• KSBB Journal
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• v.20 no.3
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• pp.220-227
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• 2005

Doxorubicin is an anthracycline-family polyketide compound with a very potent anti-cancer activity, typically produced by Streptomyces peucetius. To understand the potential target biosynthetic genes critical for the doxorubicin everproduction, a doxorubicin-specific DNA microarray chip was fabricated and applied to reveal the growth-phase-dependent expression profiles of biosynthetic genes from two doxorubicin-overproducing strains along with the wild-type strain. Two doxorubicin-overproducing 5. peucetius strains were generated via over-expression of a dnrl (a doxorubicin-specific positive regulatory gene) and a doxA (a gene involved in the conversion from daunorubicin to doxorubicin) using a streptomycetes high expression vector containing a strong ermE promoter. Each doxorubicin-overproducing strain was quantitatively compared with the wild-type doxorubicin producer based on the growth-phase-dependent doxorubicin productivity as well as doxorubicin biosynthetic gene expression profiles. The doxorubicin-specific DNA microarray chip data revealed the early-and-steady expressions of the doxorubicin-specific regulatory gene (dnrl), the doxorubicin resistance genes (drrA, drrB, drrC), and the doxorubicin deoxysugar biosynthetic gene (dnmL) are critical for the doxorubicin overproduction in S. peucetius. These results provide that the relationship between the growth-phase-dependent doxorubicin productivity and the doxorubicin biosynthetic gene expression profiles should lead us a rational design of molecular genetic strain improvement strategy.

• YAKHAK HOEJI
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• v.34 no.2
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• pp.139-146
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• 1990

Streptococcus sp. TR-1 which has inducible resistance to MLS antibiotics was isolated from soil samples in Korea. Streptococcus sp. TR-1 was cultured in Lysis broth, then a plasmid was isolated by modified Elliker method. Bacillus subtilis UOTO277 was transformed with that plasmid. This result showed that the plasmid has the gene relating with inducible resistance to MLS antibiotics. It was named pMB4 and its size was determined about 2.4 Kb by results of digestion with various restriction enzymes. Restriction endonuclease cleavage site map of pMB4 plasmid was made by double digestion of the plasmid. pMB4 plasmid has different restriction endonuclease site map from the other plasmids that have been discovered in Streptococcus sp. so far. And it could be identified that pMB4 plasmid does not have homology with ermK of Bacillus licheniformis EMR but has homology with ermC of Staphylococcus aureus from the results of Southern hybridization.

• Korean Journal of Microbiology
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• v.46 no.2
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• pp.148-151
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• 2010

The aim of this study was to investigate the erythromycin resistance patterns of Enterococci sp. present in cow milk. A total 110 erythromycin resistant Enterococci were isolated from milk samples; E. faecalis (n=101), E. avium (n=7), and E. durans (n=2). The minimum inhibitory concentration of erythromycin against 110 Enterococci were determined. The 66.3% of Entercocci (n=73) showed high level resistance (${\geq}64$ mg/ml). Among 110 isolates, 86.3% (n=95) showed $cMLS_B$ phenotype and 13.6% (n=15) showed $iMLS_B$ The aim of this study was to investigate the erythromycin resistance patterns of Enterococci sp. present in cow milk. A total 110 erythromycin resistant Enterococci were isolated from milk samples; E. faecalis (n=101), E. avium (n=7), and E. durans (n=2). The minimum inhibitory concentration of erythromycin against 110 Enterococci were determined. The 66.3% of Entercocci (n=73) showed high level resistance (${\geq}64$ mg/ml). Among 110 isolates, 86.3% (n=95) showed $cMLS_B$ phenotype and 13.6% (n=15) showed $iMLS_B$ phenotype. All of isolates have erm(B) determinant, 75.45% (n=83) have mef(A) an efflux system determinant. The majority of Enetrcococci isolated from raw milk samples in northern area of Gyeonggi-Do showed high level of resistance to erythromycin.

• 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.

• Biotechnology and Bioprocess Engineering:BBE
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• v.11 no.2
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• pp.116-120
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• 2006

We constructed four recombinant plasm ids to enhance the production of clavulanic acid (CA) in Streptomyces clavuligerus NRRL3585: (1) pIBRHL1, which includes ccaR, a pathway-specific regulatory gene involved in cephamycin C and CA biosynthesis; (2) pIBRHL2, containing claR, again a regulatory gene, which controls the late steps of CA biosynthesis; (3) pGIBR containing afsR-p, a global regulatory gene from Streptomyces peucetius; and (4) pKS, which harbors all of the genes (ccaR/ claR/ afsR-p). The plasmids were expressed in S. clavuligerus NRRL3585 along with the $ermE^*$ promoter. All of them enhanced the production of CA; 2.5-fold overproduction for pIBRHL1, 1.5-fold for pIBRHL2, 1.6-fold for pGIBR, and 1.5-fold for pKS compared to the wild type.

