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Journal of Bacteriology, December 2009, p. 7333-7342, Vol. 191, No. 23
0021-9193/09/$08.00+0 doi:10.1128/JB.00975-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Nitric Oxide Signaling in Pseudomonas aeruginosa Biofilms Mediates Phosphodiesterase Activity, Decreased Cyclic Di-GMP Levels, and Enhanced Dispersal
,
Nicolas Barraud,1
David Schleheck,1,
Janosch Klebensberger,1
Jeremy S. Webb,1,
Daniel J. Hassett,2
Scott A. Rice,1 and
Staffan Kjelleberg1*
School of Biotechnology and Biomolecular Sciences and Centre for Marine Bio-Innovation, University of New South Wales, Sydney, New South Wales 2052, Australia,1 Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 452672
Received 24 July 2009/ Accepted 22 September 2009
Bacteria in biofilms often undergo active dispersal events and revert to a free-swimming, planktonic state to complete the biofilm life cycle. The signaling molecule nitric oxide (NO) was previously found to trigger biofilm dispersal in the opportunistic pathogen Pseudomonas aeruginosa at low, nontoxic concentrations (N. Barraud, D. J. Hassett, S. H. Hwang, S. A. Rice, S. Kjelleberg, and J. S. Webb, J. Bacteriol. 188:7344-7353, 2006). NO was further shown to increase cell motility and susceptibility to antimicrobials. Recently, numerous studies revealed that increased degradation of the secondary messenger cyclic di-GMP (c-di-GMP) by specific phosphodiesterases (PDEs) triggers a planktonic mode of growth in eubacteria. In this study, the potential link between NO and c-di-GMP signaling was investigated by performing (i) PDE inhibitor studies, (ii) enzymatic assays to measure PDE activity, and (iii) direct quantification of intracellular c-di-GMP levels. The results suggest a role for c-di-GMP signaling in triggering the biofilm dispersal event induced by NO, as dispersal requires PDE activity and addition of NO stimulates PDE and induces the concomitant decrease in intracellular c-di-GMP levels in P. aeruginosa. Furthermore, gene expression studies indicated global responses to low, nontoxic levels of NO in P. aeruginosa biofilms, including upregulation of genes involved in motility and energy metabolism and downregulation of adhesins and virulence factors. Finally, site-directed mutagenesis of candidate genes and physiological characterization of the corresponding mutant strains uncovered that the chemotaxis transducer BdlA is involved in the biofilm dispersal response induced by NO.
* Corresponding author. Mailing address: School of Biotechnology and Biomolecular Sciences and Centre for Marine Bio-Innovation, Biological Sciences Building, University of New South Wales, Sydney, NSW 2052, Australia. Phone: 61 (2) 9385 2102. Fax: 61 (2) 9385 1779. E-mail: s.kjelleberg{at}unsw.edu.au
Published ahead of print on 2 October 2009.
Supplemental material for this article may be found at http://jb.asm.org/.
Present address: Department of Biology, University of Konstanz, D-78457 Konstanz, Germany.
Present address: School of Biological Sciences, University of Southampton, Southampton SO16 7PX, United Kingdom.
Journal of Bacteriology, December 2009, p. 7333-7342, Vol. 191, No. 23
0021-9193/09/$08.00+0 doi:10.1128/JB.00975-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
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