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Infection and Immunity, March 2007, p. 1079-1088, Vol. 75, No. 3
0019-9567/07/$08.00+0     doi:10.1128/IAI.01143-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Isolation and Characterization of Biofilm Formation-Defective Mutants of Staphylococcus aureus{triangledown}

Patrick H. Tu Quoc,1 Pierre Genevaux,2 Maria Pajunen,3 Harri Savilahti,3,4 Costa Georgopoulos,2 Jacques Schrenzel,1 and William L. Kelley1*

Division of Infectious Diseases, University Hospital of Geneva, 24 rue Micheli du Crest, CH-1211 Geneva 14, Switzerland,1 Department of Microbiology and Molecular Medicine, CMU-University of Geneva, 1 rue Michel-Servet CH-1211 Geneva 14, Switzerland,2 Program in Cellular Biotechnology, Institute of Biotechnology, Viikki Biocenter, FIN-00014, University of Helsinki, Finland,3 Division of Genetics and Physiology, Department of Biology, FIN-20014, University of Turku, Finland4

Received 20 July 2006/ Returned for modification 5 October 2006/ Accepted 28 November 2006

Staphylococcus aureus produces biofilm and this mode of colonization facilitates infections that are often difficult to treat and engender high morbidity and mortality. We have exploited bacteriophage Mu transposition methods to create an insertional mutant library in a highly biofilm-forming S. aureus clinical isolate. Our screen identified 38 insertions in 23 distinct genes together with one intergenic region that significantly reduced biofilm formation. Nineteen insertions were mapped in loci not previously known to affect biofilm in this organism. These include insertions in codY, srrA, mgrA, and fmtA, a putative DEAD-box helicase, two members of the zinc-metallo-ß lactamase/ß-CASP family, and a hypothetical protein with a GGDEF motif. Fifteen insertions occurred in the icaADBC operon, which produces intercellular adhesion antigen (PIA) and is important for biofilm formation in many strains of S. aureus and Staphylococcus epidermidis. Obtaining a high proportion of independent Em-Mu disruptions in icaADBC demonstrated both the importance of PIA for biofilm formation in this clinical strain and the strong validation of the screening procedure that concomitantly uncovered additional mutants. All non-ica mutants were further analyzed by immunoblotting and biochemical fractionation for perturbation of PIA and wall teichoic acid. PIA levels were diminished in the majority of non-ica insertional mutants. Three mutant strains were chosen and were functionally complemented for restored biofilm formation by transformation with plasmids carrying the cloned wild-type gene under the control of a xylose-inducible promoter. This is a comprehensive collection of biofilm-defective mutants that underscores the multifactorial genetic program underlying the establishment of biofilm in this insidious pathogen.

* Corresponding author. Mailing address: Service of Infectious Diseases, University Hospital of Geneva, 24 rue Micheli-du-Crest, CH-1211 Geneva 14, Switzerland. Phone: 41 22 372 9819. Fax: 41 22 372 9830. E-mail: William.Kelley{at}hcuge.ch.

{triangledown} Published ahead of print on 11 December 2006.

Editor: V. J. DiRita

Infection and Immunity, March 2007, p. 1079-1088, Vol. 75, No. 3
0019-9567/07/$08.00+0     doi:10.1128/IAI.01143-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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