Establishment of Tn5096-Based Transposon Mutagenesis in Gordonia polyisoprenivorans
Establishment of Tn5096-Based Transposon Mutagenesis in Gordonia polyisoprenivorans
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基于 Tn5096 的 Gordonia polyisoprenivoran 转座子诱变的建立
DOI：
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发表时间：
2005
影响因子：
4.4
通讯作者：
A. Steinbüchel
中科院分区：
文献类型：
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作者：
Q. Banh;M. Arenskötter;A. Steinbüchel
摘要研究了转座子Tn5、Tn10、Tn611和Tn5096在多异丙叉大头藻VH2菌株中的转座特性。使用Tn5或Tn10均未获得插入突变体。携带Tn611的温敏质粒pCG79已整合到多异戊二烯吸虫的染色体中，但插入的突变体很不稳定，经常恢复为野生型表型。相反，通过Tn5096介导的转座子诱变获得了各种稳定的突变体。通过将含有Tn5096的pMA5096作为一个整体整合到基因组中，获得了营养缺陷型、类胡萝卜素生物合成缺陷突变株和橡胶利用缺陷突变株和/或高度支化的类异戊二烯烃利用突变株。从分离的约25,000个突变体中，pMA5096的插入位点随后被定位在20个独立突变体中，这些基因可能与上述代谢途径或可能的调节蛋白有关。对pMA5096介导的聚顺-1，4-异戊二烯生物降解缺陷突变体的基因分析没有发现与最近发现的催化聚顺-1，4-异戊二烯初始裂解的酶的基因同源。一个橡胶阴性突变体在mcr中被破坏，编码一个α-甲基酰基辅酶A消旋酶。该突变株对聚顺-1，4-异戊二烯和高度支化的异戊二烯类碳氢化合物均有降解缺陷。
ABSTRACT The transposons Tn5, Tn10, Tn611, and Tn5096 were characterized regarding transposition in Gordonia polyisoprenivorans strain VH2. No insertional mutants were obtained employing Tn5 or Tn10. The thermosensitive plasmid pCG79 harboring Tn611 integrated into the chromosome of G. polyisoprenivorans; however, the insertional mutants were fairly unstable und reverted frequently to the wild-type phenotype. In contrast, various stable mutants were obtained employing Tn5096-mediated transposon mutagenesis. Auxotrophic mutants, mutants defective or deregulated in carotenoid biosynthesis, and mutants defective in utilization of rubber and/or highly branched isoprenoid hydrocarbons were obtained by integration of plasmid pMA5096 harboring Tn5096 as a whole into the genome. From about 25,000 isolated mutants, the insertion loci of pMA5096 were subsequently mapped in 20 independent mutants in genes which could be related to the above-mentioned metabolic pathways or to putative regulation proteins. Analyses of the genotypes of pMA5096-mediated mutants defective in biodegradation of poly(cis-1,4-isoprene) did not reveal homologues to recently identified genes coding for enzymes catalyzing the initial cleavage of poly(cis-1,4-isoprene). One rubber-negative mutant was disrupted in mcr, encoding an α-methylacyl-coenzyme A racemase. This mutant was defective in degradation of poly(cis-1,4-isoprene) and also of highly branched isoprenoid hydrocarbons.
影响因子：
2.9
作者：
Krebs, C;Agar, JN;Johnson, MK
通讯作者：
Johnson, MK