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Journal of Bacteriology, May 2003, p. 2700-2710, Vol. 185, No. 9
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.9.2700-2710.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Use of In-Biofilm Expression Technology To Identify Genes Involved in Pseudomonas aeruginosa Biofilm Development{dagger}

Antonio Finelli, Claude V. Gallant, Keith Jarvi, and Lori L. Burrows*

Centre for Infection and Biomaterials Research, Hospital for Sick Children,1 Division of Urology, Department of Surgery, University of Toronto, Toronto, Ontario, Canada2

Received 8 November 2002/ Accepted 8 January 2003

Mature Pseudomonas aeruginosa biofilms form complex three-dimensional architecture and are tolerant of antibiotics and other antimicrobial compounds. In this work, an in vivo expression technology system, originally designed to study virulence-associated genes in complex mammalian environments, was used to identify genes up-regulated in P. aeruginosa grown to a mature (5-day) biofilm. Five unique cloned promoters unable to promote in vitro growth in the absence of purines after recovery from the biofilm environment were identified. The open reading frames downstream of the cloned promoter regions were identified, and knockout mutants were generated. Insertional mutation of PA5065, a homologue of Escherichia coli ubiB, was lethal, while inactivation of PA0240 (a porin homologue), PA3710 (a putative alcohol dehydrogenase), and PA3782 (a homologue of the Streptomyces griseus developmental regulator adpA) had no effect on planktonic growth but caused defects in biofilm formation in static and flowing systems. In competition experiments, mutants demonstrated reduced fitness compared with the parent strain, comprising less than 0.0001% of total biofilm cells after 5 days. Therefore, using in-biofilm expression technology, we have identified novel genes that do not affect planktonic growth but are important for biofilm formation, development, and fitness.

* Corresponding author. Mailing address: Centre for Infection and Biomaterials Research, 7142A Elm Wing, Hospital for Sick Children Research Institute, 555 University Ave., Toronto, ON, Canada M5G 1X8. Phone: (416) 813-6293. Fax: (416) 813-6461. E-mail: lori.burrows{at}sickkids.ca.

{dagger} For a commentary on this article, see page 2687 in this issue.

Journal of Bacteriology, May 2003, p. 2700-2710, Vol. 185, No. 9
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.9.2700-2710.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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