• Microbiology and Biotechnology Letters
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• v.19 no.4
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• pp.372-379
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• 1991

A rapid and simple quantitative assay method for aminoglycoside-3'- phosphotransferase (APH(3')) derived from E. coli ATCC 21990 was developed using the thin layer chromatographic densitometry, 3'-phosphorylated kanamycin B (3'-PKMB), product of APH (3') reaction, was separated from reaction mixtures by developing on the silica gel TLC plate with chloroform-methanol-ammonia water (3:4:3). The quantity of the 3'-PKMB was measured by densitometry after color development by ninhydrin method. Densitometric TLC assay for APH (3') was showed a good quantitative result and reproducibility. Sensitivity of this assay was 1.56 nmol of 3'-PKMB and could be analyzed many samples at same time. This method may be applicable for the analysis of inactivating enzymes of aminoglycoside antibiotics.

• Microbiology and Biotechnology Letters
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• v.19 no.5
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• pp.491-496
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• 1991

To maximize the production of aminoglycoside-3'-phosphotransferase of E. coli ATCC 21990 carrying R factor which encodes aminoglycoside-3'-phosphotransferase (APH(3')) phosphorylating the 3'-hydroxyl group of aminoglycoside, mutants M1 and M2, media composition and several factors affecting the enzyme production during fermentation were studied. Although the specific activity of APH(3') produced by a mutant M1 was increased as much as four times than that of E. coii ATCC 21990, the growth rate was decreased. The increase of the enzyme production was obtained by increased biomass during fermentation. A mutant M2 was obtained to increase the cell growth rate. Mutant M2 cells were cultivated with optimal media and pure oxygen gas in a fed-batch mode of fermentor operation. The specific activity of APH(3') was decreased, but total enzyme activity of APH(3') was increased as much as two point five times than that of mutant MI.

• Journal of Microbiology and Biotechnology
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• v.23 no.11
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• pp.1529-1535
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• 2013

Tuberculosis is a worldwide epidemic disease caused by Mycobacterium tuberculosis, with an estimated one-third of the human population currently affected. Treatment of this disease with aminoglycoside antibiotics has become less effective owing to antibiotic resistance. Recent determination of the crystal structure of the M. tuberculosis Rv3168 protein suggests a structure similar to that of Enterococcus faecalis APH(3')-IIIa, and that this protein may be an aminoglycoside phosphotransferase. To determine whether Rv3168 confers antibiotic resistance against kanamycin, we performed dose-response antibiotic resistance experiments using kanamycin. Expression of the Rv3168 protein in Escherichia coli conferred antibiotic resistance against $100{\mu}M$ kanamycin, a concentration that effected cell growth arrest in the parental E. coli strain and an E. coli strain expressing the $Rv3168^{D249A}$ mutant, in which the catalytic Asp249 residue was mutated to alanine. Furthermore, we detected phosphotransferase activity of Rv3168 against kanamycin as a substrate. Moreover, docking simulation of kanamycin into the Rv3168 structure suggests that kanamycin fits well into the substrate binding pocket of the protein, and that the phosphorylation-hydroxyl-group of kanamycin was located at a position similar to that in E. faecalis APH(3')-IIIa. On the basis of these results, we suggest that the Rv3168 mediates kanamycin resistance in M. tuberculosis, likely through phosphotransferase targeting of kanamycin.

• Journal of Microbiology and Biotechnology
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• v.29 no.3
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• pp.367-372
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• 2019

