• Korean Journal of Microbiology
• /
• v.51 no.1
• /
• pp.39-47
• /
• 2015

Secondary metabolism in actinomycetes has been known to be controlled by a small molecule, ${\gamma}$-butyrolactone autoregulator, the binding of which to each corresponding receptor leads to the regulation of the transcriptional expression of the secondary metabolites. We expected that expression of an autoregulator receptor or a pleiotropic regulator in a non-host was to be gained insight of effective production of new metabolic materials. In order to study the function of the receptor protein (seaR), which is isolated from Saccharopolyspora erythraea, we introduced the seaR gene to Streptomyces coelicolor A3(2) as host strains. An effective transformation procedure for S. coelicolor A3(2) was established based on transconjugation by Escherichia coli ET12567/pUZ8002 with a ${\varphi}C31$-derived integration vector, pSET152, which contained int, oriT, attP and $ermEp^*$ (erythromycin promotor). Therefore, the pEV615 was introduced into S. coelicolor A3(2) by conjugation and integrated at the attB locus in the chromosome of the recipients by the ${\varphi}C31$ integrase (int) function. Exconjugant of S. coelicolor A3(2) containing the seaR gene was confirmed by PCR and transcriptional expression of the seaR gene in the transformant was analyzed by RT-PCR. In case of S. coelicolor A3(2), a phenotype microarray was used to analyze the phenotype of transformant compared with wild type by seaR expression. After that, in order to confirm the accuracy of the results obtained from the phenotype microarray, an antimicrobial susceptibility test was carried out. This test indicated that sensitivity of the transformant was higher than wild type in tetracycline case. These results indicated that some biosynthesis genes or resistance genes for tetracycline biosynthesis in transformant might be repressed by seaR expression. Therefore, subsequent experiments, analysis of transcriptional pattern of genes for tetracycline production or resistance, are needed to confirm whether biosynthesis genes or resistance genes for tetracycline are repressed or not.

• Korean Journal of Microbiology
• /
• v.45 no.3
• /
• pp.286-290
• /
• 2009

The transcription of mRNA of avian influenza virus is regulated temporally during infection. Therefore, the measurement of transcript level in host cells should be performed before viral release from host cells because errors can occur in the analysis of the transcript levels if the viruses released from the infected cells re-infect cells. In this study, the timing of viral release was determined by measuring the level of viral RNA from viruses released from H9N2-infected chicken fibroblast cell line UMNSAH/DF-1 by semi-quantitative RT-PCR. The viral genomic RNA was isolated together with mouse total RNA which was added to the collected medium as carrier to monitor the viral RNA recovery and to use its GAPDH as an internal control for normalizing reverse transcription reaction as well as PCR reaction. It was found that viral release of H9N2 in the chicken fibroblast cell line UMNSAH/DF-1 took between 16 and 20 h after infection. We measured all 8 viral mRNA levels. Of the 8 transcripts, 7 species of viral mRNAs (each encoding HA, NA, PB1, PB2, NP, M, NS, respectively) except PA mRNA showed robust amplification, indicating these mRNA can be used as targets for amplification to measure transcript levels. These results altogether suggest that the method in this study can be used for screening antiviral materials against viral RNA polymerase as a therapeutic target.

• Korean Journal of Microbiology
• /
• v.44 no.3
• /
• pp.237-243
• /
• 2008

Chemical and microbial characteristics of bacterial populations were investigated in a quercus and pine humus forest soil. Soil pH was $5.3\pm0.4$ and $4.1\pm0.9$ from each sample of a quercus and pine humus forest soil; C/N ratio of humus forest soil was $17.84\pm4.6%$ and $21.76\pm8%$, respectively. Total organic acid was investigated as 69.57 mM/g dry soil and 53.72 mM/g dry soil in each humus forest soil. Glutamine, pyruvate, succinate, lactic acid and acetic acid of pine humus forest soil were $1.5\sim4.5$ times higher than those of quercus humus forest soil. As we evaluated phylogenetic characteristics of bacterial populations by 16S rRNA-ARDRA analysis with DNA extracted from each humus forest soil. Based on the 16S rRNA sequences, 44 clone from ARDRA groups of quercus humus forest soil were classified into 7 phyla: ${\alpha},{\beta},{\gamma},{\delta}$-Proteobacteria, Acidobacteria, Actinobacteria, and Firmicutes. Thirty-two clone from ARDRA groups of pine humus forest soil were classified into 8 phyla: ${\alpha},{\beta},{\gamma}$-Proteobacteria, Acidobacteria, Bacteroides, Verrucomicrobia, Planctomycetes, and Gemmatomonadetes. According to PCA (Principal Component Analysis) based on 16S rRNA base sequence, there were three main groups of bacteria. All clone of Cluster I were originated from quercus humus forest soil, while 67% clone of Cluster II and 63% clone of Clusters III were separated from pine humus forest soil.

