205 research outputs found
Code for: A genetically encoded biosensor to monitor dynamic changes of c-di-GMP with high temporal resolution
No full text<div>
<div>This repository contains the code used for analyzing the single-cell microscopy data described in the following publication:</div>
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<div>A genetically encoded biosensor to monitor dynamic changes of c-di-GMP with high temporal resolution</div>
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<div>Andreas Kaczmarczyk, Simon van Vliet, Roman Peter Jakob, Raphael Dias Teixeira, Inga Scheidat, Alberto Reinders, Alexander Klotz, Timm Maier, Urs Jenal</div>
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<div>Biozentrum, University of Basel, 4056 Basel, Switzerland</div>
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<div>Correspondence to: urs.jenal[at]unibas.ch, andreas.kaczmarczyk[at]unibas.ch</div>
</div>
Identification and analysis of Clp protease substrates in "C. crescentus"
No full textProteolysis is an irreversible regulatory mechanism by which cells can remove a
protein whose function is no longer required or if its presence in a particular cellular
compartment and/or at a certain time is harmful to the cell. Degradation of
cytoplasmic proteins is energy dependent, and in prokaryotic cells is carried out by
five ATP-dependent proteases, namely, ClpXP, ClpAP, FtsH, Lon and HslUV
(Gottesman and Maurizi 1992). Protein degradation has been shown to be of crucial
importance for a variety of cellular processes such as stress response, DNA damage
repair and cell cycle progression (Jenal and Fuchs 1998; Jenal and Hengge-Aronis
2003; Straus et al. 1988; Sutton et al. 2000).
A critical event occurring during the cell cycle progression of C. crescentus is the
degradation of the essential master cell cycle regulator, CtrA, during the G1-to-S
phase transition (Quon et al. 1996). CtrA belongs to the response regulator
superfamily of proteins and aside from directly controlling the expression of about
100 genes (Laub et al. 2002; Laub et al. 2000) it suppresses DNA replication
initiation by binding to several sites in the origin of replication (Quon et al. 1998).
CtrA is degraded in a ClpXP-dependent manner as depletion of ClpXP results in its
stabilisation. Furthermore, in the absence of ClpXP, cells are arrested at the G1 phase
of the cell cycle, become filamentous and lose viability (Jenal and Fuchs 1998). A
similar phenotype is observed in cells expressing a stable and constitutively active
variant of CtrA (Domian et al. 1997). Inhibition of CtrA degradation alone does not
cause a G1 cell cycle block, suggesting that the G1 arrest observed in cells depleted
for ClpXP is not due to CtrA stabilisation. This suggests that the ClpXP-dependent degradation of one or several additional proteins is essential for cell cycle progression
and survival.
The primary aim of this work was to identify novel substrates of the ClpXP protease,
particularly those whose timed degradation is critical for G1-to-S phase transition.
This task is of crucial importance as previous work by Grünenfelder et al. (2001) has
shown that a large fraction of the cell’s proteins is rapidly degraded and differentially
synthesised during the cell cycle. It is likely that a subset of these proteins is involved
in cell cycle progression and control. In the first part of this thesis (Chapter 3) we
created a vector that allowed the conditional expression of the dominant negative
allele of clpX, clpXATP, for use in a global proteomics screen to identify ClpXP
substrates. In this screen, proteins that became stable upon disruption of ClpX activity
were to be identified. The rationale behind our generating the conditional clpXATP
allele was to create a system wherein ClpX activity can be rapidly disrupted in cells.
The original ClpX depletion strain created by Jenal and Fuchs (1998) required at least
four hours before the ClpX protein is undetectable in cells. Since the result of ClpX
depletion is ultimately cell death, use of this mutant in a proteomics screen would
make it difficult to distinguish between proteins stabilised as a direct effect of ClpX
depletion and those stabilised as a consequence of cell deterioration.
We found that expression of clpXATP, which has mutations in the walker A motif of
the ATPase domain, results in rapid CtrA stabilisation, cell elongation and cell death.
We propose that this is due to ClpXATP monomers inactivating ClpX through the
formation of mixed oligomers with ClpXwt monomers. As this study was in progress,
the crystal structure of ClpX from H. pylori was solved revealing that the residues we mutated in ClpXATP contact the ATP moiety and may be involved in ATP hydrolysis
(Kim and Kim 2003). Thus, our results indicate that the presence of ClpXATP disrupts
ClpX activity by preventing the assembly of hexameric rings, disturbing ATP binding
and/or inhibiting ATP hydrolysis by the mixed ClpX hexamers. Although the nature
and oligomeric state of the mixed oligomers is not clear from our results, previous
work with clpA alleles with similar mutations in the walker A motif has demonstrated
that mixed hexamers do form and that monomer swapping readily occurs (Seol et al.
