IHB OpenIR  > 鱼类生物学及渔业生物技术研究中心  > 期刊论文
Type I restriction-modification system and its resistance in electroporation efficiency in Flavobacterium columnare
Li, N.; Zhang, L. Q.; Zhang, J.; Liu, Z. X.; Huang, B.; Zhang, S. H.; Nie, P.; Nie, P (reprint author), Chinese Acad Sci, Stare Key Lab Freshwater Ecol & Biotechnol, Inst Hydrobiol, Wuhan 430072, Hubei Province, Peoples R China.
2012-11-09
Source PublicationVETERINARY MICROBIOLOGY
ISSN0378-1135
Volume160Issue:1-2Pages:61-68
AbstractFlavobacterium 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.; 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.
SubtypeArticle
KeywordRestriction Modification System Type i R-m System Flavobacterium Columnare Electroporation Efficiency
Department[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
DOI10.1016/j.vetmic.2012.04.045
WOS HeadingsScience & Technology ; Life Sciences & Biomedicine
Funding OrganizationNational Basic Research Program of China (973 Program) [2009CB118703] ; National Basic Research Program of China (973 Program) [2009CB118703] ; National Basic Research Program of China (973 Program) [2009CB118703] ; National Basic Research Program of China (973 Program) [2009CB118703]
Indexed BySCI
Language英语
WOS Research AreaMicrobiology ; Veterinary Sciences
WOS SubjectMicrobiology ; Veterinary Sciences
WOS IDWOS:000310418300009
WOS KeywordCATFISH ICTALURUS-PUNCTATUS ; CHANNEL CATFISH ; DNA METHYLTRANSFERASES ; GENETIC DIVERSITY ; RNA-GENE ; FISH ; SEQUENCE ; PSYCHROPHILUM ; FLEXIBACTER ; PLASMID
Funding OrganizationNational Basic Research Program of China (973 Program) [2009CB118703] ; National Basic Research Program of China (973 Program) [2009CB118703] ; National Basic Research Program of China (973 Program) [2009CB118703] ; National Basic Research Program of China (973 Program) [2009CB118703]
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Document Type期刊论文
Identifierhttp://ir.ihb.ac.cn/handle/342005/17224
Collection鱼类生物学及渔业生物技术研究中心_期刊论文
Corresponding AuthorNie, P (reprint author), Chinese Acad Sci, Stare Key Lab Freshwater Ecol & Biotechnol, Inst Hydrobiol, Wuhan 430072, Hubei Province, Peoples R China.
AffiliationChinese Acad Sci, Stare Key Lab Freshwater Ecol & Biotechnol, Inst Hydrobiol, Wuhan 430072, Hubei Province, Peoples R China
Recommended Citation
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.
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