Deactivation of aminoglycosides by their modifying enzymes, including a number of aminoglycoside O-phosphotransferases, is the most ubiquitous resistance mechanism in aminoglycoside-resistant pathogens. Nonetheless, in a couple of biosynthetic pathways for gentamicins, fortimicins, and istamycins, phosphorylation of aminoglycosides seems to be a unique and initial step for the creation of a natural defensive structural feature such as a 3',4'-dideoxy scaffold. Our aim was to elucidate the biochemical details on the beginning of these C3',4'-dideoxygenation biosynthetic steps for aminoglycosides. The biosynthesis of istamycins must surely involve these 3',4'-didehydroxylation steps, but much less has been reported in terms of characterization of istamycin biosynthetic genes, especially about the phosphotransferase-encoding gene. In the disruption and complementation experiments pointing to a putative gene, istP, in the genome of wild-type Streptomyces tenjimariensis, the function of the istP gene was proved here to be a phosphotransferase. Next, an in-frame deletion of a known phosphotransferase-encoding gene forP from the genome of wild-type Micromonospora olivasterospora resulted in the appearance of a hitherto unidentified fortimicin shunt product, namely 3-O-methyl-FOR-KK1, whereas complementation of forP restored the natural fortimicin metabolite profiles. The bilateral complementation of an istP gene (or forP) in the ${\Delta}forP$ mutant (or ${\Delta}istP$ mutant strain) successfully restored the biosynthesis of 3',4'-dideoxy fortimicins and istamycins, thus clearly indicating that they are interchangeable launchers of the biosynthesis of 3',4'-dideoxy types of 1,4-diaminocyclitol antibiotics.

• Korean Journal of Veterinary Service
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• v.35 no.1
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• pp.1-8
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• 2012

To detect the virulence genes (invA and spvC) and antimicrobial resistance genes, polymerase chain reaction (PCR) was carried out using total 67 strains of S. Schwarzengrund isolated from pigs. As results, invA was detected from all 67 strains of S. Schwarzengrund, however, spvC was not at all. All 12 strains with ampicillin resistance, 15 strains with chloramphenicol resistance, 9 strains with kanamycin resistance, 1 strain with sulfamethoxazole/trimethoprim resistance, and 66 (98.5%) of 67 strains with tetracycline resistance carried TEM (${\beta}$-lactamase $bla_{TEM}$), cmlA (nonenzymatic chloramphenicol resistance), aphA1-Iab (aminoglycoside phosphotransferase), sulII (dihydropteroate synthase), and tetA (class A tetracycline resistance), respectively. All 63 strains with streptomycin resistance carried 3 aminoglycoside resistance genes, including aadA (aminoglycoside adenyltransferase), strA, and strB (streptomycin phosphotransferase). With respect to prevalence of antibiotic resistance genes occurred in S. Schwarzengrund, genes for strB (46.0%); strA and strB (30.2%); aadA, strA, and strB (9.5%); strA (7.9%); aadA and strB (3.2%); and aadA (3.2%) were detected by PCR.

• Journal of Microbiology
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• v.44 no.6
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• pp.671-673
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• 2006

A new 4.87 kb Escherichia-Pseudomonas shuttle vector has been constructed by inserting a 1.27 kb DNA fragment with a replication origin of a Pseudomonas plasmid pRO1614 into the 3.6 kb E. coli plasmid pBGS18. This vector, designated pJH1, contains an aminogly-coside phosphotransferase gene (aph) from Tn903, a lacZ' gene for $\alpha$-complementation and a versatile multiple cloning site possessing unique restriction sites for EcoRI, SacI, KpnI, SmaI, BamHI, XbaI, SalI, BspMI, PstI, SphI, and HindIII. When pJH1 was transformed into E. coli DHS${\alpha}$ and into P. putida HK-6, it was episomally and stably maintained in both strains. In addition, the enhanced green fluorescent protein (EGFP) gene which was transcriptionally cloned into pJH1 rendered E. coli cells fluorescence when its transformants were illuminated at 488 nm.

• Journal of Microbiology and Biotechnology
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• v.6 no.3
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• pp.219-220
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• 1996

The pKH2 isolated from the multidrug-resistant Staphylococcus aureus SA2 is a 40.98-kb plasmid and mediates resistance to ampicillin, clindamycin, erythromycin, kanamycin, and streptomycin. The 3.4-kb HindIII fragment conferring kanamycin resistance was cloned from the pKH2 into pBluescriptII $KS^+$ and partial sequence determination of that fragment was carried out. Sequence analysis revealed that the kanamycin resistance gene which encoded aminoglycoside 3'-phosphotransferase was linked to the streptothricin resistance gene. But a nonsense mutation was found in the streptothricin resistance gene and this mutation resulted in a truncated protein of streptothricin acetyltransferase. Homology comparison with nucleotide sequence databases revealed that the 3.4-kb HindIII fragment of pKH2 had been derived not from S. aureus but from Gram-negative Campylobacter coli.

