Journal of Microbiology

Journal Articles

Effects of Phosphorus‑dissolving Dark Septate Endophytes on the Growth of Blueberry: Qixin Luo , Rui Hou , Xiaojing Shang , Si Li; J. Microbiol. 2023;61(9):837-851. Published online October 5, 2023; DOI: https://doi.org/10.1007/s12275-023-00080-2

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Dark septate endophytes (DSEs) are widely distributed and improve plant growth. DSEs secrete large amounts of enzymes to mineralize insoluble phosphorus in soil and convert it into soluble phosphorus, promoting plant uptake of phosphorus. However, the effects of DSEs with phosphate-solubilizing ability on host plants need further study. In this study, phosphorusdissolving DSEs were screened for growth-promoting effects. We isolated, identified and characterized three DSE species (Thozetella neonivea, Pezicula ericae and Hyaloscyphaceae sp.) showing phosphate-solubilizing ability. The impact of single, dual or triple inoculation of DSEs on blueberry plant characteristics was studied. Their effects on colonization intensity, seedling biomass, nutrients in plants and soil, and activities of plant resistance enzymes and soil enzymes were markedly upregulated relative to the control (P < 0.05). The available phosphorus and acid phosphatase levels in different combinations were significantly increased. These findings indicate that the application of the three DSEs may be valuable in facilitating the cultivation of blueberry with a higher biomass and improved plant quality.

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Needle in a Haystack: Culturing Plant‐Beneficial Helotiales Lineages From Plant Roots
Pauline Bruyant, Jeanne Doré, Laurent Vallon, Yvan Moënne‐Loccoz, Juliana Almario
Environmental Microbiology.2025;[Epub] CrossRef
Acidomelania saccharicola sp. nov., a new species of dark septate endophytes in Helotiales, with potential of controlling Fusarium wilt of banana
Qian Nong, Yan Zhang, Yanyan Long, Yanlu Chen, Liping Qin, Shanyu Lin, Fenghua Zeng, Ling Xie
Biological Control.2025; 206: 105781. CrossRef
The Three-Dimensional Structure of the Genome of the Dark Septate Endophyte Exophiala tremulae and Its Symbiosis Effect on Alpine Meadow Plant Growth
Chu Wu, Junjie Fan, Die Hu, Honggang Sun, Guangxin Lu, Yun Wang, Yujie Yang
Journal of Fungi.2025; 11(4): 246. CrossRef
Growth-Promoting Effects of Dark Septate Endophytes Fungus Acrocalymma on Tomato (Solanum lycopersicum)
Xiaoxiao Feng, Ying Jin, Zhupeiqi Zhong, Yongli Zheng, Huiming Wu
Journal of Fungi.2025; 11(7): 510. CrossRef
Inoculation dose and strain identity shape dark septate endophyte effects on plant-soil nutrient stoichiometry in ecological restoration
Shiwei Guo, Mingyi Li, Roujia Kang, Wennian Xu, Haoji Jia, Dong Xia, Daxiang Liu
Applied Soil Ecology.2025; 216: 106523. CrossRef
Dark septate endophytes promote the growth of Cynodon dactylon under drought stress and enhance its potential for use in the ecological restoration of slopes
Haoji Jia, Qiming Geng, Mingyi Li, Ran Wang, Fuhao Wang, Yuxin Deng, Wennian Xu, Daxiang Liu
Frontiers in Plant Science.2025;[Epub] CrossRef
Diversity and Functional Roles of Root-Associated Endophytic Fungi in Two Dominant Pioneer Trees Reclaimed from a Metal Mine Slag Heap in Southwest China
Bo Bi, Yuqing Xiao, Xiaonan Xu, Qianqian Chen, Haiyan Li, Zhiwei Zhao, Tao Li
Microorganisms.2024; 12(10): 2067. CrossRef
Short-term organic fertilizer substitution increases sorghum yield by improving soil physicochemical characteristics and regulating microbial community structure
Mengen Nie, Guangqian Yue, Lei Wang, Yizhong Zhang
Frontiers in Plant Science.2024;[Epub] CrossRef

Transposon insertion site sequencing (TIS) of Pseudomonas aeruginosa: Hongbaek Cho; J. Microbiol. 2021;59(12):1067-1074. Published online December 4, 2021; DOI: https://doi.org/10.1007/s12275-021-1565-y

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Transposon insertion site sequencing (TIS) is a technique that determines the insertion profile of a transposon mutant library by massive parallel sequencing of transposon-genomic DNA junctions. Because the transposon insertion profile reflects the abundance of each mutant in the library, it provides information to assess the fitness contribution of each genetic locus of a bacterial genome in a specific growth condition or strain background. Although introduced only about a dozen years ago, TIS has become an important tool in bacterial genetics that provides clues to study biological functions and regulatory mechanisms. Here, I describe a protocol for generating high density transposon insertion mutant libraries and preparing Illumina sequencing samples for mapping the transposon junctions of the transposon mutant libraries using Pseudomonas aeruginosa as an example.

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Transposon insertion sequencing analysis reveals conditional essential genes for infection in Pseudomonas plecoglossicida
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Optimizing phage-based mutant recovery and minimizing heat effect in the construction of transposon libraries in Staphylococcus aureus
Sally W. Yousief, Nader Abdelmalek, Bianca Paglietti
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Meiyi Ge, Jian Luo, Yi Wu, Guobo Shen, Xi Kuang
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Optimization of Transposon Mutagenesis Methods in Pseudomonas antarctica
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Construction of high-density transposon mutant library of Staphylococcus aureus using bacteriophage ϕ11
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[Protocol]Construction of a multicopy genomic DNA library and its application for suppression analysis: Hongbaek Cho; J. Microbiol. 2019;57(12):1041-1047. Published online November 22, 2019; DOI: https://doi.org/10.1007/s12275-019-9417-8

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Suppression analysis is used for the identification of new genes and genetic interactions when there is a notable phenotype available for genetic selection or screening. A random genomic DNA library constructed on a multi-copy plasmid is a useful tool for suppression analysis when one expects that an overdose of a few genes will suppress the phenotype. These libraries have been successfully used to determine the function of a gene by revealing genes whose functions are related to the gene of interest. They have also been used to identify the targets of chemical or biological agents by increasing the number of unaffected target gene products in a cell. In this article, I will discuss important considerations for constructing multicopy genomic DNA libraries. The protocol provided in this paper should be a useful guide for constructing genomic DNA libraries in many bacterial species for which multi-copy plasmids are available.

