Sunwook Jeong

Spirosoma pulveris sp. nov., a bacterium isolated from a dust sample collected at Chungnam province, South Korea

Strain JSH 5-14T, a Gram-negative, non-motile, and curved rod-shaped bacterium, was isolated from a dust sample collected at Nonsan, Chungnam province, South Korea, and was characterized to determine its taxonomic position. Phylogenetic analysis based on the 16S rRNA gene sequence of strain JSH 5-14T revealed that it belongs to the genus Spirosoma, family Cytophagaceae, class Cytophagia. The highest degree of sequence similarities of strain JSH 5-14T were found with Spirosoma liguale DSM 74T (97.8%) and Spirosoma endophyticum EX 36T (96.2%). The predominant fatty acids were summed feature 3 (composed of C16:1ω7c/C16:1ω6c) and C16:1ω5c. The major polar lipid was phosphatidylethanolamine, and the predominant respiratory quinone was MK-7. Based on the phylogenetic, chemotaxonomic, and phenotypic data, we propose the strain JSH 5-14T (=KCTC 42550T =JCM 30688T =KEMB 9004-165T) should be classified as a type strain of a novel species, for which the name Spirosoma pulveris sp. nov., is proposed.

Deinococcus actinosclerus sp. nov., a novel bacterium isolated from soil, South Korea.

Three Gram-stain-positive, catalase- and oxidase-positive coccus- or rod-shaped bacterial strains, designated BM2T, BM4 and BM5, were isolated from soil in South Korea. They showed strong resistance to gamma radiation with a D10 value of 9 kGy but weak UVC resistance. The 16S rRNA sequences of strains BM2T, BM4 and BM5 represent a novel subline within the genus Deinococcus in the family Deinococcaceae. The 16S rRNA gene sequences of strains BM2T, BM4 and BM5 were indistinguishable and showed 98.1-87.3 % similarity with other species of the genus Deinococcus. Strain BM2T exhibited relatively high levels of DNA-DNA hybridization with BM4 (87 ± 0.8 %) and BM5 (92 ± 1.2 %). Meanwhile, it showed a low level of DNA-DNA hybridization ( < 30 %) with other closely related species of the genus Deinococcus. The strains showed the typical chemotaxonomic characteristics of the genus Deinococcus, with the presence of menaquinone 8 as the respiratory quinone; the major fatty acids were summed feature 3 (composed of C16 : 1ω7c/C16 : 1ω6c), C15 : 1ω6c and C16 : 0. The DNA G+C content of strain BM2T was 69.7 mol%. The polar lipid profile included major amounts of phosphatidylglycerol, phosphatidylcholine and an unknown aminolipid. On the basis of phenotypic and genotypic properties, and phylogenetic distinctiveness, strains BM2T, BM4 and BM5 should be classified in a novel species in the genus Deinococcus, for which the name Deinococcus actinosclerus sp. nov. is proposed. The type strain is BM2T ( = KEMB 5401-184T = JCM 30700T); reference strains are BM4 ( = JCM 30701) and BM5 ( = JCM 30702).

Engineering Synthetic Multistress Tolerance in Escherichia coli by Using a Deinococcal Response Regulator, DR1558

ABSTRACT Cellular robustness is an important trait for industrial microbes, because the microbial strains are exposed to a multitude of different stresses during industrial processes, such as fermentation. Thus, engineering robustness in an organism in order to push the strains toward maximizing yield has become a significant topic of research. We introduced the deinococcal response regulator DR1558 into Escherichia coli (strain Ec-1558), thereby conferring tolerance to hydrogen peroxide (H2O2). The reactive oxygen species (ROS) level in strain Ec-1558 was reduced due to the increased KatE catalase activity. Among four regulators of the oxidative-stress response, OxyR, RpoS, SoxS, and Fur, we found that the expression of rpoS increased in Ec-1558, and we confirmed this increase by Western blot analysis. Electrophoretic mobility shift assays showed that DR1558 bound to the rpoS promoter. Because the alternative sigma factor RpoS regulates various stress resistance-related genes, we performed stress survival analysis using an rpoS mutant strain. Ec-1558 was able to tolerate a low pH, a high temperature, and high NaCl concentrations in addition to H2O2, and the multistress tolerance phenotype disappeared in the absence of rpoS. Microarray analysis clearly showed that a variety of stress-responsive genes that are directly or indirectly controlled by RpoS were upregulated in strain Ec-1558. These findings, taken together, indicate that the multistress tolerance conferred by DR1558 is likely routed through RpoS. In the present study, we propose a novel strategy of employing an exogenous response regulator from polyextremophiles for strain improvement.