• Parasites, Hosts and Diseases
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• v.59 no.2
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• pp.179-182
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• 2021

Human infection with Taenia asiatica or a hybrid between Taenia saginata and T. asiatica has not been reported in Cambodia. We detected for the first time a hybrid form between T. saginata and T. asiatica in Preah Vihear Province, Cambodia. An adult tapeworm specimen, i.e., 75 cm long strobila without scolex, was expelled from a 27-year-old man after praziquantel medication and purging. It was morphologically indistinguishable between T. saginata and T. asiatica. Several proglottids were molecularly analyzed to confirm the tapeworm species. The mitochondrial gene encoding cytochrome c oxidase subunit 1 (cox1) and nuclear genes encoding elongation factor-1α (ef1) and ezrin-radixin-moesin (ERM)-like protein (elp) were sequenced, and a single-allele analysis was performed to confirm the haploid genotype. The results revealed that our sample showed a discrepancy between the mitochondrial and 2 nuclear genes. It possessed homozygous sequences typical of T. saginata at cox1 and ef1 loci. However, it was heterozygous at the elp locus, with 1 allele in T. asiatica (elpA) and 1 in T. saginata (elpC), which indicates that it is a hybrid between T. saginata and T. asiatica. The present results confirmed the presence of a hybrid between T. saginata and T. asiatica in Cambodia and strongly suggest the existence of also 'pure' T. asiatica in Cambodia.

• Journal of Microbiology and Biotechnology
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• v.33 no.2
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• pp.211-218
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• 2023

A cryptic plasmid (pTH32) was characterized from Tetragenococcus halophilus 32, an isolate from jeotgal, Korean traditional fermented seafood. pTH32 is 3,198 bp in size with G+C content of 35.84%, and contains 4 open reading frames (ORFs). orf1 and orf2 are 456 bp and 273 bp in size, respectively, and their translation products showed 65.16% and 69.35% similarities with RepB family plasmid replication initiators, respectively, suggesting the rolling-circle replication (RCR) mode of pTH32. orf3 and orf4 encodes putative hypothetical protein of 186 and 76 amino acids, respectively. A novel Tetragenococcus-Escherichia coli shuttle vector, pMJ32E (7.3 kb, Em^r), was constructed by ligation of pTH32 with pBluescript II KS(+) and an erythromycin resistance gene (ErmC). pMJ32E successfully replicated in Enterococcus faecalis 29212 and T. halophilus 31 but not in other LAB species. A pepA gene, encoding aminopeptidase A (PepA) from T. halophilus CY54, was successfully expressed in T. halophilus 31 using pMJ32E. The transformant (TF) showed higher PepA activity (49.8 U/mg protein) than T. halophilus 31 cell (control). When T. halophilus 31 TF was subculturd in MRS broth without antibiotic at 48 h intervals, 53.8% of cells retained pMJ32E after 96 h, and only 2.4% of cells retained pMJ32E after 14 days, supporting the RCR mode of pTH32. pMJ32E could be useful for the genetic engineering of Tetragenococcus and Enterococcus species.

• 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.

• Journal of Fisheries and Marine Sciences Education
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• v.28 no.1
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• pp.59-68
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• 2016

For comparison of tetracycline-resistant ($Tc^R$) genes, we obtained 21 and 14 $Tc^R$ Staphylococcus spp. from marine environment and human patient, respectively. Although all isolates from human were identified as Staphylococcus aureus, higher proportion of $Tc^R$ isolates (12 out of 14) from human were utilizing tet(M) gene compared to that of $Tc^R$ isolates (6 out of 21) from marine environment. Additionally, collaborated utilization of tet(M) and erm(A) in $Tc^R-Em^R$ S. aureus in human patient, but not in $Tc^R$ Staphylococcus spp. isolates from marine environment was also characterized. Based on the nucleotide sequence of transposon related to $Tc^R$ gene, we confirmed the origin of tet(M) gene in $Tc^R$ Staphylococci isolated from marine environments and human are derived from Tn916/1545-like and Tn5801 transposon, respectively. It is the first report showing the presence of Tn5801 in all $Tc^R$ S. aureus carrying tet(M) in human patient. Alignment of the fully sequenced tet(M) from marine environmental isolates was also agreed with the determined transposons by showing the genomic mosaic structure composed with three genomic parts from Tn916/1545 and unknown transposons. Genetic characteristics of these tet(M) in environmental isolates were similar to each other but different from those in isolates from human showing only tet(M) from Tn916/5801 type. It may imply the presence of less dramatic communication of antibiotic resistant genes between Staphylococci isolated from marine environment and human.

• Journal of Microbiology
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• v.45 no.5
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• pp.447-452
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• 2007

In this study, we compared the phenotypic and genotypic characteristics of 138 MRSA isolates obtained from adult and pediatric patients (adult, 50; children, 88). The resistance rates against gentamicin, clindamycin, and ciprofloxacin were much higher in the adult MRSA isolates than in the pediatric MRSA isolates. The ermC gene, which is responsible for inducible clindamycin resistance, was detected in 52(59.1%) of the 88 pediatric MRSA isolates but in only 5(10.0%) of the 50 adult MRSA isolates. MRSA isolates of clonal type ST5 with an integration of SCCmec type II/II variants was the most predominant clone among the adult isolates, while clonal type ST72 with an integration of SCCmec IV/IVA was the most predominant clone among the pediatric MRSA isolates. Staphylococcal enterotoxin A and toxic shock syndrome toxin-1 were prevalent among the adult MRSA isolates but not among the pediatric MRSA isolates. The results of this study demonstrated remarkable differences between adult and pediatric MRSA isolates in terms of their antimicrobial susceptibility profiles, SCCmec type, multilocus sequence type, staphylococcal toxin genes, and erythromycin resistance genes.