• Korean Journal of Microbiology
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• v.28 no.2
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• pp.104-108
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• 1990

The chromosomal DNA of Agrobacterium tumefaciens contains the genes required for bacterial attachment to plant cell which is an essential atage in crown gall tumorigenesis by Ti-plasmid. In order to clone the genes, Agrobacterium tumefaciens strain A5512 was mutagenized by transposon Tn5 and two Agrobacterium tumefaciens mutants which are attachment-defective and nontumorigenic were isolated. From one of the two mutants, a chromosomal virulence region which was required for attachment to the plant cells was cloned.

• Korean Journal of Biological Psychiatry
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• v.6 no.1
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• pp.67-73
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• 1999

The cAMP-dependent protein kinase(PKA) is an intracellular enzyme with serine-threonine kinase activity that plays a key role in cell growth, differentiation, and apoptosis in eukaryotes. In order to understand the PKA signal transduction pathway regulating cell life cycle and identify its role, we focused on the characterization of up-/down-regulated genes by PKA using the differential display polymerase chain reaction. Seven differentially expressed sequence tags(DEST) have been obtained. Among these DESTs, 2 DESTs were homologous to the sequence of genes from BLAST search result. KC1-5 DEST that was up-regulated in A123.7 cells was highly corresponded to mouse apoptosis-related gene(MA-3) or mouse mRNA for topoisomerase inhibitor suppressed(TIS). MA-3 was induced in various types of apoptosis, specially in NGF-deprived apoptotic PC12 cells. TIS was down-regulated in the RVC lymphoma cells incubated with topoisomerase inhibitor that induces DNA strand breakages. PG1-1 DEST that was highly expressed in PC12 cells was corresponded to transposon Tn10 3'-end. Tnansposon Tn10 was up-regulated in differentiated myeloblastic ML-1 cells by 12-O-tetradecanoylphorbol-13-acetate. This study illuminates that MA-3/TIS was down-regulated by PKA activity, and transposon Tn10 was up-regulated by it.

• Korean Journal of Microbiology
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• v.30 no.4
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• pp.286-290
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• 1992

The present study was performed to isolate the fluorescent pseudomonads from Kwang-Ju soil and to construct a mutant strain defective in siderophore biosynthesis. The siderophore-secreting pseudomonads were screened on Blue agar (Chrome Azuol S agar) plates and one strain of them was designated to Pseudominas fluorescens (P. fluorescens) PY002. To construct a mutant defective in siderophore biosynthesis, P. fluorescens PY002 was randomly mutagenized with a transposon Tn5. The location of Tn5 integrated into chromosomal of the mutants strain was determined by Southern blot analysis. The mutagenized strain showed non-fluorescent on a King's B agar plate and were defective in iron (III) acquisition ability.

• Korean Journal of Microbiology
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• v.32 no.1
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• pp.12-19
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• 1994

KEM1 is known to control the spindle pole body or microtubule function, probably in response to the cellular nutritional conditions in Saccharomyces cerevisiae. Transposon insertions were performed in the cloned KEM1 gene using mini-Tn10-LUK element carrying E. coli ${\beta}$-galactosidase structural gene. A collection of ranfom Tn10-LUK insertions defined an approximately 3.5 kb region required for the KEM1 function. From this collection functional KEM1::lacZ protein fusions were identified. Indirect immunofluorescence using anti-${\beta}$-galacatosidase antibodies localized the KEM1::lacZ fusion protein to the periphery of the nucleus.

• The Plant Pathology Journal
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• v.36 no.5
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• pp.491-496
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• 2020

An understanding of the contribution of secondary metabolites (SMs) to the antagonistic and biocontrol activities of bacterial biocontrol agents serves to improve biocontrol potential of the strain. In this study, to evaluate the contribution of each SM produced by Pseudomonas fluorescens NBC275 (Pf275) to its antifungal and biocontrol activity, we combined in silico analysis of the genome with our previous study of transposon (Tn) mutants. Thirteen Tn mutants, which belonged to 6 biosynthetic gene clusters (BGCs) of a total 14 BGCs predicted by the antiSMASH tool were identified by the reduction of antifungal activity. The biocontrol performance of Pf275 was significantly dependent on 2,4-diacetylphloroglucinol and pyoverdine. The clusters that encode for arylpolyene and an unidentified small linear lipopeptide influenced antifungal and biocontrol activities. To our knowledge, our study identified the contribution of SMs, such as a small linear lipopeptide and arylpolyene, to biocontrol efficacy for the first time.

