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Urs Jenal

Publications and source records attributed to Urs Jenal.

At least 19 recordsLinked to original sources
PubMed2006

Genetic analysis of a novel pathway for D-xylose metabolism in Caulobacter crescentus.

Genetic data suggest that the oligotrophic freshwater bacterium Caulobacter crescentus metabolizes D-xylose through a pathway yielding alpha-ketoglutarate, comparable to the recently described L-arabinose degradation pathway of Azospirillum brasilense. Enzymes of the C. crescentus pathway, including an NAD(+)-dependent xylose dehydrogenase, are encoded in the xylose-inducible xylXABCD operon (CC0823-CC0819).

Arabinose↗
PubMed2006

Allosteric control of cyclic di-GMP signaling.

Cyclic di-guanosine monophosphate is a bacterial second messenger that has been implicated in biofilm formation, antibiotic resistance, and persistence of pathogenic bacteria in their animal host. Although the enzymes responsible for the regulation of cellular levels of c-di-GMP, diguanylate cyclases (DGC) and phosphodiesterases, have been identified recently, little information is available on the molecular mechanisms involved in controlling the activity of these key enzymes or on the specific interactions of c-di-GMP with effector proteins. By using a combination of genetic, biochemical, and modeling techniques we demonstrate that an allosteric binding site for c-di-GMP (I-site) is responsible for non-competitive product inhibition of DGCs. The I-site was mapped in both multi- and single domain DGC proteins and is fully contained within the GGDEF domain itself. In vivo selection experiments and kinetic analysis of the evolved I-site mutants led to the definition of an RXXD motif as the core c-di-GMP binding site. 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 DGCs 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 and makes DGCs a valuable target for drug design to develop new strategies against biofilm-related diseases.

Allosteric Site↗
PubMed2006

Holdfast formation in motile swarmer cells optimizes surface attachment during Caulobacter crescentus development.

The adhesive holdfast is required for irreversible surface anchoring of Caulobacter crescentus cells. The holdfast is synthesized early during swarmer cell development and, together with pili and a functional flagellum, contributes to optimal attachment during cell differentiation. We present evidence that the timing of holdfast formation in swarmer cells is regulated posttranslationally and is dependent on the diguanylate cyclase PleD.

Bacterial Adhesion↗
PubMed2006

Mechanisms of cyclic-di-GMP signaling in bacteria.

Cyclic-di-GMP is a ubiquitous second messenger in bacteria. The recent discovery that c-di-GMP antagonistically controls motility and virulence of single, planktonic cells on one hand and cell adhesion and persistence of multicellular communities on the other has spurred interest in this regulatory compound. Cellular levels of c-di-GMP are controlled through the opposing activities of diguanylate cyclases and phosphodiesterases, which represent two large families of output domains found in bacterial one- and two-component systems. This review concentrates on structural and functional aspects of diguanylate cyclases and phosphodiesterases, and on their role in transmitting environmental stimuli into a range of different cellular functions. In addition, we examine several well-established model systems for c-di-GMP signaling, including Pseudomonas, Vibrio, Caulobacter, and Salmonella.

Bacteria↗
PubMed2005

Identification and characterization of a cyclic di-GMP-specific phosphodiesterase and its allosteric control by GTP.

Cyclic diguanylic acid (c-di-GMP) is a global second messenger controlling motility and adhesion in bacterial cells. Synthesis and degradation of c-di-GMP is catalyzed by diguanylate cyclases (DGC) and c-di-GMP-specific phosphodiesterases (PDE), respectively. Whereas the DGC activity has recently been assigned to the widespread GGDEF domain, the enzymatic activity responsible for c-di-GMP cleavage has been associated with proteins containing an EAL domain. Here we show biochemically that CC3396, a GGDEF-EAL composite protein from Caulobacter crescentus is a soluble PDE. The PDE activity, which 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 (K(D) 4 microM) and operates by lowering the K(m) 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.

Allosteric Regulation↗
PubMed2004

Structural basis of activity and allosteric control of diguanylate cyclase.

