative stress agents such as reactive oxygen species and reactive nitrogen species. Therefore, to determine whether the transposition of ISs was induced in D. geothermalis by both gamma irradiation and DBD plasma radiation, we selected non-pigmented mutants with disrupted target genes encoding carotenoid biosynthesis enzymes such as a phytoene synthase (dgeo_0523) and a phytoene desaturase (dgeo_0524). Different DNA-binding protein-deficient mutants exhibited novel transposition of ISs. Dps (dgeo_0257), OxyR (dgeo_1888), and the LysR (dgeo_2840) family regulator, in addition to cystine importer-disrupted and -overexpressed mutants (dgeo_1986–87 and dgeo_1985R) and wild-type D. geothermalis were tested in this study. Active IS transposition was not detected in two wild-type control species (Deinococcus radiodurans and Deinococcus radiopugnans) after phenotypic selection in gamma irradiation. Our finding demonstrated that gamma irradiation triggers the transposition of particular IS elements, especially ISDge2 and ISDge3 of the IS1 family, ISDge5 of the IS701 family, and ISDge6 of the IS5 family in wild-type strain and the Δdgeo_0257, Δdgeo_1986–87, Δdgeo_1985R, and Δdgeo_2840 mutants. Furthermore, DBD plasma radiation triggered the transposition of ISDge11 of the IS4 family in the wild-type strain; ISDge6 of the IS5 family on Δdgeo_0257, Δdgeo_1888 and Δdgeo_2840; ISDge5 of the IS701 family on Δdgeo_0257 strain.

Genome based reclassification of Deinococcus swuensis as a heterotypic synonym of Deinococcus radiopugnans.

Deinococcus species are widely studied due to their utility in bioremediation of sites contaminated with radioactive elements. In the present study, we re-evaluated the taxonomic placement of two species of the genus Deinococcus namely D. swuensis DY59T and D. radiopugnans ATCC 19172T based on whole genome analyses. The 16S rRNA gene analysis revealed a 99.58% sequence similarity between this species pair that is above the recommended threshold value for species delineation. These two species also clustered together in both the 16S rRNA gene and core genome based phylogenies depicting their close relatedness. Furthermore, more than 98% of genes were shared between D. swuensis DY59T and D. radiopugnans ATCC 19172T. Interestingly, D. swuensis DY59T and D. radiopugnans ATCC 19172T shared high genome similarity in different genomic indices. They displayed an average nucleotide identity value of 97.63%, an average amino acid identity value of 97% and a digital DNA-DNA hybridization value equal to 79.50%, all of which are well above the cut-off for species delineation. Altogether, based on these evidences, D. swuensis DY59T and D. radiopugnans ATCC 19172T constitute a single species. Hence, as per the priority of publication, we propose that Deinococcus swuensis Lee et al. 2015 should be reclassified as a later heterotypic synonym of Deinococcus radiopugnans.

Radio-protective and antioxidative activities of astaxanthin from newly isolated radio-resistant bacterium Deinococcus sp. strain WMA-LM9

A radio-resistant bacterium, designated as strain WMA-LM9, was isolated from desert soil. 16S rRNA gene sequencing indicated that the bacterium belongs to genus Deinococcus with maximum similarity to Deinococcus radiopugnans. Deinococcus sp. strain WMA-LM9 was found to be resistant to a ultraviolet (UV) dose of 5 × 103 J/m2, hydrogen peroxide (50 mM) and mitomycin C (10 μg/ml). A carotenoid pigment was extracted using chloroform/methanol/acetone (7:5:3) and purified by high-performance liquid chromatography on a C18 analytical column. The compound was characterised as mono-esterified astaxanthin by 1H, 13C nuclear magnetic resonance and mass spectrometry. It was tested for antioxidant activity, total flavonoids and phenolic content, radioprotective potential in correlation to the prevention of protein oxidation and DNA strand breaks in vitro. The carotenoid pigment showed a very potent antioxidant activity and significantly stronger scavenging ability against superoxides, with an IC50 (concentration causing 50% inhibition of the desired activity) of 41.6 μg/ml. The total phenolic and flavonoid contents were 12.1 and 7.4 μg in terms of gallic acid and quercetin equivalents per milligram of dried mass, respectively. astaxanthin also showed a higher inhibitory action against oxidative damage to collagen, elastin and bovine serum albumin than did β-carotene. The carotenoid also inhibited breaks to DNA strands, as indicated by the results of the DNA damage prevention assay. We conclude that astaxanthin from Deinococcus sp. strain WMA-LM9 has protective effects against radiation-mediated cell damage, and it also protects cellular protein and DNA against oxidative stress and other anti-oxidant activities.

