• The Plant Pathology Journal
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• 제20권3호
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• pp.229-233
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• 2004

The bacterial leaf blight, which is caused by Xantho-monas oryzae pv. oryzae, is the most damaging and intractable disease of rice. To identify the genes involved in the virulence mechanism of transposon TnS complex, which possesses a linearized transposon and transposase, was successfully introduced into X. oryzae pv. oryzae by electroporation. The transposon mutants were selected and confirm the presence of transposition in X. oryzae pv. oryzae by the PCR amplification of transposon fragments and the Southern hybridization using these mutants. Furthermore, transposon insertion sites in the mutant bacterial chromosome were deter-mined by direct genomic DNA sequencing using transposon-specific primers with ABI 3100 Genetic Analyzer. Efficiency of transposition was influenced mostly by the competence status of X. oryzae pv. oryzae cells and the conditions of electroporation. These results indicated that the insertion mutagenesis strategy could be applied to define function of uncharacterized genes in X. oryzae pv. oryzae.

• Journal of Microbiology and Biotechnology
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• 제19권3호
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• pp.217-228
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• 2009

Mobile genetic segments, or transposons, are also referred to as jumping genes as they can shift from one position in the genome to another, thus inducing a chromosomal mutation. According to the target site-specificity of the transposon during a transposition event, the result is either the insertion of a gene of interest at a specific chromosomal site, or the creation of knockout mutants. The former situation includes the integration of conjugative transposons via site-specific recombination, several transposons preferring a target site of a conserved AT-rich sequence, and Tn7 being site-specifically inserted at attTn7, the downstream of the essential glmS gene. The latter situation is exploited for random mutagenesis in many prokaryotes, including IS (insertion sequence) elements, mariner, Mu, Tn3 derivatives (Tn4430 and Tn917), Tn5, modified Tn7, Tn10, Tn552, and Ty1, enabling a variety of genetic manipulations. Randomly inserted transposons have been previously employed for a variety of applications such as genetic footprinting, gene transcriptional and translational fusion, signature-tagged mutagenesis (STM), DNA or cDNA sequencing, transposon site hybridization (TraSH), and scanning linker mutagenesis (SLM). Therefore, transposon-mediated genetic engineering is a valuable discipline for the study of bacterial physiology and pathogenesis in living hosts.

• 한국식물병리학회지
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• 제11권3호
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• pp.265-270
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• 1995

Transposon mutation of Xanthomonas campestris pv. vesicatoria (Xcv) was induced by using transposon omegon ($\Omega$)-Km (Tn $\Omega$Km), which was confirmed by resistance to kanamycin (KMr), and nonpathogenic mutants were selected through the inoculation test on pepper plants. The mutagenesis frequency was about 6$\times$10-8, and 53 out of 2,000 Kmr bacterial colonies tested were nonpathogenic to the pepper cultivar Cheung-Hong. Optimum conditions for the Tn $\Omega$Km mutagenesis of Xcv were Luria Bertani (LB) broth medium for culture of Xcv, yeast extract-dextrose-CaCO3 (YDC) agar medium for selection of Tn $\Omega$Km-mediated mutants, and over 1 to 2 in the ratio of the donor (Escherichia coli S17-1 with the plasmid pJFF350 $\Omega$Km) and the recipient (Xcv) in the culture for the mutagenesis. One of the 4 nonpathogenic mutants (WNP1, WNP3, WNP4 and WNP5), which had been reconfirmed through the inoculation on pepper cv. Dabokgun, showed no differences in the production of exoenzymes such as protease and polygalacturonase and extracellular polysaccharides in vitro and the bacterial growth rate from those of the wild type of Xcv.

• Journal of Microbiology and Biotechnology
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• 제5권2호
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• pp.110-113
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• 1995

Transposon Tn5 was used to induce random insertional mutations in Bradyrhizobium japonicum, a soybean endosymbiont. By genomic Southern blot analysis, transposition events were found to have occurred randomly throughout the B. japonicum genome. After screening 3, 626 mutants by auxotrophy test, a histidine auxotroph was isolated. Upon plant infection test, the His mutant showed a 3~4 day delay in nodule formation.

• 미생물학회지
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• 제26권1호
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• pp.20-26
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• 1988

Three $NIf^{-}$ -mutants were isolated from Enterbacter agglomerans 339 through the transposon umtagenesis using a RP4-mobilising system for its nif-gene characterization. All mutants hadn't acetylene-reduction ability. Then we confirmed that Tn5 was inserted into all conserved nif-plasmids through the Southern Hybridization.

