• Korean Journal of Microbiology
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• v.40 no.3
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• pp.237-243
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• 2004

The aim of this study is to investigate the detection rate of putative periodontopathogens, Porphyromonas gingivalis, Tannerella forsythia, and Actiobacillus actinomycetemcomitans, related to cardiovascular diseases(CVD). Plaques were sampled from 15 subjects (4 sites of denture base and/or tooth) with sterilized explorers and were transported in IX PBS. The detection of periodontopathogens was performed by polymerase chain reaction with species-specific primers based on 16S rDNA. The PCR products were cloned into pGEM-T easy vector and its nucleotides were sequenced in order to confirm the specificity. Our data showed that the detection rate of P. gingivalis and T forsythia in denture base of edentulous patients was 25％ and 75％, respectively. And the detection rate of P. gingivalis and T.forsythia in denture base of patient having one more tooth was 91％. The results indicate that plaque of denture base may serve as reservoirs of oral bacteria related to CVD.

• Korean Journal of Microbiology
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• v.51 no.3
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• pp.256-262
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• 2015

In order to study the effect of the receptor protein (SeaR), which is isolated from Saccharopolyspora erythraea, we introduced the SeaR gene to Streptomyces virginiae as host strains. An effective transformation procedure for S. virginiae was established based on transconjugation by Escherichia coli ET12567/pUZ8002 with a ${\varphi}C31$-derived integration vector, pSET152, which contained int, oriT, attP, and $ermEp^{\ast}$ (erythromycin promotor). Therefore, the pEV615 was introduced into S. virginiae by conjugation and integrated at the attB locus in the chromosome of the recipients by the ${\varphi}C31$ integrase (int) function. Transformants of S. virginiae containing the SeaR gene were confirmed by PCR and transcriptional expression of the SeaR gene in the transformants was analyzed by RT-PCR, respectively. And, we examined the production time of virginiamycins in the culture media of both the transformants and the wild type. The production time of virginiamycins in the wild type and transformants was the same. When 100 ng/ml of synthetic $VB-C_6$ was added to the state of 6 or 8 hour cultivation of wild type and transformants, respectively, the virginiamycins production was induced, meaning that the virginiamycins production in the wild type was detected 2 h early than transformants. From these results, SeaR expression was also affected to virginiamycins production in transformants derived from S. virginiae. In this study, we showed that the SeaR protein worked as a repressor in transformants.

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

• KSBB Journal
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• v.23 no.6
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• pp.519-524
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• 2008

Monacolin-K is a strong anti-hypercholesterolemic agent produced by Monascus sp. via polyketide pathway. High-yielding mutants of monacolin-K were developed through rational screening strategies adopted based on understanding of monacolin-K biosynthetic pathway. Through the experiments for investigating various amino acids as putative precursors for the monacolin-K biosynthesis, it was found that production level of monacolin-K was remarkably increased when optimum amount of cysteine was supplemented into the production medium. We suggested that these phenomena might be related to the special roles of SAM (S-adenosyl methionine), a putative methyl group donor in the biosynthetic pathway of monacolin-K, demonstrating close interrelationship between SAM-synthesizing primary metabolism and monacolin-K synthesizing secondary metabolism. Namely, increase in the intracellular amount of SAM derived from the putative precursor, cysteine which was extracellularly supplemented into the production medium might contribute to the significant enhancement in the monacolin-K biosynthetic capability of the highly mutated producers. On the basis of these assumptions derived from the above fermentation results, we decided to construct efficient expression vectors harboring SAM synthetase gene (metK) cloned from A. nidulans, with the hope that increased intracellular level of SAM could lead to further enhancement in the monacolin-K production through overcoming a rate-limiting step associated with monacolin-K biosynthesis. Hence, in order to overcome the plausible rate-limiting step associated with monacolin-K biosynthesis by increasing intracellular level of SAM, we transformed the producer mutants with an efficient expression vector harboring gpdA promoter of the producer microorganism, and metK gene. Notably, from the resulting various transformants, we were able to screen a very high-yielding transformant which showed approximately 3.3 fold higher monacolin-K productivity than the parallel nontransformed mutants in shake flask cultures performed under the identical fermentation conditions.