• Korean Journal of Microbiology
• /
• v.44 no.3
• /
• pp.244-250
• /
• 2008

To evaluate bacteriological water quality, samples were taken from drinking water dispensers placed at S company (S-C) and U highschool (U-H) in Ulsan. The medians of heterotrophic plate counts (HPCs) were 53 CFU/ml for the 74 water samples of S-C and 80 CFU/ml for the 36 cold water samples of U-H, and 38% of the S-C and 42% of the U-H samples showed HPC bacterial concentrations higher than 100 CFU/ml. Coliform bacteria were detected from one sample of S-C. To determine the major source of bacterial contamination, water samples were taken daily for $6\sim8$ days from the bottled water containers as well as the faucets of an experimental water dispenser. While the average HPCs in the bottled water containers were 33 CFU/ml for the first and 132 CFU/ml for the 2nd analysis, the HPC concentration in the cold water samples was 1,022 CFU/ml for the 2nd analysis. These results suggest that the majority of bacteria detected in the cold water samples were originated from the biofilms on the surface of water passages within the water dispensers. There was no significant increase in HPC bacterial concentrations within the bottled water container after installation on the water dispenser. We could isolate and tentatively identify 3 genera 6 species of Gram-positive and 7 genera 7 species of Gram-negative bacteria from the plate count agar plates of U-H samples. Among the isolates, 72% were observed as Gram-positive, and Micrococcus spp. was the most abundant with 54% of the total, followed by Sphingomonas paucimobilis with 16%. It appears that most of the HPC bacteria detected in water dispensers originate from indoor airborne bacteria, which may play important roles in the formation of biofilms on the surface of water passages within the water dispensers.

• Korean Journal of Microbiology
• /
• v.52 no.3
• /
• pp.344-351
• /
• 2016

A marine bacterium, designated as strain 50C-3, was isolated from a seawater sample collected from the East Sea of South Korea. The strain is a Gram-negative, aerobic, yellow colored polar-flagellated bacterium that grows at $20-50^{\circ}C$ and pH 5.5-8.5. Optimal growth occurred at $40-50^{\circ}C$, at pH 6.5-7.5, and in the presence of 2% (w/v) NaCl. Based on 16S rRNA gene sequence similarity, the isolate was considered to represent a member of the genus Ruegeria. The result of this analysis showed that strain 50C-3 shared 99.4% and 96.98% sequence similarity with Ruegeria intermedia CC-GIMAT-$2^T$ and Ruegeria lacuscaerulensis ITI-$1157^T$, respectively. Furthermore, strain 50C-3 showed clear differences from related strains in terms of several characteristics such as motility, carbon utilization, enzyme production, etc. The DNA G+C content was 66.7 mol%. Chemotaxonomic analysis indicated ubiquinone-10 (Q-10) as the predominant respiratory quinone. Based on phenotypic, chemotaxonomic, and phylogenetic characteristics, the isolate represents a novel variant of the Ruegeria intermedia CC-GIMAT-$2^T$, for which we named Ruegeria sp. 50C-3 (KCTC23890=DSM25519). Strain 50C-3 did not produce cellulase and agarase, but produced alkaline phosphatase, ${\alpha}$-galactosidase, and ${\beta}$-galactosidase. The three enzymes showed stable activities even at $50^{\circ}C$ and thus regarded as thermostable enzymes. Especially, the ${\beta}$-galactosidase activity enhanced by 1.9 times at $50^{\circ}C$ than that at $37^{\circ}C$, which may be very useful for industrial application.

