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Journal of Bacteriology, August 2008, p. 5178-5189, Vol. 190, No. 15
0021-9193/08/$08.00+0     doi:10.1128/JB.00375-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

A Staphylococcal GGDEF Domain Protein Regulates Biofilm Formation Independently of Cyclic Dimeric GMP

Linda M. Holland,^1, Sinéad T. O'Donnell,^1, Dmitri A. Ryjenkov,² Larissa Gomelsky,² Shawn R. Slater,³ Paul D. Fey,³ Mark Gomelsky,² and James P. O'Gara^1*

School of Biomolecular and Biomedical Science, Ardmore House, University College Dublin, Belfield, Dublin 4, Ireland,¹ Department of Molecular Biology, University of Wyoming, Laramie, Wyoming,² Departments of Pathology and Microbiology and Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska³

Received 14 March 2008/ Accepted 16 May 2008

Cyclic dimeric GMP (c-di-GMP) is an important biofilm regulator that allosterically activates enzymes of exopolysaccharide biosynthesis. Proteobacterial genomes usually encode multiple GGDEF domain-containing diguanylate cyclases responsible for c-di-GMP synthesis. In contrast, only one conserved GGDEF domain protein, GdpS (for GGDEF domain protein from Staphylococcus), and a second protein with a highly modified GGDEF domain, GdpP, are present in the sequenced staphylococcal genomes. Here, we investigated the role of GdpS in biofilm formation in Staphylococcus epidermidis. Inactivation of gdpS impaired biofilm formation in medium supplemented with NaCl under static and flow-cell conditions, whereas gdpS overexpression complemented the mutation and enhanced wild-type biofilm development. GdpS increased production of the icaADBC-encoded exopolysaccharide, poly-N-acetyl-glucosamine, by elevating icaADBC mRNA levels. Unexpectedly, c-di-GMP synthesis was found to be irrelevant for the ability of GdpS to elevate icaADBC expression. Mutagenesis of the GGEEF motif essential for diguanylate cyclase activity did not impair GdpS, and the N-terminal fragment of GdpS lacking the GGDEF domain partially complemented the gdpS mutation. Furthermore, heterologous diguanylate cyclases expressed in trans failed to complement the gdpS mutation, and the purified GGDEF domain from GdpS possessed no diguanylate cyclase activity in vitro. The gdpS gene from Staphylococcus aureus exhibited similar characteristics to its S. epidermidis ortholog, suggesting that the GdpS-mediated signal transduction is conserved in staphylococci. Therefore, GdpS affects biofilm formation through a novel c-di-GMP-independent mechanism involving increased icaADBC mRNA levels and exopolysaccharide biosynthesis. Our data raise the possibility that staphylococci cannot synthesize c-di-GMP and have only remnants of a c-di-GMP signaling pathway.

Published ahead of print on 23 May 2008.

L.M.H. and S.T.O. contributed equally to this work.