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Applied and Environmental Microbiology, April 2002, p. 2008-2017, Vol. 68, No. 4
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.4.2008-2017.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Statistical Analysis of Pseudomonas aeruginosa Biofilm Development: Impact of Mutations in Genes Involved in Twitching Motility, Cell-to-Cell Signaling, and Stationary-Phase Sigma Factor Expression

Arne Heydorn,1 Bjarne Ersbøll,2 Junichi Kato,3 Morten Hentzer,1 Matthew R. Parsek,4 Tim Tolker-Nielsen,1 Michael Givskov,1 and Søren Molin1*

Section of Molecular Microbiology,1 Informatics and Mathematical Modelling, The Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark,2 Department of Fermentation Technology, Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8527, Japan,3 Environmental Engineering Group, Department of Civil Engineering, Northwestern University, Evanston, Illinois4

Received 27 September 2001/ Accepted 17 January 2002

Four strains of Pseudomonas aeruginosa (wild type, {Delta}pilHIJK mutant, lasI mutant, and rpoS mutant) were genetically tagged with the green fluorescent protein, and the development of flow chamber-grown biofilms by each of them was investigated by confocal laser scanning microscopy. The structural developments of the biofilms were quantified by the computer program COMSTAT (A. Heydorn, A. T. Nielsen, M. Hentzer, C. Sternberg, M. Givskov, B. K. Ersbøll, and S. Molin, Microbiology 146:2395-2407, 2000). Two structural key variables, average thickness and roughness, formed the basis for an analysis of variance model comprising the four P. aeruginosa strains, five time points (55, 98, 146, 242, and 314 h), and three independent rounds of biofilm experiments. The results showed that the wild type, the {Delta}pilHIJK mutant, and the rpoS mutant display conspicuously different types of temporal biofilm development, whereas the lasI mutant was indistinguishable from the wild type at all time points. The wild type and the lasI mutant formed uniform, densely packed biofilms. The rpoS mutant formed densely packed biofilms that were significantly thicker than those of the wild type, whereas the {Delta}pilHIJK mutant formed distinct microcolonies that were regularly spaced and almost uniform in size. The results are discussed in relation to the current model of P. aeruginosa biofilm development.

* Corresponding author. Mailing address: Section of Molecular Microbiology, BioCentrum-DTU, The Technical University of Denmark, Building 301, DK-2800 Kgs. Lyngby, Denmark. Phone: 45 45 25 25 13. Fax: 45 45 93 28 09. E-mail: soren.molin{at}biocentrum.dtu.dk.

Applied and Environmental Microbiology, April 2002, p. 2008-2017, Vol. 68, No. 4
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.4.2008-2017.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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