• Journal of Food Hygiene and Safety
• /
• v.24 no.2
• /
• pp.174-181
• /
• 2009

The consumption of "ready-to-eat" agricultural products is recently increasing and the safety of these agricultural products is forefront of public concerns. The 120 samples of paprikas, strawberries and tomatoes, which are the representative exported agricultural products, were purchased at the department stores and discount stores in Daejeon. And we determined the microbiological and parasitological contamination level of these agricultural products using culture media, multiplex PCR, commercial bacterial detection kit and microscopy, and also evaluated the decontamination method. Mean counts of total aerobic bacteria from these agricultural products ranged from $1.3{\times}10^4$ CFU/g to $1.8{\times}10^5$ CFU/g, and mean counts of coliforms ranged from $1.4{\times}10^3$ CFU/g to $9.6{\times}10^3$ CFU/g. There was no significant difference in the level of bacterial contamination between the agricultural products from department stores and the ones from discount stores. Strawberry showed the highest contamination level for the bacteria and we also found the unidentified parasite eggs. Enterobacter cloacae was the most frequently isolated bacteria strain, but no food poisoning pathogenic bacteria except Staphylococcus aureus was isolated from the products by multiplex PCR. Compared to unwashed products, tab water-washed ones showed 80% decrease of the counts of total aerobic bacteria on the agricultural products, and the rates decreased more by incorporating detergent or ultrasonic wave treatment. We concluded that the biological contamination levels among paprikas, strawberries and tomatoes were the highest in strawberries, but there were not significant difference according to distribution systems.

• Korean Journal of Microbiology
• /
• v.46 no.3
• /
• pp.240-247
• /
• 2010

Pseudomonas aeruginosa is a Gram (-) opportunistic human pathogen causing a wide variety of infections on lung, urinary tract, eyes, and burn wound sites and quorum sensing (QS), a cell density-sensing mechanism plays an essential role in Pseudomonas pathogenesis. In order to investigate the importance of QS in the Pseudomonas infections of Korean patients, we isolated 189 clinical strains of P. aeruginosa from the patients in Pusan Paik Hospital, Busan, South Korea. The QS signal production of these clinical isolates was measured by signal diffusion assay on solid media using reporter strains. While most clinical strains (79.4%) produced the QS signals as similar level as a wild type strain, PAO1 did, where LasR, the initial QS signal sensor-regulator was fully activated, a minority of them (4.2%) produced much less QS signals at the level to which LasR failed to respond. Similarly, while 72.5% of the clinical isolates produced QS signals enough to activate QscR, an another QS signal sensor-regulator, some few of them (9%) produced the QS signals at much lower level where QscR was not activated. For further analysis, we selected 74 clinical strains that were obtained from the patients under suspicion of Pseudomonas infection and investigated the total protease activity that is considered important for virulence. Interestingly, significant portion of them showed very low protease activity (44.6%) or no detectable protease activity (12.2%). When the biofilm-forming ability that is considered very important in chronic infection was examined, most isolates showed lower biofilm-forming activity than PAO1. Similarly, significant portion of clinical isolates showed reduced motility (reduced swarming activity in 51.4% and reduced twitching activity in 41.9%), or non-detectable motility (swarming-negative in 28.4% and twitching-negative in 28.4%). Our result showed that the clinical isolates that produced QS signals at the similar level to wild type could have significantly reduced activities in the protease production, biofilm formation, and motility, and some clinical isolates had unique patterns of motility, biofilm formation, and protease production that are not correlated to their QS activity.

