Identification and molecular characterization of a cyclic-di-GMP effector protein, PlzA (BB0733): additional evidence for the existence of a functional cyclic-di-GMP regulatory network in the Lyme disease spirochete,Borrelia burgdorferi

Abstract

The Borrelia burgdorferi Rrp1 protein is a diguanylate cyclase that controls a regulon consisting of ~10% of the total genome. Because Rrp1 lacks a DNA-binding domain, its regulatory capability is most likely mediated through the production of bis-(3′–5′)-cyclic dimeric GMP (c-di-GMP). C-di-GMP binds to and activates the regulatory activity of proteins that harbor a PilZ domain. The occurrence of a PilZ domain within a protein is not in and of itself sufficient to convey c-di-GMP binding, as other structural aspects of the protein are important in the interaction. In this study, we have assessed the expression and c-di-GMP binding ability of the sole PilZ domain-containing protein of B. burgdorferi B31, PlzA. PlzA was determined to be upregulated by tick feeding and to be expressed during mammalian infection. The gene is highly conserved and present in all Borrelia species. Analyses of recombinant PlzA demonstrated its ability to bind c-di-GMP and site-directed mutagenesis revealed that this interaction is highly specific and dependent on Arg residues contained within the PilZ domain. In summary, this study is the first to identify a c-di-GMP effector molecule in a spirochete and provides additional evidence for the existence of a complete c-di-GMP regulatory network in the Lyme disease spirochete, B. burgdorferi.

Cyclic di-GMP: the First 25 Years of a Universal Bacterial Second Messenger

“…The C terminus of G. xylinus BcsA, the PilZ domain protein YcgR from E. coli (52), the DgrA protein from C. crescentus (51), and the Vibrio cholerae PilZ domain proteins PlzC and PlzD (147) were shown to bind c-di-GMP in vitro with high specificities, demonstrating that the PilZ domain was indeed the long-sought-after c-di-GMP receptor. The ability of PilZ domains to bind c-di-GMP in vitro has now been demonstrated for PilZ domain proteins of numerous bacterial species (147)(148)(149).…”

“…Furthermore, PilZ domain proteins were found to adopt different oligomeric states (Fig. 1), from monomeric to tetrameric (82,83,87,152) (51,52,148,(154)(155)(156), polysaccharide synthesis and translocation (18,149,157), and DNA binding (120,158), are described in this review. It is clear that the PilZ domain functions as a versatile module that can regulate diverse activities in a c-di-GMP-dependent manner.…”

Molecular Signaling Mechanisms of the Periopathogen, Treponema denticola

“…The importance of c-di-GMP in spirochetal biology is highlighted by recent studies of B. burgdorferi (Ryjenkov et al, 2005;Rogers et al, 2009;Freedman et al, 2009;Sultan et al, 2010). B. burgdorferi encodes a single diguanylate cyclase designated as response regulatory protein 1 (Rrp1).…”

“…The effector mechanisms of c-di-GMP are just beginning to be defined. C-di-GMP has been shown to bind to PilZ domain-containing proteins, inducing conformational changes that modulate protein activity (Ryjenkov et al, 2006;Freedman et al, 2009). C-di-GMP has also been shown to bind to proteins that lack obvious PilZ domains (Chin et al, 2010).…”

“…Strain 35405 harbors proteins with GGDEF, EAL, and PilZ domains ( Table 2). One of the PilZ domain proteins (TDE0214) has significant homology to the PlzA protein of B. burgdorferi (Freedman et al, 2009). C-di-GMP has the potential to regulate numerous processes that are central to the ability of T. denticola to survive and thrive in the subgingival crevice.…”