• Microbiology and Biotechnology Letters
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• v.17 no.3
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• pp.183-187
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• 1989

The bacteriophage Mu and transposon Tn5 containing plasmid pJB4JI-transferred transposon Tn5 to Caulobuter crescentus. When several thousand of transposon Tn5 insertion mutants were examined, we found auxotrophic and motility mutants at frequencies of 2％ and 3％, respectively. Transposition of transposon Tn5 was analyzed by the reverse field electrophoresis and Southern hybridization. The results indicated that transposon Tn5 was randomly inserted to Caulobuter crescentus chromosome but the plasmid vector, pJB4JI, was not maintained.

• Journal of Microbiology and Biotechnology
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• v.19 no.3
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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.

• Korean Journal of Microbiology
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• v.26 no.4
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• pp.305-311
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• 1988

The spectinomycin resistant strain of slow growing R. japonicum R-168 was selected to be participated in conjugation with E. coli WA803/pGS9. Tn5 was introduced from suicide vector pGS9 into R. japonicum R-168 $spr^{r}$ chromosome at the frequency of $1.0\times 10^{-5}-5.0\times 10^{-7}$ and the transconjugante were selected on the yeast extract-mannitol plate containing kanamycin ($50{\mu}$g/ml) and spectinomycin ($100{\mu}$g/ml) after 8-9 days incubation. All transconjugants we tested were found to contain Tn 5 DNA on their genome, which was confirmed by Southern hybridization experiments. R. japonicum RNa75, which had been selected through plant test, was found to be defective in symbiotic nitrogen fixing ability and the production of leghemoglobin in soybean nodules formed by the inoculation of this mutant. In addition, this mutant strain hardly developed nitrogenase activity asymbiotically in contrast with the wild type strain, indicating that some nitrogen fixing gene might be blocked in this strain and the production of leghemoglobin could be decreased by the interference in nitrogen fixing genes.

• Microbiology and Biotechnology Letters
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• v.19 no.1
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• pp.25-30
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• 1991

The crystal protein gene (cp) of Bacillus tizuringienszs subsp. liuvstaki (B.t.k.) HI173 was subcloned into HanzHI site of central region (Tn5-cp) or BglII site of IS50L region (IS50L-cp) in Tn5, and transposed into the chromosomal DNA of five strains of root-colonizing Pseudomonas. The expression of cp gene in Acwiomoncrs transconjugants was demonstrated by immunoblot analysis and bioassay against larvae of the Hyphantria cunea.

• KSBB Journal
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• v.21 no.2
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• pp.90-95
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• 2006

Escherichia coli harboring pAC-LYCO4 and pDdxs was used for lycopene production. Three wild type strains of E. coli OW1, MG1655, and W3110 were compared with DH5${\alpha}$ used before for lycopene production. Lycopene productivity of E. coli MG1655 was similar to DH5${\alpha}$ and the highest among those wild type strain. Therefore, MG1655 strain was used for random transposon and NTG mutagenesis to increase lycopene productivity. Through transposon mutation, five transposon mutants with increased lycopene productivity were obtained. It was found that genes knocked out by transposon insertion were treB in Tn1 mutant, B2436 in Tn2 mutant, and rfaH in Tn3, 4, and 5 mutants. Lycopene productivity was the highest in Tn4 mutant among the Tn mutants, which was 6-fold and 8-fold higher in lycopene concentration and content, respectively, in comparison with those obtained with wild type strain. NTG4 mutant was acquired with NTG mutation. The highest lycopene productivity of 6 mg/L and 4 mg/g DCW was obtained from the NTG4 mutant when arabinose of 0.013 mM was added for induction of dxs, rate-limiting gene of MEP pathway. The lycopene productivity of NTG4 mutant was increased 18-fold and 12-fold in lycopene concentration and content, respectively when comparing with the wild type strain.

• Journal of Microbiology and Biotechnology
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• v.5 no.2
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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.

• Microbiology and Biotechnology Letters
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• v.31 no.1
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• pp.25-31
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• 2003

Delftia acidovorans 51-A isolated from river water degrades aniline. In order to clone genes involved in aniline degradation, transposon Tn5-B20 was inserted into the strain 51-A to generate a mutant strain 10-4-2 that cannot utilize aniline as a carbon source. The mutant strain was not an auxotroph but could not degrade aniline. Southern hybridization analysis indicated that the transposon was inserted into the mutant bacterial DNA as a single copy. Flanking DNA fragment of Tn5-B2O insertion was cloned and sequenced. DNA sequence analysis revealed three ORFs encoding TdnQ, TdnT, and TdnA 1 that arc responsible for catechol formation from aniline through oxidative deamination. The analysis also confirmed that Tn5-B2O was inserted at the immediate downstream of tdnA1. The result suggests that the transposon insertion behind tdirA1 disrupted the pathway of the catechol formation from aniline, resulting in the mutant phenotype, which cannot degrade aniline. A large plasmid over 100-kb in size was detected from D. acidovorans 51-A and Southern hybridization analysis with Tn5-B2O probe showed that the transposon was inserted on the plasmid named pTDN51. Our results indicated that the tdn genes on pTDN51 of D. acidovorans 51-A are involved in aniline degradation.

• The Plant Pathology Journal
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• v.20 no.3
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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.

• Korean Journal of Microbiology
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• v.26 no.1
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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.

• Korean Journal Plant Pathology
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• v.11 no.3
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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.