• Korean Journal of Microbiology
• /
• v.41 no.1
• /
• pp.24-37
• /
• 2005

This study was conducted to analyze the molecular epidemiological properties and to select the most efficient and reliable PCR method on 116 of Staphylococcus aureus (S. aureus) isolates from Korean cattle, black goat, pig, dog, chicken, mouse and also human clinical cases from hospital. The distribution patterns of SSG [species specific genes; coagulase (coa), protein A (spa), nuclease (nuc) and aroA (RsaI) gene] were analyzed by PCR method. Among the SSGs, the nuc-gene was found in all strains $(100\%)$ tested and followed by coa-gene $(87.9\%)$, spa-gene $(91.4\%)$ and aroA-gene $(26.7\%)$, in order. The genetic subtyping by RFLP method was performed on the coa [AluI] and aroA-gene [RsaI] PCR products. The mecA-gene PCR and PCR-RFLP techniques were chosen to detect and verify of MRSA strains. Only the human strains $(12.1\%)$ were detected the positive mecA-gene products (533 bp), which were divided into two specific bands [201 & 332 bp] by HhaI enzyme digestion. On coa-gene and spa-gene typing, coa-gene was typed with ten kinds of genotype and coa-3 type were determined as the most predominant genotype, while spa-gene was divided into eleven kinds of genotype and also spa-7 type were selected the most prevalent genotype based on their genetic variations. On the aroA and coa-gene subtyping by PCR-RFLP, aroA-gene products were discriminated with only seven types of genotype, while coa-gene products were further divided into an eleven genotype, respectively. In comparison of SID values of five PCR based typing methods, the coa-PCR-RFLP (SID0.894) was evaluated the most efficient and reliable tools and followed by coa-PCR (SID0.883) and aroA-PCR-RFLP (SID0.462), in order. In conclusion, we could determined that the coa-PCR-RFLP method was the most suitable genetic analysis tool for S. aureus and MRSA strains from domestic animals and humans.

• Korean Journal of Microbiology
• /
• v.38 no.2
• /
• pp.86-95
• /
• 2002

As a result of genome projects, the research to elucidate the function of a protein of interest has recently been well-recognized. In order to facilitate functional genomics, a useful mammalian gene expression vector is required. Using an infectious CDNA clone of BVDV pNADLclns-, we have developed a mammalian gene expression vector. In this study, a replication-competent full-length infectious CDNA clone containing puremycin acetyltransferase (pac) gene (pNADLclns-/pac) was successfully generated. The viral RNA replication and viral protein NS3 synthesis were examined by detecting metabollically $^{32}P$-labelled genomic viral RNA and immunoblotting with a mouse anti-NS3 antibody. To generate viral replicon as an expression vector, we examine if the viral structural genes (C, E0, El, E2) are required for viral replication by deletion analysis. As a result, all of the structural proteins are dispensable for viral replication per se, but essential for infectious viral particle formation. Based on our deletion analysis, we have generated a replication-competent BVDV viral replicon (pNADLclns-/pac/${\Delta}S$), whose structural genes are all deleted. In addition to NADLclns- /pac/${\Delta}S$, NADLclns-/ luc/${\Delta}S$ viral replicon containing luciferase gene as a reporter was constructed and fecund to be replication-compotent in HeLa and BHK cells as well as MDBK cells. Therefore, BVDV viral replicon developed in our study will be a useful tool to express a protein of interest in various mammalian cells.

• Korean Journal of Microbiology
• /
• v.52 no.2
• /
• pp.192-201
• /
• 2016

Recently, Psychrobacter sp. ArcL13 strain showing the extracellular lipase activity was isolated from the Chuckchi Sea of the Arctic Ocean. However, due to the low expression levels of the enzyme in the natural strain, the production of recombinant lipase is crucial for various applications. Identification of the gene for the enzyme is prerequisite for the production of the recombinant protein. Therefore, in the present study, a novel lipase gene (ArcL13-Lip) was isolated from Psychrobacter sp. ArcL13 strain by gene prospecting using PCR, and its complete nucleotide sequence was determined. Sequence analysis showed that ArcL13-Lip has high amino acid sequence similarity to lipases from bacteria of some Psychrobacter genus (84-90%) despite low nucleotide sequence similarity. The lipase gene was cloned into the bacterial expression plasmid and expressed in E. coli. SDS-PAGE analysis of the cells showed that ArcL13-Lip was expressed as inclusion bodies with a molecular mass of about 35 kDa. Refolding was achieved by diluting the unfolded protein into refolding buffers containing various additives, and the highest refolding efficiency was seen in the glucose-containing buffer. Refolded ArcL13-Lip showed high hydrolytic activity toward p-nitrophenyl caprylate and p-nitrophenyl decanoate among different p-nitrophenyl esters. Recombinant ArcL13-Lip displayed maximal activity at $40^{\circ}C$ and pH 8.0 with p-nitrophenyl caprylate as a substrate. Activity assays performed at various temperatures showed that ArcL13-Lip is a cold-active lipase with about 40% and 73% of enzymatic activity at $10^{\circ}C$ and $20^{\circ}C$, respectively, compared to its maximal activity at $40^{\circ}C$.