Sangyong Lim

Deinococcus daejeonensis sp. nov., isolated from sludge in a sewage disposal plant

A Gram-stain-positive, strictly aerobic, spherical, non-motile red-pigmented bacterial strain, designated MJ27(T), was isolated from a sludge sample of the Daejeon sewage disposal plant in South Korea. A polyphasic approach was used to study the taxonomic position of strain MJ27(T). Strain MJ27(T) shared highest 16S rRNA gene sequence similarity with Deinococcus grandis DSM 3963(T) (98.8 %), Deinococcus caeni Ho-08(T) (97.5 %) and Deinococcus aquaticus PB314(T) (96.6 %.); levels of sequence similarity with the type strains of other Deinococcus species were less than 96.0 %. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain MJ27(T) belonged to the clade formed by members of the genus Deinococcus in the family Deinococcaceae. The G+C content of the genomic DNA of strain MJ27(T) was 67.6 mol%. The chemotaxonomic characteristics of strain MJ27(T) were typical of members of the genus Deinococcus, with MK-8 as the predominant respiratory quinone, C(16:1)ω7c, C(15:1)ω6c, C(16:0) and C(15:0) as major fatty acids (>12 %), ornithine as the diamino acid in the cell-wall peptidoglycan and resistance to gamma radiation [D(10) (dose required to reduce the bacterial population by tenfold) >9 kGy]. The low levels of DNA-DNA relatedness reported here (5.3±1.5-29.2±2.3 %) indicate that strain MJ27(T) represents a species that is separate from its closest relatives in the genus Deinococcus. On the basis of phylogenetic inference, fatty acid profile and other phenotypic properties, strain MJ27(T) is considered to represent a novel species of the genus Deinococcus, for which the name Deinococcus daejeonensis sp. nov. is proposed. The type strain is MJ27(T) ( = KCTC 13751(T) = JCM 16918(T)).

Mutational analysis of OGG1, MYH, MTH1 in FAP, HNPCC and sporadic colorectal cancer patients: R154H OGG1 polymorphism is associated with sporadic colorectal cancer patients

MYH, OGG1 and MTH1 are members of base excision repair (BER) families, and MYH germline mutations were recently identified in patients with multiple adenomas or familial adenomatous polyposis (FAP). A total of 20 APC-negative Korean FAP patients were analyzed for OGG1, MYH and MTH1 germline mutations. A total of 19 hereditary nonpolyposis colorectal cancer (HNPCC), 86 suspected HNPCC, and 246 sporadic colorectal cancer cases were investigated for OGG1 and MYH mutations. A total of 14 R154H OGG1 polymorphisms were identified in hereditary, sporadic colorectal cancers, and normal controls. For the case-control analysis of OGG1 R154H, a total of 625 hereditary or sporadic colorectal cancer patients and 527 normal controls were screened. R154H was a rare polymorphism associated with sporadic colorectal cancer patents (OR: 3.586, P= 0.053). R154H does not segregate with cancer phenotypes. Upon examining the possibility of recessive inheritance of R154H, we could not identify any complementary mutations in OGG1, MYH or MTH1. Samples with R154H were further screened for mutations of K-ras, β-catenin, APC, p53, BRAF and the microsatellite instability (MSI) status. Eight somatic mutations were identified in these genes and G:C to T:A transversion mutations were not dominant in samples harboring R154H. This result raises the possibility that OGG1 R154H may function as a low/moderate-penetrance modifier for colorectal cancer development.

Mlc regulation of Salmonella pathogenicity island I gene expression via hilE repression

The global regulator Mlc is a repressor of several genes and operons that are involved in sugar uptake and metabolism. A Salmonella enterica serovar Typhimurium mlc mutant showed reduced levels of invasion and cytotoxicity compared to the wild-type, and exhibited reduced expression levels of hilD, hilA and invF, which are regulatory genes in the Salmonella pathogenicity island 1 (SPI1). However, the effects of Mlc on hilD expression and bacterial invasiveness were not seen in the hilE mutant, and hilE expression was increased in the mlc mutant, which suggests that Mlc exerts positive effects on the expression of SPI1 genes by reducing the expression of HilE, which is known to down-regulate the expression of SPI1 genes through direct interaction with HilD. We found that the two known promoters of hilE were not modulated by Mlc, and we identified a third promoter, designated P3, which was repressed by Mlc. The gel mobility shift assay and footprinting analysis revealed that Mlc repressed hilE in a direct manner by binding to two distinct sites in the hilE P3 promoter region. The specific down-regulation of hilD observed in the presence of Mlc regulon-inducible sugars, such as glucose and mannose, could not be detected in the mlc mutant. Based on these results, we propose that Mlc functions to sense the availability of sugars and is linked to virulence gene regulation by its ability to control hilE expression in Salmonella.