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Identification and characterization of the lipoprotein N -acyltransferase in Bacteroides
Krista M. Armbruster, Jiawen Jiang, Mariana G. Sartorio, Nichollas E. Scott, Jenna M. Peterson, Jonathan Z. Sexton, Mario F. Feldman, Nicole M. Koropatkin
Proceedings of the National Academy of Sciences.2024;[Epub] CrossRef

Review

MINIREVIEW] Modern and Simple Construction of Plasmid: Saving Time and Cost: Hideki Nakayama , Nobuo Shimamoto; J. Microbiol. 2014;52(11):891-897. Published online October 31, 2014; DOI: https://doi.org/10.1007/s12275-014-4501-6

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Construction of plasmids has been occupying a significant fraction of laboratory work in most fields of experimental biology. Tremendous effort was made to improve the traditional method for constructing plasmids, in which DNA fragments digested with restriction enzymes were ligated. However, the traditional method remained to be a standard protocol more than 40 years. At last, several recent inventions are rapidly and completely replacing the traditional method, because they are far quicker with less cost, and requiring less material. We here introduce three such methods that cover up most of the cases. Moreover, they are complementary with each other. Our lab protocols are provided for “no strain, no pain” construction of plasmids.

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Insight on flavinylation and functioning factor in Type B succinate dehydrogenase from Gram-positive bacteria
Yusuke Shiota, Tomoyuki Kosaka
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Tumor biology experimental course design based on the integration of molecular biology and metabolomics
Xinliang Zhu, Ting Tang
Cogent Education.2024;[Epub] CrossRef
Truncation-Free Genetic Code Expansion with Tetrazine Amino Acids for Quantitative Protein Ligations
Alex J. Eddins, Riley M. Bednar, Subhashis Jana, Abigail H. Pung, Lea Mbengi, Kyle Meyer, John J. Perona, Richard B. Cooley, P. Andrew Karplus, Ryan A. Mehl
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Akira Muto, Simon Goto, Daisuke Kurita, Chisato Ushida, Hyota Himeno
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Tomoyuki Kosaka, Mutsumi Goda, Manami Inoue, Toshiharu Yakushi, Mamoru Yamada
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Natural killer cells unleashed: Checkpoint receptor blockade and BiKE/TriKE utilization in NK-mediated anti-tumor immunotherapy
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In vivo Assembly in Escherichia coli of Transformation Vectors for Plastid Genome Engineering
Yuyong Wu, Lili You, Shengchun Li, Meiqi Ma, Mengting Wu, Lixin Ma, Ralph Bock, Ling Chang, Jiang Zhang
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Journal Article

NOTE] Comparison of the Genetic Structures Surrounding qnrA1 in Korean Enterobacter cloacae and Chinese Escherichia coli Strains Isolated in the Early 2000s: Evidence for qnrA Mobilization via Inc HI2 Type Plasmid: Sang Hoon Han , Young Ah Kim , Minggui Wang , Yangsoon Lee , Hae-Sun Chung , Jong Hwa Yum , Dongeun Yong , Kyungwon Lee , June Myung Kim; J. Microbiol. 2012;50(1):166-169. Published online February 27, 2012; DOI: https://doi.org/10.1007/s12275-012-1350-z

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Abstract PDF: The flanking genetic structure of qnrA1 in Korean Enterobacter cloacae was identical to that of the Chinese Escherichia coli strain, the first qnrA1-carrying strain reported in Asia. Analysis of restriction enzyme sites and Southern blot hybridization results showed that qnrA1 was transferred between E. cloacae and E. coli via Inc HI2 type plasmid.

Research Support, Non-U.S. Gov'ts

NOTE] Complete Sequence and Organization of the Sphingobium chungbukense DJ77 pSY2 Plasmid: Sun-Mi Yeon , Young-Chang Kim; J. Microbiol. 2011;49(4):684-688. Published online September 2, 2011; DOI: https://doi.org/10.1007/s12275-011-1262-3

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Sphingobium chungbukense DJ77 is capable of metabolizing priority chemicals of human health concern such as polycyclic aromatic hydrocarbons (PAHs), extracellular polysaccharide (EPS), and antibiotics. Here, we report the complete DNA and genetic organization of the plasmid pSY2 from strain DJ77. A DNA sequence analysis revealed that pSY2 comprises 18,779 bp encoding 22 open reading frames (ORFs) with 59.5% G+C content. The ORFs on pSY2 were classified into DNA replication, conjugative function, transposition, plasmid stability/partition, and other functional groups (transport, fatty acid biosynthesis, stress, and growth rate regulation). Three ORFs on pSY2 were hypothetical proteins.

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Biochemical characterization and molecular docking analysis of novel esterases from Sphingobium chungbukense DJ77
Woo-Ri Shin, Hyun-Ju Um, Young-Chang Kim, Sun Chang Kim, Byung-Kwan Cho, Ji-Young Ahn, Jiho Min, Yang-Hoon Kim
International Journal of Biological Macromolecules.2021; 168: 403. CrossRef
Molecular Docking Analysis and Biochemical Evaluation of Levansucrase from Sphingobium chungbukense DJ77
Soo Youn Lee, Woo-Ri Shin, Simranjeet Singh Sekhon, Jin-Pyo Lee, Young-Chang Kim, Ji-Young Ahn, Yang-Hoon Kim
ACS Combinatorial Science.2018; 20(7): 414. CrossRef
Degradative plasmids from sphingomonads
Andreas Stolz
FEMS Microbiology Letters.2014; 350(1): 9. CrossRef

Molecular Analysis of a Prolonged Spread of Klebsiella pneumoniae Co-producing DHA-1 and SHV-12 β-Lactamases: Young Kyung Yoon , Hye Won Cheong , Hyunjoo Pai , Kyoung Ho Roh , Jeong Yeon Kim , Dae Won Park , Jang Wook Sohn , Seung Eun Lee , Byung Chul Chun , Hee Sun Sim , Min Ja Kim; J. Microbiol. 2011;49(3):363-368. Published online June 30, 2011; DOI: https://doi.org/10.1007/s12275-011-0491-9

