• Journal of Microbiology and Biotechnology
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• 제15권2호
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• pp.376-383
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• 2005

Alcaligenes sp. JMP228 carrying 2,4­dichlorophenoxyacetic acid (2,4-D) degradative plasmid pJP4 was inoculated into natural soil, and transfer of the plasmid pJP4 to indigenous soil bacteria was investigated with and without 2,4-D amendment. Plasmid pJP4 transfer was enhanced in the soils treated with 2,4-D, compared to the soils not amended with 2,4-D. Several different transconjugants were isolated from the soils treated with 2,4-D, while no indigenous transconjugants were obtained from the unamended soils. Inoculation of the soils with both the donor Alcaligenes sp. JMP228/pJP4 and a recipient Burkholderia cepacia DBO 1 produced less diverse transconjugants than the soils inoculated with the donor alone. Repetitive extragenic palindromic-polymerase chain reaction (REP-PCR) analysis of the transconjugants exhibited seven distinct genomic DNA fingerprints. Analysis of 16S rDNA sequences indicated that the transconjugants were related to members of the genera Burkholderia and Pandoraea. Denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA genes revealed that inoculation of the donor caused clear changes in the bacterial community structure of the 2,4-D­amended soils. The new 16S rRNA gene bands in the DGGE profile corresponded with the 16S rRNA genes of 2,4-D­degrading transconjugants isolated from the soil. The results indicate that introduction of the 2,4-D degradative plasmid as Alcaligenes sp. JMP228/pJP4 has a substantial impact on the bacterial community structure in the 2,4-D-amended soil.

• Journal of Plant Biotechnology
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• 제38권2호
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• pp.105-116
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• 2011

Transgenic zoysiagrass (Zoysia japonica Steud.) expressing the bar gene inserted in the plant genome has been generated previously through Agrobacterium tumefaciens-mediated transformation. The GM zoysiagrass (event: JG21) permits efficient management of weed control of widely cultivated zoysiagrass fields, reducing the frequency and cost of using various herbicides for weed control. Now we have carried out the environmental risk assessment of JG21 prior to applying to the governmental regulatory agency for the commercial release of the GM turf grass outside of test plots. The morphological phenotypes, molecular analysis, weediness and gene flow from each test plot of JG21 and wild-type zoysiagrasses have been evaluated by selectively analyzing environmental effects. There were no marked differences in morphological phenotypes between JG21 and wild-type grasses. The JG21 retained its stable integration in the host plant in T1 generation, exhibiting a 3:1 segregation ratio according to the Mendelian genetics. We confirmed the copy number (1) of JG21 by using Southern blot analysis, as the transgenic plants were tolerant to ammonium glufosinate throughout the culture period. From cross-fertilization and gene flow studies, we found a 9% cross-pollination rate at the center of JG21 field and 0% at distances over 3 m from the field. The JG21 and wild-type zoysiagrass plants are not considered "weed" because zoysiagrasses generally are not dominant and do not spread into weedy areas easily. We assessed the horizontal gene transfer (HGT) of the transgene DNA to soil microorganisms from JG21 and wild-type plants. The bar gene was not detected from the total genomic DNA extracted from each rhizosphere soil of GM and non-GM Zoysia grass fields. Through the monitoring of JG21 transgene's unintentional release into the environment, we found no evidence for either pollen mediated gene flow of zoysiagrass or seed dispersal from the test field within a 3 km radius of the natural habitat.

• Journal of Microbiology
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• 제45권3호
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• pp.193-198
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• 2007

A marine bacterium was isolated from Mai Po Nature Reserve of Hong Kong and identified as Vibrio cholerae MP-1. It contains a small plasmid designated as pVC of 3.8 kb. Four open reading frames (ORFs) are identified on the plasmid, but none of them shows homology to any known protein. Database search indicated that a 440 bp fragment is 96% identical to a fragment found in a small plasmid of another V. cholerae. Further experiments demonstrated that a 2.3 kb EcoRI fragment containing the complete ORF1, partial ORF4 and their intergenic region could self-replicate. Additional analyses revealed that sequence upstream of ORF1 showed the features characteristic of theta type replicons. Protein encoded by ORF1 has two characteristic motifs existed in most replication initiator proteins (Rep): the leucine zipper (LZ) motif located at the N-terminal region and the alpha helix-turn-alpha helix motif (HTH) located at the C-terminal end. The results suggest that pVC replicates via the theta type mechanism and is likely a novel type of theta replicon.