1995; Singh and Maurizi 1994).
Through the global comparison of protein stability between wild-type and clpXATP
expressing cells, we found nine proteins to be stabilised as a result of ClpX
inactivation. These include CtrA and CheD, both previously identified as ClpXP
substrates using genetic means (Jenal and Fuchs 1998; M.R.K. Alley, unpublished).
Target validation confirmed that CtrA, CheD and the product of the CC2323 gene
were degraded in a ClpXP-dependent manner.
CC2323 is a protein of unknown function whose orthologues are found exclusively in
alpha proteobacteria. CC2323 expression was previously found to be regulated by
GcrA, a cell cycle regulator that inversely oscillates with CtrA (Holtzendorff et al.
2004). We found that CC2323 synthesis is limited to the late S-, and G2- phase of the
cell cycle and that its product is rapidly degraded. As a result, the CC2323 protein is
only present when it is actively synthesised and is therefore absent in SW and ST
cells. Our results indicate that CC2323 may be degraded by ClpXP and that its levels
during the cell cycle are controlled only through its regulated expression. Although
CC2323 was found to be non-essential for growth, our results indicate that its overproduction is deleterious for cell growth and survival. Thus, it appears that either
high levels of CC2323, or its undesirable presence in certain cellular compartments
and/or phases of the cell cycle, have negative effects on the cells. Future analysis will
aim to address the reasons why CC2323 overproduction is harmful to cells and why
its cellular concentration appears stringently controlled during the cell cycle at the
levels of both expression and proteolysis.
In the second part of this thesis (Chapter 4) we defined SsrA-tagged proteins as
additional targets of the ClpXP protease in C. crescentus, and conducted a functional
examination of the SsrA tag. The SsrA is a protein tag that is attached to proteins
under a variety of conditions, including starvation, and targets them for degradation
by ATP-dependent proteases. In E. coli, ClpXP is the main protease that is
responsible for SsrA-tagged substrate degradation (Gottesman et al. 1998). We
constructed several fusions between FlbD, a transcriptional regulator of late flagellar
genes, and the SsrA to determine if in C. crescentus, as in E. coli, ClpXP degrades
SsrA tagged substrates. FlbD-SsrA was found to be highly unstable but was stabilised
upon induction of the clpXATP allele. Similarly, FlbD-SsrA was stabilised when ClpP
was depleted from cells. This indicated that ClpXP is responsible for the rapid
turnover of SsrA-tagged proteins in C. crescentus.
SsrA-tagged FlbD variants were then used to genetically dissect the SsrA degradation
pathway. We found that cells bearing FlbD-SsrA were non-motile due to the rapid
degradation of FlbD and consequent lack of flagellar gene expression. To identify
mutations, cis or trans, that stabilised FlbD-SsrA, a selection for motile suppressors
was carried out. Our hypothesis was that cells which regained motility would have stabilised FlbD through mutations in the SsrA tag or in an accessory component. Only
two suppressors were isolated that contained amino acid substitutions in the SsrA tag,
indicating that these are important residues for recognition by ClpX.
The remainder of the motile suppressors contained deletion or insertion frame-shifts
by which the identity of the FlbD C-terminus was completely altered and the SsrA tag
removed. In most cases, this resulted in FlbD stabilisation. However, transfer of one
of those alleles into a clean genetic background suggested that the flbD allele alone is
not able to restore motility. From this we concluded that FlbD variants with an altered
C-terminus were non-functional and that a second mutation in trans must have
occurred to restore motility. Consistent with this, FlbD fused to a stable variant of
SsrA (FlbD-SsrADDD) did not support motility. Motile suppressors of strains carrying
FlbD-SsrADDD had retained the nature of their SsrA tag, again suggesting that
mutations in trans had restored motility. Those could map to components that either
regulate the activity of FlbD or interact with it. It will be interesting to map these
mutations as they may provide useful information about FlbD and its regulation of
flagellar assembly in C. crescentus. The challenge for future work will be to map the
second site mutation(s) and to define the exact contributions of cis- and transmutations
for FlbD stability and/or activity.