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The study investigated molecular mechanisms for prolonged nosocomial spread of multidrug-resistant Klebsiella pneumoniae co-producing plasmid-mediated AmpC β-lactamase DHA-1 and extended-spectrum β-lactamase SHV-12. Forty-eight clinical isolates of K. pneumonia, resistant to the extended-spectrum cephalosporins, were collected in a 750-bed university hospital over a year. The isolates were characterized for PCR-based β-lactamase genotypes, isoelectric focusing and pulsed-field gel electrophoresis (PFGE) profiles. Resistance transfer was performed by plasmid conjugation and confirmed by a duplex-PCR and Southern hybridization. On β-lactamase typing, the strains producing only the DHA-1 enzyme (n=17) or co-producing DHA-1 and SHV-12 enzymes (n=15) were predominant. Judging from a one year-distribution of PFGE profiles, the co-producer was spread primarily with single clonal expansion of the PFGE-type A with subtypes (n=14), whereas the strains producing only DHA-1 enzyme were spread simultaneously with the PFGE-type A (n=11) and other PFGE types (n=6). Transconjugants of the co-producers were confirmed to harbor either both blaDHA-1 and blaSHV-12 or only the blaDHA-1. In conclusion, this study indicated that the persistent nosocomial spread of multidrug-resistant K. pneumoniae strains was primarily associated with expansion of a clone harboring both the blaDHA-1 and blaSHV-12 or the blaDHA-1 only, and to a lesser extent with the horizontal transfer of the resistant plasmids. Our observations have clinical implication for the control and prevention of nosocomial dissemination of multidrug-resistant K. pneumoniae strains.

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High Prevalence of CTX-M-15-Type Extended-Spectrum β-Lactamase Among AmpC β-Lactamase-Producing Klebsiella pneumoniae Isolates Causing Bacteremia in Korea
Min Kyeong Cha, Cheol-In Kang, So Hyun Kim, Doo Ryeon Chung, Kyong Ran Peck, Nam Yong Lee, Jae-Hoon Song
Microbial Drug Resistance.2018; 24(7): 1002. CrossRef
Role of piperacillin/tazobactam as a carbapenem-sparing antibiotic for treatment of acute pyelonephritis due to extended-spectrum β-lactamase-producing Escherichia coli
Young Kyung Yoon, Jong Hun Kim, Jang Wook Sohn, Kyung Sook Yang, Min Ja Kim
International Journal of Antimicrobial Agents.2017; 49(4): 410. CrossRef
Emergence of serotype K1 Klebsiella pneumoniae ST23 strains co-producing the plasmid-mediated AmpC beta-lactamase DHA-1 and an extended-spectrum beta-lactamase in Korea
Hae Suk Cheong, Doo Ryeon Chung, Chaeyoeng Lee, So Hyun Kim, Cheol-In Kang, Kyong Ran Peck, Jae-Hoon Song
Antimicrobial Resistance & Infection Control.2016;[Epub] CrossRef
Effects of Group 1 versus Group 2 Carbapenems on the Susceptibility of Acinetobacter baumannii to Carbapenems: A Before and After Intervention Study of Carbapenem-Use Stewardship
Young Kyung Yoon, Kyung Sook Yang, Seung Eun Lee, Hyun Jeong Kim, Jang Wook Sohn, Min Ja Kim, Mark Alexander Webber
PLoS ONE.2014; 9(6): e99101. CrossRef

Characterization of Plasmid pSY3 in Sphingobium chungbukense DJ77: Sun-Mi Yeon , Young-Chang Kim; J. Microbiol. 2009;47(6):796-800. Published online February 4, 2010; DOI: https://doi.org/10.1007/s12275-009-0329-x

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This study determined the complete nucleotide sequence of the plasmid pSY3 from Sphingobium chungbukense DJ77. It was 35,735 bp long with a G+C content of 61.9%. Forty open reading frames (ORFs) were found. We predicted these ORFs would encode proteins associated with plasmid replication, conjugative transfer, transposition of genes, plasmid stability/partition, hypothetical protein, and some other functions. Genes for biodegradation were not found. No other plasmid homologous to pSY3 in the overall nucleotide sequence or gene organization could be found in the NCBI database.

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Sphingomonas sediminicola Is an Endosymbiotic Bacterium Able to Induce the Formation of Root Nodules in Pea (Pisum sativum L.) and to Enhance Plant Biomass Production
Candice Mazoyon, Bertrand Hirel, Audrey Pecourt, Manuella Catterou, Laurent Gutierrez, Vivien Sarazin, Fréderic Dubois, Jérôme Duclercq
Microorganisms.2023; 11(1): 199. CrossRef
Complete sequence and organization of the Sphingobium chungbukense DJ77 pSY2 plasmid
Sun-Mi Yeon, Young-Chang Kim
The Journal of Microbiology.2011; 49(4): 684. CrossRef

Cloning and Molecular Characterization of a Novel Rolling-Circle Replicating Plasmid, pK1S-1, from Bacillus thuringiensis subsp. kurstaki K1: Ming Shun Li , Jong Yul Roh , Xueying Tao , Zi Niu Yu , Zi Duo Liu , Qin Liu , Hong Guang Xu , Hee Jin Shim , Yang-Su Kim , Yong Wang , Jae Young Choi , Yeon Ho Je; J. Microbiol. 2009;47(4):466-472. Published online September 9, 2009; DOI: https://doi.org/10.1007/s12275-009-0020-2