Acceptor Specificity of Amylosucrase from Deinococcus radiopugnans and Its Application for Synthesis of Rutin Derivatives.

The transglycosylation activity of amylosucrase (ASase) has received significant attention owing to its use of an inexpensive donor, sucrose, and broad acceptor specificity, including glycone and aglycone compounds. The transglycosylation reaction of recombinant ASase from Deinococcus radiopugnans (DRpAS) was investigated using various phenolic compounds, and quercetin-3-O-rutinoside (rutin) was found to be the most suitable acceptor molecule used by DRpAS. Two amino acid residues in DRpAS variants (DRpAS Q299K and DRpAS Q299R), assumed to be involved in acceptor binding, were constructed by site-directed mutagenesis. Intriguingly, DRpAS Q299K and DRpAS Q299R produced 10-fold and 4-fold higher levels of rutin transglycosylation product than did the wild-type (WT) DRpAS, respectively. According to in silico molecular docking analysis, the lysine residue at position 299 in the mutants enables rutin to more easily position inside the active pocket of the mutant enzyme than in that of the WT, due to conformational changes in loop 4.

Sec Pathway Influences the Growth of Deinococcus radiodurans

The release of extracellular DNA molecules (eDNA) contributes to various biological processes, such as biofilm formation, virulence, and stress tolerance. The quantity of eDNA released by bacteria is usually regulated by extracellular nucleases that are secreted by different systems. In this study, we show that high concentrations of eDNA inhibit the growth of two strains of Deinococcaceae, Deinococcus radiodurans, and Deinococcus radiopugnans, but have no effect on other selected organisms, such as Escherichia coli. In D. radiodurans, an extracellular nuclease was shown to be secreted through the Sec pathway. Disruption of one member of this pathway, SecD/F, inhibited cell growth, suggesting that the Sec pathway plays an important role in growth rate. However, the Sec pathway mutant exhibited a greater deficiency in growth rate compared with the extracellular nuclease mutant, indicating that the pathway not only secretes the extracellular nuclease, but has other unknown functions as well.

Molecular cloning and expression of amylosucrase from highly radiation-resistant Deinococcus radiopugnans

Amylosucrase (AS), a glucosyltransferase of the glycoside hydrolase 13 family, does not require a nucleotide-activated sugar as a glucosyl-donor. A gene (drpas) of Deinococcus radiopugnans encoding a putative AS was identified and cloned for characterization. The amino acid sequence revealed that drpas exhibited 76 and 74% identities with AS genes from D. geothermalis and D. radiodurans, respectively. Recombinant DRpAS exhibited AS activities. The ratios of hydrolysis, polymerization, and isomerization reactions were 5.8:82.7:11.5 at 40°C. The DRpAS was highly thermostable and produced more polymerization products than AS from D. radiodurans although the optimum growth temperature of both strains is 30°C.

Deinococcus swuensis sp. nov., a gamma-radiation-resistant bacterium isolated from soil