• Archives of Pharmacal Research
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• v.5 no.2
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• pp.87-91
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• 1982

In order to develop an analytical method for amikacin and butirosin in presence of their parent antibiotics, kanamycin A and ribostamycin, high-performance liquid chromatographic technique and microbioassay method were evaluated and compared. Using high performance liquid chromatography, two acylated antibiotics, amikacin and butirosin was partially separated from their parent antibiotics, to provide a qualitative analytical method. In microbioassay using Pseudomonas aeruginosa TI-13, a producer of aminoglycoside-3-phosphotransferase I, only acylated antibiotics were selectively analyzed when paper disc-susceptibility assay was used. The standard curve showed a good correlation between the response and odse in semilogarithmic plat with correlation coefficients above 0.96, and analytical deviation from expected dose was within 10%.

• Biotechnology and Bioprocess Engineering:BBE
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• v.5 no.1
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• pp.1-6
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• 2000

For the rapid selection of higher recombinant hirudin producing strain in a methylotrophic yeast Hansenula polymorpha, a multiple gene integration and dose-dependent selection vector, based on a telomere-associated ARS and a bacterial aminoglycoside 3-phosphotransferase (aph) gene, was adopted. Two hirudin expression cassettes (HV1 and HV2) were constructed using the MOX promoter of H. polymorpha and the mating factor $\alpha$ secretion signal of S. cerevisiae. Multiple integrants of a transforming vector containing hirudin expression cassettes were easily selected by using an antibiotic, G418. Hirudin expression level and integrated plasmid copy number of the tested transformants increased with increasing the concentration of G418 used for selection. The expression level of HV1 was consistently higher than that of HV2 under the similar conditions, suggesting that the gene context might be quite important for the high-level gene expression in H. polymorpha. The highest hirudin producing strain selected in this study produced over 96 mg/L of biologically active hirudin in a 500-mL flask and 165 mg/L in a 5-L fermentor.

• Biomedical Science Letters
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• v.18 no.1
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• pp.79-82
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• 2012

Acinetobacter infections are of great concern in clinical settings because of multi-drug resistance (MDR) and high mortality of the infected patients. The MDR Acinetobacter baumannii has emerged as a significant infectious agent in hospitals worldwide. The purpose of this study was to determine for molecular characterization of MDR A. baumannii clinical isolates obtained from the Wonju Christian Hospital in Gangwon province of Korea. A total of seventy nonduplicate A. baumannii isolates were collected from the Wonju Christian Hospital in Korea from March to April in 2011. All of the MDR A. baumannii isolates were encoded by $bla_{OXA-23-like}$ gene and all isolates with the $bla_{OXA-23-like}$ gene had the upstream element ISAba1 to promote increased gene expression and subsequent resistance to carbapenem. 16S rRNA methylase gene (armA) was detected in 44 clinical isolates which were resistant to amikacin, and phosphotransferase genes encoding aac(3)-Ia and aac(6')-Ib were the most prevalent. A combination of 16S rRNA methylase and aminoglycoside-modifying enzyme genes (armA, aac(3)-Ia, aac(6')-Ib, and aph(3')-Ia) were found in 31 isolates. The sequencing results for the quinolone resistance-determining region (QRDR) of gyrA and parC revealed the presence of Ser (TCA) 83 Leu (TTA) and Ser (TCG) 80 Leu (TTG) substitutions in the respective enzymes for all MDR. Molecular typing for MDR A. baumannii could be helpful in confirming the identification of a common source or cross-contamination. This is an important step in enabling epidemiological tracing of these strains.