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Abstract PDF: Bacillus thuringiensis, an entomopathogenic bacterium belonging to the B. cereus group, harbors numerous extra-chromosomal DNA molecules whose sizes range from 2 to 250 kb. In this study, we used a plasmid capture system (PCS) to clone three small plasmids from B. thuringiensis subsp. kurstaki K1 which were not found in B. thuringiensis subsp. kurstaki HD-1, and determined the complete nucleotide sequence of plasmid pK1S-1 (5.5 kb). Of the six putative open reading frames (ORF2-ORF7) in pK1S-1, ORF2 (MobK1) showed approximately 90% aa identity with the Mob-proteins of pGI2 and pTX14-2, which are rolling circle replicating group VII (RCR group VII) plasmids from B. thuringiensis. In addition, a putative origin of transfer (oriT) showed 95.8% identity with those of pGI2 and pTX14-2. ORF3 (RepK1) showed relatively low aa identity (17.8~25.2%) with the Rep protein coded by RCR plasmids, however. The putative double- strand origin of replication (dso) and single-strand origin of replication (sso) of pK1S-1 exhibited approximately 70% and 64% identities with those of pGI2 and pTX14-2. ORF6 and 7 showed greater than 50% similarities with alkaline serine protease, which belongs to the subtilase family. The other 2 ORFs were identified as hypothetical proteins. To determine the replicon of pK1S-1, seven subclones were contructed in the B. thuringiensis ori-negative pHT1K vector and were electroporated into a plasmid cured B. thuringiensis strain. The 1.6 kb region that included the putative ORF3 (Rep1K), dso and ORF4, exhibited replication ability. These findings identified pK1S-1 as a new RCR group VII plasmid, and determined its replication region.

Journal Article

Mobilization Functions of the Bacteriocinogenic Plasmid pRJ6 of Staphylococcus aureus: Marcus Livio Varella Coelho , Hilana Ceotto , Danielle Jannuzzi Madureira , Ingolf F. Nes , Maria do Carmo de Freire Bastos; J. Microbiol. 2009;47(3):327-336. Published online June 26, 2009; DOI: https://doi.org/10.1007/s12275-009-0044-7

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Plasmid pRJ6 is the first known bacteriocinogenic mobilizable (Mob) plasmid of Staphylococcus aureus. Its Mob region is composed of four mob genes (mobCDAB) arranged as an operon, a genetic organization uncommon among S. aureus Mob plasmids. oriTpRJ6 was detected in a region of 431 bp, positioned immediately upstream of mobC. This region, when cloned into pCN37, was able to confer mobilization to the recombinant plasmid only in the presence of pRJ6. The entire Mob region, including oriTpRJ6, is much more similar to Mob regions from several coagulase-negative staphylococci plasmids, although some remarkable similarities with S. aureus Mob plasmids can also be noted. These similarities include the presence within oriTpRJ6 of the three mcb (MobC binding sites), firstly described in pC221 and pC223, an identical nick site also found in these same plasmids, and a nearly identical srapC223 site (sequence recognized by MobA). pRJ6 was successfully transferred to S. epidermidis by conjugation in the presence of the conjugative plasmid pGO1. Altogether these findings suggest that pRJ6 might have been originally a coagulase-negative staphylococci plasmid that had been transferred successfully to S. aureus.

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Comprehensive Approaches for the Search and Characterization of Staphylococcins
Rosa Fernández-Fernández, Carmen Lozano, Rine Christopher Reuben, Laura Ruiz-Ripa, Myriam Zarazaga, Carmen Torres
Microorganisms.2023; 11(5): 1329. CrossRef
Molecular characterization of aureocin 4181: a natural N-formylated aureocin A70 variant with a broad spectrum of activity
Selda Loase Salustiano Marques-Bastos, Marcus Lívio Varella Coelho, Hilana Ceotto-Vigoder, Patrícia Carlin Fagundes, Gabriela Silva Almeida, Dag A. Brede, Ingolf F. Nes, Maria Aparecida Vasconcelos de Paiva Brito, Maria do Carmo de Freire Bastos
Brazilian Journal of Microbiology.2020; 51(4): 1527. CrossRef
Nisin and lysostaphin activity against preformed biofilm of Staphylococcus aureus involved in bovine mastitis
H. Ceotto-Vigoder, S.L.S. Marques, I.N.S. Santos, M.D.B. Alves, E.S. Barrias, A. Potter, D.S. Alviano, M.C.F. Bastos
Journal of Applied Microbiology.2016; 121(1): 101. CrossRef
Origin-of-transfer sequences facilitate mobilisation of non-conjugative antimicrobial-resistance plasmids inStaphylococcus aureus
Frances G. O'Brien, Karina Yui Eto, Riley J. T. Murphy, Heather M. Fairhurst, Geoffrey W. Coombs, Warren B. Grubb, Joshua P. Ramsay
Nucleic Acids Research.2015; 43(16): 7971. CrossRef
The gene cluster of aureocyclicin 4185: the first cyclic bacteriocin of Staphylococcus aureus
Amina Potter, Hilana Ceotto, Marcus Lívio Varella Coelho, Allan J. Guimarães, Maria do Carmo de Freire Bastos
Microbiology .2014; 160(5): 917. CrossRef
Immunity to the Staphylococcus aureus leaderless four-peptide bacteriocin aureocin A70 is conferred by AurI, an integral membrane protein
Marcus Lívio Varella Coelho, Bruna Gonçalves Coutinho, Olinda Cabral da Silva Santos, Ingolf F. Nes, Maria do Carmo de Freire Bastos
Research in Microbiology.2014; 165(1): 50. CrossRef
Identification of new staphylococcins with potential application as food biopreservatives
Andreza Freitas de Souza Duarte, Hilana Ceotto, Marcus Lívio Varella Coelho, Maria Aparecida Vasconcelos Brito, Maria do Carmo de Freire Bastos
Food Control.2013; 32(1): 313. CrossRef
Aureocin A70 production is disseminated amongst genetically unrelated Staphylococcus aureus involved in bovine mastitis
H. Ceotto, R.C. da Silva Dias, J. dos Santos Nascimento, M.A.V. de Paiva Brito, M. Giambiagi-deMarval, M. do Carmo de Freire Bastos
Letters in Applied Microbiology.2012; 54(5): 455. CrossRef
Genes Involved in Immunity to and Secretion of Aureocin A53, an Atypical Class II Bacteriocin Produced by Staphylococcus aureus A53
Janaína dos Santos Nascimento, Marcus Lívio Varella Coelho, Hilana Ceotto, Amina Potter, Luana Rocha Fleming, Zhian Salehian, Ingolf F. Nes, Maria do Carmo de Freire Bastos
Journal of Bacteriology.2012; 194(4): 875. CrossRef
Major Families of Multiresistant Plasmids from Geographically and Epidemiologically Diverse Staphylococci
Julia E S Shearer, Joy Wireman, Jessica Hostetler, Heather Forberger, Jon Borman, John Gill, Susan Sanchez, Alexander Mankin, Jacqueline LaMarre, Jodi A Lindsay, Kenneth Bayles, Ainsley Nicholson, Frances O’Brien, Slade O Jensen, Neville Firth, Ronald A S
G3 Genes|Genomes|Genetics.2011; 1(7): 581. CrossRef
Bacteriocin production by Staphylococcus aureus involved in bovine mastitis in Brazil
Hilana Ceotto, Janaína dos Santos Nascimento, Maria Aparecida Vasconcelos de Paiva Brito, Maria do Carmo de Freire Bastos
Research in Microbiology.2009; 160(8): 592. CrossRef