• Journal of Microbiology and Biotechnology
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• 제33권3호
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• pp.277-287
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• 2023

Since the first discovery of antibiotics, introduction of new antibiotics has been coupled with the occurrence of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs). Rapid dissemination of ARB and ARGs in the aquatic environments has become a global concern. ARB and ARGs have been already disseminated in the aquatic environments via various routes. Main hosts of most of ARGs were found to belong to Gammaproteobacteria class, including clinically important potential pathogens. Transmission of ARGs also occurs by horizontal gene transfer (HGT) mechanisms between bacterial strains in the aquatic environments, resulting in ubiquity of ARGs. Thus, a few of ARGs and MGEs (e.g., strA, sul1, int1) have been suggested as indicators for global comparability of contamination level in the aquatic environments. With ARB and ARGs contamination, the occurrence of critical pathogens has been globally issued due to their widespread in the aquatic environments. Thus, active surveillance systems have been launched worldwide. In this review, we described advancement of methodologies for ARGs detection, and occurrence of ARB and ARGs and their dissemination in the aquatic environments. Even though numerous studies have been conducted for ARB and ARGs, there is still no clear strategy to tackle antibiotic resistance (AR) in the aquatic environments. At least, for consistent surveillance, a strict framework should be established for further research in the aquatic environments.

• 디지털융복합연구
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• 제13권12호
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• pp.313-318
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• 2015

본 연구에서는 세균의 항균제 내성기전을 연구하기 위해 일개의 대학병원에서 분리된 Enterobacter cloacae를 대상으로 extended spectrum ${\beta}$-lactamase (ESBL) 및 16S rRNA methyltransferase 유전자를 검출하고 항균제 감수성 양상을 조사하였다. 대상균주 중 총 8 균주가 CTX-M-15형 ESBL을 생성하는 것으로 확인되었으며 이 균주들 중 3 균주는 16S rRNA methyltransferase의 한 종류인 armA 유전자도 동시에 가지고 있는 것으로 나타났다. CTX-M-15형 ESBL 유전자와 armA 유전자를 동시에 가지고 있는 E. cloacae는 3세대 cephalosporin 계열 및 aminoglycoside 계열의 항균제 뿐 만 아니라 fluoroquinolone 계열의 항균제에도 내성을 보였다. 더구나 이러한 항균제 내성 유전자들은 플라스미드를 통해 다른 세균으로 전달 될 수 있어 다제내성 세균의 출현 및 확산을 촉진 할 수 있다. 따라서 E. cloacae를 대상으로 지속적인 항균제 내성 유전자를 모니터링 하는 것은 항균제 내성 확산방지를 위해 중요할 것으로 사료된다.

• 한국균학회소식:학술대회논문집
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• 한국균학회 2014년도 춘계학술대회 및 임시총회
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• pp.27-28
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• 2014

