C-di-GMP acts as a cell cycle oscillator to drive chromosome replication

Cyclic di-GMP (c-di-GMP) is an omnipresent bacterial second Messenger molecule which has been recognized as a central regulator of Lifestyle transitions. Generally, high levels of c-di-GMP promote a biofilm associated, surface attached lifestyle, while low levels of c-di-GMP favor a single cell, motile lifestyle. A wide range of c-di-GMP effector proteins are known which control various cellular functions. It has long been assumed that c-di-GMP is involved in the regulation of cell cycle progression. In this work the role of c-di-GMP on the G1-S transition is described in the aquatic bacterium Caulobacter crescentus. C. crescentus is an ideal model organism since G1-S transition is developmentally linked to the swarmer to stalked cell transition and therefore easily observable. Moreover, c-di-GMP influences several processes at the swarmer to stalked cell transition. Thus, disturbing the c-di-GMP-dependent processes causes specific phenotypes. In the first part of this work, the effect of c-di-GMP on core components of the C. crescentus cell cycle control machinery is assessed. It is described that the essential histidine kinase CckA (Cell cycle kinase A) is regulated by c-di-GMP. Binding of CckA to c-di-GMP activates the phosphatase activity of CckA and leads to dephosphorylation of the transcription factor CtrA (Central transcriptional activator A) which ultimately initiates chromosome replication. Furthermore it is shown that c-di-GMP is required in the predivisional cell to establish a CckA-dependent CtrA phosphorylation gradient. The second part describes the mechanism by which c-di-GMP activates CckA phosphatase activity. It was possible to isolate several mutations in CckA which specifically target certain activities of CckA and thereby give an insight into the intramolecular signaling mechanisms. Additionally, a recently solved Crystal structure of CckA in complex with c-di-GMP will increase our understanding of the activation of phosphatse activity. The third part of this work deals with the regulation of several histidine kinases by a single domain response regulator. The single domain response Regulator MrrA (Multifunctional response regulator A) is shown to be a central part of the C. crescentus stress response pathway. MrrA is phorphorylated by two cognate histidine kinases and additionally acts as a repressor of one of the kinases. The downstream target of MrrA is the histidine kinase LovK which is the main activator of the general stress response. It is demonstrated that phosphorylated MrrA is an allosteric activator of LovK. Taken together this work increases the understanding of how c-di-GMP regulates cell cycle progression and additionally gives insight into the modes of regulation of histidine kinases

Cyclic di-GMP mediates a histidine kinase/phosphatase switch by noncovalent domain cross-linking

Histidine kinases are key components of regulatory networks in bacteria. Although many of these enzymes are bifunctional, mediating both phosphorylation and dephosphorylation of downstream targets, the molecular details of this central regulatory switch are unclear. We showed recently that the universal second messenger cyclic di-guanosine monophosphate (c-di-GMP) drives Caulobacter crescentus cell cycle progression by forcing the cell cycle kinase CckA from its default kinase into phosphatase mode. We use a combination of structure determination, modeling, and functional analysis to demonstrate that c-di-GMP reciprocally regulates the two antagonistic CckA activities through noncovalent cross-linking of the catalytic domain with the dimerization histidine phosphotransfer (DHp) domain. We demonstrate that both c-di-GMP and ADP (adenosine diphosphate) promote phosphatase activity and propose that c-di-GMP stabilizes the ADP-bound quaternary structure, which allows the receiver domain to access the dimeric DHp stem for dephosphorylation. In silico analyses predict that c-di-GMP control is widespread among bacterial histidine kinases, arguing that it can replace or modulate canonical transmembrane signaling

Cyclic di-GMP: second messenger extraordinaire

Cyclic dinucleotides (CDNs) are highly versatile signalling molecules that control various important biological processes in bacteria. The best-studied example is cyclic di-GMP (c-di-GMP). Known since the late 1980s, it is now recognized as a near-ubiquitous second messenger that coordinates diverse aspects of bacterial growth and behaviour, including motility, virulence, biofilm formation and cell cycle progression. In this Review, we discuss important new insights that have been gained into the molecular principles of c-di-GMP synthesis and degradation, which are mediated by diguanylate cyclases and c-di-GMP-specific phosphodiesterases, respectively, and the cellular functions that are exerted by c-di-GMP-binding effectors and their diverse targets. Finally, we provide a short overview of the signalling versatility of other CDNs, including c-di-AMP and cGMP-AMP (cGAMP)

Multilayered control of chromosome replication in Caulobacter crescentus.

