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Journal of Bacteriology, August 2008, p. 5710-5719, Vol. 190, No. 16
0021-9193/08/$08.00+0     doi:10.1128/JB.00596-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Precise Determination, Cross-Recognition, and Functional Analysis of the Double-Strand Origins of the Rolling-Circle Replication Plasmids in Haloarchaea{triangledown}

Ligang Zhou,1,2 Meixian Zhou,1,{dagger} Chaomin Sun,1,{ddagger} Jing Han,1,2 Qiuhe Lu,1,2 Jian Zhou,1 and Hua Xiang1*

State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences,1 Graduate University of Chinese Academy of Sciences, Beijing 100101, People's Republic of China2

Received 30 April 2008/ Accepted 9 June 2008

The precise nick site in the double-strand origin (DSO) of pZMX201, a 1,668-bp rolling-circle replication (RCR) plasmid from the haloarchaeon Natrinema sp. CX2021, was determined by electron microscopy and DSO mapping. In this plasmid, DSO nicking occurred between residues C404 and G405 within a heptanucleotide sequence (TCTC/GGC) located in the stem region of an imperfect hairpin structure. This nick site sequence was conserved among the haloarchaeal RCR plasmids, including pNB101, suggesting that the DSO nick site might be the same for all members of this plasmid family. Interestingly, the DSOs of pZMX201 and pNB101 were found to be cross-recognized in RCR initiation and termination in a hybrid plasmid system. Mutation analysis of the DSO from pZMX201 (DSOZ) in this hybrid plasmid system revealed that: (i) the nucleotides in the middle of the conserved TCTCGGC sequence play more-important roles in the initiation and termination process; (ii) the left half of the hairpin structure is required for initiation but not for termination; and (iii) a 36-bp sequence containing TCTCGGC and the downstream sequence is essential and sufficient for termination. In conclusion, these haloarchaeal plasmids, with novel features that are different from the characteristics of both single-stranded DNA phages and bacterial RCR plasmids, might serve as a good model for studying the evolution of RCR replicons.

* Corresponding author. Mailing address: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Datun Road, Chaoyang District, Beijing 100101, People's Republic of China. Phone and fax: (86) 10-6480-7472. E-mail: xiangh{at}sun.im.ac.cn

{triangledown} Published ahead of print on 20 June 2008.

{dagger} Present address: Departments of Pediatric Dentistry and Microbiology, University of Alabama at Birmingham Schools of Medicine and Dentistry, Birmingham, AL 35244.

{ddagger} Present address: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720.

Journal of Bacteriology, August 2008, p. 5710-5719, Vol. 190, No. 16
0021-9193/08/$08.00+0     doi:10.1128/JB.00596-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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