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Journal of Bacteriology, January 2005, p. 37-44, Vol. 187, No. 1
0021-9193/05/$08.00+0     doi:10.1128/JB.187.1.37-44.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Timing and Localization of Rhamnolipid Synthesis Gene Expression in Pseudomonas aeruginosa Biofilms

Yannick Lequette and E. P. Greenberg*

Department of Microbiology and W. M. Keck Microbial Communities and Cell Signalling Program, Carver College of Medicine, University of Iowa, Iowa City, Iowa

Received 19 May 2004/ Accepted 16 August 2004

Pseudomonas aeruginosa biofilms can develop mushroom-like structures with stalks and caps consisting of discrete subpopulations of cells. Self-produced rhamnolipid surfactants have been shown to be important in development of the mushroom-like structures. The quorum-sensing-controlled rhlAB operon is required for rhamnolipid synthesis. We have introduced an rhlA-gfp fusion into a neutral site in the P. aeruginosa genome to study rhlAB promoter activity in rhamnolipid-producing biofilms. Expression of the rhlA-gfp fusion in biofilms requires the quorum-sensing signal butanoyl-homoserine lactone, but other factors are also required for expression. Early in biofilm development rhlA-gfp expression is low, even in the presence of added butanoyl-homoserine lactone. Expression of the fusion becomes apparent after microcolonies with a depth of >20 µm have formed and, as shown by differential labeling with rfp or fluorescent dyes, rhlA-gfp is preferentially expressed in the stalks rather than the caps of mature mushrooms. The rhlA-gfp expression pattern is not greatly influenced by addition of butanoyl-homoserine lactone to the biofilm growth medium. We propose that rhamnolipid synthesis occurs in biofilms after stalks have formed but prior to capping in the mushroom-like structures. The differential expression of rhlAB may play a role in the development of normal biofilm architecture.

* Corresponding author. Mailing address: 540 EMRB, Roy and Lucille Carver College of Medicine, University of Iowa, Iowa City, IA 52242. Phone: (319) 335-7775. Fax: (319) 335-7949. E-mail: everett-greenberg{at}uiowa.edu.

Journal of Bacteriology, January 2005, p. 37-44, Vol. 187, No. 1
0021-9193/05/$08.00+0     doi:10.1128/JB.187.1.37-44.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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