• Journal of Microbiology and Biotechnology
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• 제16권3호
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• pp.450-456
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• 2006

Pseudomonas fluorescens MC07 is a growth-promoting rhizobacterium that suppresses mycelial growth in fungi such as Rhizoctonia solani, Pythium ultimum, Fusarium oxysporum, and Phytophthora capsici. To determine the role of the bacterium's antifungal activity in disease suppression, we screened 2,500 colonies generated by Tn5lacZ insertions, and isolated a mutant 157 that had lost antifungal activity. The EcoRI fragment carrying Tn5lacZ was cloned into pBluescript II SK(+) and used as a probe to isolate wild-type clones from a genomic library of the parent strain, MC07. Two overlapping cosmid clones, pEH4 and pEH5, that had hybridized with the mutant clone were isolated. pEH4 conferred antifungal activity to the heterologous host P.fluorescens strain 1855.344, whereas pEH5 did not. Through transposon mutagenesis of pEH4 and complementation analyses, we delineated the 14.7-kb DNA region that is responsible for the biosynthesis of an antifungal compound. DNA sequence analysis of the region identified 11 possible open reading frames (ORF), ORF1 through ORF11. A BLAST search of each putative protein implied that the proteins may be involved in an antifungal activity similar to polyketides.

• Journal of Microbiology and Biotechnology
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• 제31권8호
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• pp.1060-1068
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• 2021

Community-associated Methicillin-Resistant Staphylococcus aureus (CA-MRSA) is notorious as a leading cause of soft tissue infections. Despite several studies on the Agr regulator, the mechanisms of action of Agr on the virulence factors in different strains are still unknown. To reveal the role of Agr in different CA-MRSA, we investigated the LACΔagr mutant and the MW2Δagr mutant by comparing LAC (USA300), MW2 (USA400), and Δagr mutants. The changes of Δagr mutants in sensitivity to oxacillin and several virulence factors such as biofilm formation, pigmentation, motility, and membrane properties were monitored. LACΔagr and MW2Δagr mutants showed different oxacillin sensitivity and biofilm formation compared to the LAC and MW2 strains. Regardless of the strain, the motility was reduced in Δagr mutants. And there was an increase in the long chain fatty acid in phospholipid fatty acid composition of Δagr mutants. Other properties such as biofilm formation, pigmentation, motility, and membrane properties were different in both Δagr mutants. The Agr regulator may have a common role like the control of motility and straindependent roles such as antibiotic resistance, biofilm formation, change of membrane, and pigment production. It does not seem easy to control all MRSA by targeting the Agr regulator only as it showed strain-dependent behaviors.

• Journal of Microbiology and Biotechnology
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• 제29권8호
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• pp.1299-1309
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• 2019

As an opportunistic bacterial pathogen, Pseudomonas aeruginosa PAO1 contains two phenazine-producing gene operons, phzA1B1C1D1E1F1G1 (phz1) and phzA2B2C2D2E2F2G2 (phz2), each of which is independently capable of encoding all enzymes for biosynthesizing phenazines, including phenazine-1-carboxylic acid and its derivatives. Other previous study reported that the RpoS-deficient mutant SS24 overproduced pyocyanin, a derivative of phenazine-1-carboxylic acid. However, it is not known how RpoS mediates the expression of two phz operons and regulates pyocyanin biosynthesis in detail. In this study, with deletion of the rpoS gene in the $PA{\Delta}phz1$ mutant and the $PA{\Delta}phz2$ mutant respectively, we demonstrated that RpoS exerted opposite regulatory roles on the expression of the phz1and phz2 operons. We also confirmed that the phz1 operon played a critical role and especially biosynthesized much more phenazines than the phz2 operon when the rpoS gene was knocked out in P. aeruginosa. By constructing the translational reporter fusion vector lasR'-'lacZ and the chromosomal fusion mutant $PA{\Delta}lasR::lacZ$, we verified that RpoS deficiency caused increased expression of lasR, a transcription regulator gene in a first quorum sensing system (las) that activates overexpression of the phz1 operon, suggesting that in the absence of RpoS, LasR might act as an intermediate in overproduction of phenazine biosynthesis mediated by the phz1 operon in P. aeruginosa.

