中国科学院机构知识库网格
Chinese Academy of Sciences Institutional Repositories Grid
Type I restriction-modification system and its resistance in electroporation efficiency in Flavobacterium columnare

文献类型:期刊论文

作者Li, N.; Zhang, L. Q.; Zhang, J.; Liu, Z. X.; Huang, B.; Zhang, S. H.; Nie, P.
刊名VETERINARY MICROBIOLOGY
出版日期2012-11-09
卷号160期号:1-2页码:61-68
关键词Restriction modification system Type I R-M system Flavobacterium columnare Electroporation efficiency
ISSN号0378-1135
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.
中文摘要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. 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.
WOS标题词Science & Technology ; Life Sciences & Biomedicine
类目[WOS]Microbiology ; Veterinary Sciences
研究领域[WOS]Microbiology ; Veterinary Sciences
关键词[WOS]CATFISH ICTALURUS-PUNCTATUS ; CHANNEL CATFISH ; DNA METHYLTRANSFERASES ; GENETIC DIVERSITY ; RNA-GENE ; FISH ; SEQUENCE ; PSYCHROPHILUM ; FLEXIBACTER ; PLASMID
收录类别SCI
资助信息National Basic Research Program of China (973 Program) [2009CB118703]
语种英语
WOS记录号WOS:000310418300009
公开日期2013-01-09
源URL[http://ir.ihb.ac.cn/handle/342005/17224]  
专题水生生物研究所_鱼类生物学及渔业生物技术研究中心_期刊论文
作者单位Chinese Acad Sci, Stare Key Lab Freshwater Ecol & Biotechnol, Inst Hydrobiol, Wuhan 430072, Hubei Province, Peoples R China
推荐引用方式
GB/T 7714
Li, N.,Zhang, L. Q.,Zhang, J.,et al. Type I restriction-modification system and its resistance in electroporation efficiency in Flavobacterium columnare[J]. VETERINARY MICROBIOLOGY,2012,160(1-2):61-68.
APA Li, N..,Zhang, L. Q..,Zhang, J..,Liu, Z. X..,Huang, B..,...&Nie, P..(2012).Type I restriction-modification system and its resistance in electroporation efficiency in Flavobacterium columnare.VETERINARY MICROBIOLOGY,160(1-2),61-68.
MLA Li, N.,et al."Type I restriction-modification system and its resistance in electroporation efficiency in Flavobacterium columnare".VETERINARY MICROBIOLOGY 160.1-2(2012):61-68.

入库方式: OAI收割

来源:水生生物研究所

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