In the third and final part of this thesis (Grünenfelder et al. 2004), we examined cell
cycle-dependent FliF degradation. FliF forms the MS ring that anchors the flagellum
in the inner membrane. Degradation of FliF at the G1-to-S phase transition coincides
with flagellar ejection and was hypothesised to be the committing step of this
developmental process (Grünenfelder et al. 2003; Jenal and Shapiro 1996). We found that the non-essential ClpAP protease is required for the degradation of FliF as SW
cells differentiate into ST cells. To define the nature of the ClpAP degradation signal,
we conducted a high resolution mutational analysis of the FliF C-terminus. We found
that though the degradation signal of FliF resides in the last 28 residues of the protein,
no primary sequence appears to govern its turnover. Instead, our results indicate a
requirement for hydrophobic residues at the C-terminus of FliF
Second messenger mediated spatiotemporal control of cell cycle and development
No full textDuring the biphasic life cycle of Caulobacter crescentus motile, free-living swarmer
cells differentiate into sessile, surface attached stalked cells. The swarmer cell is
replication inert and is unable to divide. During the swarmer-to-stalked cell
differentiation, degradation of CtrA, a master regulator that blocks replication initiation,
leads the onset of chromosome replication. After this obligate cell differentiation step,
which is mainly regulated by the degradation of the master cell cycle regulator CtrA,
stalked cells immediately initiate their chromosome replication. Recently, dynamic colocalization
of CtrA and its protease ClpXP to cell pole was proposed as a timing
mechanism for cell cycle-dependent CtrA degradation.
We have identified the response regulator PopA as an essential regulator for CtrA
sequestration to the incipient stalked cell pole and for subsequent CtrA degradation by
the nearby ClpXP protease complex. Time laps fluorescence microscopy of PopA-GFP
showed that PopA itself dynamically sequesters to the cell poles during the C.
crescentus cell cycle. While PopA sequestration to the flagellated pole depends on
PodJ, a swarmer pole specificity factor, localization to the incipient stalked pole
depends on the C-terminal GGDEF output domain of PopA. We demonstrate that in
contrast to most GGDEF domain proteins, PopA lacks diguanylate cyclase activity.
Instead, PopA functions as cyclic di-GMP effector protein, which specifically binds the
bacterial second messenger at a conserved binding site (I-site) within the GGDEF
domain. An intact PopA I-site is required for PopA sequestration to the incipient stalked
pole as well as for CtrA degradation during the cell cycle. PopA directs CtrA to the
ClpXP occupied cell pole via a direct interaction with an adaptor protein, RcdA. Based
on this we postulate that c-di-GMP bound PopA facilitates the dynamic distribution of
CtrA to the cell pole where it s degraded by ClpXP. This is the first report that links cdi-
GMP to protein dynamics and cell cycle control in bacteria.
In addition to its prominent role in cell cycle control, PopA was identified as novel
component of the complex regulatory network that orchestrates polar development in
C. crescentus. PopA, together with PleD and DgcB, two active diguanylate cyclases,
controls cell motility, holdfast formation and surface attachment. Our data suggest that
PopA interferes with PleD and DgcB to coordinate cell motility, stalk biogenesis,
holdfast formation and finally surface attachment. Based on this, we propose that PopA is a bifunctional protein, involved in control and coordination of C. crescentus cell
cycle and development
The discovery of SycO reveals a new function for type three secretion effector chaperones
No full textThe Type Three Secretion (T3S) system is a device used by many Gram-negative pathogens that allows bacteria to deliver effector proteins straight into the eukaryotic cell cytosol. These effectors interfere with various signaling pathways to subvert the host cell functions. The secretion machinery of the T3S system consist of a basal body spanning the bacterial inner and outer membrane followed by a stiff hollow needle outside the bacterium. The fully assembled secretion apparatus constitute a continuous hollow conduit that connects the bacteria to the eukaryotic target cell. After cell contact, virulence proteins -called effectors- are injected directly into the cytosol of the host cell via the T3S apparatus. Several effectors of the T3S system require the assistance of specific cytosolic chaperones to be efficiently exported. There are three classes of T3S chaperones. Effector proteins are assisted by Class I chaperones. Although Class I chaperones are well characterized, their main function is still a matter of controversy. In this thesis, we demonstrate that orf155 encodes a specific chaperone for the effector YopO that we called SycO. We showed that SycO enhances YopO secretion