Expression and Mutational Analysis of DinB- Like Protein DR0053 in Deinococcus radiodurans

In order to understand the mechanism governing radiation resistance in Deinococcus radiodurans, current efforts are aimed at identifying potential candidates from a large repertoire of unique Deinococcal genes and protein families. DR0053 belongs to the DinB/YfiT protein family, which is an over-represented protein family in D. radiodurans. We observed that dr0053 transcript levels were highly induced in response to gamma radiation (γ-radiation) and mitomycin C (MMC) exposure depending on PprI, RecA and the DrtR/S two-component signal transduction system. Protein profiles demonstrated that DR0053 is a highly induced protein in cultures exposed to 10 kGy γ-radiation. We were able to determine the transcriptional start site of dr0053, which was induced upon irradiation, and to assign the 133-bp promoter region of dr0053 as essential for radiation responsiveness through primer extension and promoter deletion analyses. A dr0053 mutant strain displayed sensitivity to γ-radiation and MMC exposure, but not hydrogen peroxide, suggesting that DR0053 helps cells recover from DNA damage. Bioinformatic analyses revealed that DR0053 is similar to the Bacillus subtilis protein YjoA, which is a substrate of bacterial protein-tyrosine kinases. Taken together, the DNA damage-inducible (din) gene dr0053 may be regulated at the transcriptional and post-translational levels.

PprM, a Cold Shock Domain-Containing Protein from Deinococcus radiodurans, Confers Oxidative Stress Tolerance to Escherichia coli

Escherichia coli is a representative microorganism that is frequently used for industrial biotechnology; thus its cellular robustness should be enhanced for the widespread application of E. coli in biotechnology. Stress response genes from the extremely radioresistant bacterium Deinococcus radiodurans have been used to enhance the stress tolerance of E. coli. In the present study, we introduced the cold shock domain-containing protein PprM from D. radiodurans into E. coli and observed that the tolerance to hydrogen peroxide (H2O2) was significantly increased in recombinant strains (Ec-PprM). The overexpression of PprM in E. coli elevated the expression of some OxyR-dependent genes, which play important roles in oxidative stress tolerance. Particularly, mntH (manganese transporter) was activated by 9-fold in Ec-PprM, even in the absence of H2O2 stress, which induced a more than 2-fold increase in the Mn/Fe ratio compared with wild type. The reduced production of highly reactive hydroxyl radicals (·OH) and low protein carbonylation levels (a marker of oxidative damage) in Ec-PprM indicate that the increase in the Mn/Fe ratio contributes to the protection of cells from H2O2 stress. PprM also conferred H2O2 tolerance to E. coli in the absence of OxyR. We confirmed that the H2O2 tolerance of oxyR mutants reflected the activation of the ycgZ-ymgABC operon, whose expression is activated by H2O2 in an OxyR-independent manner. Thus, the results of the present study showed that PprM could be exploited to improve the robustness of E. coli.

PprM is necessary for up-regulation of katE1, encoding the major catalase of Deinococcus radiodurans, under unstressed culture conditions

Deinococcus radiodurans is a poly-extremophilic organism, capable of tolerating a wide variety of different stresses, such as gamma/ultraviolet radiation, desiccation, and oxidative stress. PprM, a cold shock protein homolog, is involved in the radiation resistance of D. radiodurans, but its role in the oxidative stress response has not been investigated. In this study, we investigated the effect of pprM mutation on catalase gene expression. pprM disruption decreased the mRNA and protein levels of KatE1, which is the major catalase in D. radiodurans, under normal culture conditions. A pprM mutant strain (pprMMT) exhibited decreased catalase activity, and its resistance to hydrogen peroxide (H2O2) decreased accordingly compared with that of the wild-type strain. We confirmed that RecG helicase negatively regulates katE1 under normal culture conditions. Among katE1 transcriptional regulators, the positive regulator drRRA was not altered in pprM-, while the negative regulators perR, dtxR, and recG were activated more than 2.5-fold in pprMMT. These findings suggest that PprM is necessary for KatE1 production under normal culture conditions by down-regulation of katE1 negative regulators.

Deinococcus seoulensis sp. nov., a bacterium isolated from sediment at Han River in Seoul, Republic of Korea.

Strain 16F1ET was isolated from a 3-kGy-irradiated sediment sample collected at Han River in Seoul, Republic of Korea. Cells of this strain were observed to be Gram-positive, pililike structure, and short rod shape, and colonies were red in color. The strain showed the highest degree of 16S rRNA gene sequence similarity to Deinococcus aquaticus PB314T (98.8%), Deinococcus depolymerans TDMA-24T (98.1%), Deinococcus caeni Ho-08T (98.0%), and Deinococcus grandis DSM 3963T (97.0%). 16S rRNA gene sequence analysis identified this strain as a member of the genus Deinococcus (Family: Deinococcaceae). The genomic DNA G+C content of strain 16F1ET was 66.9 mol%. The low levels of DNA-DNA hybridization (< 56.2%) with the species mentioned above identified strain 16F1ET as a novel Deinococcus species. Its oxidase and catalase activities as well as the production of acid from glucose were positive. Growth of the strain was observed at 10–37°C (optimum: 20–30°C) and pH 4–10 (optimum: pH 7–8). The cells tolerated less than 5% NaCl and had low resistance to gamma radiation (D10 < 4 kGy). Strain 16F1ET possessed the following chemotaxonomic characteristics: C16:0, C15:1ω6c, and C16:1ω7c as the major fatty acids; phosphoglycolipid as the predominant polar lipid; and menaquinone-8 as the predominant respiratory isoprenoid quinone. Based on the polyphasic evidence, as well as the phylogenetic, genotypic, phenotypic, and chemotaxonomic characterization results, strain 16F1ET (=KCTC 33793T =JCM 31404T) is proposed to represent the type strain of a novel species, Deinococcus seoulensis sp. nov.

