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Journal of Bacteriology, September 2000, p. 4822-4828, Vol. 182, No. 17
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Analysis of Genetic Elements Controlling Staphylococcus aureus lrgAB Expression: Potential Role of DNA Topology in SarA Regulation

David F. Fujimoto, Eric W. Brunskill,dagger and Kenneth W. Bayles*

Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, Moscow, Idaho 83844-3052

Received 29 March 2000/Accepted 16 June 2000

Penicillin-induced killing and murein hydrolase activity in Staphylococcus aureus are dependent on a variety of regulatory elements, including the LytSR two-component regulatory system and the virulence factor regulators Agr and Sar. The LytSR effects on these processes can be explained, in part, by the recent finding that a LytSR-regulated operon, designated lrgAB, affects murein hydrolase activity and penicillin tolerance. To examine the regulation of lrgAB expression in greater detail, we performed Northern blot and promoter fusion analyses. Both methods revealed that Agr and Sar, like LytSR, positively regulate lrgAB expression. A mutation in the agr locus reduced lrgAB expression approximately sixfold, while the sar mutation reduced lrgAB expression to undetectable levels. cis-acting regulatory elements involved in lrgAB expression were identified by fusing various fragments of the lrgAB promoter region to the xylE reporter gene and integrating these constructs into the chromosome. Catechol 2,3-dioxygenase assays identified DNA sequences, including an inverted repeat and intrinsic bend sites, that contribute to maximal lrgAB expression. Confirmation of the importance of the inverted repeat was achieved by demonstrating that multiple copies of the inverted repeat reduced lrgAB promoter activity, presumably by titrating out a positive regulatory factor. The results of this study demonstrate that lrgAB expression responds to a variety of positive regulatory factors and suggest that specific DNA topology requirements are important for optimal expression.

* Corresponding author. Mailing address: Department of Microbiology, Molecular Biology and Biochemistry, College of Agriculture, University of Idaho, Moscow, ID 83844-3052. Phone: (208) 885-7164. Fax: (208) 885-6518. E-mail: kbayles{at}uidaho.edu.

dagger Present address: The Childrens' Hospital Research Foundation, Division of Developmental Biology, Cincinnati, OH 45229.

Journal of Bacteriology, September 2000, p. 4822-4828, Vol. 182, No. 17
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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