Recent discoveries suggest that a novel second messenger, bis-(3'-->5')-cyclic di-GMP (c-diGMP), is extensively used by bacteria to control multicellular behavior. Condensation of two GTP to the dinucleotide is catalyzed by the widely distributed diguanylate cyclase (DGC or GGDEF) domain that occurs in various combinations with sensory and/or regulatory modules. The crystal structure of the unorthodox response regulator PleD from Caulobacter crescentus, which consists of two CheY-like receiver domains and a DGC domain, has been solved in complex with the product c-diGMP. PleD forms a dimer with the CheY-like domains (the stem) mediating weak monomer-monomer interactions. The fold of the DGC domain is similar to adenylate cyclase, but the nucleotide-binding mode is substantially different. The guanine base is H-bonded to Asn-335 and Asp-344, whereas the ribosyl and alpha-phosphate moieties extend over the beta2-beta3-hairpin that carries the GGEEF signature motif. In the crystal, c-diGMP molecules are crosslinking active sites of adjacent dimers. It is inferred that, in solution, the two DGC domains of a dimer align in a two-fold symmetric way to catalyze c-diGMP synthesis. Two mutually intercalated c-diGMP molecules are found tightly bound at the stem-DGC interface. This allosteric site explains the observed noncompetitive product inhibition. We propose that product inhibition is due to domain immobilization and sets an upper limit for the concentration of this second messenger in the cell.

Allosteric Regulation↗
PubMed2004

Cell cycle-dependent dynamic localization of a bacterial response regulator with a novel di-guanylate cyclase output domain.

Pole development is coordinated with the Caulobacter crescentus cell cycle by two-component signaling proteins. We show that an unusual response regulator, PleD, is required for polar differentiation and is sequestered to the cell pole only when it is activated by phosphorylation. Dynamic localization of PleD to the cell pole provides a mechanism to temporally and spatially control the signaling output of PleD during development. Targeting of PleD to the cell pole is coupled to the activation of a C-terminal guanylate cyclase domain, which catalyzes the synthesis of cyclic di-guanosine monophosphate. We propose that the local action of this novel-type guanylate cyclase might constitute a general regulatory principle in bacterial growth and development.

Bacterial Proteins↗
PubMed2004

Cyclic di-guanosine-monophosphate comes of age: a novel secondary messenger involved in modulating cell surface structures in bacteria?

The cyclic nucleotide cyclic di-guanosine-monophosphate (c-diGMP) was recognized in the 1980s as a signaling compound that is involved in controlling the condensation of glucose moieties into cellulose polymers. More recent data from several different bacterial species now suggest that c-diGMP might have a general role as secondary messenger in modulating bacterial growth on surfaces by regulating cellular adhesion components and preparing cells for cell-cell and cell-surface interactions.

Bacteria↗
PubMed2004

Identification of the protease and the turnover signal responsible for cell cycle-dependent degradation of the Caulobacter FliF motor protein.

Flagellar ejection is tightly coupled to the cell cycle in Caulobacter crescentus. The MS ring protein FliF, which anchors the flagellar structure in the inner membrane, is degraded coincident with flagellar release. Previous work showed that removal of 26 amino acids from the C terminus of FliF prevents degradation of the protein and interferes with flagellar assembly. To understand FliF degradation in more detail, we identified the protease responsible for FliF degradation and performed a high-resolution mutational analysis of the C-terminal degradation signal of FliF. Cell cycle-dependent turnover of FliF requires an intact clpA gene, suggesting that the ClpAP protease is required for removal of the MS ring protein. Deletion analysis of the entire C-terminal cytoplasmic portion of FliF C confirmed that the degradation signal was contained in the last 26 amino acids that were identified previously. However, only deletions longer than 20 amino acids led to a stable FliF protein, while shorter deletions dispersed over the entire 26 amino acids critical for turnover had little effect on stability. This indicated that the nature of the degradation signal is not based on a distinct primary amino acid sequence. The addition of charged amino acids to the C-terminal end abolished cell cycle-dependent FliF degradation, implying that a hydrophobic tail feature is important for the degradation of FliF. Consistent with this, ClpA-dependent degradation was restored when a short stretch of hydrophobic amino acids was added to the C terminus of stable FliF mutant forms.