in vitro and is required for translocation of YopO into infected cells. By pulldown assay we demonstrated that residues 20 to 77 of YopO are required and sufficient for SycO binding. Using crosslinking experiments and size exclusion chromatography analysis, we determined the stoichiometry of purified SycO and YopO-SycO complexes. SycO alone forms dimers in solution and the YopO-SycO complex has a 1:2 stoichiometry. These results suggested that SycO is a typical chaperone of the Class I. YopO is a serine/theronine kinase that interacts with Rho and Rac and disrupts the cytoskeleton of the target cells. YopO has been shown to localize at the cell plasma-membrane. By transfection of YopO-EGFP hybrid proteins into HEK293T cells, we demonstrated that the chaperone-binding domain (CBD) coincides with the membrane localization domain of YopO. Nevertheless, the CBD was not needed for the kinase activity of YopO. By ultracentrifugation, we also showed that the CBD causes YopO aggregation in the bacteria, when SycO does not cover it. Further, we show that the CBD of YopE and YopT also caused aggregation in the bacteria in the absence of SycE and SycT respectively. YopE, YopT and T3S effectors in other systems also act at the membrane of the eukaryotic host cell. We propose a new hypothesis concerning the role of T3S chaperones. The sub-cellular localization domain of effectors is aggregation-prone and creates the need for a chaperone inside bacteria. We propose that masking such aggregation-prone localization domains may be a general function for type III effector chaperones
Supplementary Movie 1 from: A genetically encoded biosensor to monitor dynamic changes of c-di-GMP with high temporal resolution
No full text<p>This record contains<strong> Supplementary Movie 1</strong> from:</p>
<p><strong>A genetically encoded biosensor to monitor dynamic changes of c-di-GMP with high temporal resolution</strong></p>
<p>Andreas Kaczmarczyk, Simon van Vliet, Roman Peter Jakob, Raphael Dias Teixeira, Inga Scheidat, Alberto Reinders, Alexander Klotz, Timm Maier, Urs Jenal</p>
<p>Biozentrum, University of Basel, 4056 Basel, Switzerland</p>
<p>Correspondence to: urs.jenal[at]unibas.ch, andreas.kaczmarczyk[at]unibas.ch</p>
<p> </p>
Analysis of cyclic di-GMP signaling components in "caulobacter crescentus" behavior and cell cycle control
No full textCell cycle progression and polar morphogenesis in Caulobacter crescentus are coordinated by the interplay of multiple proteins in time and space. One major regulatory factor is the second messenger cyclic di-GMP (c-di-GMP) therefore especially the activities of enzymes that are responsible for synthesis and breakdown of this small molecule are tightly regulated. The swarmer cell specific population in the early phase of the cell cycle contains low levels of c-di-GMP due to the action of the phosphodiesterase PdeA. During the course of cell cycle progression, PdeA is degraded and thereby the activity of the diguanylate cyclase (DGC) DgcB is released. At the same time a second DGC, PleD, is activated by a phosphorylation relay, to elevate c-di-GMP levels necessary for cell development. The two proteins DgcB and PleD are the main cyclases in C. crescentus contributing to the intracellular c-di-GMP pool. Cells lacking both DGCs have severe defects affecting cell morphology and cell cycle progression. However, a residual c-di-GMP concentration is still detectable in the pleD dgcB double mutant presumingly due to the activity of other DGCs of C. crescentus.
This work addressed the question, which additional GGDEF domain proteins reveal DGC activity and contribute to the c-di-GMP content in C. crescentus cells. This work presented here shows that two additional cyclases, BipB and BipC (bifunctional proteins B and C), are involved in c-di-GMP signaling. Both enzymes belong to the group of so-called composite proteins harboring a GGDEF and EAL domain, encoding for opposing catalytic activities, respectively. Single deletions of either bipB or bipC showed no phenotype. However, in combination with the deletion of pleD and dgcB, no c-di-GMP could be detected. The lack of c-di-GMP resulted in miss-localization of the effector protein PopA that is involved in the degradation of the replication inhibitor CtrA. Therefore, CtrA is stabilized in those cells leading to elongated cell morphology. These phenotypes resemble the phenotypes of a strain lacking all predicted DGCs (gutted strain, GS). To measure specifically low levels of c-di-GMP a strain was used lacking DGCs and in addition all PDEs (really gutted strain, rGS) to avoid immediate degradation in the GS. Introduction of either bipB or bipC in the rGS reverted the strain to a wild-type phenotype, e.g. motility and popA localization, indicating a DGC phenotype in vivo. However, in the presence of different PDEs like in the GS neither bipB nor bipC were able to revert the phenotype to wild-type suggesting weak DGC activity of both enzymes.