Research Support, Non-U.S. Gov'ts

Class 1 and Class 2 Integrons and Plasmid-Mediated Antibiotic Resistance in Coliforms Isolated from Ten Rivers in Northern Turkey: Osman Birol Ozgumus , Cemal Sandalli , Ali Sevim , Elif Celik-Sevim , Nuket Sivri; J. Microbiol. 2009;47(1):19-27. Published online February 20, 2009; DOI: https://doi.org/10.1007/s12275-008-0206-z

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Abstract PDF: We aimed to determine the molecular mechanisms of antibiotic resistance in coliforms isolated from ten rivers in northern region of Turkey. A total of 183 isolates were tested for antimicrobial susceptibility by disk diffusion and agar dilution methods. Resistance to ampicillin, streptomycin, trimethoprim, tetracycline, and chloramphenicol was detected in 58%, 51.9%, 24%, 28.4%, and 12.5%, respectively. Twelve (6.5%) phylogenetically distant organisms were detected to harbor self-transmissible plasmids ranging 52 to >147 kb in sizes. Resistances to ampicillin, tetracycline, trimethoprim, streptomycin, and nalidixic acid were commonly transferable traits. Transferable nalidixic acid-resistant strains harbored qnrS gene, which was the first report of plasmid-mediated quinolone resistance in bacteria of environmental origin in Turkey. Fourteen and five coliforms harbored class 1 and class 2 integrons, respectively, and some of them were located on transferable plasmids. Sequence analyses of variable regions of the class 1 and 2 integrons harbored various gene cassettes, dfrA1, dfr2d, dfrA7, dfrA16, dfrA17, aadA1, aadA5, blaOXA-30, and sat1. A gene cassette array, dfrA16 has been demonstrated for the first time in a Citrobacter koseri isolate. Class 1 and class 2-bearing strains were clustered in different groups by BOX-PCR fingerprinting. Rivers in the northern Turkey may act as receptacle for the multi-drug resistant enterobacteria and can serve as reservoirs of the antimicrobial resistance determinants in the environment. The actual risk to public health is the transfer of resistance genes from the environmental bacteria to human pathogens.

Prevalence of Tetracycline Resistance Genes in Greek Seawater Habitats: Theodora L. Nikolakopoulou , Eleni P. Giannoutsou , Adamandia A. Karabatsou , Amalia D. Karagouni; J. Microbiol. 2008;46(6):633-640. Published online December 24, 2008; DOI: https://doi.org/10.1007/s12275-008-0080-8

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The presence of selected tetracycline resistance (TcR) genes was studied in different Greek seawater habitats, originated from wastewater treatment facilities, fishfarm, and coastal environments. The methods employed included assessment of the presence of twelve gene clusters by PCR, followed by hybridization with specific probes, in habitat extracted DNA, TcR bacteria, and exogenous isolated plasmids conferring TcR. The direct DNA-based analysis showed that tet(Α) and tet(K) genes were detected in all habitats, whilst tet(C) and tet(E) were present in fishfarm and wastewater effluent samples and tet(M) was detected in fishfarm and coastal samples. Resistance genes tet(A), tet(C), tet(K), and tet(M) were detected in 60 of the 89 isolates screened. These isolates were identified by fatty acid methyl ester analysis (FAME) as Stenotrophomonas, Acinetobacter, Pseudomonas, Bacillus, and Staphylococcus strains. The presence of the TcR genes in 15% of the bacterial isolates coincided with the presence of IncP plasmids. A habitat-specific dissemination of IncP alpha plasmids in wastewater effluent isolates and of IncP beta plasmids in fishfarm isolates was observed. Exogenous isolation demonstrated the presence of plasmids harbouring TcR genes in all the habitats tested. Plasmids were shown to carry tet(A), tet(C), tet(E), and tet(K) genes. It is concluded that TcR genes are widespread in the seawater habitats studied and often occur on broad host range plasmids that seem to be well disseminated in the bacterial communities.

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Teresa Cardona-Cabrera, Carmen González-Azcona, Paula Eguizábal, Olga Alexandrou, Giorgos Catsadorakis, Panagiotis Azmanis, Carmen Lozano, Ursula Höfle, Carmen Torres
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pVC, a Small Cryptic Plasmid from the Environmental Isolate of Vibrio cholerae MP-1: Ruifu Zhang , Yanling Wang , Pak Chow Leung , Ji-Dong Gu; J. Microbiol. 2007;45(3):193-198.; DOI: https://doi.org/2543 [pii]

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Abstract PDF: 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.

Evidence of Indigenous NAH Plasmid of Naphthalene Degrading Pseudomonas putida PpG7 Strain Implicated in Limonin Degradation: Moushumi Ghosh , Abhijit Ganguli , Meenakshi Mallik; J. Microbiol. 2006;44(5):473-479.; DOI: https://doi.org/2451 [pii]

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Abstract PDF: A well characterized naphthalene-degrading strain, Pseudomonas putida PpG7 was observed to utilize limonin, a highly-oxygenated triterpenoid compound as a sole source of carbon and energy. Limonin concentrations evidenced a 64% reduction over 48 h of growth in batch cultures. Attempts were made to acquire a plasmid-less derivative via various methods (viz. Ethidium Bromide, SDS, elevated temperature & mitomycin C), among which the method involving mitomycin C (20 μg/ml) proved successful. Concomitant with the loss of plasmid in P. putida PpG7 strain, the cured derivative was identified as a lim- phenotype. The lim+ phenotype could be conjugally transferred to the cured derivative. Based on the results of curing with mitomycin C, conjugation studies and presence of ndo gene encoding naphthalene 1,2 dioxygenase, it was demonstrated that genes for the limonin utilization were encoded on an 83 kb indigenous transmissible Inc. P9 NAH plasmid in Pseudomonas putida PpG7 strain.

