• Archives of Pharmacal Research
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• 제21권3호
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• pp.310-314
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• 1998

DW-116 is a new fluoroquinolone antimicrobial agent with a broad spectrum. In order to elucidate the resistance mechanism to DW-116 in Acinetobacter spp. bacteria, total chromosomal DNA was isolated from 10 strains of Acinetobacter spp. resistant to DW-116. Quinolone resistance determinant region (QRDR) of DNA gyrase gene was amplified by PCR. The 345 bp nucleotide fragment yielded was inserted into pKF 3 which was used as the vector. Comparisons of the DNA sequences of 8 strains with that of the wild type strain revealed a Ser-83 to Leu mutation in mutants and all ten strains contained one silent mutation$(T{\rightarrow}G)$in QRDR. From Acinetobacter MB4-8 strain, DNA gyrase was isolated and purified, through novobiocin-sepharose, heparin-sepharose affinity column chromatography. The enzyme was composed of two subunits and the molecular mass of subunits A and B were 75.6 and 51.9 kDa, respectively. The supercoiling activity of the reconstituted DNA gyrase composed of subunit A from Acinetobacter MB4-8 and subunit B from E. coli was not inhibited by $128{\mu}\textrm{g}$ml of ciprofloxacin. It might be said that one of the resistance mechanisms to DW-116 in Acinetohacter MB4-8 was subunit A alteration of DNA gyrase.

• Archives of Pharmacal Research
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• 제18권3호
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• pp.173-178
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• 1995

Ciprofloxacin resistance mechanisms were studied by investigating the inhibitory effect of ciprofloxacin on the gyrase-mediated DNA supercoiling and the intracellular accumulation of ciprofloxacin in clinical isolates of Pseudomonas aeruginosa. A higher amount of ciprofloxacin was required to inhibit the gyrases purified from the ciprofloxacin-resistant strains than that from the sensitive strain. Reconstitution of heterologous gyrase subunits from different strains revealed alterations in the A and/or the B subunits of gyrase in these strains. In addition, the resistant strains accumulated approximately a half amount of ciprofloxacin inside the cells, compared to the sensitive strain. However, when the active efflux was blocked by carbonyl cyanide m-chlorophenyl hydrazone treatment, intracellular concentration of ciprofloxacin was elevated about 4-7 fold in these strains, while the sensitive strain was not significantly affected by this treatment, indicating that the ciprofloxacin-resistant strains developed a drug efflux system. Interestingly, these resistant strains expressed an envelope protein of approximately 51 kD. These studies suggest that alterations in the gyrase as well as the active drug-efflux system conferred dual ciprofloxacin resistance mechanisms to these clinical isolates of P. aeruginosa.

• Molecules and Cells
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• 제20권3호
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• pp.392-400
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• 2005

The genes encoding the DNA gyrase A (GyrA) and B subunits (GyrB) of Methylovorus sp. strain SS1 were cloned and sequenced. gyrA and gyrB coded for proteins of 846 and 799 amino acids with calculated molecular weights of 94,328 and 88,714, respectively, and complemented Escherichia coli gyrA and gyrB temperature sensitive (ts) mutants. To analyze the role of type II topoisomerases in the intrinsic quinolone resistance of methylotrophic bacteria, the sequences of the quinolone resistance-determining regions (QRDRs) in the A subunit of DNA gyrase and the C subunit (ParC) of topoisomerase IV (Topo IV) of Methylovorus sp. strain SS1, Methylobacterium extorquens AM1 NCIB 9133, Methylobacillus sp, strain SK1 DSM 8269, and Methylophilus methylotrophus NCIB 10515 were determined. The deduced amino acid sequences of the QRDRs of the ParCs in the four methylotrophic bacteria were identical to that of E. coli ParC. The sequences of the QRDR in GyrA were also identical to those in E. coli GyrA except for the amino acids at positions 83, 87, or 95. The $Ser^{83}$ to Thr substitution in Methylovorus sp. strain SS1, and the $Ser^{83}$ to Leu and $Asp^{87}$ to Asn substitutions in the three other methylotrophs, agreed well with the minimal inhibitory concentrations of quinolones in the four bacteria, suggesting that these residues play a role in the intrinsic susceptibility of methylotrophic bacteria to quinolones.

