• Journal of Microbiology
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• v.42 no.2
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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.

• Journal of Life Science
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• v.14 no.1
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• pp.21-25
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• 2004

To identify genes involved in the decolorization of both crystal violet and malachite green, we isolated random mutants generated by transposon insertion in triphenylmethane-decolorizing bacterium, Citrobacter sp. The resulting mutant bank yielded 14 mutants with complete defect in color removal capability of both crystal violet and malachite green. Southern hybridization with a Tn5 fragment as a probe showed a single hybridized band in 5 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. From comparison with a sequence database, putative protein products encoded by cmg genes were identified as follows. cmg 2 is MaIC protein in maltose transport system; cmg 6 is transcriptional regulator (LysR-type): cmg 12 is a putative oxidoreductase. The sequences deduced from two cmg genes, cmg 8 and cmg 11, showed no significant similarity to any protein with a known function. Therefore, these results indicate that these two cmg genes encode unidentified proteins responsible for decolorization of both crystal violet and malachite green.

• KSBB Journal
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• v.11 no.1
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• pp.115-123
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• 1996

Two kinds of mutants were isolated to clone a cluster of genes essential for zooglan biosynthesis. Zoogloea ramigera 115 strains produce capsular polysaccharide. To achieve conjugation in strain 115 and to facilitate recovery of product, a capsule non-forming strain was isolated via successive centrifugation and screening. The other kind of mutants devoid of or producing altered exopolysaccharides were obtained using classical transposon(Tn5) technique and screened for altered colony morphology and celluflour binding properties. Complementation of these mutants was achieved with Z. ramigera 115 slime gene library constructed in a broad host range cosmid vector and helper plasmid by triparental conjugation.

• Microbiology and Biotechnology Letters
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• v.26 no.5
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• pp.393-399
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• 1998

Enterobacter sp.B54 which shows antagonistic activity to Phytophthora capsici on potato dextrose agar was selected among 112 strains isolated from Korean soil. After Tn5 lac-induced mutants were obtained through Pl :: Tn5 lac mutagenesis, 2 mutants for loss of antibiosis and 1 mutant for increased antibiosis were screened by using in vitro fungal inhibition assay. When the 3 mutants affected in antibiosis were analyzed by southern hybridization with pRZ102 (ColEl :: Tn5) as a probe, its results suggest that Tn5 lac was randomly inserted into different chromosomal sites in these mutants.

• Journal of Microbiology and Biotechnology
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• v.4 no.3
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• pp.165-170
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• 1994

An efficient and convenient method of introducing transposable elements into acetic acid bacteria was developed by the method of conjugal transfer. The ampicillin-resistant strain, Acetobacter sp. HA, was selected to be conjugated with two E. coli strains, WA803 containing pGS9 and AC8001 harboring pJB4JI. The Tn5 containing suicide vector pGS9 or pJB4JI, was transferred from E. coli to Acetobacter sp. HA and kanamycin-ampicillin-resistant transconjugants obtained at high frequencies. The conjugal frequencies of pGS9 and pJB4JI were 6.20$\times$$l0^{-1} and 2.79$\times$l0{-1}$ per recipient, respectively. The transfer method was applied on four different strains of Acetobacter. The conjugal transfer frequencies ranged from 2.00$\times$$l0^{-2} to 4.45$\times$l0^{-8}$ per recipient in the three strains. Some transconjugants tested were found to contain Tn5 DNA in their genomes and this was confirmed by Southem blot analysis. This is the first study which shows that Tn5 mutagenesis can be applied to successfully isolate mutants of Acetobacter genus.

• Korean Journal of Microbiology
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• v.27 no.4
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• pp.323-333
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• 1989

Plasmid DNA molecules containing strong promoter upstream from IS50L or IS50R, the two insertion sequences that flank Tn5, were constructed to amplify the synthesis of Tn5-encoded polypeptides. When proteins made by cells that contain these plasmids were analyzed on polyacrylamide gels, enhanced synthesis of IS50R polypeptides could be detected. Synthesis of this polypeptide apparently is initiated within the large open reading frame of this element. In addition, the stability of IS50L-and IS50R-encoded polypeptides was analyzed. It was found that IS50L polypeptides are relatively unstable in vivo. This instability could account for the observed inability of this element to promote transposition.

