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Applied and Environmental Microbiology, September 2004, p. 5557-5568, Vol. 70, No. 9
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.9.5557-5568.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Isolation and Characterization of a Rolling-Circle-Type Plasmid from Rhodococcus erythropolis and Application of the Plasmid to Multiple-Recombinant-Protein Expression

Nobutaka Nakashima1 and Tomohiro Tamura1,2*

Proteolysis and Protein Turnover Research Group, Research Institute of Genome-Based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), Tsukisamu-Higashi, Toyohira-ku,1 Laboratory of Molecular Environmental Microbiology, Graduate School of Agriculture, Hokkaido University, Kita-ku, Sapporo, Japan2

Received 25 December 2003/ Accepted 12 May 2004

We isolated, sequenced, and characterized the cryptic plasmid pRE8424 from Rhodococcus erythropolis DSM8424. Plasmid pRE8424 is a 5,987-bp circular plasmid; it carries six open reading frames and also contains cis-acting elements, specifically a single-stranded origin and a double-stranded origin, which are characteristic of rolling-circle-replication plasmids. Experiments with pRE8424 derivatives carrying a mutated single-stranded origin sequence showed that single-stranded DNA intermediates accumulated in the cells because of inefficient conversion from single-stranded DNA to double-stranded DNA. This result indicates that pRE8424 belongs to the pIJ101/pJV1 family of rolling-circle-replication plasmids. Expression vectors that are functional in several Rhodococcus species were constructed by use of the replication origin from pRE8424. We previously reported a cryptic plasmid, pRE2895, from R. erythropolis, which may replicate by a {theta}-type mechanism, like ColE2 plasmids. The new expression vectors originating from pRE8424 were compatible with those derived from pRE2895. Coexpression experiments with these compatible expression vectors indicated that the plasmids are suitable for the simultaneous expression of multiple recombinant proteins.

* Corresponding author. Mailing address: Proteolysis and Protein Turnover Research Group, Research Institute of Genome-Based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517, Japan. Phone: 81-11-857-8938. Fax: 81-11-857-8980. E-mail: t-tamura{at}aist.go.jp.

Applied and Environmental Microbiology, September 2004, p. 5557-5568, Vol. 70, No. 9
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.9.5557-5568.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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  • Mitani, Y., Meng, X., Kamagata, Y., Tamura, T. (2005). Characterization of LtsA from Rhodococcus erythropolis, an Enzyme with Glutamine Amidotransferase Activity. J. Bacteriol. 187: 2582-2591 [Abstract] [Full Text]  


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