Deinococcus persicinus, a new species of radiation-resistant bacteria from soil.

Two Gram-stain-negative, oxidase-negative, catalase-positive, aerobic and coccus-shaped bacterial strains, KSY3-6T and JSH6-18, were isolated from soil in South Korea. Strains KSY3-6T and JSH6-18 showed high resistance to gamma-ray and UVC irradiation. The 16S rRNA gene sequences of strains KSY3-6T and JSH6-18 showed a novel subline within the genus Deinococcus in the family Deinococcaceae. They shared 94.8-86.4 % nucleotide similarities with other species of the genus Deinococcus. Strain KSY3-6T exhibited high DNA-DNA hybridization values with JSH6-18 (77±0.8 %). The two strains showed typical chemotaxonomic characteristics of the genus Deinococcus, including the presence of menaquinone 8 (MK-8) as predominant respiratory quinone and C16 : 0, C17 : 0cyclo and summed feature 3 (C16 : 1ω7c/C16: 1ω6c) as major fatty acids. The G+C content of the DNA of strains KSY3-6T and JSH6-18 was 62.0 and 62.4 mol%, respectively. Polar lipids in strains KSY3-6T and JSH6-18 were mainly phosphoglycolipids. Based on their phenotypic and genotypic properties, strains KSY3-6T and JSH6-18 should be classified as representatives of a novel species in the genus Deinococcus, for which the name Deinococcus persicinus sp. nov. is proposed. The type strain is KSY3-6T (=KCTC 33787T=JCM 31313T). The reference strain is JSH6-18 (=KCTC 33788=JCM 31312).

Oxidative stress response of Deinococcus geothermalis via a cystine importer

A cystine-dependent anti-oxidative stress response is characterized in Deinococcus geothermalis for the first time. Nevertheless, the same transcriptional directed Δdgeo_1985F mutant strain was revealed to have an identical phenotype to the wild-type strain, while the reverse transcriptional directed Δdgeo_1985R mutant strain was more resistant to oxidative stress at a certain concentration of H2O2 than the wild-type strain. The wild-type and mutant strains expressed equal levels of superoxide dismutase and catalase under H2O2-induced stress. Although the expression levels of the general DNA-damage response-related genes recA, pprA, ddrA, and ddrB were up-regulated by more than five-fold in the wild-type strain relative to the Δdgeo_1985R mutant strain, the mutant strain had a higher survival rate than the wild-type under H2O2 stress. The Δdgeo_1985R mutant strain highly expressed a cystine-transporter gene (dgeo_1986), at levels 150-fold higher than the wild-type strain, leading to the conclusion that this cystine transporter might be involved in the defensive response to H2O2 stress. In this study, the cystine transporter was identified and characterized through membrane protein expression analysis, a cystine-binding assay, and assays of intracellular H2O2, cysteine, and thiol levels. The genedisrupted mutant strain of the cystine importer revealed high sensitivity to H2O2 and less absorbed cystine, resulting in low concentrations of total thiol. Thus, the absorbed cystine via this cystine-specific importer may be converted into cysteine, which acts as a primitive defense substrate that non-enzymatically scavenges oxidative stress agents in D. geothermalis.

Deinococcus sedimenti sp. nov. isolated from river sediment

A novel Gram-positive, oval-shaped, non-motile bacterium designated strain 16F1LT was isolated from sediment collected from the Han River in Seoul, Republic of Korea. Based on the 16S rRNA gene sequence (1,448 bp), this strain was identified as a member of the genus Deinococcus that belongs to the class Deinococci. Similarities in the 16S rRNA gene sequence were shown with Deinococcus daejeonensis MJ27T (99.0%), D. grandis DSM 3963T (98.1%), D. radiotolerans C1T (97.5%), and D. caeni Ho-08T (97.2%). Strain 16F1LT was classified as a different genomic species from closely related Deinococcus members, based on less than 70% DNA-DNA relatedness. Genomic DNA G+C content of strain 16F1LT was 67.2 mol%. Strain 16F1LT was found to grow at temperatures of 10–37°C (optimum 25°C) and pH 7–8 (optimum pH 7) on R2A medium, and was catalase-positive and oxidase-negative. Strain 16F1LT showed resistance to gamma radiation (D10 > 2 kGy). In addition, this strain had the following chemotaxonomic characteristics: the major fatty acids were C15:1ω6c and C16:1ω7c; the polar lipid profile contained phosphoglycolipids, unknown aminophospholipids, an unknown aminoglycolipid, unknown aminolipids, an unknown glycolipid, an unknown phospholipid, and an unknown polar lipid; the major quinone was MK-8. Phylogenetic, genotypic, phenotypic, and chemotaxonomic characteristics indicated that strain 16F1LT represents a novel species within the genus Deinococcus, for which the name Deinococcus sedimenti sp. nov. is proposed. The type strain is 16F1LT (=KCTC 33796T =JCM 31405T).