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Applied and Environmental Microbiology, February 2009, p. 1110-1120, Vol. 75, No. 4
0099-2240/09/$08.00+0     doi:10.1128/AEM.02416-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

MucR, a Novel Membrane-Associated Regulator of Alginate Biosynthesis in Pseudomonas aeruginosa

Iain D. Hay, Uwe Remminghorst, and Bernd H. A. Rehm^*

Institute of Molecular Biosciences, Massey University, Private Bag 11222, Palmerston North, New Zealand

Received 21 October 2008/ Accepted 9 December 2008

Alginate biosynthesis by Pseudomonas aeruginosa was shown to be regulated by the intracellular second messenger bis-(3'-5')-cyclic-dimeric-GMP (c-di-GMP), and binding of c-di-GMP to the membrane protein Alg44 was required for alginate production. In this study, PA1727, a c-di-GMP-synthesizing enzyme was functionally analyzed and identified to be involved in regulation of alginate production. Deletion of the PA1727 gene in the mucoid alginate-overproducing P. aeruginosa strain PDO300 resulted in a nonmucoid phenotype and an about 38-fold decrease in alginate production; thus, this gene is designated mucR. The mucoid alginate-overproducing phenotype was restored by introducing the mucR gene into the isogenic mucR mutant. Moreover, transfer of the MucR-encoding plasmid into strain PDO300 led to an about sevenfold increase in alginate production, wrinkly colony morphology, increased pellicle formation, auto-aggregation, and the formation of highly structured biofilms as well as the inhibition of swarming motility. Outer membrane protein profile analysis showed that overproduction of MucR mediates a strong reduction in the copy number of FliC (flagellin), required for flagellum-mediated motility. Translational reporter enzyme fusions with LacZ and PhoA suggested that MucR is located in the cytoplasmic membrane with a cytosolic C terminus. Deletion of the proposed C-terminal GGDEF domain abolished MucR function. MucR was purified and identified using tryptic peptide fingerprinting and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Overall, experimental evidence was provided suggesting that MucR specifically regulates alginate biosynthesis by activation of alginate production through generation of a localized c-di-GMP pool in the vicinity of Alg44.

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* Corresponding author. Mailing address: Institute of Molecular Biosciences, Massey University, Private Bag 11222, Palmerston North, New Zealand. Phone: 64 6 350 5515. Fax: 64 6 350 5688. E-mail: b.rehm{at}massey.ac.nz

Published ahead of print on 16 December 2008.

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Applied and Environmental Microbiology, February 2009, p. 1110-1120, Vol. 75, No. 4
0099-2240/09/$08.00+0     doi:10.1128/AEM.02416-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Hay, I. D., Gatland, K., Campisano, A., Jordens, J. Z., Rehm, B. H. A. (2009). Impact of Alginate Overproduction on Attachment and Biofilm Architecture of a Supermucoid Pseudomonas aeruginosa Strain. Appl. Environ. Microbiol. 75: 6022-6025 [Abstract] [Full Text]