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

Two gamma- and UVC-resistant bacterial strains, designated JSH3-1T and 9-2-2, were isolated from garden soil in South Korea. Cells were Gram-stain-positive, aerobic, non-motile and spherical. A polyphasic approach was used to study the taxonomic properties of strains JSH3-1T and 9-2-2. Phylogenetic analysis based on nearly full-length 16S rRNA gene sequences of strains JSH3-1T and 9-2-2 indicated highest similarity with Deinococcus radiomollis PO-04-20-132T (94.7 and 94.9 %, respectively); levels of sequence similarity with the type strains of other Deinococcus species were less than 94.0 %. Strains JSH3-1T and 9-2-2 shared relatively high 16S rRNA gene sequence similarity (98.7 %) and had a high DNA reassociation value of 81±0.5 %. Meanwhile, they showed low levels of DNA reassociation (<25 %) with other closely related species of the genus Deinococcus. The two strains showed chemotaxonomic features typical of the genus Deinococcus, with the presence of menaquinone 8 as the respiratory quinone. The predominant fatty acids were iso-C17 : 0, iso-C13 : 0 and anteiso-C13 : 0. The polar lipids comprised phosphoglycolipid, aminophospholipid, glycolipid and unknown aminolipids. The DNA G+C contents of strains JSH3-1T and 9-2-2 were 62.0 and 61.9 mol%, respectively. On the basis of their phenotypic and genotypic characteristics, and phylogenetic distinction, strains JSH3-1T (=KCTC 33790T=JCM 31311T) and 9-2-2 (=KCTC 33789=JCM 31310) should be classified within a novel species of the genus Deinococcus, for which the name Deinococcus ruber sp. nov. is proposed.

Deinococcus radioresistens sp. nov., a UV and gamma radiation-resistant bacterium isolated from mountain soil.

Two Gram-negative, non-motile, short rod-shaped bacterial strains, designated as 8A(T) and 28A, were isolated from Mount Deogyusan, Jeonbuk Province, South Korea. The isolates were analyzed by a polyphasic approach, revealing variations in their phenotypic characters but high DNA-DNA hybridisation values reciprocally, confirming that they belong to the same species. Both the isolates also showed a high resistance to UV compared with Deinococcus radiodurans, and a gamma-radiation resistance similar to other members of the genus Deinococcus. Phylogenetic analysis with the 16S rRNA gene sequences of closely related species indicated their similarities were below 97 %. Chemotaxonomic data showed the most abundant fatty acids to be C16:1ω7c and C16:0. The strains can be distinguished from closely related species by the production of esterase (C4) and α-galactosidase, and by their ability to assimilate L-alanine, L-histidine and N-acetyl-D-glucosamine. Based on the phenotypic, phylogenetic, and chemotaxonomic data, the isolates represent a novel species of the genus Deinococcus, for which the name Deinococcus radioresistens sp. nov. is proposed. The type strain is 8A(T) (KEMB 9004-109(T) = JCM 19777(T)), and a second strain is 28A (KEMB 9004-113 = JCM 19778).

FrnE, a cadmium-inducible protein in Deinococcus radiodurans, is characterized as a disulfide isomerase chaperone in vitro and for its role in oxidative stress tolerance in vivo.

Deinococcus radiodurans R1 exposed to a lethal dose of cadmium shows differential expression of a large number of genes, including frnE (drfrnE) and some of those involved in DNA repair and oxidative stress tolerance. The drfrnE::nptII mutant of D. radiodurans showed growth similar to that of the wild type, but its tolerance to 10 mM cadmium and 10 mM diamide decreased by ~15- and ~3-fold, respectively. These cells also showed nearly 6 times less resistance to gamma radiation at 12 kGy and ~2-fold-higher sensitivity to 40 mM hydrogen peroxide than the wild type. In trans expression of drFrnE increased cytotoxicity of dithiothreitol (DTT) in the dsbA mutant of Escherichia coli. Recombinant drFrnE showed disulfide isomerase activity and could maintain insulin in its reduced form in the presence of DTT. While an equimolar ratio of wild-type protein could protect malate dehydrogenase completely from thermal denaturation at 42 °C, the C22S mutant of drFrnE provided reduced protection to malate dehydrogenase from thermal inactivation. These results suggested that drFrnE is a protein disulfide isomerase in vitro and has a role in oxidative stress tolerance of D. radiodurans possibly by protecting the damaged cellular proteins from inactivation.

