attB site disruption in marine Actinomyces sp M048 via DNA transformation of a site-specific integration vector

doi:10.1042/BA20070124

IOCAS-IR > 实验海洋生物学重点实验室

	attB site disruption in marine Actinomyces sp M048 via DNA transformation of a site-specific integration vector
	Hou, Yan-Hua 1,2; Wang, Quan-Fu 2; Ding, Ling 1; Li, Fu-Chao 1; Qin, Song 1
	2008-05-01
发表期刊	BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY
ISSN	0885-4513
卷号	50 期号:Part 1 页码:11-16
文章类型	Article
摘要	An efficient conjugation method has been developed for the marine Actinomyces sp. isolate M048 to facilitate the genetic manipulation of the chandrananimycin biosynthesis gene cluster. A phi C31-derived integration vector pIJ8600 containing oriT and attP fragments was introduced into strain M048 by bi-parental conjugation from Escherichia coli ET12567 to strain M048. Transformation efficiency was (6.38 +/- 0.41) x 10(-5) exconjugants per recipient spore. Analysis of eight exconjugants showed that the plasmid pIJ8600 was stably integrated at a single chromosomal site (attB) of the Actinomyces genome. The DNA sequence of the attB was cloned and shown to be conserved. The results of antimicrobial activity analysis indicated that the insertion of plasmid pIJ8600 seemed to affect the biosynthesis of antibiotics that could strongly inhibit the growth of E. coli and Mucor miehei (Tu284). HPLC-MS analysis of the extracts indicated that disruption of the attB site resulted in the complete abolition of chandrananimycin A-C production, proving the identity of the gene cluster. Instead of chandrananimycins, two bafilomycins were produced through disruption of the attB site from the chromosomal DNA of marine Actinomyces sp. M048.; An efficient conjugation method has been developed for the marine Actinomyces sp. isolate M048 to facilitate the genetic manipulation of the chandrananimycin biosynthesis gene cluster. A phi C31-derived integration vector pIJ8600 containing oriT and attP fragments was introduced into strain M048 by bi-parental conjugation from Escherichia coli ET12567 to strain M048. Transformation efficiency was (6.38 +/- 0.41) x 10(-5) exconjugants per recipient spore. Analysis of eight exconjugants showed that the plasmid pIJ8600 was stably integrated at a single chromosomal site (attB) of the Actinomyces genome. The DNA sequence of the attB was cloned and shown to be conserved. The results of antimicrobial activity analysis indicated that the insertion of plasmid pIJ8600 seemed to affect the biosynthesis of antibiotics that could strongly inhibit the growth of E. coli and Mucor miehei (Tu284). HPLC-MS analysis of the extracts indicated that disruption of the attB site resulted in the complete abolition of chandrananimycin A-C production, proving the identity of the gene cluster. Instead of chandrananimycins, two bafilomycins were produced through disruption of the attB site from the chromosomal DNA of marine Actinomyces sp. M048.
关键词	Antimicrobial Activity Attb Site Of Actinomyces Strain M048 Bafilomycin Chandrananimycin Dna Transformation Marine Actinomyces
DOI	10.1042/BA20070124
URL	查看原文
收录类别	SCI
语种	英语
WOS记录号	WOS:000255677400002
引用统计	被引频次：2[WOS] [WOS记录] [WOS相关记录]
文献类型	期刊论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/6278
专题	实验海洋生物学重点实验室
作者单位	1.Acad Sinica, Inst Oceanol, Qingdao 266071, Peoples R China 2.Harbin Inst Technol, Sch Ocean, Weihai 264209, Peoples R China
第一作者单位	中国科学院海洋研究所
推荐引用方式 GB/T 7714	Hou, Yan-Hua,Wang, Quan-Fu,Ding, Ling,et al. attB site disruption in marine Actinomyces sp M048 via DNA transformation of a site-specific integration vector[J]. BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY,2008,50(Part 1):11-16.
APA	Hou, Yan-Hua,Wang, Quan-Fu,Ding, Ling,Li, Fu-Chao,&Qin, Song.(2008).attB site disruption in marine Actinomyces sp M048 via DNA transformation of a site-specific integration vector.BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY,50(Part 1),11-16.
MLA	Hou, Yan-Hua,et al."attB site disruption in marine Actinomyces sp M048 via DNA transformation of a site-specific integration vector".BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY 50.Part 1(2008):11-16.

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