Adenosine Triphosphatases↗
PubMed2003

Proteome of Caulobacter crescentus cell cycle publicly accessible on SWICZ server.

Here we present the Swiss-Czech Proteomics Server (SWICZ), which hosts the proteomic database summarizing information about the cell cycle of the aquatic bacterium Caulobacter crescentus. The database provides a searchable tool for easy access of global protein synthesis and protein stability data as examined during the C. crescentus cell cycle. Protein synthesis data collected from five different cell cycle stages were determined for each protein spot as a relative value of the total amount of [(35)S]methionine incorporation. Protein stability of pulse-labeled extracts were measured during a chase period equivalent to one cell cycle unit. Quantitative information for individual proteins together with descriptive data such as protein identities, apparent molecular masses and isoelectric points, were combined with information on protein function, genomic context, and the cell cycle stage, and were then assembled in a relational database with a world wide web interface (http://proteom.biomed.cas.cz), which allows the database records to be searched and displays the recovered information. A total of 1250 protein spots were reproducibly detected on two-dimensional gel electropherograms, 295 of which were identified by mass spectroscopy. The database is accessible either through clickable two-dimensional gel electrophoretic maps or by means of a set of dedicated search engines. Basic characterization of the experimental procedures, data processing, and a comprehensive description of the web site are presented. In its current state, the SWICZ proteome database provides a platform for the incorporation of new data emerging from extended functional studies on the C. crescentus proteome.

Bacterial Proteins↗
PubMed2003

Regulation by proteolysis in bacterial cells.

Regulation by proteolysis plays a major role in bacterial stress responses, the cell cycle and development. Key regulators of these processes are subject to conditional proteolysis that depends on complex cellular information processing. This information includes temporal and spatial cues, and recent research has revealed a striking potential for multiple signal integration.

Adenosine Triphosphatases↗
PubMed2003

Role of the GGDEF regulator PleD in polar development of Caulobacter crescentus.

Several members of the two-component signal transduction family have been implicated in the control of polar development in Caulobacter crescentus: PleC and DivJ, two polarly localized histidine sensor kinases; and the response regulators DivK and PleD. The PleD protein was shown previously to be required during the swarmer-to-stalked cell transition for flagellar ejection and efficient stalk biogenesis. Here, we present data indicating that PleD also controls the onset of motility and a cell density switch immediately preceding cell division. Constitutively active alleles of pleD or wspR, an orthologue from Pseudomonas fluorescens, almost completely suppressed C. crescentus motility and inhibited the increase in swarmer cell density during cell differentiation. The observation that these alleles also had a dominant-negative effect on motility in a pleC divJ and a pleC divK mutant background indicated that PleD is located downstream of the other components in the signal transduction cascade, which controls the activity of the flagellar motor. In addition, the presence of a constitutive pleD or wspR allele resulted in a doubling of the average stalk length. Together, this is consistent with a model in which the active form of PleD, PleD approximately P, negatively controls aspects of differentiation in the late predivisional cell, whereas it acts positively on polar development during the swarmer-to-stalked cell transition. In agreement with such a model, we found that DivJ, which localizes to the stalked pole during cell differentiation, positively controlled the in vivo phosphorylation status of PleD, and the swarmer pole-specific PleC kinase modulated this status in a negative manner. Furthermore, domain switch experiments demonstrated that the WspR GGDEF output domain from P. fluorescens is active in C. crescentus, favouring a more general function for this novel signalling domain over a specific role such as DNA or protein interaction. Possible roles for PleD and its C-terminal output domain in modulating the polar cell surface of C. crescentus are discussed.

Alleles↗
PubMed2003

Role of the cytoplasmic C terminus of the FliF motor protein in flagellar assembly and rotation.

