title: Construction, properties, and application of the pCB5 plasmid, a novel conjugative shuttle vector with a Cupriavidus basilensis origin of replication.
abstract: 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.
authors: College of Life Sciences, Zhejiang University, 310058, Hangzhou, Zhejiang, People's Republic of China.
authors: Sheng
authors: S
authors: Wang
authors: College of Life Sciences, Zhejiang University, 310058, Hangzhou, Zhejiang, People's Republic of China.
authors: Mei-Nan
authors: MN
authors: Zhang
authors: College of Life Sciences, Zhejiang University, 310058, Hangzhou, Zhejiang, People's Republic of China.
authors: Na-Ling
authors: NL
authors: Bai
authors: Polar Research Institute of China, 200136, Shanghai, People's Republic of China.
authors: Hai-Tao
authors: HT
authors: Ding
authors: College of Life Sciences, Zhejiang University, 310058, Hangzhou, Zhejiang, People's Republic of China.
authors: Xu-Fen
authors: XF
authors: Zhu
authors: College of Life Sciences, Zhejiang University, 310058, Hangzhou, Zhejiang, People's Republic of China. yhzhao225@zju.edu.cn.
authors: Yu-Hua
authors: YH
authors: Zhao
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular; D049921:Cupriavidus / Q000235:genetics*; D004925:Escherichia / Q000235:genetics; D018628:Gene Dosage; D005818:Genetic Engineering; D005822:Genetic Vectors; D060847:Metabolic Engineering; D009876:Operon; D010647:Phenyl Ethers / Q000378:metabolism; D010957:Plasmids / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011401:Promoter Regions, Genetic; D018741:Replication Origin; D017422:Sequence Analysis, DNA
chemical_list: D010647:Phenyl Ethers; C031538:phenyl ether
keywords: Copy number; Cupriavidus basilensis; Shuttle vector; pCB5