• 식물병연구
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• 제30권3호
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• pp.268-277
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• 2024

Erwinia pyrifoliae is a gram-negative bacterial pathogen that commonly causes black shoot blight in pear and apple tree. Although the pathogenicity of this bacterial species is very similar to E. amylovora, there is no specific explanation of its pathogenic genes and mechanisms. In this study, our investigation into E. pyrifoliae pathogenicity involved generating seven YKB12327 mutant strains using Tn5 transposon mutagenesis. Observations revealed weakened growth rate and loss of pathogenicity in these mutants. Whole-genome sequencing and alignment analysis identified transposon insertions within the coding sequences of five strains and in the intergenic region of two strains. Annotation analysis elucidated genes directly or indirectly associated with pathogenicity. Notably, mutant strain MT16 displayed a transposon insertion mutation in the cyclic-di-GMP phosphodiesterase (pdeF) gene, a key player in bacterial signaling, governing microbial behavior and adaptation to environmental changes. Our findings provide insights into the genetic regulation of E. pyrifoliae pathogenicity, suggesting potential avenues for further research aimed at understanding and controlling this bacterial pathogen by targeting pdeF to mitigate apple black shoot blight disease.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2005년도 International Meeting of the Microbiological Society of Korea
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• pp.129-131
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• 2005

• KSBB Journal
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• 제21권2호
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• pp.90-95
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• 2006

라이코펜 생산성을 향상시키기 위하여 몇 가지 대장균 균주들을 대상으로 생산성 시험을 한 결과 MG1655 균주가 가장 우수하였다. 대장균 MG1655 균주의 염색체 DNA 내에 존재하는 특정 유전자의 기능이 상실되었을 때 라이코펜 생산성이 증가되는 변이체를 선발하기 위해 transposon 돌연변이를 수행하였으며, 5종의 우수 transposon 변이체를 얻었다. 특히, Tn4 변이체는 야생형 MG1655에 비해서 라이코펜 생산량은 6배, 균체 내 함량은 7배로 가장 높았다. Transposon 삽입에 의해 결손된 유전자를 파악하기 위해 inverse PCR을 통해 염기서열을 확인하였으며, 결손된 유전자는 rfaH, treB, B2436으로 규명되었다. 또한 특정 유전자 기능의 상실뿐만 아니라 기능의 강화나 변화 등에 의한 라이코펜 생산성이 증가된 변이체를 선발하기 위해 NTG 돌연변이를 수행하였으며, 4개의 우수한 NTG 변이체를 얻었다. 이들 중에 NTG4 변이체가 가장 높은 라이코펜 생산량을 보였다. 특히, NTG4 변이체는 발현유도물질인 arabinose의 소량(0.013mM) 첨가 시에 라이코펜 농도, 6 mg/L와 균체 내 함량 4 mg/g DCW로 최대의 라이코펜 생산성을 보였고, 이것은 야생형 MG1655에 비해서 각각 18배와 12배 높은 생산성이다.

• Journal of Microbiology
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• 제42권2호
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• pp.139-142
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• 2004

To identify genes involved in the decolorization of brilliant green, we isolated random mutants generated by transposon insertion in brilliant green-decolorizing bacterium, Citrobacter sp. The resulting mutant bank yielded 19 mutants with a complete defect in terms of the brilliant green color removing ability. Southern hybridization with a Tn5 fragment as a probe showed a single hybridized band in 7 mutants and these mutants appeared to have insertions at different sites of the chromosome. Tn5-inserted genes were isolated and the DNA sequence flanking Tn5 was determined. By comparing these with a sequence database, putative protein products encoded by bg genes were identified as follows: bg 3 as a LysR-type regulatory protein; bg 11 as a MalG protein in the maltose transport system; bg 14 as an oxidoreductase; and bg 17 as an ABC transporter. The sequences deduced from the three bg genes, bg 2, bg 7 and bg 16, showed no significant similarity to any protein with a known function, suggesting that these three bg genes may encode unidentified proteins responsible for the decolorization of brilliant green.

• 농업과학연구
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• 제50권2호
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• pp.283-289
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• 2023

Erwinia amylovora , which causes fire blight disease on apple and pear trees, is one of the most important phytopathogens because of its devastating impact. Currently, the only way to effectively control fire blight disease is through the use of antibiotics such as streptomycin, kasugamycin, or oxytetracycline. However, problems with the occurrence of resistant strains due to the overuse of antibiotics are constantly being raised. It is therefore necessary to develop novel disease control methods through an advanced understanding of the pathogenesis mechanism of E. amylovora . To better understand the pathogenesis of E. amylovora , we investigated unknown virulence factors by random mutagenesis and screening. Random mutants were generated by Tn5 transposon insertion, and the pathogenicity of the mutants was assessed by inoculation of the mutants on apple fruitlets. A total of 17 avirulent mutants were found through screening of 960 random mutants. Among them, 14 mutants were already reported as non-pathogenic strains, while three mutants, TS3128_M2899 (ΔSUFU ), TS3128_M2939 (ΔwcaG ), and TS3128_M3747 (ΔrecB ), were not reported. Further study of the association between E. amylovora pathogenicity and these 3 novel genes may provide new insight into the development of control methods for fire blight disease.