The EAL Domain Protein VieA Is a Cyclic Diguanylate Phosphodiesterase

Abstract

The newly recognized bacterial second messenger 3,5-cyclic diguanylic acid (cyclic diguanylate (c-di-GMP)) has been shown to regulate a wide variety of bacterial behaviors and traits. Biosynthesis and degradation of c-di-GMP have been attributed to the GGDEF and EAL protein domains, respectively, based primarily on genetic evidence. Whereas the GGDEF domain was demonstrated to possess diguanylate cyclase activity in vitro, the EAL domain has not been tested directly for c-di-GMP phosphodiesterase activity. This study describes the analysis of c-di-GMP hydrolysis by an EAL domain protein in a purified system. The Vibrio cholerae EAL domain protein VieA has been shown to inversely regulate biofilmspecific genes (vps) and virulence genes (ctxA), presumably by decreasing the cellular pool of c-di-GMP. VieA was maximally active at neutral pH, physiological ionic strength, and ambient temperatures and demonstrated c-di-GMP hydrolytic activity with a K m of 0.06 M. VieA was unable to hydrolyze cGMP. The putative metal coordination site of the EAL domain, Glu 170 , was demonstrated to be necessary for VieA activity. Furthermore, the divalent cations Mg 2؉ and Mn 2؉ were necessary for VieA activity; conversely, Ca 2؉and Zn 2؉ were potent inhibitors of the VieA phosphodiesterase. Calcium inhibition of the VieA EAL domain provides a potential mechanism for regulation of c-di-GMP degradation.

PilZ Domain Proteins Bind Cyclic Diguanylate and Regulate Diverse Processes in Vibrio cholerae

“…In the classical biotype of V. cholerae, it has been shown that expression of the PDEA VieA positively regulates virulence gene expression and negatively regulates vps expression (8,13,17). This information supports the model that c-di-GMP assists in the transition from environment to host via modulation of gene expression.…”

“…Some DGCs contain a feedback-inhibitory site (I-site) that binds dimeric c-di-GMP (16), and thus the I-site comprises one type of c-di-GMP binding domain. PDEAs bind c-di-GMP and degrade it via linearization to pGpG; the linear intermediate is further hydrolyzed to two GMP molecules by a second, presumably ubiquitous phosphodiesterase B (16,17). Thus, PDEAs must also contain a c-di-GMP binding site, although this site has yet to be defined.…”

“…Thus, PDEAs must also contain a c-di-GMP binding site, although this site has yet to be defined. The DGC and PDEA activities have been attributed to proteins containing GGDEF and EAL domains, respectively, each named for conserved amino acid residues (17)(18)(19)(20)(21)(22). In addition, the HD-GYP domain proteins have recently been shown to degrade c-di-GMP (23).…”

Cell–cell signal-dependent dynamic interactions between HD-GYP and GGDEF domain proteins mediate virulence in Xanthomonas campestris

“…Although it is established that the enzymatic and regulatory activities of the HD-GYP domain depend upon the HD dyad, the role of other conserved residues, including the GYP motif, is unknown. Two other protein domains are implicated in the synthesis and degradation of cyclic di-GMP; the GGDEF domain is a diguanylate cyclase involved in cyclic di-GMP synthesis, whereas the EAL domain is a second cyclic di-GMP phosphodiesterase (11)(12)(13)(14)(15). Previously we used yeast two-hybrid (Y2H) analysis to show that the HD-GYP domain of RpfG of X. axonopodis pv.…”

Genetic reductionist approach for dissecting individual roles of GGDEF proteins within the c-di-GMP signaling network in Salmonella

“…The only known effector domain that synthesizes c-di-GMP (designated as diguanylate cyclase activity, DGC) is the GGDEF domain. Two domains, EAL and HD-GYP, hydrolyze c-di-GMP (designated as phosphodiesterase activity, PDE) into linear 5Ј-phosphoguanylyl-(3Ј-5Ј)-guanosine (pGpG) or guanosine monophosphate (GMP), respectively (7)(8)(9)(10)(11)(12)(13). Many DGC proteins also contain an RxxD conserved motif 5 amino acids upstream from the GGDEF motif.…”