Twenty-six FliF monomers assemble into the MS ring, a central motor component of the bacterial flagellum that anchors the structure in the inner membrane. Approximately 100 amino acids at the C terminus of FliF are exposed to the cytoplasm and, through the interaction with the FliG switch protein, a component of the flagellar C ring, are essential for the assembly of the motor. In this study, we have dissected the entire cytoplasmic C terminus of the Caulobacter crescentus FliF protein by high-resolution mutational analysis and studied the mutant forms with regard to the assembly, checkpoint control, and function of the flagellum. Only nine amino acids at the very C terminus of FliF are essential for flagellar assembly. Deletion or substitution of about 10 amino acids preceding the very C terminus of FliF resulted in assembly-competent but nonfunctional flagella, making these the first fliF mutations described so far with a Fla(+) but Mot(-) phenotype. Removal of about 20 amino acids further upstream resulted in functional flagella, but cells carrying these mutations were not able to spread efficiently on semisolid agar plates. At least 61 amino acids located between the functionally relevant C terminus and the second membrane-spanning domain of FliF were not required for flagellar assembly and performance. A strict correlation was found between the ability of FliF mutant versions to assemble into a flagellum, flagellar class III gene expression, and a block in cell division. Motile suppressors could be isolated for nonmotile mutants but not for mutants lacking a flagellum. Several of these suppressor mutations were localized to the 5' region of the fliG gene. These results provide genetic support for a model in which only a short stretch of amino acids at the immediate C terminus of FliF is required for flagellar assembly through stable interaction with the FliG switch protein.

Amino Acid Sequence↗
PubMed2002

The Caulobacter cell cycle: timing, spatial organization and checkpoints.

In Caulobacter crescentus, a complex regulatory network integrates temporal and spatial information to control the ordered progression of the cell cycle, and to synchronize cell proliferation with development. Periodicity includes the timed synthesis, activation or destruction of key regulatory proteins, which activate a large number of genes at the appropriate time of the cell cycle. Checkpoints serve to couple cell division and polar development to the replication and segregation state of the chromosome. Asymmetrically positioned regulatory components are involved in the sequential positioning of polar organelles. New work sheds light on the spatial organization of cellular components involved in cell cycle progression and polar differentiation, and starts to define the molecular nature of checkpoints involved in cell cycle control and development.

Caulobacter↗
PubMed2002

The FtsH protease is involved in development, stress response and heat shock control in Caulobacter crescentus.

The ftsH gene of Caulobacter crescentus has been isolated and identified as a component of the general stress response of this organism. In C. crescentus, ftsH expression is transiently induced after temperature upshift and in stationary phase. Consistent with this, mutants deprived of the FtsH protease are viable at normal growth conditions, but are highly sensitive to elevated temperature, increased salt concentration or the presence of antibiotics. Overexpression of ftsH resulted in an increased salt but not thermotolerance, emphasizing the importance of the FtsH protease in stress response. Mutants lacking FtsH were unable to undergo morphological and physiological adaptation in stationary phase and, upon starvation, experienced a more pronounced loss of viability than cells containing FtsH. In addition, cells lacking FtsH had an increased cellular concentration of the heat shock sigma factor sigma32, indicating that, as in Escherichia coli, the FtsH protease is involved in the control of the C. crescentus heat shock response. In agreement with this, transcription of the heat-induced sigma32-dependent gene dnaK was derepressed at normal temperature when FtsH was absent. In contrast, the groEL gene, which is controlled in response to heat stress by both sigma32 and a HcrA/CIRCE mechanism, was not derepressed in an ftsH mutant. Finally, FtsH is involved in C. crescentus development and cell cycle control. ftsH mutants were unable to synthesize stalks efficiently and had a severe cell division phenotype. In the absence of FtsH, swarmer cells differentiated into stalked cells faster than when FtsH was present, even though the entire cell cycle was longer under these conditions. Thus, directly or indirectly, the FtsH protease is involved in the inherent biological clock mechanism, which controls the timing of cell differentiation in C. crescentus.

ATP-Dependent Proteases↗