Isolation and Characterization of a Rhodococcus Species Strain Able to Grow on ortho- and para-Xylene: Jung Yeon Jang , Dockyu Kim , Hyun Won Bae , Ki Young Choi , Jong-Chan Chae , Gerben J. Zylstra , Young Min Kim , Eungbin Kim; J. Microbiol. 2005;43(4):325-330.; DOI: https://doi.org/2258 [pii]

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Abstract PDF: Rhodococcus sp. strain YU6 was isolated from soil for the ability to grow on o-xylene as the sole carbon and energy source. Unlike most other o-xylene-degrading bacteria, YU6 is able to grow on p-xylene. Numerous growth substrate range experiments, in addition to the ring-cleavage enzyme assay data, suggest that YU6 initially metabolizes o- and p-xylene by direct aromatic ring oxidation. This leads to the formation of dimethylcatechols, which was further degraded largely through meta-cleavage pathway. The gene encoding meta-cleavage dioxygenase enzyme was PCR cloned from genomic YU6 DNA using previously known gene sequence data from the o-xylene-degrading Rhodococcus sp. strain DK17. Subsequent sequencing of the 918-bp PCR product revealed a 98% identity to the gene, encoding methylcatechol 2,3-dioxygenase from DK17. PFGE analysis followed by Southern hybridization with the catechol 2,3-dioxygenase gene demonstrated that the gene is located on an approximately 560-kb megaplasmid, designated pJYJ1 <br>

Journal Articles

Plasmid Profiling and Curing of Lactobacillus Strains Isolated from the Gastrointestinal Tract of Chicken: Sieo Chin Chin , Norhani Abdullah , Tan Wen Siang , Ho Yin Wan; J. Microbiol. 2005;43(3):251-256.; DOI: https://doi.org/2217 [pii]

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Abstract PDF: In this study, we assessed the susceptibility of 12 Lactobacillus strains, all of which had been isolated from the gastrointestinal tracts of chicken, to three antibiotics (chloramphenicol, erythromycin and tetracycline) used commonly as selective markers in transformation studies of lactic acid bacteria. Among these strains, 17%, 58%, and 25% were found to exhibit a high degree of resistance to 200 mg/ml of tetracycline, erythromycin, and chloramphenicol, respectively. Seven of the 12 Lactobacillus strains exhibiting resistance to at least 50 mg/ml of chloramphenicol or erythromycin, and five strains exhibiting resistance to at least 50 mg/ml of tetracycline, were subsequently subjected to plasmid curing with chemical curing agents, such as novobiocin, acriflavin, SDS, and ethidium bromide. In no cases did the antibiotic resistance of these strains prove to be curable, with the exception of the erythromycin resistance exhibited by five Lactobacillus strains (L. acidophilus I16 and I26, L. fermentum I24 and C17, and L. brevis C10). Analysis of the plasmid profiles of these five cured derivatives revealed that all of the derivatives, except for L. acidophilus I16, possessed profiles similar to those of wild-type strains. The curing of L. acidophilus I16 was accompanied by the loss of 4.4 kb, 6.1 kb, and 11.5 kb plasmids.

Stabilities of Artificially Transconjugated Plasmids for the Bioremediation of Cocontaminated Sites: Kyung Pyo Yoon; J. Microbiol. 2005;43(2):196-203.; DOI: https://doi.org/2161 [pii]

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Abstract PDF: Here, we attempted to evaluate the activity of artificially transconjugated multiple plasmids in “designer biocatalysts” for the bioremediation of cocontaminated sites under nonselective conditions. We observed profound losses in the percent survivals of artificially transconjugated plasmid activity (66 - 78% loss immediately after freeze-drying, 99.45 - 99.88% loss by the end of 6 months storage) in reconstituted Pseudomonas sp. KM12TC. Such unpredictable high losses of this particular plasmid appeared to clearly be a deleterious effect. However, even after 6 months of storage, the cells remained able to degrade 95% of phenol within 9 days, and the full efflux of ^73As, as compared to that of the non-freeze-dried cells, was successfully achieved 4 to 9 days later. These results indicate that “stable designer biocatalysts” can remain viable, even after freeze-drying and 6 months of storage.

Effect of Temperature on Persistence of Recombinant Plasmid pCU103 in Different Waters: Kwak, Myong Ja , Kim, Chi Kyung , Kim, Young Chang , Lim, Jae Yun , Kim, Young Soo , Lee, Ki Sung , Min, Kyung Hee; J. Microbiol. 1995;33(3):178-183.

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Abstract PDF: The recombinant plasmid of pCU103 constructed by cloning pcbCD genes in pBluescript SK(+) was studied for the effect of temperature on its persistence in different waters by the methods of electrophoresis, Southern hybridization, quantification, and transformation. The plasmid was very rapidly degraded out in non-sterile FW water without regards to water temperature, probably due to the effect of biochemical factor such as nucleases. The pCU103 was most persistent at 4℃ in any water environments, moderately persistant at 15℃, but least stable at 30℃ such results could be explained by the facts that hydrogen bonds in double-stranded plasmid DNAs become unstable and that nucleases are activated by increasing temperature. The intact structure of pCU1-3 was generally observed by gel electrophoresis under the conditions which the plasmid should be 2.0 ng/㎕ or higher in concentration and that about 10² CFU/ml or more transformant cells should be recovered.

Effects of Genetically Different 2.4-D-degradative Plasmids on Degradation Phenotype and Competitiveness of Soil Microorganisms: Hong, Seok Myeong , Ahn, Young Joon , Park, Yong Keun , Min, Kyung Hee , Kim, Chi Kyung , Ka, Jong Ok; J. Microbiol. 1995;33(3):208-214.