Strain DY59(T), a Gram-positive non-motile bacterium, was isolated from soil in South Korea, and was characterized to determine its taxonomic position. Phylogenetic analysis based on the 16S rRNA gene sequence of strain DY59(T) revealed that the strain DY59(T) belonged to the family Deinococcaceae in the class Deinococci. The highest degree of sequence similarities of strain DY59(T) were found with Deinococcus radiopugnans KACC 11999(T) (99.0%), Deinococcus marmoris KACC 12218(T) (97.9%), Deinococcus saxicola KACC 12240(T) (97.0%), Deinococcus aerolatus KACC 12745(T) (96.2%), and Deinococcus frigens KACC 12220(T) (96.1%). Chemotaxonomic data revealed that the predominant fatty acids were iso-C15:0 (19.0%), C16:1 ω7c (17.7%), C15:1 ω6c (12.6%), iso-C17:0 (10.3%), and iso-C17:1 ω9c (10.3%). A complex polar lipid profile consisted of a major unknown phosphoglycolipid. The predominant respiratory quinone is MK-8. The cell wall peptidoglycan contained D-alanine, L-glutamic acid, glycine, and L-ornithine (di-amino acid). The novel strain showed resistance to gamma radiation, with a D10 value (i.e. the dose required to reduce the bacterial population by 10-fold) in excess of 5 kGy. Based on the phylogenetic, chemotaxonomic, and phenotypic data, strain DY59(T) (=KCTC 33033(T) =JCM 18581(T)) should be classified as a type strain of a novel species, for which the name Deinococcus swuensis sp. nov. is proposed.

Development of a Qualitative Dose Indicator for Gamma Radiation Using Lyophilized Deinococcus

The feasibility of using Deinococcus showing strong resistance to both desiccation and ionizing radiation as a dose indicator of gamma radiation exposure was evaluated. Three Deinococcus strains having different levels of radiation resistance, Deinococcus radiodurans (DRD), Deinococcus radiopugnans (DRP), and the DRD pprI mutant (DRM), were selected to develop an appropriate dose indicator for a broad range of exposures. DRD, DRP, and DRM cultures with different numbers of cells [~10(7) to 10(3) colony forming units (CFU)/100 microliter] were lyophilized and subjected to various doses of gamma radiation to determine a critical dose that inhibited bacterial growth completely. Finally, a combination of DRD at ~10(7) and ~10(6) CFU, DRP at ~10(5) CFU, and DRM at ~10(4) CFU successfully indicated exposure to 5, 10, 20, and 30 kGy of gamma radiation, respectively. This study shows the possibility of developing a qualitative indicator of radiation exposure using Deinococcus.

Development of versatile shuttle vectors for Deinococcus grandis

To develop new shuttle vectors for Deinococcus species, the nucleotide sequence of the small cryptic plasmid pUE30 from Deinococcus radiopugnans ATCC19172 was determined. The 2467-bp plasmid possesses two open reading frames, one encoding 88 amino acid residues (Orf1) and the other encoding 501 amino acid residues (Orf2). The predicted amino acid sequence encoded by Orf1 exhibits similarity to the N-terminal regions of replication proteins encoded by repABC-type plasmids of a-proteobacteria . On the other hand, the predicted amino acid sequence encoded by Orf2 exhibits similarity to replication proteins encoded by plasmids of D. radiodurans SARK and Thermus species. Hybrid plasmids consisting of pUE30 and pKatCAT5, which replicates in E. coli with a chloramphenicol resistance determinant, were shown to autonomously replicate in D. grandis ATCC43672. Deletion analysis revealed that Orf2 was necessary for replication of the plasmids in D. grandis. On the other hand, a DNA fragment encompassing the Orf1-coding region was involved in the instability of the plasmid in D. grandis. An expression plasmid that possesses the D. radiodurans minimal groE promoter was constructed, and a firefly luciferase gene was successfully expressed in D. grandis. The D. grandis host-vector system developed in this study should prove useful in the bioremediation of radioactive waste and for the investigation of DNA repair mechanisms.

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.

Deinococcus ficus sp. nov., isolated from the rhizosphere of Ficus religiosa L.

