• Korean Journal of Veterinary Research
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• v.29 no.2
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• pp.59-67
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• 1989

This paper deals with the genetic properties of R plasmids in Salmonella originated from pigs and cattle. The plasmid DNA was examined for incompatibility, stability and fertility inhibition(Fi), and gel electrophoresis was performed for isolation of plasmid DNA. The results obtained were summerized as follows: 1. Among the 66 conjugative R plasmids from 44 pigs and 22 cattle, 61 R plasmids (92.4%) were $Fi^-$, whereas the remainder were $Fi^+$. 2. The Inc groups of 66 R plasmids were determined with 7 standard plasmids. Twenty-six R plasmids were classified into Inc group $I{\alpha}$, H1, H2 or F1, 40 R plasmids being not classified with standard plasmids used, and the Inc group $I{\alpha}$ (57.7%) was most frequent. 3. Inc groups $I{\alpha}$, H1, and F1 were identified in strains from swine, Inc groups H2 and F1 from cattle. 4. The plasmid DNA profiles in 16 Salmonella isolated from pigs and cattle were confirmed as being 1 to 10 fragments by the gel eletrophoresis. Their molecular weight ranged 1.0 to 90 megadalton. 5. The molecular weight of conjugative plasmids ranged 1.0 to 80 megadalton in 4 Salmonella (P-4, P-5, P-7 and P-8) isolated from pigs.

• Journal of Microbiology and Biotechnology
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• v.30 no.12
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• pp.1862-1869
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• 2020

The spread of plasmid-mediated colistin resistance has posed a serious threat to public health owing to its effects on the emergence of pandrug-resistant bacteria. In this study, we investigated the prevalence and characteristics of mcr-1-positive Escherichia coli isolated from retail meat samples in Korea. In total, 1,205 E. coli strains were isolated from 3,234 retail meat samples in Korea. All E. coli strains were subjected to antimicrobial susceptibility testing and were examined for the presence of mcr-1 gene. All mcr-1-positive E. coli (n = 10, 0.8%) from retail meat were subjected to pulse-field gel electrophoresis (PFGE) and whole-genome sequencing (WGS). The transferability of mcr-1 gene was determined by conjugation assays. The mcr-1-positive strains exhibited diverse clonal types. Our mcr-1 genes were located in plasmids belonged to the IncI2 (n = 1) and IncX4 (n = 8) types, which were reported to be prevalent in Asia and worldwide, respectively. Most mcr-1 genes from mcr-1-positive strains (9/10) were transferable to the recipient strain and the transfer frequencies ranged from 2.4 × 10-3 to 9.8 × 10-6. Our data suggest that the specific types of plasmid may play an important role in spreading plasmid-mediated colistin resistance in Korea. Furthermore, our findings suggest that the retail meat may be an important tool for disseminating plasmid-mediated colistin resistance.

• Annals of Laboratory Medicine
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• v.38 no.6
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• pp.555-562
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• 2018

Background: The emerging mobile colistin resistance gene, mcr-1, is an ongoing worldwide concern and an evaluation of clinical isolates harboring this gene is required in Korea. We investigated mcr-1-possessing Enterobacteriaceae among Enterobacteriaceae strains isolated in Korea, and compared the genetic details of the plasmids with those in Escherichia coli isolates from livestock. Methods: Among 9,396 Enterobacteriaceae clinical isolates collected between 2010 and 2015, 1,347 (14.3%) strains were resistant to colistin and those were screened for mcr-1 by PCR. Colistin minimum inhibitory concentrations (MICs) were determined by microdilution, and conjugal transfer of the mcr-1-harboring plasmids was assessed by direct mating. Whole genomes of three mcr-1-positive Enterobacteriaceae clinical isolates and 11 livestock-origin mcr-1-positive E. coli isolates were sequenced. Results: Two E. coli and one Enterobacter aerogenes clinical isolates carried carried IncI2 plasmids harboring mcr-1, which conferred colistin resistance (E. coli MIC, 4 mg/L; E. aerogenes MIC, 32 mg/L). The strains possessed the complete conjugal machinery except for E. aerogenes harboring a truncated prepilin peptidase. The E. coli plasmid transferred more efficiently to E. coli than to Klebsiella pneumoniae or Enterobacter cloacae recipients. Among the three bacterial hosts, the colistin MIC was the highest for E. coli owing to the higher mcr-1-plasmid copy number and mcr-1 expression levels. Ten mcr-1-positive chicken-origin E. coli strains also possessed mcr-1-harboring IncI2 plasmids closely related to that in the clinical E. aerogenes isolate, and the remaining one porcine-origin E. coli possessed an mcr-1-harboring IncX4 plasmid. Conclusions: mcr-1-harboring IncI2 plasmids were identified in clinical Enterobacteriaceae isolates. These plasmids were closely associated with those in chicken-origin E. coli strains in Korea, supporting the concept of mcr-1 dissemination between humans and livestock.

