Principles of c-di-GMP signaling : characterization of a second messenger system orchestrating bacterial life style

Bacteria are able to switch between two mutually exclusive lifestyles, motile single cells and
sedentary multicellular communities, known as biofilms, that colonize surfaces. Recent studies
demonstrated that the global bacterial second messenger c-di-GMP orchestrates the
developmental transition between both lifestyles. In a wide variety of bacterial species high
intracellular c-di-GMP levels provoke excretion of protective and adhesive exopolymeric
substances and inhibit flagella and pili based cell motility. Synthesis and degradation of c-di-GMP
is catalyzed by diguanylate cyclases (DGC’s) and c-di-GMP-specific phosphodiesterases (PDE),
respectively. Although the enzymes responsible for the synthesis of c-di-GMP have been recently
identified, little information is available on general regulatory principles of the c-di-GMP signaling
circuitry. Here we present genetic and biochemical approaches in combination with structural
analysis to elucidate the molecular mechanisms of signal transduction, signal modulation and
signal inactivation.
In (Christen and Christen et al 2007, PNAS) we describe the isolation of several c-di-GMP binding
proteins from Caulobacter crescentus by affinity chromatography. One of these proteins, DgrA, is a
PilZ homolog involved in mediating c-di-GMP-dependent control of C. crescentus cell motility.
Biochemical and structural analysis of DgrA and homologs from C. crescentus, Salmonella
typhimurium and Pseudomonas aeruginosa identified this protein family as the first class of specific
diguanylate receptors. Our studies suggested a general mechanism for c-di-GMP binding and
signal transduction whereby increased concentrations of c-di-GMP are sensed by DgrA through
direct binding and induce conformational changes of the diguanylate receptor that block motility by
interfering with motor function rather than flagellar assembly.
In (Christen and Christen et al 2006, JBC) we demonstrate that an allosteric binding site for c-di-
GMP (I-site) is responsible for non-competitive product inhibition of DGC’s. The I-site was mapped
in both multi- and single domain DGC proteins and shown to be fully contained within the GGDEF
domain itself. In vivo evolution experiments combined with kinetic analysis of the obtained I-site
mutants led to the definition of an RXXD motif as the core allosteric binding site for c-di-GMP.
Based on these results and based on the observation that the I-site is conserved in a majority of
known and potential DGC proteins, we propose that product inhibition of DGC’s is of fundamental
importance for c-di-GMP signaling and cellular homeostasis. The definition of the I-site binding
pocket provides an entry point into unraveling the molecular mechanisms of ligand-protein
interactions involved in c-di-GMP signaling, makes DGC's a valuable target for drug design and
offers new strategies against biofilm-related diseases.
In (Christen et al 2005, JBC) we show biochemically that CC3396, a GGDEF-EAL composite
protein from C. crescentus, is a soluble PDE. The PDE activity, rapidly converts c-di-GMP into the
linear dinucleotide pGpG is confined to the C-terminal EAL domain of CC3396, depends on the
presence of Mg2+ ions and is strongly inhibited by Ca2+ ions. Remarkably, the associated GGDEF
domain, which contains an altered active site motif (GEDEF), lacks detectable DGC activity.
Instead, this domain is able to bind GTP and in response activates the PDE activity in the
neighboring EAL domain. PDE activation is specific for GTP (KD 4 μM) and operates by lowering
the KM for c-di-GMP of the EAL domain to a physiologically significant level (420 nM). Mutational
analysis suggested that the substrate-binding site (A-site) of the GGDEF domain is involved in the
GTP-dependent regulatory function, arguing that a catalytically inactive GGDEF domain has
retained the ability to bind GTP and in response can activate the neighboring EAL domain. Based
on this we propose that the c-di-GMP-specific PDE activity is confined to the EAL domain, that
GGDEF domains can either catalyze the formation of c-di-GMP or can serve as regulatory domains
and that c-di-GMP-specific phosphodiesterase activity is coupled to the cellular GTP level in
bacteria.
In addition to the contribution in understanding the c-di-GMP signaling circuitry we characterized in
(Stephens et al 2007, JBac) the metabolic enzymes and regulators of D-xylose catabolism in C.
crescentus by genetic and biochemical methods. A saturated transposon screen was used to
define the xylXABCD operon consisting of five genes, essential for xylose degradation.
Subsequently biochemical and bioinformatical approaches were applied to provide enzymatic
functions and predict possible conversion pathways for xylose catabolism. We demonstrated that
the xylXABCD operon is tightly control via a LacI like repressor and defined determinants of the
xylose operator, critical for negative control of xylXABCD transcription.