• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2006년도 International Meeting of the Microbiological Society of Korea
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• pp.105-107
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• 2006

Two kinds of nucleoside hydrolases (NHs) encoded by rih1 and rih2 were cloned from Corynebacterium ammoniagenes using deoD- and gsk-defective Escherichia coli. Sequence analysis revealed that NH 1 was a protein of 337 aa with a deduced molecular mass of 35,892 Da, whereas NH 2 consisted of 308 aa with a calculated molecular mass of 32,310 Da. Experiments with crude extracts of IPTG-induced E. coli CGSC 6885(pTNU23) and 6885(pTNI12) indicated that the Rihl enzyme could catalyse the hydrolysis of uridine and cytidine and showed pyrimidine-specific ribonucleoside hydrolase activity. Rih2 was able to hydrolyse both purine and pyrimidine ribonucleosides with the following order of activity-inosine>adenosine>uridine>guanosine>xanthosine>cytidine-and was classified in the non-specific NHs family. rih1 and rih2 deletion mutants displayed a decrease in cell growth on minimal medium supplemented with pyrimidine and purine/pyrimidine nucleosides, respectively, compared with the wild-type strain. Growth of each mutant was substantially complemented by introducing rih1 and rih2, respectively. Furthermore, disruption of both rih1 and rih2 led to the inability of the mutant to utilize purine and pyrimidine nucleosides as sole carbon source on minimal medium. These results indicated that rih1 and rih2 play major roles in the salvage pathways of nucleosides in this micro-organism.

• Journal of Microbiology and Biotechnology
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• 제12권2호
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• pp.296-300
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• 2002

Bacterial cell surface components are the major factors responsible for pathogenesis and bioremediation. In particular, the surface of a Gram-negative bacterium cell has a variety of components compared to that of a Gram-positive cell. In our previous study, we isolated an isogenic mutant of Bradyrhizobium japonicum, which exhibited altered cell surface characteristics, including an increased hydrophobicity. Polyacrylamide gel electrophoretic analysis of the lipopolysaccharide (LPS) in the mutant demonstrated that the O-polysaccharide part was completely absent. Meanwhile, a gel permeation chromatographic analysis of the exopolysaccharide (EPS) in the mutant demonstrated that it was unaltered. Since LPSs are known to have several anion groups that interact with various cation groups and metal ions, the mutant provided an opportunity to examine the direct role of LPS in metal binding by B. japonicum. Using atomic absorption spectrophotometry, it was clearly demonstrated that LPS was involved in metal binding. The binding capacity of the LPS mutant to various metal ions $(Cd^{2+},\;Cu^{2+},\;Pb^{2+},\;and\;Zn^{2+})$ was 50-70% lower than that of the wild-type strain. Also, through an EPS analysis and desorption experiment, it was found that EPS and centrifugal force had no effect on the metal binding. Accordingly, it would appear that LPS molecules on B. japonicum effect the properties, which precipitate more distinctly metal-rich mineral phase.

• Journal of Microbiology and Biotechnology
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• 제17권2호
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• pp.234-243
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• 2007

Vibrio vulnificus is an opportunistic pathogen that causes septicemia in humans. To identify the genes associated with its pathogenicity, in vivo expression technology (IVET) was used to select genes specifically expressed in a host, yet not significantly in vitro. Random lacZ-fusions in the genome of V vulnificus strain MO6-24/O were constructed using an IVET vector, pSG3, which is a suicide vector containing promoterless-aph and -lacZ as reporter genes. A total of ${\sim}18,000$ resulting library clones were then intraperitoneally injected into BALB/c mice using a colony forming unit (CFU) of $1.6{\times}10^6$. Two hours after infection, kanamycin was administered at $200{mu}g$ per gram of mouse weight. After two selection cycles, 11 genes were eventually isolated, which were expressed only in the host. Among these genes, VV20781 and VV21007 exhibiting a homology to a hemagglutinin gene and tolC, respectively, were selected based on having the highest frequency. When compared to wild-type cells, mutants with lesions in these genes showed no difference in the rate of growth rate, yet a significant decrease in cytotoxicity and the capability to form a biofilm.