For BipB bifunctional enzyme activity could be demonstrated in vitro and in vivo, whereas the DGC and the PDE activities were present at the same time. The cyclase activity of BipB is substrate inhibited via c-di-GMP binding to the inhibitory site motif RxxD. Based on these finding we propose that BipB is a bifunctional protein contributing under the applied conditions with BipC, PleD and DgcB to intracellular c-di-GMP levels in C. crescentus.
The c-di-GMP signaling circuit involves not only cyclases and phosphodiesterases, which produce c-di-GMP upon an environmental stimulus but also effector proteins that bind c-di-GMP and therefore transmit the signal into an intracellular response. Knowing different c-di-GMP binding proteins would allow understanding c-di-GMP output systems. Therefore, a biochemical screen was carried out using c-di-GMP linked to a capture compound to specifically isolate c-di-GMP binding proteins. Among the novel identified proteins a group clusters next to chemotaxis genes. One of the hits is CmcA (named after its involvement in c-di-GMP dependent motor control), a single domain response regulator lacking the conserved phosphorylation site (aspartate) necessary for the function of a RR. Deletion of cmcA results in an increase in motility. To transmit the chemotactic signal CheY proteins interact directly with the flagellar apparatus. Therefore, the localization pattern of CmcA in different flagellar mutants was determined showing polar localization dependent on the MS-ring forming protein FliF. This localization pattern is missing in c-di-GMP deficient cells. From these results, we concluded that CmcA regulates motility in a c�di-GMP dependent manner
Insights into the activation mechanism of PopA, a cyclic di-GMP effector protein involved in cell cycle and development of "Caulobacter Crescentus"
No full textIn Caulobacter crescentus, a complex network integrating cyclic di-GMP and Phosphorylation-dependent signals controls the proteolysis of key regulatory proteins to drive cell cycle and polar morphogenesis. The c-di-GMP input is processed by the effector protein PopA. Upon binding of c-di-GMP, PopA is sequestered to the old cell pole where it recruits the replication and cell division inhibitors CtrA and KidO and mediates their destruction by the polar ClpXP protease prior to entry into S-phase. In addition to its role at the stalked cell pole, PopA localizes to the opposite cell pole in dependence of the general topology factor PodJ where it exerts a yet unknown function.
Here we address the activation and polar sequestration mechanism of PopA guided by an existing activation model for the highly homologous c-di-GMP signaling protein PleD. PopA and PleD do not only share an identical domain organization (Rec1-Rec2-GGDEF), but also show similar spatio-temporal behavior during the cell cycle. While PleD is activated and targeted to the old cell pole via phosphorylation-induced dimerization, we show that PopA stalked pole function is phosphorylation-independent and requires c-di-GMP binding as a primary input signal for activation and polar localization. c-di-GMP binds to conserved primary and secondary I-sites within the PopA GGDEF domain and we show that intact binding sites are required for PopA positioning and function. This suggests that c-di-GMP-dependent crosslinking of adjacent GGDEF domains contributes to the localization of an active PopA dimer to the cell pole. Consistent with this, we demonstrate that the GGDEF domain encodes the polar localization signal(s), while the N-terminal receiver domains serve as interaction platform for downstream components that are actively recruited by PopA.
Among these downstream factors is RcdA, a small mediator protein that interacts with the first PopA receiver domain and helps to recruit and degrade CtrA and KidO. In a screen for additional components of the PopA pathway we identify two novel proteins that directly interact with PopA, CC1462 and CC2616. CC1462 is a ClpXP substrate that requires PopA for polar positioning and subsequent degradation during swarmer-to-stalked cell transition. Although located in a flagellar gene cluster, deletion of CC1462 did not affect flagellar assembly and function. Its cellular role as well as the significance of its cell cycle-dependent degradation requires further studies. CC2616, the second PopA interaction partner, is not proteolytically processed and thus belongs to another class of PopA-dependent substrates. CC2616 is annotated as guanine deaminase, which is predicted to catalyze the conversion from guanine to xanthine thereby irreversibly removing guanine based nucleotides from a cellular pool. A CC2616 deletion leads to increased attachment and decreased motility, a phenocopy of strains with elevated c-di-GMP levels. It is not clear whether CC2616 indeed has deaminase activity or whether it has adopted a novel function.