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Abstract PDF: The effects of various 2, 4-D-degradative plasmids on the axenic growth patterns, the degradation phenotypes, and the competitiveness of different host bacteria were evaluated in liquid cultures; the organisms and plasmids used were Alcaligenes eutrophus JMP134/pJP4, Alcaligenes paradoxus/p2811, Pseudomonas pickettii/p712, pJP4, and p712 or p 2811 exhibited very different restriction fragment profiles in restriction endonuclease digests. These plasmids were transferred to the recipients (P. cepacia and Alcaligenes JMP228) at relatively high frequencies ranging from 8.9 × 10^-3 to 1.6 × 10^-5 per donor cell. In the axenic liquid cultures the fast-growing strains, such as P. pseudomallei/p745 and P. cepacia/pJP4, exhibited short lag periods, high specific growth rates, and high relative fitness coefficients, while the slow-growing strains, such as P. pickettii/p712 and A. paradoxus/p2811, had long lag periods, low specific growth rates, and low relative fitness coefficients. Depending on the type of plasmid containing the genes for the 2, 4-D pathway, some transconjugants exhibited intermediate growth patterns between the fast-growing strains and the slow-growing strains. The plasmid and plasmid-host interactions determined specific growth rate and lag time, respectively, which were shown to be principal determinants of competitiveness among the strains, but relative fitness coefficient derived from the axenic culture was not always predictive for the mixed culture condition.

Structural and functional stability of the genetic recombinant plasmid pCU103 in different water environments: Kim, Chi Kyung , Kwak, Myoung Ja , Lee, Sung Gie; J. Microbiol. 1996;34(3):241-247.

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Abstract PDF: The stbility of the genetically engineered microorganisms and their recombinant plasmids released in natural environments has been regarded as one of the molecular ecological topics. In this study, the recombinant plasmids pCU103 in which the pcbCD genes involved in biodegradation of biphenyl and 4-chlorobiphenyl were cloned in pBluescript SK(+) vector, were examined for their structural and functional stability in different waters at 15℃ by the methods of electrophoresis, Southern hybridization, quantification with fluorescent dye, and transformation. The recombinant plamids maintained their stabilities for about 30 days in sterilized distilled water (SDW), 15 days in autoclaved creek water (AW), 25 days in filtered and autoclaved non-sterible creek water (FAW), 4 days in Luria-Bertani (LB) broth, and less than one day in filtered non-sterile creek water (FW). The covalently closed circular (CCC) form of the plasmid was decreased and open circular (OC) form was increased as a function of incubation time, and then linear (L) form was produced to be ultimately degraded out. The degradation rates of the plasmid were proportionally correlated to trophic level of the water, and the biological factor such as DNases was found to be one of the most critical factors affecting structural and functional stability of the plasmid in non-sterile natural water.

Fate of genetically engineered 2,4-D-Degrading microorganisms in natural soils and waters: Hong, Seok Myeong , Lee, Yin Won , Kim, Chi Kyung , Ka, Jong Ok; J. Microbiol. 1996;34(4):320-326.

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Abstract PDF: To analyze the effects of host versus plasmid on survival of 2,4-degrading bacteria in environmental samples, strains Pseudomonas cepacia/pJP4, Alcaligenes JMP228/pJP4, P. cepacia/p712, and Alcaligenes JMP228/p712 were separately inoculated into samples of field soil, paddy soil, lake water, and river water, and then the changes of their populations were measured. The strains used contained a 2,4-D degradative plasmid, either pJP4 conferring fast-growing property to the host or p712 conferring slow-growing property, and were resistant to antibiotics such that the inoculated strains could be enumerated against the indigenous microbial populations. In sterile environmental samples, these strains were stably maintained at the levels used for inoculation, except in sterile paddy soil where Alcaligenes JMP228 strains died drapidly. In natural soil samples for four strains declined steadily with time, but in natural water samples their populations fell rapidly at the early phase and then remained almost constant. When the environmental samples were treated with 2, 4-D, P. cepacia/pJP4 and P. cepacia/p712 maintained significant numbers, while Alcaligenes JMP228/pJP4 and Alcaligenes JMP228/p712 declined significantly in most of the samples. The results indicated that the survivability of genetically modified microorganisms could vary depending on the environments and that their abundance in the environments under 2,4-D selection was markedly influenced by the nature of the 2,4-D degradative plasmid as well as type of the host strain.

A plasmid vector faciliting gene expression in both yeast and mammalian cells: Lee , Tae Ho; J. Microbiol. 1997;35(2):149-151.

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Abstract PDF: A plasmid vector with combined features of yeast shuttle vector and mammalian expression vector was constructed to facilitate expression of cloned gene in both cell-types. All necessary elements required for plasmid maintenance and selection in E. coli, yeast and mammalian cells were size-economically arranged in this plasmid. The human cytomegalovirus (CMV) immediate early promoter and yeast GAL1 promoter were sequentially placed in front of the gene to be expressed. The synthetic splicing donor and acceptor sequences were inserted into the immediate upstream and downstream of the GAL1 promotor, allowing the CMV promotor to direct the expression of a given gene in mammalian cell environment by splicing out the interfering GAL1 promotor sequence. When the resulting vector containing LacZ as a gene was introduced into yeast and mammalian cells, both cells efficiently produced β-galactosidase, dimonstrating its dual host usage.

The Genetic Organization of the Linear Mitochondrial Plasmid mlp1 from Pleurotus ostreatus NFFA2: Kim, Eun Kyung , Youn, Hye Sook , Koo, Yong Bom , Roem Jung Hye; J. Microbiol. 1997;35(4):264-270.

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Abstract PDF: The structure of plasmid mlp1, a linear 10.2kb mitochondrial plasmid of Pleurotus ostreatus NFF A2 was determined by restriction enzyme mapping and partial sequencing. The plasmid encodes at least two proteins; a putative RNA polymerase showing homology to yeast mitochondrial RNA polymerase and to viral-encoded RNA polymerases, and a putative DNA polymerase showing significant homology to the family B thpe DNA polymerases. It also contains terminal inverted repeat sequences at both ends which are longer than 274 bp. A 1.6 kb EcoRI restriction fragment of m1p1 containing the putative RNA polymerase gene did not hybridize to the nuclear or motochondrial genomes from P. ostreatus, suggesting that it may encode plasmidspecific RNA polymerase. The gene fragment also did not hybridize with the RNA polymerase gene (RPO41) from Saccaromyces cerevisiae. The relationship between genes in m1p1 and those in another linear plasmid pC1K1 of Claviceps purpurea was examined by DNA hybridization. The result indicates that the genes for DNA and RNA polymerases are not closely related with those in C. purpurea.