• Journal of Microbiology and Biotechnology
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• 제26권6호
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• pp.1148-1161
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• 2016

Lactobacilli are universally recognized as probiotics that are widely used in the adjuvant treatment of inflammatory diseases, such as vaginitis and enteritis. With the overuse of antibiotics in recent years, the lactobacilli in the human body are killed, which could disrupt the microecological balance in the human body and affect health adversely. In this work, cephalosporin-resistant Lactobacillus casei RL20 was obtained successfully from the feces of healthy volunteers, which possessed a stable genetic set. However, the shortage of lactic acid (72.0 g/l at 48 h) by fermentation did not meet the requirement for its use in medicine. To increase the production of lactic acid, the functional genes pfk and glk were introduced into the wild strain. A yield of 144.2 g/l lactic acid was obtained in the transgenic L. casei RL20-2 after fermentation for 48 h in 1 L of basic fermentation medium with an initial glucose concentration of 100 g/l and increasing antibacterial activity. These data suggested that L. casei RL20-2 that exhibited a high yield of lactic acid may be a potential probiotic to inhibit the spread of bacterial infectious diseases and may be used for vaginitis therapy.

• Journal of Microbiology and Biotechnology
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• 제22권10호
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• pp.1324-1329
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• 2012

Phosphomannomutase (ManB) is involved in the biosynthesis of GDP-mannose, which is vital for numerous processes such as synthesis of carbohydrates, production of alginates and ascorbic acid, and post-translational modification of proteins. Here, we discovered that a deletion mutant of manB (BG101) in Streptomyces coelicolor (S. coelicolor) showed higher sensitivity to bacteriostatic chloramphenicol (CM) than the wild-type strain (M145), along with decreased production of CM metabolites. Deletion of manB also decreased the mRNA expression level of drug efflux pumps (i.e., cmlR1 and cmlR2) in S. coelicolor, resulting in increased sensitivity to CM. This is the first report on changes in antibiotic sensitivity to CM by deletion of one glycolysis-related enzyme in S. coelicolor, and the results suggest different approaches for studying the antibiotic-resistant mechanism and its regulation.

• 한국균학회지
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• 제38권1호
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• pp.48-53
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• 2010

팽이버섯의 다양한 유전자의 기능을 연구하기 위한 방법으로 팽이 자실체의 주름조직을 사용하여 A. tumefaciens을 매개로 한 형질전환을 수행하였다. Hygromycin 항생제 저항성 유전자를 포함하고 있는 AGL-1 pBGgHg를 팽이버섯의 자실체 조직에 도입시킴으로서 형질전환체들을 얻을 수 있었다. 형질전환체 선발은 첫 번째로 hygromycin 선발 배지에서 선발한 뒤 gpd-FH, hph-R primer를 이용하여 PCR로 확인 할 수 있었다. hygromycin은 30 ug/ml에서 선발 효율이 가장 높았다. 형질전환체 중 일부는 갈색 자실체를 갖는 wild type과는 달리 백색 자실체를 나타내었다. 형질전환체의 southern blot결과 외래유전자가 팽이버섯 내부에 하나 또는 그 이상의 다양한 copy 수로 삽입되어 있음을 알 수 있었다. 또한 삽입된 유전자의 주변 염기서열에 대한 정보는 Inverse PCR을 통해 알 수 있었다.

• Journal of Microbiology
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• 제37권4호
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• pp.248-255
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• 1999

We have analyzed the MRE3/REC114 gene of Saccharomyces cerevisiae, previously detected in isolation of mutants defective in meiotic recombination. We cloned the MRE3/REC114 gene by complementation of the meiotic recombination defect and it has been mapped to chormosome XIII. The DNA sequence analysis revealed that the MRE3 gene is identical to the REC114 gene. The upstream region of the MRE3/REC114 gene contains a T_4C site, a URS (upstream repression sequence) and a TR (T-rich) box-like sequence, which reside upstream of many meiotic genes. Coincidentally, northern blot analysis indicated that the three sizes of MRE3/REC114 transcripts, 3.4, 1.4 and 1.2 kb, are induced in meiosis. A less abundant transcript of 1.4 kb is detected in both mitotic and meiotic cells, suggesting that it is needed in mitosis as well as meiosis. To examine the role of the MRE3/REC114 gene, we constructed mre3 disruption mutants. Strains carrying an insertion or null deletion of the MRE3/REC114 gene showed slow growth in nutrient medium and the doubling time of these cells increased approximately by 2-fond compared to the wild-type strain. Moreover, the deletion mutant (${\delta}$mre3) displayed no meiotically induced recombination and no viable spores. The mre3/rec114 spore lethality can be suppressed by spo13, a mutation that causes cells to bypass reductional division. The double-stranded breaks (DSBs) which are involved in initiation of meiotic recombination were not detected in the analysis of meiotic chromosomal DNA from the mre3/rec114 disruptant. From these results we suggest that the MRE3/REC114 gene product is essential in normal growth and in early meiotic stages involved in meiotic recombination.

