Construction,properties, and application of the pCB5 plasmid,a novel conjugative shuttle vector with a Cupriavidus basilensis origin of replication

Cupriavidus basilensis is a species with diverse metabolic capabilities, including degradation of xenobiotics and heavy metal resistance. Although the genomes of several strains of this species have been sequenced, no plasmid has yet been constructed for genetic engineering in this species. In this study, we identified a novel plasmid, designated pWS, from C. basilensis WS with a copy number of 1–3 per cell and a length of 2150 bp. pWS contained three protein-coding genes, among which only rep was required for plasmid replication. Rep showed no homology with known plasmid replication initiators. Unlike most plasmids, pWS did not have a cis-acting replication origin outside the region of rep. The minimal replicon of pWS was stable in C. basilensis WS without selection. A conjugative C. basilensis/Escherichia coli shuttle vector, pCB5, was constructed using the minimal replicon of pWS. Interestingly, the copy number of pCB5 was flexible and could be manipulated. Enhancing the expression level of Rep in pCB5 by either doubling the promoter or coding region of rep resulted in doubling of the plasmid copy number. Moreover, replacing the native promoter of rep with the lac promoter increased the copy number by over fivefold. Finally, using two different β-galactosidase reporting systems constructed with pCB5, we successfully demonstrated the different regulatory patterns of bph and dmp operons during diphenyl ether (DE) degradation in C. basilensis WS. Thus, this shuttle vector provided an efficient tool for DNA cloning and metabolic engineering in C. basilensis.

     

我国东北地区发现姬鼩鼱分布

2014年在黑龙江省横道河子地区(44°48′44″N,129°02′04″E,海拔约740 m)采集到1只鼩鼱(标本编号为CH5)。2015年在内蒙古自治区达赉湖地区(48°37′20″N,117°53′17″E,海拔约720 m)采集2只鼩鼱(标本编号为DE7和DE12)。这些新获标本经鉴定为姬鼩鼱(Sorex minutissimus)。《小鼩鼱(食虫目:鼩鼱科)辽宁省新纪录》文中的标本(080910,090920)经重新鉴定也为姬鼩鼱。利用mt DNA的Cyt b基因全序列构建系统进化树,结果揭示,小鼩鼱聚为一支,姬鼩鼱聚为另一支,新获标本(CH5、DE7、DE12)和待厘定标本(080910、090920)都聚在姬鼩鼱一支,进一步支持形态学鉴定结果。2015年采集的姬鼩鼱为内蒙古自治区新纪录,而《小鼩鼱(食虫目:鼩鼱科)辽宁省新纪录》文中的小鼩鼱(Sorex minutus)(标本号:080910,090920)更正为辽宁省姬鼩鼱新纪录。