is a Gram-negative bacterium that can readily adapt to a variety of environmental conditions (Stover et al.
). The preferred mode of growth of P. aeruginosa
is in a densely populated multi-cellular community called a biofilm, a state in which the bacteria are encapsulated in a matrix that serves to protect the bacteria from host defences and provides resistance to antibiotics (Ryder et al.
). The matrix is composed of exopolysaccharides, proteins and nucleic acids (Allesen-Holm et al.
; Friedman & Kolter, 2004a
is capable of forming three different types of exopolysaccharides encoded by three separate gene clusters: alginate, Psl and Pel (Ohman, 1986
; Friedman & Kolter, 2004b
; Jackson et al.
; Stover et al.
). Expression of the pel
gene cluster has been linked to biofilm growth (Colvin et al.
). Pel was discovered by screening a transposon mutant library for the lack of a specific type of biofilm which forms at the air–liquid interface of a standing culture and is known as a pellicle (Friedman & Kolter, 2004b
). The genetic locus responsible for this phenotype has been identified and annotated as the pelABCDEFG
operon (Friedman & Kolter, 2004b
). While the composition of the pellicle remains mostly unknown, the matrix material may contain the O-antigen of lipopolysaccharide and cyclic glucans in addition to the Pel polysaccharide (Coulon et al.
). Carbohydrate and linkage analyses suggest that the Pel polysaccharide is rich in glucose (Friedman & Kolter, 2004b
The protein product of the pelD
gene, PelD, has been identified as an essential regulator of pellicle formation and thus has been proposed as a possible target for therapeutic intervention (Lee et al.
). PelD is a 51 kDa putative inner membrane protein which was predicted using the Phyre
server (Kelley & Sternberg, 2009
) to contain four transmembrane (TM) helices at the N-terminus followed by tandem-arranged cytoplasmic GAF and GGDEF domains (Fig. 1
Figure 1 (a) Schematic diagram of the domain organization of PelD. The boundaries of the transmembrane (TM) segments and the GAF and GGDEF domains are denoted above the schematic and were predicted using HMMTOP (Tusnády & Simon, 2001 (more ...)
Studies in the last decade on the bacterial secondary messenger bis-(3′,5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) have significantly increased our understanding of bacterial signalling. This molecule has been shown to be involved in the transition from the planktonic state to the sessile biofilm state, with higher cellular concentrations of c-di-GMP enhancing biofilm formation in multiple Gram-negative species (Tamayo et al.
). Cyclic di-GMP receptors of known function are involved in exopolysaccharide synthesis, motility, transcription and subcellular or cell-surface protein localization (Schirmer & Jenal, 2009
; Newell et al.
). The formation of c-di-GMP is catalyzed by GGDEF-domain-containing diguanylate cyclases and is degraded by a family of enzymes called phosphodiesterases, which contain EAL or HD-GYP domains (Ryan et al.
). GGDEF domains are named after the sequence of the amino acids that define the active site of diguanylate cyclases. These enzymes catalyze the formation of c-di-GMP through the cyclization of two guanosine triphosphate (GTP) molecules and are regulated by allosteric inhibition through the primary inhibition site (Ip
site) characterized by the Rxx
D motif (De et al.
). The GGDEF domain in PelD has been shown to be degenerate as it lacks the characteristic residues required for catalytic function, but the protein is still capable of binding c-di-GMP. Three conserved residues, Arg367, Asp370 and Arg402, thought to resemble the Ip
site of the GGDEF domain found in Caulobacter crescentus
PleD (Chan et al.
) have been implicated in this binding and have been shown to be required for PEL polysaccharide production in vivo
. These findings have led to the hypothesis that PelD acts as a c-di-GMP receptor and that c-di-GMP binding may provide a means of coupling PEL biosynthesis to export (Lee et al.
PelD is also predicted to contain a GAF domain, a domain that has been implicated in a diverse array of functions (Aravind & Ponting, 1997
). The name of this domain is derived from cG
MP phosphodiesterases, A
denylyl cyclases and F
hlA, the first three protein families in which it was discovered. GAF domains are typically involved in ligand binding and/or protein–protein interactions; however, in bacteria GAF domains have also been shown to be associated with gene regulation (Aravind & Ponting, 1997
). The role of the GAF domain in PelD has not yet been determined, but conceivably it could act as the signalling module and stimulate PEL production either through dimerization or possibly by interaction with one of the other Pel proteins in response to c-di-GMP binding.
To gain insight into the mechanism of PEL biosynthesis and export, and to determine the role of PelD in this process, we have initiated structural studies of PelD; here, we describe the overexpression, purification and crystallization of a soluble fragment of PelD encompassing the GAF and GGDEF domains.