Deinococcus humi sp. nov., isolated from soil

A Gram-staining-positive, strictly aerobic, spherical, non-motile, red-pigmented bacterium, designated strain MK03(T), was isolated from a soil sample collected in South Korea. The taxonomic position of the novel strain was investigated using a polyphasic approach. In phylogenetic analyses based on 16S rRNA gene sequences, strain MK03(T) was placed in a clade formed by members of the genus Deinococcus in the family Deinococcaceae and appeared to be most closely related to Deinococcus aerolatus 5516T-9(T) (97.4% sequence similarity), Deinococcus marmoris AA-63(T) (97.2%), Deinococcus radiopugnans ATCC 19172(T) (97.2%) and Deinococcus saxicola AA-1444(T) (96.9%). The genomic DNA G+C content of the novel strain was 64.5 mol%. The chemotaxonomic characteristics of strain MK03(T) were typical of members of the genus Deinococcus: MK-8 was identified as the predominant respiratory quinine, the major fatty acids were C(16:1)ω7c, C(15:1)ω6c, C(16:0) and C(15:0, )ornithine was found to be the diamino acid in the cell-wall peptidoglycan and the novel strain showed resistance to gamma radiation, with a D(10) value (i.e. the dose required to reduce the bacterial population by 10-fold) in excess of 9 kGy. In hybridization experiments, only low DNA-DNA relatedness values (11.6-34.5%) were recorded between the novel strain and its closest relatives in the genus Deinococcus. Based on the phylogenetic, chemotaxonomic, phenotypic and DNA-DNA relatedness data, strain MK03(T) represents a novel species of the genus Deinococcus, for which the name Deinococcus humi sp. nov. is proposed. The type strain is MK03(T) ( = KCTC 13619(T)  = JCM 17915(T)).

LsrR-Mediated Quorum Sensing Controls Invasiveness of Salmonella typhimurium by Regulating SPI-1 and Flagella Genes

Bacterial cell-to-cell communication, termed quorum sensing (QS), controls bacterial behavior by using various signal molecules. Despite the fact that the LuxS/autoinducer-2 (AI-2) QS system is necessary for normal expression of Salmonella pathogenicity island-1 (SPI-1), the mechanism remains unknown. Here, we report that the LsrR protein, a transcriptional regulator known to be involved in LuxS/AI-2-mediated QS, is also associated with the regulation of SPI-1-mediated Salmonella virulence. We determined that LsrR negatively controls SPI-1 and flagella gene expressions. As phosphorylated AI-2 binds to and inactivates LsrR, LsrR remains active and decreases expression of SPI-1 and flagella genes in the luxS mutant. The reduced expression of those genes resulted in impaired invasion of Salmonella into epithelial cells. Expression of SPI-1 and flagella genes was also reduced by overexpression of the LsrR regulator from a plasmid, but was relieved by exogenous AI-2, which binds to and inactivates LsrR. These results imply that LsrR plays an important role in selecting infectious niche of Salmonella in QS dependent mode.

Proteome analysis of Salmonella enterica serovar Typhimurium fis mutant

Salmonella enterica serovar Typhimurium is an enteric pathogen and a principal cause of gastroenteritis in humans. The factor-for-inversion stimulation protein (Fis) is known to play a pivotal role in the expression of Salmonella pathogenicity island (SPI)-1 genes in addition to various cellular processes such as recombination, replication, and transcription. In order to understand Fis function in pathogenicity of Salmonella, we performed two-dimensional gel electrophoresis and identified proteins whose expression pattern is affected by Fis using mass spectrometry. The results revealed various proteins that can be grouped according to their respective cellular functions. These groups include the genes involved in the metabolism of sugar, flagella synthesis, translation, and SPI expression. Changes in SPI expression suggest the possibility that regulation of genes in SPI-2 as well as SPI-1 is affected by Fis.

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.

A comparative evaluation of radiation-induced DNA damage using real-time PCR : Influence of base composition

Abstract Lim, S., Yoon, H., Ryu, S., Jung, J., Lee, M. and Kim, D. A Comparative Evaluation of Radiation-Induced DNA Damage using Real-Time PCR: Influence of Base Composition. Radiat. Res. 165, 430–437 (2006). To study the radiosensitivity of DNA segments at the open reading frame (gene) level, real-time PCR was used to analyze DNA damages induced by ionizing radiation. After irradiation (1, 3 and 5 kGy) of genomic DNA purified from Salmonella typhimurium, real-time PCR based on SYBR Green fluorescence and melting temperature was performed using various primer sets targeting the rfbJ, rfaJ, rfaB, hilD, ssrB, pipB, sopD, pduQ, eutG, oadB, ccmB and ccmA genes. The ccmA and ccmB genes, which existed as two copies on the chromosome and had a high GC content (∼70%), showed much lower radiosensitivities than the other genes tested, particularly at 5 kGy; this distinctive feature was seen only when the genes were located on the chromosome, regardless of copy number. Our results reinforce the concept that gene sensitivity to ionizing radiation depends on the base composition and/or the spatial localization of the gene on the chromosome.