• Restorative Dentistry and Endodontics
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• 제39권4호
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• pp.288-295
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• 2014

Objectives: This study was designed to evaluate the synergistic antibacterial effect of xylitol and ursolic acid (UA) against oral biofilms in vitro. Materials and Methods: S. mutans UA 159 (wild type), S. mutans KCOM 1207, KCOM 1128 and S. sobrinus ATCC 33478 were used. The susceptibility of S. mutans to UA and xylitol was evaluated using a broth microdilution method. Based on the results, combined susceptibility was evaluated using optimal inhibitory combinations (OIC), optimal bactericidal combinations (OBC), and fractional inhibitory concentrations (FIC). The anti-biofilm activity of xylitol and UA on Streptococcus spp. was evaluated by growing cells in 24-well polystyrene microtiter plates for the biofilm assay. Significant mean differences among experimental groups were determined by Fisher's Least Significant Difference (p < 0.05). Results: The synergistic interactions between xylitol and UA were observed against all tested strains, showing the FICs < 1. The combined treatment of xylitol and UA inhibited the biofilm formation significantly and also prevented pH decline to critical value of 5.5 effectively. The biofilm disassembly was substantially influenced by different age of biofilm when exposed to the combined treatment of xylitol and UA. Comparing to the single strain, relatively higher concentration of xylitol and UA was needed for inhibiting and disassembling biofilm formed by a mixed culture of S. mutans 159 and S. sobrinus 33478. Conclusions: This study demonstrated that xylitol and UA, synergistic inhibitors, can be a potential agent for enhancing the antimicrobial and anti-biofilm efficacy against S. mutans and S. sobrinus in the oral environment.

• Journal of Microbiology and Biotechnology
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• 제31권9호
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• pp.1191-1199
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• 2021

Enteropathogenic Escherichia coli (EPEC), which belongs to the attaching and effacing diarrheagenic E. coli strains, is a major causative agent of life-threatening diarrhea in infants in developing countries. Most EPEC isolates correspond to certain O serotypes; however, many strains are non-typeable. Two EPEC strains, EPEC001 and EPEC080, which could not be serotyped during routine detection, were isolated. In this study, we conducted an in-depth characterization of their putative O-antigen gene clusters (O-AGCs) and also performed constructed mutagenesis of the O-AGCs for functional analysis of O-antigen (OAg) synthesis. Sequence analysis revealed that the occurrence of O-AGCs in EPEC001 and E. coli O132 may be mediated by recombination between them, and EPEC080 and E. coli O2/O50 might acquire each O-AGC from uncommon ancestors. We also indicated that OAg-knockout bacteria were highly adhesive in vitro, except for the EPEC001 wzy derivative, whose adherent capability was less than that of its wild-type strain, providing direct evidence that OAg plays a key role in EPEC pathogenesis. Together, we identified two EPEC O serotypes in silico and experimentally, and we also studied the adherent capabilities of their OAgs, which highlighted the fundamental and pathogenic role of OAg in EPEC.

• Parasites, Hosts and Diseases
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• 제57권4호
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• pp.369-377
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• 2019

Artemisinin-based combination therapy (ACT) resistance is widespread throughout the Greater Mekong Subregion. This raises concern over the antimalarial treatment in Thailand since it shares borders with Cambodia, Laos, and Myanmar where high ACT failure rates were reported. It is crucial to have information about the spread of ACT resistance for efficient planning and treatment. This study was to identify the molecular markers for antimalarial drug resistance: Pfkelch13 and Pfmdr1 mutations from 5 provinces of southern Thailand, from 2012 to 2017, of which 2 provinces on the Thai- Myanmar border (Chumphon and Ranong), one on Thai-Malaysia border (Yala) and 2 from non-border provinces (Phang Nga and Surat Thani). The results showed that C580Y mutation of Pfkelch13 was found mainly in the province on the Thai-Myanmar border. No mutations in the PfKelch13 gene were found in Surat Thani and Yala. The Pfmdr1 gene isolated from the Thai-Malaysia border was a different pattern from those found in other areas (100% N86Y) whereas wild type strain was present in Phang Nga. Our study indicated that the molecular markers of artemisinin resistance were spread in the provinces bordering along the Thai-Myanmar, and the pattern of Pfmdr1 mutations from the areas along the international border of Thailand differed from those of the non-border provinces. The information of the molecular markers from this study highlighted the recent spread of artemisinin resistant parasites from the endemic area, and the data will be useful for optimizing antimalarial treatment based on regional differences.