• Journal of Microbiology and Biotechnology
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• 제19권11호
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• pp.1456-1463
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• 2009

Vibrio parahaemolyticus possessing urease-positive property is relatively rare, but such strains consistently exhibit the TDH-related hemolysin (TRH) gene. In this study, we examined the effects of incubation temperature on urease activity expression, using the TH3996 and AQ4673 strains where the enzyme activity is known to be temperature-dependent and -independent, respectively. In the TH3996 strain, $\beta$-galactosidase activity was 4.4-fold lower after $30^{\circ}C$ cultivation than after $37^{\circ}C$ in a ureR-lacZ fusion strain, but temperature dependency was not found in ureD- or nikA-lacZ fusion strains. However, ureR-, ureD-, and nikA-lacZ fusions of the AQ4673 strain was not influenced by incubation temperature. We compared the promoter sequences of ureR between the above two strains. Intriguingly, we detected mismatches of two nucleotides between the two strains located at positions -66 and -108 upstream of the methionine initiation codon for UreR. Additionally, urease activity was not affected by culture temperature at either $30^{\circ}C$ or $37^{\circ}C$ by allelic introduction of the AQ4673 ureR gene into the TH3996 ureR deletion mutant. Taken together, our study demonstrates that the transcriptional factor UreR is involved in the temperature dependency of urease activity, and two nucleotides within the ureR promoter region are of particular importance for the urease activity dependency of V. parahaemolyticus.

• The Plant Pathology Journal
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• 제35권4호
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• pp.351-361
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• 2019

In our previous study, pyrrolnitrin produced in Pseudomonas chlororaphis G05 plays more critical role in suppression of mycelial growth of some fungal pathogens that cause plant diseases in agriculture. Although some regulators for pyrrolnitrin biosynthesis were identified, the pyrrolnitrin regulation pathway was not fully constructed. During our screening novel regulator candidates, we obtained a white conjugant G05W02 while transposon mutagenesis was carried out between a fusion mutant $G05{\Delta}phz{\Delta}prn::lacZ$ and E. coli S17-1 (pUT/mini-Tn5Kan). By cloning and sequencing of the transposon-flanking DNA fragment, we found that a vfr gene in the conjugant G05W02 was disrupted with mini-Tn5Kan. In one other previous study on P. fluorescens, however, it was reported that the deletion of the vfr caused increased production of pyrrolnitrin and other antifungal metabolites. To confirm its regulatory function, we constructed the vfr-knockout mutant $G05{\Delta}vfr$ and $G05{\Delta}phz{\Delta}prn::lacZ{\Delta}vfr$. By quantifying ${\beta}-galactosidase$ activities, we found that deletion of the vfr decreased the prn operon expression dramatically. Meanwhile, by quantifying pyrrolnitrin production in the mutant $G05{\Delta}vfr$, we found that deficiency of the Vfr caused decreased pyrrolnitrin production. However, production of phenazine-1-carboxylic acid was same to that in the wild-type strain G05. Taken together, Vfr is required for pyrrolnitrin but not for phenazine-1-carboxylic acid biosynthesis in P. chlororaphis G05.

• Journal of Microbiology
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• 제41권2호
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• pp.109-114
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• 2003

A promoter that is inducible by paraquat and menadione, the superoxide generators, independently of soxRS has been found in front of the sufABCDSE operon in Escherichia coli. Based on the observation that SufA is a holomog of IscA that functions in the assembly of iron sulfur cluster and the sufA promoter (sufAp) contains a putative Fur-binding consensus, we investigated whether this gene is regulated by Fur, a ferric uptake regulator, When examined in several sufAp-lacZ chromosomal fusion strains, sufAp was induced by EDTA, an iron chelator and a well-known Fur-inducer, The basal level of sufA expression increased dramatically in fur mutant, suggesting repression of sufAp by Fur. The derepression in fur mutant and EDTA-induction of sufA expression required nucleotides up to -61, where a putative Fur box is located. Purified Fur protein bound to the DNA fragment containing the putative Fur box between -35 and -10 promoter elements. The regulation by Fur and menadione induction of sufAp acted independently. The rpoS mutation increased sufA induction by menadione, suggesting that the stationary sigma factor RpoS acts negatively on sufA induction.

• 대한의생명과학회지
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• 제5권1호
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• pp.17-26
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• 1999

