Mechanisms of cyclic-di-GMP signaling : insight into the biochemistry, signal transduction and regulation of the bacterial second messenger cyclic-di-GMP

Christen, Matthias. Mechanisms of cyclic-di-GMP signaling : insight into the biochemistry, signal transduction and regulation of the bacterial second messenger cyclic-di-GMP. 2007, Doctoral Thesis, University of Basel, Faculty of Science.


Official URL: http://edoc.unibas.ch/diss/DissB_7774

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Bacteria are able to switch between two mutually exclusive lifestyles, motile single cells and
sedentary multicellular communities that colonize surfaces. These behavioral changes contribute to
an increased fitness in structured environments and are controlled by the ubiquitous bacterial
second messenger c-di-GMP. In response to changing environments, fluctuating levels of c-di-
GMP antagonistically affect motility and virulence of single, planktonic cells as well as cell surface
adhesins and persistence of sedentary, multicellular communities. The cellular levels of c-di-GMP
are controlled by opposing enzymatic activities of diguanylate cyclases (DGCs) and
phosphodiesterases (PDEs), which represent two large families of output domains found in
bacterial one- and two-component systems. The present work investigates structural, functional
and regulatory aspects of diguanylate cyclases and phosphodiesterases, and explores their role in
signal transduction processes transmitting environmental stimuli into a range of different cellular
functions. Furthermore we report the isolation and characterization of novel components of the cdi-
GMP signaling network mediating its output functions.
In (Christen et al. 2005, JBC 280:30829-30837), we report the finding that the c-di-GMP specific
phosphodiesterase activity resides in the widespread EAL domain. By analyzing the enzymatic
reaction products and investigating the substrate specificity of wild type and various mutant
enzymes, we demonstrate that a single EAL domain itself catalyzes, in a Mg2+ dependent manner,
the cleavage of the second messenger c-di-GMP into the linear dinucleotide pGpG. Furthermore,
we report the discovery that in a GGDEF-EAL protein a catalytic inactive GGDEF domain can bind
GTP and in response allosterically activates the EAL domain. Thus we conclude that GGDEF
domains can have either catalytic or regulatory function and suggest, that the cellular GTP pool
may serve as an input signal into c-di-GMP-mediated signal transduction.
In (Christen & Christen et al. 2006, JBC 281:32015-32024), we describe an important novel feature of
GGDEF proteins, which produce the ubiquitous bacterial signaling molecule c-di-GMP. This paper
reports the results of in depth structure-function analysis of an allosteric feedback inhibition
mechanism that generally acts to regulate diguanylate cyclase activities in bacteria. The
mechanism involves binding of the second messenger product, c-di-GMP at an inhibition site (Isite)
that is coupled via a conserved beta-strand to the active site (A-site) of the enzyme. The study
involves an array of biochemical and genetic techniques applied on various diguanylate cyclases to
establish the sequence determinants of the I-site as well as the in vivo physiological relevance of Isite
function. Allosteric product inhibition of diguanylate cyclases turns out to have fundamental
functional and physiological implications, including threshold setting for c-di-GMP production by
particular GGDEF proteins, which can contribute to precision, robustness, noise reduction and
accelerated kinetics of c-di-GMP signaling. 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, and makes DGCs a valuable target for drug design to develop new strategies
against biofilm-related diseases.
In (Christen & Christen et al. 2007, PNAS), we enlighten the signal transduction mechanism of the
bacterial second messenger c-di-GMP and demonstrate the existence of diguanylate receptor
proteins mediating its output functions. We report the biochemical purification of c-di-GMP receptor
proteins from C. crescentus crude extract and describe their physiological role in c-di-GMP
dependent repression of cell motility. A multitude of biochemical, genetic and NMR experiments
was used to characterize these effector proteins and homologs from S. enterica and P. aeruginosa
down to molecular level. In particular we used [33P] c-di-GMP UV cross linking studies to
demonstrate that these receptors specifically bind c-di-GMP in the sub micromolar range and, in
combination with NMR spectrometry, to elicit determinants for c-di-GMP binding. Furthermore, we
performed genetic suppressor analysis and epistasis experiments with receptor deletion and
pointmutants, to corroborate that the identified diguanlyate receptors from C. crescentus act in vivo
downstream of the second messenger c-di-GMP.
We further report the isolation and characterization of a C. crescentus adenylosuccinate
synthetase (PurA, CC3103), an enzyme of the purine biosynthesis pathway that has high affinity
for c-di-GMP. Using recombinant purified PurA for kinetic and ligand binding studies, we show that
c-di-GMP is a potent inhibitor of PurA activity. Initial rate kinetics revealed that c-di-GMP inhibition
is competitive with respect to GTP and noncompetitive with respect to IMP. These findings suggest
a role for c-di-GMP as regulator of the cellular nucleotide pool. We propose that c-di-GMP inhibits
the first step of the de novo biosynthesis of AMP and by that directs IMP toward guanine
biosynthesis, thereby preventing the drainage of the guanine pool.
Advisors:Jenal, Urs
Committee Members:Schirmer, Tilman and Wennemers, Helma
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Infection Biology > Molecular Microbiology (Jenal)
05 Faculty of Science > Departement Biozentrum > Growth & Development > Molecular Microbiology (Jenal)
UniBasel Contributors:Christen, Matthias and Jenal, Urs and Schirmer, Tilman and Wennemers, Helma
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:7774
Thesis status:Complete
Bibsysno:Link to catalogue
Number of Pages:174
Identification Number:
Last Modified:22 Jan 2018 15:50
Deposited On:13 Feb 2009 15:51

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