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题名: Type I restriction-modification system and its resistance in electroporation efficiency in Flavobacterium columnare
作者: Li, N.1; Zhang, L. Q.1; Zhang, J.1; Liu, Z. X.1; Huang, B.1; Zhang, S. H.1; Nie, P.1
通讯作者: Nie, P (reprint author), Chinese Acad Sci, Stare Key Lab Freshwater Ecol & Biotechnol, Inst Hydrobiol, Wuhan 430072, Hubei Province, Peoples R China.
关键词: Restriction modification system ; Type I R-M system ; Flavobacterium columnare ; Electroporation efficiency
刊名: VETERINARY MICROBIOLOGY
发表日期: 2012-11-09
DOI: 10.1016/j.vetmic.2012.04.045
卷: 160, 期:1-2, 页:61-68
收录类别: SCI
文章类型: Article
部门归属: [Li, N.; Zhang, L. Q.; Zhang, J.; Liu, Z. X.; Huang, B.; Zhang, S. H.; Nie, P.] Chinese Acad Sci, Stare Key Lab Freshwater Ecol & Biotechnol, Inst Hydrobiol, Wuhan 430072, Hubei Province, Peoples R China
WOS标题词: Science & Technology ; Life Sciences & Biomedicine
资助者: National Basic Research Program of China (973 Program) [2009CB118703]
类目[WOS]: Microbiology ; Veterinary Sciences
研究领域[WOS]: Microbiology ; Veterinary Sciences
摘要: Flavobacterium columnare, the causative agent of columnaris disease, infects freshwater fish worldwide. However, the pathogenicity of this bacterium is poorly understood due possibly to the lack of an efficient in-frame knockout technique. In order to improve electroporation efficiency, the type I restriction-modification system (R-M system) was cloned and its role in electroporation was examined in F. columnare G(4) strain. The complete sequence of type I R-M system in the bacterium, designated as Fcl, contains all three subunits of type I R-M system, named as fclM, fclS, fclR, respectively, with the identification of a hypothetical gene, fclX. Constitutive transcription of the three genes was observed in F. columnare G(4) by RT-PCR. The ORF of fclM and fclS was cloned into the plasmid pACYC184 and transformed into Escherichia colt TOP10. The resultant E. coli strain, designated as E. coli TOPmt, was transformed with the integrative plasmid pGL006 constructed for F. columnare G(4). The integrative plasmid was re-isolated from TOPmt and incubated with the lysate of F. columnare G(4). The re-isolated integrative plasmid, designated as pGL006', showed higher resistance than pGL006. With pGL006', the electroporation efficiency of the strain G(4) increased 2.6 times, while that of F. columnare G(18) was not obviously improved. Furthermore, a method to improve the electroporation efficiency of F. columnare G(4) was developed using the integrative plasmid methylated by E. coil TOPmt which contains the fclM and fclS gene of F. columnare G(4). Further analyses showed that the fcl gene cluster may be a unique type I R-M system in F. columnare G(4). It will be of significant interest to examine the composition and diversity of R-M systems in strains of F. columnare in order to set up a suitable genetic manipulation system for the bacterium. (C) 2012 Elsevier B.V. All rights reserved.
英文摘要: Flavobacterium columnare, the causative agent of columnaris disease, infects freshwater fish worldwide. However, the pathogenicity of this bacterium is poorly understood due possibly to the lack of an efficient in-frame knockout technique. In order to improve electroporation efficiency, the type I restriction-modification system (R-M system) was cloned and its role in electroporation was examined in F. columnare G(4) strain. The complete sequence of type I R-M system in the bacterium, designated as Fcl, contains all three subunits of type I R-M system, named as fclM, fclS, fclR, respectively, with the identification of a hypothetical gene, fclX. Constitutive transcription of the three genes was observed in F. columnare G(4) by RT-PCR. The ORF of fclM and fclS was cloned into the plasmid pACYC184 and transformed into Escherichia colt TOP10. The resultant E. coli strain, designated as E. coli TOPmt, was transformed with the integrative plasmid pGL006 constructed for F. columnare G(4). The integrative plasmid was re-isolated from TOPmt and incubated with the lysate of F. columnare G(4). The re-isolated integrative plasmid, designated as pGL006', showed higher resistance than pGL006. With pGL006', the electroporation efficiency of the strain G(4) increased 2.6 times, while that of F. columnare G(18) was not obviously improved. Furthermore, a method to improve the electroporation efficiency of F. columnare G(4) was developed using the integrative plasmid methylated by E. coli TOPmt which contains the fclM and fclS gene of F. columnare G(4). Further analyses showed that the fcl gene cluster may be a unique type I R-M system in F. columnare G(4). It will be of significant interest to examine the composition and diversity of R-M systems in strains of F. columnare in order to set up a suitable genetic manipulation system for the bacterium. (C) 2012 Elsevier B.V. All rights reserved.
关键词[WOS]: CATFISH ICTALURUS-PUNCTATUS ; CHANNEL CATFISH ; DNA METHYLTRANSFERASES ; GENETIC DIVERSITY ; RNA-GENE ; FISH ; SEQUENCE ; PSYCHROPHILUM ; FLEXIBACTER ; PLASMID
语种: 英语
WOS记录号: WOS:000310418300009
ISSN号: 0378-1135
Citation statistics:
内容类型: 期刊论文
URI标识: http://ir.ihb.ac.cn/handle/342005/17224
Appears in Collections:鱼类生物学及渔业生物技术研究中心_期刊论文

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作者单位: 1.Chinese Acad Sci, Stare Key Lab Freshwater Ecol & Biotechnol, Inst Hydrobiol, Wuhan 430072, Hubei Province, Peoples R China

Recommended Citation:
Li, N.; Zhang, L. Q.; Zhang, J.; Liu, Z. X.; Huang, B.; Zhang, S. H.; Nie, P..Type I restriction-modification system and its resistance in electroporation efficiency in Flavobacterium columnare,VETERINARY MICROBIOLOGY,2012,160(40910):61-68
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