Mar (multiple-antibiotic-resistance) 대장균의 marRAB 돌연변이가 그람음성 세균들 사이로 전파 될수 있는지를 알아보기 위해서, 염색체 위의 marRAB 돌연변이를 $\lambda$placMu9 (Km$^{r}$)를 이용하여 대장균의 다른 균주내의 pUC19 (Lac$^{+}$, Ap$^{r}$)의 클로닝 사이트, S. typhimurium 및 P. aeruginosa의 염색체 위로 각각 전달시킨 후, Mar phenotype, Km$^{r}$, Lac$^{-}$, 혹은 Ap$^{r}$ 균주를 선택한 다음, salicylate (SAL)의 유도 (induction) 유무에 따른 항생제 내성검사를 실시하였다. pUC19의 보유 유무에 관계없이 대조군인 야생형 대장균 JM109, NM522에 비해서, pUC19::marRAB 돌연변이를 가진 JM109 혹은 NM522의 대부분 균주들은 항생제 내성 수준들이 훨씬 더 높았으며, Mar표현형의 유도도 SAL에 의해서 훨씬 더 높게 나타났다. 하지만 일부 소수의 항생제에 대해서는 예외적으로 내성 수준이 더 낮게 나타났는데, 야생형 JM109 균주에서는 chloramphenicol (Cm)과 tetracycline (Tc)이 , pUC19::marRAB 돌연변이를 가진 NM522 균주 에서는 Tc와 ciprofloxacin (Cp)에 대한 내성 수준이 각각 더 낮게 나타났다. S. typhimurium 및 P. aeruginosa에 대한 같은 실험의 결과도 야생형 대장균의 경우와 거의 대부분 일치하였다. 형질전환 (transformation)과 형질도입 (transduction)의 방법을 주로 사용한 이 실험의 결과는, marRAB 돌연변이가 그람음성 세균들 사이로 전파될 수 있음을 간접적으로 암시한다.

• 미생물학회지
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• 제30권5호
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• pp.371-376
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• 1992

플라스미드 pKM101 과 이의 돌연변이체 pSL4 는 UV 및 MMS 에 대해 높은 치사 저항성과 돌연변이률을 나타낸다. pKM101 과 pSLA 의 mucB 유전자의 일부분과 mucA 유전자를 lacZ fusion 벡터인 pMC874 에 subcloning 시켜 플라스미드 pBH31 과 pBH30 을 선별하였고 이 플라스미드들을 $recA^{+}lexA^{-}$, $recA^{-}와lexA^{+}$, 균주에 각각 도입하였다. $recA^{+}lexA^{+}$ 균주에서 pSLA 의 muc 유전자를 포함하는 pBH30 의 $\beta$-galactosidase활성은 pKM101 의 muc 유전자가 연결된 pBH31 보다 높았고 $recA^{-}$ 와 $lexA^{-}$ 돌연변이주에서는 UV 조사의 유무에 관계없이 $\beta$-galactosidase를 유도하지 못하였지만 $recA^{-}$ 균주에서는 UV 조사를 하지 않았을 때 pBH30 의 $\beta$-galactosidase가 pBH31 보다 약간 높게 유도되었다. 이러한 결과는 pKM101 과 pSLA 의 기능적 차이가 두 플라스미드의 Muc 단백질의 구조적 차이라고 생삭할 수 있지만, muc 유전자의 조절부위가 돌연변이되너 LexA repessor 가 작용하는 부위의 변화에 의한 것이라고도 생각햐ㄹ 수 있었다. 또한, umuC-lacZ 유전자를 가지는 pBH100 를 construction 하여 umu oeron 의 발현은 UV 조사에 의해 유도되며 recA 와 lexA 유전자에 의해 조절됨을 알 수 있었다.

• BMB Reports
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• 제32권4호
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• pp.405-408
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• 1999

The transactivation potential of HIV-1 Vpu was identified from the yeast two-hybrid screening process. The helix II domain of HIV-1 Vpu protein and mutant Vpu protein lacking the transmembrane domain exhibited transactivation of the LacZ and Leu2 reporter genes carrying LexA upstream activating sequences, but full-length HIV-1 Vpu and the helix I domain of HIV-1 Vpu did not. The helix II domain of HIV-1 Vpu consists of a number of acidic amino acids, and is especially rich in glutamic acid, a characteristic of many transcription factors. This result suggests that protein-protein interaction may occur through the acidic helix II domain of HIV-1 Vpu.

• Journal of Microbiology and Biotechnology
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• 제18권8호
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• pp.1439-1444
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• 2008

The recombinant E. coli $\Delta$6 mutant (galR, glpK, gldA, IdhA, lacI, tpiA) was used to produce 1,3-propanediol (PD) from glucose. The 1,3-PD production increased with feedback control of the glucose concentration using fed-batch fermentation. The maximum 1,3-PD concentration produced was 43 g/l after 60 h of fermentation. Glycerol production was minimized when controlling the glucose concentration at less than 1 g/l. The expression levels of seven enzymes related to the 1,3-PD production metabolism were compared during the cell growth phase and 1,3-PD production phase, and their expression levels all increased during 1,3-PD production, with the exception of alcohol dehydrogenase.