• 대한의생명과학회지
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• 제13권2호
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• pp.141-148
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• 2007

We determined the sequences of the quinolone resistance-determining region (QRDR) of gyrA and gyrB for 21 clinical strains of Enterobacteriaceae resistant to ciprofloxacin, norfloxacin and levofloxacin. The clinical strains were isolated from the specimens of three general hospitals in Busan. In the present study, we found mutations in type II topoisomerase (DNA gyrase) genes for all strains. We confirmed that some genera of Enterobacteriaceae of clinical specimen exhibited decreased sensitivity to fluroquinolone due to changes in Ser-83$\rightarrow$Leu and Asp-87$\rightarrow$Asn types on gyrA and alterations in Glu-465$\rightarrow$Arg and Ser-492$\rightarrow$Asn type on gyrB. All the twenty-one strains had a missense mutation in gyrA (codon 83 and 87). Three of them had an additional mutation in gyrB (codon 465 or 492), but one of them had an additional mutation in gyrB (codon 426, 427, 491, 495 and 496). The strains which had two mutations in type II topoisomerase genes (gyrA and gyrB) were significantly more resistant to fluoroquinolones than those with a single mutation in gyrA (mean MICs of ciprofloxacin: $\geq8\mu$g/ml, mean MICs of levofloxacin: $\geq16\mu$g/ml). Interestingly, the examination of silent nucleotide changes n the gyrA and gyrB genes revealed six different patterns of DNA polymorphism, respectively. Fifteen strains of the twenty-one strains bearing the gyrase A mutation shared the same polymorphism and eleven strains of the twenty-one strains bearing the gyrase B mutation shared the same polymorphism.

• The Plant Pathology Journal
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• 제32권2호
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• pp.102-111
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• 2016

Olive culture is very important in the Mediterranean Basin. A severe outbreak of Olive Quick Decline Syndrome (OQDS) caused by Xylella fastidiosa infection was first noticed in 2013 on olive trees in the southern part of Apulia region (Lecce province, southern Italy). Studies were carried out for detection and diversity evaluation of the Apulian strain of Xylella fastidiosa. The presence of the pathogen in olive samples was detected by PCR amplifying the 16S rDNA, gyrase B subunit (gyrB) and HL hypothetical protein genes and single nucleotide polymorphisms (SNPs) assessment was performed to genotype X. fastidiosa. Twelve SNPs were recorded over gyrB and six SNPs were found for HL gene. Less variations were detected on 16S rDNA gene. Only gyrB and HL provided sufficient information for dividing the Apulian X. fastidiosa olive strains into subspecies. Using HL nucleotide sequences was possible to separate X. fastidiosa into subspecies pauca and fastidiosa. Whereas, nucleotide variation present on gyrB gene allowed separation of X. fastidiosa subsp. pauca from the other subspecies multiplex and fastidiosa. The X. fastidiosa strain from Apulia region was included into the subspecies pauca based on three genes phylogenetic analyses.

• 대한의생명과학회지
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• 제10권4호
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• pp.507-514
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• 2004

The Pseudomonas aeruginosa isolated from the clinical specimens has a mutation on the QRDR (quinolone resistance determining region). There were obvious mutations in both gyrA and parC gene which are major targets of quinolone. Simultaneous mutations were found two sites or more on these genes in all of ten strains. GyrB or parE gene had only silent mutation without converted amino acids. We confirmed that P. aeruginosa from clinical specimens exhibited decreased sensitivity to fluroquiolone due to changed Thr-83→lle and Asp-87→Asn types on gyrA and altered Ser-87→Leu type on parC. This is the first finding that a new Met-93→Thr type on parC as well as mutations on gyrB or parE genes differed from existing patterns. This study showed more mutations of gyrA rather than parC, suggesting that change of Type Ⅳ topoisomerase is more serious than that of type Ⅱ (DNA gyrase).

• 대한임상검사과학회지
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• 제48권2호
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• pp.74-81
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• 2016

Fluoroquinolone 계 항생제의 광범위한 사용으로 인해 이 약제에 대한 내성 Ureaplasma 종의 분리 비율이 높아지고 있다. Fluoroquinolone 계 항생제 내성은 주로 DNA gyrase와 topoisomerase IV 유전자의 돌연변이로 인해 발생하는 것으로 알려져 있다. DNA gyrase는 A와 B 2개의 소단위로 이루어져 있으며, gyrA와 gyrB 유전자에 의해 암호화되어 있고, Topoisomerase IV는 parC와 parE 유전자에 의해 암호화되어 있다. 본 연구가 진행된 서울의 1개 3차 병원에서 2012년부터 2013년까지 1년동안 Ureaplasma 종의 fluoroquinolone 계 항생제인 OFL과 CIP의 항생제검사 감수성 결과를 분석한 결과 내성과 중등도를 합산할 경우 66.08%, 92.69%로 매우 높은 내성 비율을 보였다. 이에 Ureaplasma 종을 OFL과 CIP에 대한 감수성을 기준으로 4개 그룹으로 분류하여 gyrA, gyrB, parC, parE 유전자의 돌연변이 여부를 검사하여 항생제 내성과의 관련성을 밝히고자 하였다. 그 중 parC 유전자의 돌연변이 빈도가 높아 topoisomerase IV의 돌연변이가 fluoroquinolone 계 약제에 대한 내성과 밀접한 관련이 있음을 확인할 수 있었다. 본 연구를 통해 GyrB의 Asn481Ser, ParC의 Phe149Leu, Asp150Met, Asp151Ile, Ser152Val, ParE의 Pro446Ser, Arg448Lys을 추가로 발견할 수 있었다. 최근 fluoroquinolone 계 항생제의 사용이 증가하고 있기 때문에 추후 Ureaplasma 종의 fluoroquinolone 계 항생제 내성에 대한 지속적인 모니터링이 필수적일 것으로 사료되며, 이와 관련한 유전자의 돌연변이 양상과의 상관관계를 분석하여 기존 배양검사의 단점을 보완할 수 있는 분자 진단학적 검사법의 추가적인 분석이 필요할 것으로 사료된다.

