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Journal of Bacteriology, February 2005, p. 1441-1454, Vol. 187, No. 4
0021-9193/05/$08.00+0     doi:10.1128/JB.187.4.1441-1454.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

A Three-Component Regulatory System Regulates Biofilm Maturation and Type III Secretion in Pseudomonas aeruginosa{dagger}

Sherry L. Kuchma, John P. Connolly, and George A. O'Toole*

Department of Microbiology and Immunology, Dartmouth Medical School, Hanover, New Hampshire

Received 10 September 2004/ Accepted 4 November 2004

Biofilms are structured communities found associated with a wide range of surfaces. Here we report the identification of a three-component regulatory system required for biofilm maturation by Pseudomonas aeruginosa strain PA14. A transposon mutation that altered biofilm formation in a 96-well dish assay originally defined this locus, which is comprised of genes for a putative sensor histidine kinase and two response regulators and has been designated sadARS. Nonpolar mutations in any of the sadARS genes result in biofilms with an altered mature structure but do not confer defects in growth or early biofilm formation, swimming, or twitching motility. After 2 days of growth under flowing conditions, biofilms formed by the mutants are indistinguishable from those formed by the wild-type (WT) strain. However, by 5 days, mutant biofilms appear to be more homogeneous than the WT in that they fail to form large and distinct macrocolonies and show a drastic reduction in water channels. We propose that the sadARS three-component system is required for later events in biofilm formation on an abiotic surface. Semiquantitative reverse transcription-PCR analysis showed that there is no detectable change in expression of the sadARS genes when cells are grown in a planktonic culture versus a biofilm, indicating that this locus is not itself induced during or in response to biofilm formation. DNA microarray studies were used to identify downstream targets of the SadARS system. Among the genes regulated by the SadARS system are those required for type III secretion. Mutations in type III secretion genes result in strains with enhanced biofilm formation. We propose a possible mechanism for the role that the SadARS system plays in biofilm formation.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Dartmouth Medical School, Room 505, Vail Building, North College St., Hanover, NH 03755. Phone: (603) 650-1248. Fax: (603) 650-1245. E-mail: georgeo{at}dartmouth.edu.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

Journal of Bacteriology, February 2005, p. 1441-1454, Vol. 187, No. 4
0021-9193/05/$08.00+0     doi:10.1128/JB.187.4.1441-1454.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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