• 한국미생물·생명공학회지
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• 제19권4호
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• pp.372-379
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• 1991

E.coli ATCC 21990의 aminoglycoside-3'-phosphotransferase(APH(3'))의 신속하고 간편한 정량적 방법을 TLC densitometry를 이용하여 확립하였다. APH(3') 반응생산물인 3' 위치에 인산화된 kanamycin B(3'PKMB)는 silica gel plate에서 cnloroform-methanol-method-ammonia water (3:4:3) 전개용매로 반응물에서 분리되었고, 3'PKMB의 양은 ninhydrin으로 발색 후 densitometry로 측정하였다. APH(3')의 densitometric TLC assay는 좋은 정량적 결과와 재현성을 보였고, 3'-PKMB에 대한 감도는 1.56nmol이었으며 많은 시료의 분석이 한번의 실시로 가능하였다. 이 방법은 aminoglycoside 항생제의 불활화 효소 분석에 응용이 가능하리라 여겨진다.

• 한국미생물·생명공학회지
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• 제19권5호
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• pp.491-496
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• 1991

Aminoglycoside-3'-phosphotransferase(APH(3'))를 생산하는 균주인 E.coli ATCC 21990을 산업적으로 이용하기 위해서 자외선 조사 및 NTG를 처리하고 고농도의 kanamycin B에 내성을 갖는 변이주인 E.coli M1과 M2를 선별하였다. E.coli M1은 단위 균체당 효소 생산성은 높으나 생육속도가 낮아 실용적이질 못했고 주 질소원인 yeast extract를 사용했을때 E.coli M1보다 E.coli M2가 생육속도가 훨씬 빨랐으며 약 2배의 APH(3')을 얻을 수 있었고 산소 가스를 사용하였을 경우는 약 2.5배의 APH(3')을 얻었다.

• Journal of Microbiology and Biotechnology
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• 제23권11호
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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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• 제29권3호
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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.

• 한국동물위생학회지
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• 제35권1호
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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 and Biotechnology
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• 제31권2호
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• pp.250-258
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• 2021

Among various species of marine bacteria, those belonging to the genus Halomonas have several promising applications and have been studied well. However, not much information has been available on their antibiotic resistance. In our efforts to learn about the antibiotic resistance of strain Halomonas socia CKY01, which showed production of various hydrolases and growth promotion by osmolytes in previous study, we found that it exhibited resistance to multiple antibiotics including kanamycin, ampicillin, oxacillin, carbenicillin, gentamicin, apramycin, tetracycline, and spectinomycin. However, the H. socia CKY01 resistance pattern to kanamycin, gentamicin, apramycin, tetracycline, and spectinomycin differed in the presence of 10% NaCl and 1% NaCl in the culture medium. To determine the mechanism underlying this NaCl concentration-dependent antibiotic resistance, we compared four aminoglycoside resistance genes under different salt conditions while also performing time-dependent reverse transcription PCR. We found that the aph2 gene encoding aminoglycoside phosphotransferase showed increased expression under the 10% rather than 1% NaCl conditions. When these genes were overexpressed in an Escherichia coli strain, pETDuet-1::aph2 showed a smaller inhibition zone in the presence of kanamycin, gentamicin, and apramycin than the respective control, suggesting aph2 was involved in aminoglycoside resistance. Our results demonstrated a more direct link between NaCl and aminoglycoside resistance exhibited by the H. socia CKY01 strain.

• Bulletin of the Korean Chemical Society
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• 제15권7호
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• pp.568-571
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• 1994

Streptmyces fradiae NRRL B1195 and Streptomyces kanamyceticus IFO 13414 are highly resistant to the antibiotics they produce. The ribosomes of these organisms are found to be susceptible to the antibiotics, but the cell free extract of S fradiae is found to contain a phosphotransferase and an acetyltransferase which inactivate kanamycin and neomycin, and that of S. kanamyceticus an acetyltransferse which inactivates kanamycin and neomycin. The resistance of these organisms against streptomycin is found to be due to the resistant ribosomes; actually streptomycin activates their ribosomal systems for the synthesis of polyphenylalanine.

• Journal of Microbiology
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• 제44권6호
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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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• 제6권3호
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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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• 제5권2호
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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%.