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Journal of Bacteriology, March 2001, p. 1990-1996, Vol. 183, No. 6
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.6.1990-1996.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Gene Expression in Pseudomonas aeruginosa: Evidence of Iron Override Effects on Quorum Sensing and Biofilm-Specific Gene Regulation

Nikki Bollinger,1 Daniel J. Hassett,2 Barbara H. Iglewski,3 J. William Costerton,1 and Timothy R. McDermott1,4,*

Center for Biofilm Engineering,1 and Department of Land Resources and Environmental Sciences,4 Montana State University, Bozeman, Montana 59717; Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45257-05242; and Department of Microbiology and Immunology, University of Rochester School of Medicine, Rochester, New York 146423

Received 28 August 2000/Accepted 14 December 2000

Prior studies established that the Pseudomonas aeruginosa oxidative stress response is influenced by iron availability, whereas more recent evidence demonstrated that it was also controlled by quorum sensing (QS) regulatory circuitry. In the present study, sodA (encoding manganese-cofactored superoxide dismutase [Mn-SOD]) and Mn-SOD were used as a reporter gene and endogenous reporter enzyme, respectively, to reexamine control mechanisms that govern the oxidative stress response and to better understand how QS and a nutrient stress response interact or overlap in this bacterium. In cells grown in Trypticase soy broth (TSB), Mn-SOD was found in wild-type stationary-phase planktonic cells but not in a lasI or lasR mutant. However, Mn-SOD activity was completely suppressed in the wild-type strain when TSB was supplemented with iron. Reporter gene studies indicated that sodA transcription could be variably induced in iron-starved cells of all three strains, depending on growth stage. Iron starvation induction of sodA was greatest in the wild-type strain and least in the lasR mutant and was maximal in stationary-phase cells. Reporter experiments in the wild-type strain showed increased lasI::lacZ transcription in response to iron limitation, whereas the expression level in the las mutants was minimal and iron starvation induction of lasI::lacZ did not occur. Studies comparing Mn-SOD activity in P. aeruginosa biofilms and planktonic cultures were also initiated. In wild-type biofilms, Mn-SOD was not detected until after 6 days, although in iron-limited wild-type biofilms Mn-SOD was detected within the initial 24 h of biofilm establishment and formation. Unlike planktonic bacteria, Mn-SOD was constitutive in the lasI and lasR mutant biofilms but could be suppressed if the growth medium was amended with 25 µM ferric chloride. This study demonstrated that (i) the nutritional status of the cell must be taken into account when one is evaluating QS-based gene expression; (ii) in the biofilm mode of growth, QS may also have negative regulatory functions; (iii) QS-based gene regulation models based on studies with planktonic cells must be modified in order to explain biofilm gene expression behavior; and (iv) gene expression in biofilms is dynamic.

* Corresponding author. Mailing address: Land Resources and Environmental Science, Montana State University, Bozeman, MT 59717. Phone: (406) 994-2190. Fax: (406) 994-3933. E-mail: timmcder{at}montana.edu.

Journal of Bacteriology, March 2001, p. 1990-1996, Vol. 183, No. 6
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.6.1990-1996.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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