A tdcA mutation reduces the invasive ability of Salmonella enterica serovar typhimurium

We previously observed that the transcription of some flagellar genes decreased in Salmonella Typhimurium tdcA mutant, which is a gene encoding the transcriptional activator of the tdc operon. Since flagella-mediated bacterial motility accelerates the invasion of Salmonella, we have examined the effect of tdcA mutation on the invasive ability as well as the flagellar biosynthesis in S. Typhimurium. A tdcA mutation caused defects in motility and formation of flagellin protein, FliC in S. Typhimurium. Invasion assays in the presence of a centrifugal force confirmed that the defect of flagellum synthesis decreases the ability of Salmonella to invade into cultured epithelial cells. In addition, we also found that the expression of Salmonella pathogenicity island 1 (SPI1) genes required for Salmonella invasion was down-regulated in the tdcA mutant because of the decreased expression of fliZ, a positive regulator of SPI1 transcriptional activator, hilA. Finally, the virulence of a S. Typhimurium tdcA mutant was attenuated compared to a wild type when administered orally. This study implies the role of tdcA in the invasion process of S. Typhimurium.

Unraveling Fungal Radiation Resistance Regulatory Networks through the Genome-Wide Transcriptome and Genetic Analyses of Cryptococcus neoformans

ABSTRACT The basidiomycetous fungus Cryptococcus neoformans has been known to be highly radiation resistant and has been found in fatal radioactive environments such as the damaged nuclear reactor at Chernobyl. To elucidate the mechanisms underlying the radiation resistance phenotype of C. neoformans, we identified genes affected by gamma radiation through genome-wide transcriptome analysis and characterized their functions. We found that genes involved in DNA damage repair systems were upregulated in response to gamma radiation. Particularly, deletion of recombinase RAD51 and two DNA-dependent ATPase genes, RAD54 and RDH54, increased cellular susceptibility to both gamma radiation and DNA-damaging agents. A variety of oxidative stress response genes were also upregulated. Among them, sulfiredoxin contributed to gamma radiation resistance in a peroxiredoxin/thioredoxin-independent manner. Furthermore, we found that genes involved in molecular chaperone expression, ubiquitination systems, and autophagy were induced, whereas genes involved in the biosynthesis of proteins and fatty acids/sterols were downregulated. Most importantly, we discovered a number of novel C. neoformans genes, the expression of which was modulated by gamma radiation exposure, and their deletion rendered cells susceptible to gamma radiation exposure, as well as DNA damage insults. Among these genes, we found that a unique transcription factor containing the basic leucine zipper domain, named Bdr1, served as a regulator of the gamma radiation resistance of C. neoformans by controlling expression of DNA repair genes, and its expression was regulated by the evolutionarily conserved DNA damage response protein kinase Rad53. Taken together, the current transcriptome and functional analyses contribute to the understanding of the unique molecular mechanism of the radiation-resistant fungus C. neoformans. IMPORTANCE Although there are no natural environments under intense radiation, some living organisms have been found to show high radiation resistance. Organisms harboring the ability of radiation resistance have unique regulatory networks to overcome this stress. Cryptococcus neoformans is one of the radiation-resistant fungi and is found in highly radioactive environments. However, it remains elusive how radiation-resistant eukaryotic microorganisms work differentially from radiation-sensitive ones. Here, we performed transcriptome analysis of C. neoformans to explore gene expression profiles after gamma radiation exposure and functionally characterized some of identified radiation resistance genes. Notably, we identified a novel regulator of radiation resistance, named Bdr1 (a bZIP TF for DNA damage response 1), which is a transcription factor (TF) that is not closely homologous to any known TF and is transcriptionally controlled by the Rad53 kinase. Therefore, our work could shed light on understanding not only the radiation response but also the radiation resistance mechanism of C. neoformans. Although there are no natural environments under intense radiation, some living organisms have been found to show high radiation resistance. Organisms harboring the ability of radiation resistance have unique regulatory networks to overcome this stress. Cryptococcus neoformans is one of the radiation-resistant fungi and is found in highly radioactive environments. However, it remains elusive how radiation-resistant eukaryotic microorganisms work differentially from radiation-sensitive ones. Here, we performed transcriptome analysis of C. neoformans to explore gene expression profiles after gamma radiation exposure and functionally characterized some of identified radiation resistance genes. Notably, we identified a novel regulator of radiation resistance, named Bdr1 (a bZIP TF for DNA damage response 1), which is a transcription factor (TF) that is not closely homologous to any known TF and is transcriptionally controlled by the Rad53 kinase. Therefore, our work could shed light on understanding not only the radiation response but also the radiation resistance mechanism of C. neoformans.