Kobayashi Y, Forster R J, Hefford M A, Teather R M, Wakita M, Ohmiya K, Hoshino S
Faculty of Bioresources, Mie University, Tsu, Japan.
FEMS Microbiol Lett. 1995 Aug 1;130(2-3):137-43. doi: 10.1111/j.1574-6968.1995.tb07710.x.
A small cryptic plasmid, pRJF2, from Butyrivibrio fibrisolvens strain OB157 was isolated and sequenced. The plasmid is similar in organisation to the previously sequenced Butyrivibrio plasmid, pRJF1, with two open reading frames, ORF1 and ORF2, flanking a region tentatively identified as the replication origin, and a region of unknown function defined by terminal 79 bp invert repeats. The sequences of ORF1, ORF2, and the presumptive replication origin are highly conserved. The sequence between the 79, bp invert repeats is not, and is therefore presumed to be of lesser functional significance, although the 5' and 3' termini are still highly conserved. The functional importance for plasmid replication of these regions was tested by constructing potential shuttle vectors, each lacking one or more of the regions of interest. When the region between the invert repeats was deleted and replaced by the erythromycin resistance gene from pAM beta 1 together with pUC18, to produce the 7.9 kb chimaeric plasmid pYK4, the construct was successfully transformed into E. coli and B. fibrisolvens by electroporation, and was stably maintained in both hosts. Both ORF1 and ORF2 were required for successful transformation of B. fibrisolvens.
从溶纤维丁酸弧菌OB157菌株中分离并测序了一个小的隐蔽质粒pRJF2。该质粒在结构上与先前测序的丁酸弧菌质粒pRJF1相似，有两个开放阅读框ORF1和ORF2，两侧是一个初步确定为复制起点的区域，以及一个由79 bp末端反向重复序列定义的功能未知区域。ORF1、ORF2和推测的复制起点的序列高度保守。79 bp反向重复序列之间的序列则不然，因此推测其功能意义较小，尽管5'和3'末端仍然高度保守。通过构建潜在的穿梭载体来测试这些区域对质粒复制的功能重要性，每个载体都缺失一个或多个感兴趣的区域。当反向重复序列之间的区域被删除并用来自pAM beta 1的红霉素抗性基因与pUC18一起取代，以产生7.9 kb的嵌合质粒pYK4时，该构建体通过电穿孔成功转化到大肠杆菌和溶纤维丁酸弧菌中，并在两种宿主中稳定维持。溶纤维丁酸弧菌的成功转化需要ORF1和ORF2。
