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Infection and Immunity, April 2009, p. 1623-1635, Vol. 77, No. 4
0019-9567/09/$08.00+0     doi:10.1128/IAI.01036-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Interconnections between Sigma B, agr, and Proteolytic Activity in Staphylococcus aureus Biofilm Maturation

Katherine J. Lauderdale,¹ Blaise R. Boles,¹ Ambrose L. Cheung,² and Alexander R. Horswill^1*

Department of Microbiology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242,¹ Department of Microbiology and Immunology, Dartmouth Medical School, Hanover, New Hampshire 03755²

Received 19 August 2008/ Returned for modification 29 September 2008/ Accepted 22 January 2009

Staphylococcus aureus is a proficient biofilm former on host tissues and medical implants. We mutagenized S. aureus strain SH1000 to identify loci essential for ica-independent mechanisms of biofilm maturation and identified multiple insertions in the rsbUVW-sigB operon. Following construction and characterization of a sigB deletion, we determined that the biofilm phenotype was due to a lack of sigma factor B (SigB) activity. The phenotype was conserved in a sigB mutant of USA300 strain LAC, a well-studied community-associated methicillin-resistant S. aureus isolate. We determined that agr RNAIII levels were elevated in the sigB mutants, and high levels of RNAIII expression are known to have antibiofilm effects. By introducing an agr mutation into the SH1000 or LAC sigB deletion strain, S. aureus regained biofilm capacity, indicating that the biofilm phenotype was agr dependent. Protease activity is linked to agr activity and ica-independent biofilm formation, and we observed that the protease inhibitors phenylmethylsulfonyl fluoride and -macroglobulin could reverse the sigB biofilm defect. Similarly, inactivating genes encoding both the aureolysin and Spl extracellular proteases in the sigB mutant restored biofilm capacity. Due to the growing link between murein hydrolase activity and biofilm maturation, autolysin zymography was performed, which revealed an altered profile in the sigB mutant; again, the phenotype could be repaired through protease inactivation. These findings indicate that the lack of SigB activity results in increased RNAIII expression, thus elevating extracellular protease levels and altering the murein hydrolase activity profile. Altogether, our observations demonstrate that SigB is an essential regulator of S. aureus biofilm maturation.

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* Corresponding author. Mailing address: Department of Microbiology, 540F EMRB, University of Iowa, Iowa City, IA 52242. Phone: (319) 335-7783. Fax: (319) 335-8228. E-mail: alex-horswill{at}uiowa.edu

Published ahead of print on 2 February 2009.

Editor: A. Camilli

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Infection and Immunity, April 2009, p. 1623-1635, Vol. 77, No. 4
0019-9567/09/$08.00+0     doi:10.1128/IAI.01036-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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