A pale-pink strain (CC-FR2-10T) from the rhizosphere of the sacred tree Ficus religiosa L. in Taiwan was investigated by using a polyphasic taxonomic approach. The cells were Gram-positive, rod-shaped and non-spore-forming. Phylogenetic analyses using the 16S rRNA gene sequence of the isolate indicated that the organism belongs to the genus Deinococcus, the highest sequence similarities being found with Deinococcus grandis (96.1 %), Deinococcus radiodurans (94.3 %), Deinococcus radiopugnans (93.2 %), Deinococcus indicus (93.0 %), Deinococcus proteolyticus (92.5 %), Deinococcus murrayi (92.4 %) and Deinococcus geothermalis (90.7 %). The DNA-DNA relatedness with respect to D. grandis DSM 3963T was 17.9 %. Chemotaxonomic data revealed that strain CC-FR2-10T contains only menaquinone MK-8 as the respiratory quinone, unknown phosphoglycolipids as the predominant polar lipids and 16 : 1omega7c, 17 : 1omega8c and 17 : 1omega9c iso as the predominant fatty acids. The biochemical and chemotaxonomic properties demonstrate that strain CC-FR2-10T represents a novel species, for which the name Deinococcus ficus sp. nov. is proposed. The type strain is CC-FR2-10T (=CCUG 51391T [corrected] = CIP 108832T).

A ring-like nucleoid is not necessary for radioresistance in the Deinococcaceae

BackgroundTransmission electron microscopy images of Deinococcus radiodurans R1 suggest that the nucleoid of this species exists as a "ring-like" body, and have led to speculation that this structure contributes to the radioresistance of the species. Since extreme radioresistance is characteristic of six other species of Deinococcus, we have attempted to correlate nucleoid morphology and radioresistance by determining whether the genomic DNA of each of these species exhibit similar structures.ResultsThe nucleoid morphologies of seven recognized species of Deinococcus, the radioresistant bacterium Rubrobacter radiotolerans, and the more radiosensitive deinococcal relative Thermus aquaticus were evaluated using epifluorescence and deconvolution techniques. Although the nucleoids of Deinococcus murrayi, Deinococcus proteolyticus, Deinococcus radiophilus, and Deinococcus grandis have structures similar to D. radiodurans, the majority of nucleoids found in Deinococcus radiopugnans and Deinococcus geothermalis lack any specific organization. The nucleoid of R. radiotolerans consists of multiple highly condensed spheres of DNA scattered throughout the cell. The genomic DNA of Thermus aquaticus is uniformly distributed throughout the cell.ConclusionThere is no obvious relationship between the shape of a species' nucleoid and extreme radioresistance. However, the genomes of all extremely radioresistance species examined are highly condensed relative to more radiosensitive species. Whether DNA in this tightly packed configuration contributes to the radioresistance of these bacteria remains unknown, but this common structural feature appears to limit diffusion of fragments generated post-irradiation even in cells incapable of repairing strand breaks.

PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid.

A set of broad-range PCR primers for the 16S rRNA gene in bacteria were tested, along with three series of oligonucleotide probes to detect the PCR product. The first series of probes is broad in range and consists of a universal bacterial probe, a gram-positive probe, a Bacteroides-Flavobacterium probe, and two probes for other gram-negative species. The second series was designed to detect PCR products from seven major bacterial species or groups frequently causing meningitis: Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae, S. agalactiae, Escherichia coli and other enteric bacteria, Listeria monocytogenes, and Staphylococcus aureus. The third series was designed for the detection of DNA from species or genera commonly considered potential contaminants of clinical samples, including cerebrospinal fluid (CSF): Bacillus, Corynebacterium, Propionibacterium, and coagulase-negative Staphylococcus spp. The primers amplified DNA from all 124 different species of bacteria tested. Southern hybridization testing of the broad-range probes with washes containing 3 M tetramethylammonium chloride indicated that this set of probes correctly identified all but two of the 102 bacterial species tested, the exceptions being Deinococcus radiopugnans and Gardnerella vaginalis. The gram-negative and gram-positive probes hybridized to isolates of two newly characterized bacteria, Alloiococcus otitis and Rochalimaea henselii, as predicted by Gram stain characteristics. The CSF pathogen and contaminant probe sequences were compared with available sequence information and with sequencing data for 32 different species. Testing of the CSF pathogen and contaminant probes against DNA from over 60 different strains indicated that, with the exception of the coagulase-negative Staphylococcus probes, these probes provided the correct identification of bacterial species known to be found in CSF.