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Journal of Bacteriology, June 2006, p. 4404-4412, Vol. 188, No. 12
0021-9193/06/$08.00+0     doi:10.1128/JB.00030-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Replication Control of Staphylococcal Multiresistance Plasmid pSK41: an Antisense RNA Mediates Dual-Level Regulation of Rep Expression

Stephen M. Kwong,1 Ronald A. Skurray,1 and Neville Firth1,2*

School of Biological Sciences, University of Sydney, Sydney, New South Wales 2006, Australia,1 The Sydney University Biological Informatics & Technology Centre, University of Sydney, Sydney, New South Wales 2006, Australia2

Received 10 January 2006/ Accepted 6 April 2006

Replication of staphylococcal multiresistance plasmid pSK41 is negatively regulated by the antisense transcript RNAI. pSK41 minireplicons bearing rnaI promoter (PrnaI) mutations exhibited dramatic increases in copy number, approximately 40-fold higher than the copy number for the wild-type replicon. The effects of RNAI mutations on expression of the replication initiator protein (Rep) were evaluated using transcriptional and translational fusions between the rep control region and the cat reporter gene. The results suggested that when PrnaI is disrupted, the amount of rep mRNA increases and it becomes derepressed for translation. These effects were reversed when RNAI was provided in trans, demonstrating that it is responsible for significant negative regulation at two levels, with the greatest repression exerted on rep translation initiation. Mutagenesis provided no evidence for RNAI-mediated transcriptional attenuation as a basis for the observed reduction in rep message associated with expression of RNAI. However, RNA secondary-structure predictions and supporting mutagenesis data suggest a novel mechanism for RNAI-mediated repression of rep translation initiation, where RNAI binding promotes a steric transition in the rep mRNA leader to an alternative thermodynamically stable stem-loop structure that sequesters the rep translation initiation region, thereby preventing translation.

* Corresponding author. Mailing address: School of Biological Sciences, Macleay Building A12, University of Sydney, Sydney, New South Wales 2006, Australia. Phone: (61) 2 9351 3369. Fax: (61) 2 9351 4771. E-mail: nfirth{at}bio.usyd.edu.au.

Journal of Bacteriology, June 2006, p. 4404-4412, Vol. 188, No. 12
0021-9193/06/$08.00+0     doi:10.1128/JB.00030-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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