Function of mORF1 Protein as a Terminal Recognition Factor for the Linear Mitochondrial Plasmid pMLP1 from Pleurotus ostreatus: Eun-Kyoung Kim , Jung-Hye Roe; J. Microbiol. 1999;37(4):229-233.

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Abstract PDF: The mitochondrial plasmid pMLP1 from a white-rot fungus, Pleurotus ostreatus, is a double-stranded DNA containing 381 bp terminal inverted repeat (TIR) whose 5'-ends are covalently bound by terminal proteins. The plasmid contains two major open reading frames (ORFs), encoding putative DNA and RNA polymerases, and a minor ORF encoding a small, highly basic protein. To identify the DNA binding activity that recognizes the TIR region of pMLP1, gel retardation assays were performed with mitochondrial extracts. A specific protein binding to a region between 123 and 248 nt within TIR was observed. We examined whether the gene product of mORF1 bindes to this region specifically. E. coli cell extract which contains an overproduced mORF1 protein formed a complex specific to the region between 123 and 248 nt. Inclusion of mORF1 protein in the specific complex formed between P. ostreatus mitochondrial extract and TIR was confirmed by a supershift assay using polyclonal antibodies against the mORF1 protein. Our result suggest that the product of mORF1 may function as a terminal region recognition factor (TRF), recognizing an internal region in TIR.

Use of the Yeast 1.5-Hybrid System to Detect DNA-Protein-Protein Interactions: Sook-Kyung Kim , Jin Hee Han; J. Microbiol. 2000;38(2):113-116.

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Abstract PDF: Escherichia coli F plasmid partition apparatus is composed of two trans-acting proteins (SopA and SopB) and one cis-acting DNA sequence (sopC). The SopB-sopC complex has been suggested to serve a centromere-like function through its interaction with chromosomally encoded proteins which remain to be identified. In this paper, we are introducing a new yeast 1.5-hybrid system which assembles the two-hybrid and one-hybrid system as a mean to find an additional component of the F plasmid partition system, interacting with DNA (sopC)-bound SopB protein. The result indicates that this system is a promising one, capable of selecting an interacting component.

Stable Secretion Vector Derived from the RCR (rolling-circle replication) Plasmid of Bacillus mesentericus: Seung-Soo Lee , Jeong-Sun Han , In Hyung Lee , Young- Yel l Yang , Soon-Kwang Hong , Joo-Won Suh; J. Microbiol. 2002;40(2):140-145.

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Abstract PDF: The 5.8 kb pMMH1, rolling-circle replication (RCR) plasmid of the wild type soil Bacillus mesentericus was developed into a novel secretion vector system in Bacillus subtilis. The pMMH1 turned out to have a replication origin and two open reading frames (ORFs) of the putative [gamma]-GTP and type I signal peptidase (sipP). To characterize the regions necessary for plasmid stability and high copy number, five vectors (pPS, pPP, pEN, pMN, pME) were constructed by disruption or deletion of each region in pMMH1. Like pMMH1, all constructed vectors were stable over 100 generations in a non-selective medium. Since pPS was the smallest (2.3 kb)of all, it was selected for the construction of a novel secretion vector. Using the [alpha]-amylase promoter/signal sequence of B. subtilils, the novel plasmid pJSN was constructed. When [beta]-glucosidase was expressed using pJSN, we found [beta]-glucosidase activity in the medium. This result strongly suggested that plasmid pJSN can be used for the production of bioactive peptides in B. subtilis.

Cloning of Genomic DNAs of Trametes versicolor Acting as Autonomously Replicating Sequences in Saccharomyces cerevisiae: Sora An , Kyoung Phil Park , Hyoung-Tae Choi , Kyu-Joong Kim , Kyunghoon Kim; J. Microbiol. 2002;40(3):245-247.

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Abstract PDF: A genomic DNA library of the fungus Trametes versicolor was constructed in a yeast integration vector which contains the URA3 gene of the budding yeast Saccharomyces cerevisiae and the gene responsible for hygromycin B resistance, and fragments acting as autonomously replicating sequences (ARSes) in the budding yeast were identified from the genomic DNA library. Sixteen recombinant plasmids from the library transformed the budding yeast Saccharomyces cerevisiae to Ura^+ at high frequencies. They were maintained stably under selective conditions, but were gradually lost from yeast cells at different rates under nonselective conditions, indicating that they contain eukaryotic origins of DNA replication and exist as extrachromosomal plasmids. Base sequences of four ARS DNAs among the 16 cloned fragments revealed that all of the four contain at least one 11 bp [(A/T)TTTA(T/C)(A/G)TTT(A/T)] consensus sequence of the budding yeast ARS.

Structural Analysis of the fcbABC Gene Cluster Responsible for Hydrolytic Dechlorination of 4-Chlorobenzoate from pJS1 Plasmid of Comamonas sp. P08: Jeong-Soon Lee , Kyoung Lee , Jong-Ok Ka , Jong-Chan Chae , Chi-Kyung Kim; J. Microbiol. 2003;41(2):89-94.

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Abstract PDF: Bacterial strain No. P08 isolated from wastewater at the Cheongju industrial complex was found to be capable of degrading 4-chlorobenzoate under aerobic condition. P08 was identified as Comamonas sp. from its cellular fatty acid composition and 16S rDNA sequence. The fcb genes, responsible for the hydrolytic dechlorination of 4-chlorobenzoate, were cloned from the plasmid pJS1 of Comamonas sp. P08. The fcb gene cluster of comamonas sp. P08 was organized in the order fcbB-fcbA-fcbT1-fcbT2-fcbT3-fcbC. This organization of the fcb genes was very similar to that of the fcb genes carried on the chromosomal DNA of Pseudomonas sp. DJ-12. However, it differed from the fcbA-fcbB-fcbC ordering of Arthrobacter sp. SU. The nucleotide sequences of the fcbABC genes of strain P08 showed 98% and 53% identities to those of Pseudomonas sp. DJ-12 and Arthrobacter sp. SU, respectively. This suggests that the fcb genes might have been derived from Pseudomonas sp. DJ-12 to form plasmid pJS1 in Comamonas sp. P08, or that the fcb genes in strain DJ-12 were transposed from Comamonas sp. P08 plasmid.

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