• Korean Journal Plant Pathology
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• v.10 no.1
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• pp.39-46
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• 1994

Pseudomonas fluorescens Biovar III strains S-2 antagonistic to Rhizoctonia solani was subjected to Tn5 mutagenesis by the transposon vector pGS9. Ampicillin and kanamycin resistant (Ampr, Kmr) transconjugants were recovered at a frequency of 1.3$\times$10-7 per initial recipient cell, when recipient cells were washed twice in TE buffer before conjugation. Of the ca. 3000 transconjugants, a frequency of noninhibitory (Inh-), nonfluorescent (Flu-) and auxotorphic (Pro-) mutants were 0.27%, 0.47% and 0.40%, respectively. In these mutants, all Inh- mutants showed the same colony morphology as wild type, whereas all Flu- and Pro- mutants inhibited the growth of R. solani. These mutants were also susceptible to chloramphenicol, indicating only the Tn5 element, except for parts of pGS9, was integrated into the recipient genome. In a Southern blot analysis, the Tn5 element inserted into one site on the chromosome for each of the chosen mutants. However, Tn5 insertion sites of Inh-, and Pro- mutants were differed in each other. These indicate that the genes essential for R. solani inhibition, fluorescent production and auxotrophic are chromosomally located, but not linked to each other.

• Journal of Microbiology and Biotechnology
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• v.7 no.5
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• pp.360-362
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• 1997

A transposon Tn5 mutant of Rhodobacter sphaeroides 2.4.1 was isolated for its impaired ability of growth on minimal medium containing ${\beta}$-hydroxybutyric acid as a sole carbon source. The mutant, R. sphaeroides S7 showed approximately 6-fold decrease in ${\beta}$-hydroxybutyrate dehydrogenase activity compared with that of wild type. In R. sphaeroides S7 the Tn5 was located in DNA region corresponding to a 4.2-kb EcoRI DNA fragment of R. sphaeroides 2.4.1 chromosome.

• Journal of Life Science
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• v.10 no.4
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• pp.333-339
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• 2000

To identify genes involved in the decolorization of crystal violet, we isolated random mutants generated by transponson insertion in crystal violet-declorizing bacterium, Citrobacter sp. The resulting mutant bank yielded mutants with six distinct phenotypes, and Southern hybridization with a Tn5 fragment as a probe showed a single hybridized with six distinct phenotypes, and Southern hybridization with a Tn5 fragment as a probe showed a single hybridized band in the mutants Ctg 2, 5 an 6, whereas two and three bands were detected in Ctg1, 4 and 3, respectively. Tn5-inserted genes were isolated and the DNA sequence flanking Tn5 was determined. From comparison with a sequence database, putative protein product encoded by ctg 5 was identified as E. coli maltose transproter(Mal G) homolog, whereas the deduced amino acid sequence of the other ctg genes did not show any significant similarity with any DNA or protein sequency. Therefore, these results indicate that the other ctg genes except ctg 5 encode new proteins responsible for decolorization of crystal violet.

• Journal of Microbiology and Biotechnology
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• v.4 no.2
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• pp.108-112
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• 1994

The genetic organization of the urease gene cluster from an alkalophilic Bacillus pasteurii was determined by subcloning and Tn5 transposon mutagenesis of a 10.7 kilobasepair cloned fragment. A region of DNA between 5.0 and 6.0 kb in length is necessary for urease activity. In vitro transcription-translation analysis of transposon insertion mutants of the cloned urease genes demonstrated that the major ($M_r$ 67,000) and minor ($M_r$ 20,000) structural peptides of urease are encoded at one end of the urease gene cluster and at least 3 additional polypeptides are encoded by adjacent DNA sequences.