Pigment-based whole-cell biosensor system for cadmium detection using genetically engineered Deinococcus radiodurans.

In this study, a colorimetric whole-cell biosensor for cadmium (Cd) was designed using a genetically engineered red pigment producing bacterium, Deinococcus radiodurans. Based on the previous microarray data, putative promoter regions of highly Cd-inducible genes (DR_0070, DR_0659, DR_0745, and DR_2626) were screened and used for construction of lacZ reporter gene cassettes. The resultant reporter cassettes were introduced into D. radiodurans R1 to evaluate promoter activity and specificity. Among the promoters, the one derived from DR_0659 showed the highest specificity, sensitivity, and activity in response to Cd. The Cd-inducible activity was retained in the 393-bp deletion fragment (P0659-1) of the P0569 promoter, but the expression pattern of the putative promoter fragments inferred its complex regulation. The detection range was from 10 to 1 mM of Cd. The LacZ expression was increased up to 100 µM of Cd, but sharply decreased at higher concentrations. For macroscopic detection, the sensor plasmid (pRADI-P0659-1) containing crtI as a reporter gene under the control of P0659-1 was introduced into a crtI-deleted mutant strain of D. radiodurans (KDH018). The color of this sensor strain (KDH081) changed from light yellow to red by the addition of Cd and had no significant response to other metals. Color change by the red pigment synthesis could be clearly recognized in a day with the naked eye and the detection range was from 50 nM to 1 mM of Cd. These results indicate that genetically engineered D. radiodurans (KDH081) can be used to monitor the presence of Cd macroscopically.

Genome-wide response of Deinococcus radiodurans on cadmium toxicity.

Deinococcus radiodurans is extremely resistant to various genotoxic conditions and chemicals. In this study, we characterized the effect of a sublethal concentration (100 microM) of cadmium (Cd) on D. radiodurans using a whole-genome DNA microarray. Time-course global gene expression profiling showed that 1,505 genes out of 3,116 total ORFs were differentially expressed more than 2-fold in response to Cd treatment for at least one timepoint. The majority of the upregulated genes are related to iron uptake, cysteine biosynthesis, protein disulfide stress, and various types of DNA repair systems. The enhanced upregulation of genes involved in cysteine biosynthesis and disulfide stress indicate that Cd has a high affinity for sulfur compounds. Provocation of iron deficiency and growth resumption of Cd-treated cells by iron supplementation also indicates that CdS forms in iron-sulfur-containing proteins such as the [Fe-S] cluster. Induction of base excision, mismatch, and recombinational repair systems indicates that various types of DNA damage, especially base excision, were enhanced by Cd. Exposure to sublethal Cd stress reduces the growth rate, and many of the downregulated genes are related to cell growth, including biosynthesis of cell membrane, translation, and transcription. The differential expression of 52 regulatory genes suggests a dynamic operation of complex regulatory networks by Cd-induced stress. These results demonstrate the effect of Cd exposure on D. radiodurans and how the related genes are expressed by this stress.

The combination of berberine and irradiation enhances anti-cancer effects via activation of p38 MAPK pathway and ROS generation in human hepatoma cells.

Berberine, an isoquinoline plant alkaloid, has been known to generate a wide variety of biochemical and pharmacological effects. In order to elucidate the molecular mechanism for the berberine-induced enhancement of radio-sensitization, the human hepatoma HepG2 cells were treated with berberine combined with irradiation. The anti-tumor effect of gamma radiation was found to be significantly enhanced by berberine. The evidences of apoptosis, such as apoptotic DNA fragmentation and annexin V staining, were observed in the cells treated with the combination of berberine and irradiation. Additionally, the levels of reactive oxygen species (ROS) and nitric oxide (NO) were apparently elevated in the combination system. The activations of p38, Bax, and caspase-3 were also detected in the irradiated cells pretreated with berberine. The productions of ROS and annexin V staining in the cells treated with the combination of berberine and irradiation were significantly inhibited by the specific inhibitor of p38 MAPK, SB203580. The cell death induced by berberine alone or the combination of berberine and irradiation was suppressed by the anti-oxidant, N-acetyl cysteine (NAC). Taken together, the present results clearly indicate that the combination of berberine and gamma-radiation enhance the anti-cancer effects through the p38 MAPK pathway and ROS generation.