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Journal of Bacteriology, October 2009, p. 6363-6373, Vol. 191, No. 20
0021-9193/09/$08.00+0     doi:10.1128/JB.00913-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Rbf Promotes Biofilm Formation by Staphylococcus aureus via Repression of icaR, a Negative Regulator of icaADBC{triangledown}

David Cue,1 Mei G. Lei,1 Thanh T. Luong,1 Lisa Kuechenmeister,2 Paul M. Dunman,2 Sinead O'Donnell,3 Sarah Rowe,3 James P. O'Gara,3 and Chia Y. Lee1*

Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205,1 Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska 68198,2 School of Biomolecular and Biomedical Science, Ardmore House, University College Dublin, Belfield, Dublin 4, Ireland3

Received 13 July 2009/ Accepted 5 August 2009

We previously reported the identification of a gene, rbf, involved in the regulation of biofilm formation by Staphylococcus aureus 8325-4. In an effort to study the mechanism of regulation, microarrays were used to compare the transcription profiles of the wild-type strain with an rbf mutant and an rbf overexpression strain of the clinical isolate UAMS-1. Among the genes affected by rbf overexpression are those of the intercellular adhesion (ica) locus; however, expression of these genes was not affected by an rbf deletion in the chromosome. The icaADBC genes are responsible for production of poly-N-acetylglucosamine (PNAG), a major constituent of biofilm. The icaR gene encodes a negative regulator of icaADBC. In UAMS-1 carrying an Rbf-encoding plasmid, Rbf was found to repress icaR transcription with a concomitant increase in icaADBC expression and increased PNAG and biofilm production relative to isogenic strains lacking the plasmid. Sequencing of the rbf gene from UAMS-1 showed that there was a 2-bp insertion affecting the 50th codon of the rbf open reading frame, suggesting that rbf is a pseudogene in UAMS-1. This finding explains why deletion of rbf had no effect on biofilm formation in UAMS-1. To further characterize the Rbf regulation on biofilm we compared biofilm formation, icaA and icaR transcription, and PNAG production in 8325-4 and its isogenic rbf and icaR single mutants and an rbf icaR double mutant. Our results are consistent with a model wherein rbf represses synthesis of icaR, which in turn results in derepression of icaADBC and increased PNAG production. Furthermore, purified rbf did not bind to the icaR or icaA promoter region, suggesting that rbf controls expression of an unknown factor(s) that represses icaR. The role of rbf in controlling the S. aureus biofilm phenotype was further demonstrated in a clinical strain, MW2.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, 4301 W. Markham Street, Slot 511, Little Rock, AR 72205. Phone: (501) 526-7687. Fax: (501) 686-5359. E-mail: clee2{at}uams.edu

{triangledown} Published ahead of print on 14 August 2009.

Journal of Bacteriology, October 2009, p. 6363-6373, Vol. 191, No. 20
0021-9193/09/$08.00+0     doi:10.1128/JB.00913-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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