Beauveria bassiana (Cordycipitaceae, Hypocreales, Ascomycota) is an anamorphic fungus having a potential to be used as a biological control agent because it parasitizes a wide range of arthropod hosts including termites, aphids, beetles and many other insects. A number of bioactive secondary metabolites (SMs) have been isolated from B. bassiana and functionally verified. Among them, beauvericin and bassianolide are cyclic depsipeptides with antibiotic and insecticidal effects belonging to the enniatin family. Non-ribosomal peptide synthetases (NRPSs) play a crucial role in the synthesis of these secondary metabolites. NRPSs are modularly organized multienzyme complexes in which each module is responsible for the elongation of proteinogenic and non-protein amino acids, as well as carboxyl and hydroxyacids. A minimum of three domains are necessary for one NRPS elongation module: an adenylation (A) domain for substrate recognition and activation; a tholation (T) domain that tethers the growing peptide chain and the incoming aminoacyl unit; and a condensation (C) domain to catalyze peptide bond formation. Some of the optional domains include epimerization (E), heterocyclization (Cy) and oxidation (Ox) domains, which may modify the enzyme-bound precursors or intermediates. In the present study, we analyzed genomes of B. bassiana and its allied species in Hypocreales to verify the distribution of NRPS-encoding genes involving biosynthesis of beauvericin and bassianolide, and to unveil the evolutionary processes of the gene clusters. Initially, we retrieved completely or partially assembled genomic sequences of fungal species belonging to Hypocreales from public databases. SM biosynthesizing genes were predicted from the selected genomes using antiSMASH program. Adenylation (A) domains were extracted from the predicted NRPS, NRPS-like and NRPS-PKS hybrid genes, and used them to construct a phylogenetic tree. Based on the preliminary results of SM biosynthetic gene prediction in B. bassiana, we analyzed the conserved gene orders of beauvericin and bassianolide biosynthetic gene clusters among the hypocrealean fungi. Reciprocal best blast hit (RBH) approach was performed to identify the regions orthologous to the biosynthetic gene cluster in the selected fungal genomes. A clear recombination pattern was recognized in the inferred A-domain tree in which A-domains in the 1st and 2nd modules of beauvericin and bassianolide synthetases were grouped in CYCLO and EAS clades, respectively, suggesting that two modules of each synthetase have evolved independently. In addition, inferred topologies were congruent with the species phylogeny of Cordycipitaceae, indicating that the gene fusion event have occurred before the species divergence. Beauvericin and bassianolide synthetases turned out to possess identical domain organization as C-A-T-C-A-NM-T-T-C. We also predicted precursors of beauvericin and bassianolide synthetases based on the extracted signature residues in A-domain core motifs. The result showed that the A-domains in the 1st module of both synthetases select D-2-hydroxyisovalerate (D-Hiv), while A-domains in the 2nd modules specifically activate L-phenylalanine (Phe) in beauvericin synthetase and leucine (Leu) in bassianolide synthetase. antiSMASH ver. 2.0 predicted 15 genes in the beauvericin biosynthetic gene cluster of the B. bassiana genome dispersed across a total length of approximately 50kb. The beauvericin biosynthetic gene cluster contains beauvericin synthetase as well as kivr gene encoding NADPH-dependent ketoisovalerate reductase which is necessary to convert 2-ketoisovalarate to D-Hiv and a gene encoding a putative Gal4-like transcriptional regulator. Our syntenic comparison showed that species in Cordycipitaceae have almost conserved beauvericin biosynthetic gene cluster although the gene order and direction were sometimes variable. It is intriguing that there is no region orthologous to beauvericin synthetase gene in Cordyceps militaris genome. It is likely that beauvericin synthetase was present in common ancestor of Cordycipitaceae but selective gene loss has occurred in several species including C. militaris. Putative bassianolide biosynthetic gene cluster consisted of 16 genes including bassianolide synthetase, cytochrome P450 monooxygenase, and putative Gal4-like transcriptional regulator genes. Our synteny analysis found that only B. bassiana possessed a bassianolide synthetase gene among the studied fungi. This result is consistent with the groupings in A-domain tree in which bassianolide synthetase gene found in B. bassiana was not grouped with NRPS genes predicted in other species. We hypothesized that bassianolide biosynthesizing cluster genes in B. bassiana are possibly acquired by horizontal gene transfer (HGT) from distantly related fungi. The present study showed that B. bassiana is the only species capable of producing both beauvericin and bassianolide. This property led to B. bassiana infect multiple hosts and to be a potential biological control agent against agricultural pests.

• Journal of Microbiology and Biotechnology
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• 제25권8호
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• pp.1339-1348
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• 2015

Until now, considerable effort has been made to engineer novel nitrogen-fixing organisms through the transfer of nif genes from various diazotrophs to non-nitrogen fixers; however, regulatory coupling of the heterologous nif genes with the regulatory system of the new host is still not well understood. In this work, a 49 kb nitrogen fixation island from P. stutzeri A1501 was transferred into E. coli using a novel and efficient transformation strategy, and a series of recombinant nitrogen-fixing E. coli strains were obtained. We found that the nitrogenase activity of the recombinant E. coli strain EN-01, similar to the parent strain P. stutzeri A1501, was dependent on external ammonia concentration, oxygen tension, and temperature. We further found that there existed a regulatory coupling between the E. coli general nitrogen regulatory system and the heterologous P. stutzeri nif island in the recombinant E. coli strain. We also provided evidence that the E. coli general nitrogen regulator GlnG protein was involved in the activation of the nif-specific regulator NifA via a direct interaction with the NifA promoter. To the best of our knowledge, this work plays a groundbreaking role in increasing understanding of the regulatory coupling of the heterologous nitrogen fixation system with the regulatory system of the recipient host. Furthermore, it will shed light on the structure and functional integrity of the nif island and will be useful for the construction of novel and more robust nitrogen-fixing organisms through biosynthetic engineering.