• Journal of Microbiology and Biotechnology
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• v.14 no.2
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• pp.302-311
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• 2004

Comamonas testosteroni (formerly Pseudomonas sp.) NCIMB 10643 can grow on biphenyl and alkylbenzenes $(C_2-C_7)$ via 3-substituted catechols. Thus, to identify the genes encoding the degradation, transposon-mutagenesis was carried out using pAG408, a promoter-probe mini-transposon with a green fluorescent protein (GFP), as a reporter. A mutant, NT-1, which was unable to grow on alkylbenzenes and biphenyl, accumulated catechols and exhibited an enhanced expression of GFP upon exposure to these substrates, indicating that the gfp had been inserted in a gene encoding a broad substrate range catechol 2,3-dioxygenase. The genes (2,826 bp) flanking the gfp cloned from an SphI-digested fragment contained three complete open reading frames that were designated bphCDorfl. The deduced amino acid sequences of bphCDorfl were identical to 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC), 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (BphD), and OrfI, respectively, that are all involved in the degradation of biphenyl/4-chlorobiphenyl (bph) by Ralstonia oxalatica A5. The deduced amino acid sequence of the orfl revealed a similarity to those of outer membrane proteins belonging to the OmpW family. The introduction of the bphCDorfl genes enabled the NT-l mutant to grow on aromatic hydrocarbons. In addition, PCR analysis indicated that the DNA sequence and gene organization of the bph operon were closely related to those in the bph operon from Tn4371 identified in strain A5. Furthermore, strain A5 was also able to grow on a similar set of alkylbenzenes as strain NCIMB 10643, demonstrating that, among the identified aromatic hydrocarbon degradation pathways, the bph degradation pathway related to Tn4371 was the most versatile in catabolizing a variety of aromatic hydrocarbons of mono- and bicyclic benzenes.

• Korean Journal of Agricultural Science
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• v.50 no.2
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• pp.241-247
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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.

• The Plant Pathology Journal
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• v.25 no.1
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• pp.77-85
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• 2009

Pseudomonas fluorescens strain Gpf01, isolated from ginseng rhizosphere showed antiviral activity against Cucumber mosaic virus, when tested in a local host of CMV, Chenopodium amaranticolor. Transposon mutant library of Gpf01 was prepared using pGS9::Tn5 and the mutant Gpf01-RS19 was found to loose antiviral production. We developed primers from the flanking region of Tn5 and found a cosmid clone pAV1123, harboring 1.2 kb antiviral compound producing (avcf01) locus. When a sub-clone pPH9, which carried 9.3 kb region of pAV1123, was introduced into antivirus deficient P. fluorescens wild type strain B16, it exhibited antiviral activity. Using Tn3-gus mutagenesis and complementation analysis, it was found that the genes related to antiviral activity production resided in a 9.3 kb HindIII-HindIII fragment of pAV1123, indicating that the plasmid carries an essential genes promoting antiviral activity.

• Journal of Microbiology
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• v.45 no.5
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• pp.466-472
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• 2007

A total of 98 vancomycin-resistant Enterococcus faecium (VREF) isolates (58 isolates from patients and 40 isolates from poultry) were compared based on their antimicrobial susceptibility, Tn1546 element organization, and pulsed-field gel electrophoresis (PFGE) patterns. This comparison aided in determining the relationships between the groups of isolates. All the VREF isolates harbored the vanA gene; however, 29 (29.6%) of the isolates exhibited the VanB phenotype-vanA genotype. Furthermore, the VREF isolates from humans and poultry exhibited distinct antimicrobial resistance patterns. The PCR mapping of the Tn1546 elements exhibited 12 different transposon types (A to L). The VREF isolates of poultry were classified into types A to D, whereas the human isolates were classified into types E to L. A PFGE analysis demonstrated a high degree of clonal heterogeneity in both groups of isolates; however, the distinct VREF clones appeared in each group of isolates. The deletion of the vanX-vanY genes or insertion of IS1216V in the intergenic region from the vanX-vanY genes is directly associated with the incongruence of the VanB phenotype-vanA genotype in human VREF isolates. These data suggest that the VREF isolates exhibit distinct phenotypic and genotypic traits according to their origins, which suggests that no evidence exists to substantiate the clonal spread or transfer of vancomycin resistance determinants between humans and poultry.

• Journal of Microbiology and Biotechnology
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• v.16 no.3
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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.