• 한국미생물·생명공학회지
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• 제44권3호
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• pp.324-332
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• 2016

왕겨를 탄소원으로 사용하여 증균 배양을 실시함으로써 국내 사찰에서 제조된 된장으로부터 xylan 분해능 우수한 균주를 분리하였다. 분리균 YB-1301은 DNA gyrase subunit B 유전자(gyrB)의 염기서열에 근거하여 Bacillus safensis로 동정되었다. B. safensis YB-1301을 밀기울 또는 여러 종류의 xylan들이 첨가된 배지에서 배양하였을 때 xylanase의 생산성이 급격하게 증가되었다. 특히 birchwood xylan이 첨가된 LB 배지에서 플라스크 배양을 하였을 때 최대 340 U/ml 이상의 xylanase 생산성을 보였다. Xylan이 첨가되지 않은 LB 배지에서는 매우 소량의 xylanase가 균의 성장과 연계되어 항시적으로 생산되지만, xylan이 첨가된 배지에서는 정지기 생육단계에서 xylanase의 생산이 크게 유도되었다. 더구나 xylanase 생합성은 가수분해되지 않은 xylan 보다 xylan의 효소적 가수분해 산물에 의해 더 빠르게 유도되었다. 또한 B. safensis YB-1301의 배양상등액에 존재하는 xylanase는 55℃와 pH 6.5−7.0의 반응조건에서 최대활성을 나타냈다.

• 미생물학회지
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• 제35권3호
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• pp.218-225
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• 1999

Aspergillus nidulans 의 DNA 로부터 Saccharomyces cerevisiae에서 스스로 복제가능하고, 형질전환율도 삽입벡터에 비해 10\sup 4\ 배정도 높여주는 절편을 분리한 바 있다. A. nidulans에서 ANRI 의 일부를 포함한 pILJ16-4.5는 삽입벡터인 pILJ16보다 170배 정도 높은 형질전환 효율을 보였으며, S.cerevisiae와 마찬가지로 plasmid 상태로 회수가능했다. A.nidulans 페소내에서 2-3 copy 정도로 염색체와는 별개로 존재하는 것으로 나타났으며, 재형질전환능력도 있었다. ANRI은 미토콘드리아의 DNA에서 유래한 절편으로 밝고, ARS 공통절편과 유사한 절편도 11곳에 존재한다. $\Phi$X174와 SV40 DNA에서 gyrase 가 결합하는 부위의 공통편인 YRTGNYNNY도 6곳에 존재하며, ColE1에서 gyrase가 결합하는 b site와 일치하는 절편도 하나 포함하고 있으며, ABF1 결합 부위이 공통절편과 유사한 절푠$TCN_7ACG$ 도 하나 포함하고 있다. 이를 토대로 ANRI은 A.nidulans의 형질전환 후 회수가 가능한 replicating vector 개발에 이용할 수 있다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVII)
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• pp.307-311
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• 2005

해양에서 유용물질을 생산하는 미생물을 분리하기 위하여 경상도 지역의 해안에서 시료를 채취하여 총 371 균주의 해양미생물을 얻었으며 전분, CMC 및 단백질 등과 같은 기질에 대한 분해활성을 측정하여 CMC에 대한 우수한 분해 능력을 가진 30균주를 선별하였다. 이들 30 균주를 배양하여 CMCase의 생산능력이 우수한 12 균주를 선발하였다. 이들 해양미생물을 배양하고 섬유소 분해효소를 생산한다고 알려진 B. amyloliquefaciens DL-3와 CMCase의 활성을 비교하였다. 이 중 다대포에서 분리하여 명명한 A-53 균주가 가장 높은 CMCase 활성을 나타를 생산한다고 알려진 B. amyloliquefaciens DL-3와 CMCase의 활성을 비교하였다. 이 중 다대포에서 분리하여 명명한 A-53 균주가 가장 높은 CMCase 활성을 나타내었다. A-53 균주를 16S rDNA partial sequencing 및 gyrase A partial sequencing하여 동정한 결과, Bacillus subtilis subsp. subtilis로 확인되었으며 B. subtilis subsp. subtilis A-53으로 명명하였다.