• Journal of Microbiology and Biotechnology
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• 제20권8호
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• pp.1163-1178
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• 2010

Plasmids play important roles in horizontal gene transfer among Vibrionaceae, but surprisingly little is known about their replication and incompatibility systems. In this study, we successfully developed a bioinformatics-assisted strategy of experimental identification of seven Vibrio plasmid replicons. Comparative sequences analysis of the seven Vibrio plasmid replicons obtained in this study together with eight published Vibrionaceae plasmid sequences revealed replication-participating elements involved in the ColE1 mode of replication initiation and regulation. Like plasmid ColE1, these Vibrionaceae plasmids encode two RNA species (the primer RNA and the antisense RNA) for replication initiation and regulation, and as a result, the 15 Vibrionaceae plasmids were designated as ColE1-like Vibrionaceae (CLV) plasmids. Two subgroups were obtained for the 15 CLV plasmids, based on comparison of replicon organization and phylogenetic analysis of replication regions. Coexistence of CLV plasmids were demonstrated by direct sequencing analysis and Southern hybridization, strongly suggesting that the incompatibility of CLV plasmids is determined mainly by the RNA I species like the ColE1-like plasmids. Sequences resembling the conserved Xer recombination sites were also identified on the CLV plasmids, indicating that the CLV plasmids probably use the host site-specific recombination system for multimer resolution like that used by ColE1-like plasmids. All the results indicated that the 15 plasmids form a new ColE1-like group, providing a basis for the rapid characterization and classification of Vibrionaceae plasmids.

• Journal of Microbiology and Biotechnology
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• 제23권6호
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• pp.739-749
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• 2013

The seasonal and spatial diversity of picocyanobacteria (Pcy) in lakes of the Great Mazurian Lakes (GLM) system was examined by DGGE analysis of molecular markers derived from the 16S-23S internal transcribed spacer (ITS) of the ribosomal operon and the phycocyanin operon (cpcBA-IGS). The study of nine lakes, ranging from mesotrophy to hypereutrophy, demonstrated seasonal variance of Pcy. The richness and Shannon diversity index calculated on the basis of both markers were higher in spring and lower in early and late summer. No statistically significant relationships were found between the markers and trophic status of the studied lakes or Pcy abundance. There were, however, statistically significant relationships between the diversity indices and sampling time. The analysis pointed to a different distribution of the two markers. The ITS marker exhibited more unique sequences in time and space, whereas a greater role for common and ubiquitous sequences was indicated by the cpcBA-IGS data. Examination of the Pcy community structure demonstrated that communities were grouped in highly similar clusters according to sampling season/time rather than to the trophic status of the lake. Our results suggest that time is more important than trophic status in shaping the diversity and structure of Pcy communities. The seasonal changes in picocyanobacteria and differences in diversity and community structures are discussed in the context of well-established ecological hypotheses: the PEG model, intermediate disturbance hypothesis (IDH), and horizontal gene transfer (HGT).

• Journal of Microbiology and Biotechnology
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• 제26권2호
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• pp.394-401
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• 2016

Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) system, a genome editing technology, was shown to be versatile in treating several antibiotic-resistant bacteria. In the present study, we applied the CRISPR/Cas9 technology to kill extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli. ESBL bacteria are mostly multidrug resistant (MDR), and have plasmid-mediated antibiotic resistance genes that can be easily transferred to other members of the bacterial community by horizontal gene transfer. To restore sensitivity to antibiotics in these bacteria, we searched for a CRISPR/Cas9 target sequence that was conserved among >1,000 ESBL mutants. There was only one target sequence for each TEM- and SHV-type ESBL, with each of these sequences found in ~200 ESBL strains of each type. Furthermore, we showed that these target sequences can be exploited to re-sensitize MDR cells in which resistance is mediated by genes that are not the target of the CRISPR/Cas9 system, but by genes that are present on the same plasmid as target genes. We believe our Re-Sensitization to Antibiotics from Resistance (ReSAFR) technology, which enhances the practical value of the CRISPR/Cas9 system, will be an effective method of treatment against plasmid-carrying MDR bacteria.