Taken together, this work provides insight into the activation mechanism of a c-di-GMP effector protein. We propose that PopA has evolved through gene duplication from its ancestor, the catalytic PleD response regulator but has lost catalytic activity of the diguanylate cyclase domain. Moreover, PopA has adopted an inverse intra-molecular information transfer originating through c-di-GMP binding at the C-terminal GGDEF domain, which in turn activates the N-terminal receiver stem to serve as platform for downstream partner recruitment
Structural biology of bacterial response regulator proteins and their complexes with cognate ligands
No full textTwo bacterial response regulator systems were studied in this thesis. NMR
spectroscopy and X-ray crystallography were used to determine the structures of both
domains of the antibiotic sensor TipAL, as well as of the c-di-GMP receptor PA4608,
in complex with cognate ligands. By comparison with structures of the free proteins,
we found that ligand binding induced biologically relevant structural rearrangements
in all proteins studied.
The putative multidrug resistance gene tipA of the soil bacterium Streptomyces
lividans codes for the transcription factor TipAL, which comprises two domains: a
DNA-binding domain named TipAN, and a ligand-binding domain named TipAS,
which is capable of recognizing and binding a diverse group of macrocyclic
thiopeptide antibiotics. After antibiotic binding, TipAL binds to promoter DNA and
activates transcription. In order to elucidate the specificity and flexibility of antibiotic
recognition as well as the mechanism of transcriptional activation, the two domains of
TipAL were studied separately.
We have determined the structures of TipAS with bound promothiocin A or
nosiheptide antibiotics by NMR spectroscopy. The N-terminal part of the TipAS
sequence, which is flexible and unstructured in free TipAS, forms three new helices in
both complexes, burying the bound antibiotics. Considering that the newly formed
helices form the connection between the TipAS and TipAN domains, we propose that
the formation of additional secondary structure forms the basis of transcriptional
activation by TipAL after ligand binding. The TipAS complexes with promothiocin A
and nosiheptide are similar, but differ in the dynamics of the newly formed helices;
the smaller ligand, promothiocin A, appears to leave more room for movement of
TipAS.
The structure of TipAN in complex with a fragment of tipA promoter DNA was
solved by X-ray crystallography. TipAN binds to the symmetric promoter as a dimer,
which is held together by a long, antiparallel coiled coil. In contrast to homologous
proteins, TipAN does not bend and twist bound DNA, which is a prerequisite for
transcriptional activation by other proteins of the same family. This indicates that the
activated TipAS domain is required for transcriptional activation by TipAL.
C-di-GMP is a second messenger molecule that appears to be ubiquitous in, and
unique to, the bacterial kingdom. It generally controls the switch from motile, singlecell
lifestyles to surface-attached, multicellular communities such as biofilms. The
natural receptors of c-di-GMP are the PilZ domain proteins, which include PA4608 in
Pseudomonas aeruginosa. We have solved the NMR solution structure of PA4608 in
complex with c-di-GMP. C-di-GMP binds to the protein as an intercalated dimer, displacing the C-terminal 310 helix found in the apo form. The N-terminal part of
PA4608, which contains the highly conserved RxxxR motif and which is flexible and
unstructured before ligand binding, contacts the distal side of c-di-GMP
Regulation of poly-GlcNAc expression and fimbriation in uropathogenic "E. coli"
No full textThe transition of planktonic to sessile lifestyles in bacteria rests upon a tightly controlled program that gets triggered by the environmental composition and whose implementation requires multiple feedback controls. Uropathogenic E. coli (UPEC), the predominant agents of urinary tract infections, use this lifestyle switch to shift from acute to chronic, biofilm associated infections. While the acute phase of infection is dominated by the expression of virulence factors such as type I fimbriae, biofilm matrix components including PGA prevail during biofilm associated infections. In this work, factors reported to induce a lifestyle switch are used in UPECs to investigate their effects on the expression patterns of two output-systems (type I fimbriae and PGA) during lifestyles transition. It was investigated if PGA dependent biofilm formation in UPECs requires derepression of the carbon storage regulator (Csr) system. Furthermore, it was analysed if PGA dependent biofilms respond to the bacterial second messenger c-di-GMP or the alarmone ppGpp of the stringent response and if PGA contributes to UTI pathogenesis. Finally, this work aims at clarifying the role of type I fimbriae in PGA dependent attachment and investigates if the expression patterns of the two surface-exposed structures are subject to a regulatory cross-talk
Principles of c-di-GMP signaling : characterization of a second messenger system orchestrating bacterial life style
No full textBacteria 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
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