• Journal of Microbiology and Biotechnology
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• v.2 no.4
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• pp.243-247
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• 1992

The thr operon of Escherichia coli TF427, an $\alpha$-amino-$\beta$-hydroxyvaleric acid (AHV)-resistant threonine overproducer, was cloned in a pBluescriptII $KS^＋$ plasmid by complementation of E. coli mutants. All clones contained a common 8.8 kb HindIII-generated DNA fragment and complemented the thrA, thrB, and thrC mutants by showing that these clones contained the whole thr operon. This thr operon was subcloned in the plasmid vectors pBR322, pUC18, and pECCG117, an E. coli/Corynebacterium glutamicum shuttle vector, to form recombinant plasmids pBTF11, pUTF25 and pGTF18, respectively. The subcloned thr operon was shown to be present in a 6.0 kb insert. A transformant of E. coli TF125 with pBTF11 showed an 8~11 fold higher aspartokinase I activity, and 15~20 fold higher L-threonine production than TF125, an AHV-sensitive methionine auxotroph. Also, it was found that the aspartokinase I activity of E. coli TF125 harboring pBTF11 was not inhibited by threonine and its synthesis was not repressed by threonine plus isoleucine.

• Journal of Microbiology and Biotechnology
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• v.15 no.4
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• pp.880-886
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• 2005

Using carotenoid genes of Erwinia herbicola, metabolic engineering was carried out for lycopene production with the pAC-LYCO4 plasmid, which was composed of a chromosomal DNA fragment of E. herbicola containing the crtE, crtB, and crtI genes under the control of the tetracycline promoter and the ipi gene of Haematococcus pluvialis with the trc promoter. Plasmid pAC-LYCm4 was constructed for efficient expression of the four exogenous genes using a strong RBS sequence and the same tetracycline promoter. The optimized expression construct of pAC-LYCm4 increased Iycopene production three times as compared with pAC-LYCO4. pAC-LYCm5 containing ispA behind the four exogenous genes was constructed. There was no significant difference in Iycopene production and cell growth between pAC-LYCm4 and pAC-LYCm5. FPP synthase encoded by ispA was not rate-limiting for Iycopene production. Each gene of crtE, crtB, crtI, and ipi was overexpressed, using pBAD-crtE, pBAD-crtIB, and pBAD-ipiHPI, in addition to their expression from pAC-LYCm4. However, there was no increase oflycopene production with the additional overexpression of each exogenous gene. The four exogenous genes appeared to be not rate-limiting in cells harboring pAC-LYCm4. When pDdxs, pBAD24 containing dxs, was introduced into cells harboring lycopene synthetic plasmids, lycopene production of pAC-LYCO4, pAC-LYCm4, and pAC-LYCm5 was increased by 4.7-, 2.2-, and 2.2-fold, respectively. Lycopene production of pBAD-DXm4 containing crtE, crtB, crtI, ipi, and dxs was 5.2 mg/g dry cell weight with $0.2\%$ arabinose, which was 8.7-fold higher than that of the initial strain with pAC-LYC04. Therefore, the present study showed that proper regulation of a metabolically engineered pathway is important for Iycopene production.

• Journal of Microbiology and Biotechnology
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• v.15 no.1
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• pp.66-71
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• 2005

Doxorubicin is a clinically important anticancer polyketide compound that is typically produced by Streptomyces peucetius var. caesius. To improve doxorubicin productivity by S. peucetius, a doxorubicin pathway-specific regulatory gene, dnrI, was cloned into a high-copy-number plasmid containing a catechol promoter system. The S. peucetius containing the recombinant plasmid exhibited approximately 9.5-fold higher doxorubicin productivity compared with the wild-type S. peucetius. The doxorubicin productivity by this recombinant S. peucetius strain was further improved through the optimization of culture media composition. Based on the Fractional Factorial Design (FFD), cornstarch, $K_2HPO_4$, and $MgSO_4$ were identified to be the key factors influencing doxorubicin productivity. The Response Surface Method (RSM) results based on 20 independent culture conditions with varying amounts of key factors predicted the highest theoretical doxorubicin productivity of 11.1 mg/l with corn starch of 46.33 g/l, $K_2HPO_4$ of 4.63 g/l, and $MgSO_4$ of 9.26 g/l. The doxorubicin productivity of the recombinant S. peucetius strain with the RSM-based optimized culture condition was experimentally verified to be 11.46 mg/l, which was approximately 30.8-fold higher productivity compared with the wild-type S. peucetius without culture media optimization.

• Journal of Microbiology
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• v.40 no.1
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• pp.33-37
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• 2002

An enzymatic reaction for the production of D-alanine from D-aspartic acid and pyruvate as substrates by a thermostable D-amino acid aminotransferase (D-AAT) was investigated at various conditions In the temperature range of 40-70$\^{C}$ and pH range of 6.0-9.5. The D-AAT was produced with recombinant E. coli BL21, which hosted the chimeric plasmid pTLK2 harboring the D-AAT from the novel thermophilic Bacillus sp. LK-2. The enzyme reaction was shown to follow the Ping Pong Bi Bi mechanism. The K$\_$m/ values for D-aspartic acid and pyruvate were 4.38 mar and 0.72 mM, respectively. It was observed that competitive inhibition by D-alanine, the product of this reaction, was evident with the inhibition constant K$\_$i/ value of 0.1 mM. A unique feature of this reaction scheme is that the decorboxylation of oxaloacetic acid, one of the products, spontaneously produces pyruvate. Therefore, only a catalytic amount of pyruvate is necessary for the enzyme conversion reaction to proceed. A typical time-course kinetic study skewed that D-alanine up to 88 mM could be produced from 100 mM of D-aspartic acid with a molar yield of 1.0.