The environmental Alphaproteobacterium Caulobacter crescentus is a classical model to study the regulation of the bacterial cell cycle. It divides asymmetrically, giving a stalked cell that immediately enters S phase and a swarmer cell that stays in the G1 phase until it differentiates into a stalked cell. Its genome consists in a single circular chromosome whose replication is tightly regulated so that it happens only in stalked cells and only once per cell cycle. Imbalances in chromosomal copy numbers are the most often highly deleterious, if not lethal. This review highlights recent discoveries on pathways that control chromosome replication when Caulobacter is exposed to optimal or less optimal growth conditions. Most of these pathways target two proteins that bind directly onto the chromosomal origin: the highly conserved DnaA initiator of DNA replication and the CtrA response regulator that is found in most Alphaproteobacteria The concerted inactivation and proteolysis of CtrA during the swarmer-to-stalked cell transition license cells to enter S phase, while a replisome-associated Regulated Inactivation and proteolysis of DnaA (RIDA) process ensures that initiation starts only once per cell cycle. When Caulobacter is stressed, it turns on control systems that delay the G1-to-S phase transition or the elongation of DNA replication, most probably increasing its fitness and adaptation capacities

Novel Divisome-Associated Protein Spatially Coupling the Z-Ring with the Chromosomal Replication Terminus in Caulobacter crescentus

Cell division requires proper spatial coordination with the chromosome, which undergoes dynamic changes during chromosome replication and segregation. FtsZ is a bacterial cytoskeletal protein that assembles into the Z-ring, providing a platform to build the cell division apparatus. In the model bacterium; Caulobacter crescentus; , the cellular localization of the Z-ring is controlled during the cell cycle in a chromosome replication-coupled manner. Although dynamic localization of the Z-ring at midcell is driven primarily by the replication origin-associated FtsZ inhibitor MipZ, the mechanism ensuring accurate positioning of the Z-ring remains unclear. In this study, we showed that the Z-ring colocalizes with the replication terminus region, located opposite the origin, throughout most of the; C. crescentus; cell cycle. Spatial organization of the two is mediated by ZapT, a previously uncharacterized protein that interacts with the terminus region and associates with ZapA and ZauP, both of which are part of the incipient division apparatus. While the Z-ring and the terminus region coincided with the presence of ZapT, colocalization of the two was perturbed in cells lacking; zapT; , which is accompanied by delayed midcellular positioning of the Z-ring. Moreover, cells overexpressing ZapT showed compromised positioning of the Z-ring and MipZ. These findings underscore the important role of ZapT in controlling cell division processes. We propose that ZapT acts as a molecular bridge that physically links the terminus region to the Z-ring, thereby ensuring accurate site selection for the Z-ring. Because ZapT is conserved in proteobacteria, these findings may define a general mechanism coordinating cell division with chromosome organization.; IMPORTANCE; Growing bacteria require careful tuning of cell division processes with dynamic organization of replicating chromosomes. In enteric bacteria, ZapA associates with the cytoskeletal Z-ring and establishes a physical linkage to the chromosomal replication terminus through its interaction with ZapB-MatP-DNA complexes. However, because ZapB and MatP are found only in enteric bacteria, it remains unclear how the Z-ring and the terminus are coordinated in the vast majority of bacteria. Here, we provide evidence that a novel conserved protein, termed ZapT, mediates colocalization of the Z-ring with the terminus in; Caulobacter crescentus; , a model organism that is phylogenetically distant from enteric bacteria. Given that ZapT facilitates cell division processes in; C. crescentus; , this study highlights the universal importance of the physical linkage between the Z-ring and the terminus in maintaining cell integrity

Precise Timing of Transcription by c-di-GMP Coordinates Cell Cycle and Morphogenesis in Caulobacter

Bacteria adapt their growth rate to their metabolic status and environmental conditions by modulating the length of their G1 period. Here we demonstrate that a gradual increase in the concentration of the second messenger c-di-GMP determines precise gene expression during G1/S transition in Caulobacter crescentus . We show that c-di-GMP stimulates the kinase ShkA by binding to its central pseudo-receiver domain, activates the TacA transcription factor, and initiates a G1/S-specific transcription program leading to cell morphogenesis and S-phase entry. Activation of the ShkA-dependent genetic program causes c-di-GMP to reach peak levels, which triggers S-phase entry and promotes proteolysis of ShkA and TacA. Thus, a gradual increase of c-di-GMP results in precise control of ShkA-TacA activity, enabling G1/S-specific gene expression that coordinates cell cycle and morphogenesis

Analysis of Brevundimonas subvibrioides developmental signaling systems reveals inconsistencies between phenotypes and c-di-GMP levels

© 2019 American Society for Microbiology. All Rights Reserved. The DivJ-DivK-PleC signaling system of Caulobacter crescentus is a signaling network that regulates polar development and the cell cycle. This system is conserved in related bacteria, including the sister genus Brevundimonas. Previous studies had shown unexpected phenotypic differences between the C. crescentus divK mutant and the analogous mutant of Brevundimonas subvibrioides, but further characterization was not performed. Here, phenotypic assays analyzing motility, adhesion, and pilus production (the latter characterized by a newly discovered bacteriophage) revealed that divJ and pleC mutants have phenotypes mostly similar to their C. crescentus homologs, but divK mutants maintain largely opposite phenotypes than expected. Suppressor mutations of the B. subvibrioides divK motility defect were involved in cyclic di-GMP (c-di-GMP) signaling, including the diguanylate cyclase dgcB, and cleD which is hypothesized to affect flagellar function in a c-di-GMP dependent fashion. However, the screen did not identify the diguanylate cyclase pleD. Disruption of pleD in B. subvibrioides caused no change in divK or pleC phenotypes, but did reduce adhesion and increase motility of the divJ strain. Analysis of c-di-GMP levels in these strains revealed incongruities between c-di-GMP levels and displayed phenotypes with a notable result that suppressor mutations altered phenotypes but had little impact on c-di-GMP levels in the divK background. Conversely, when c-di-GMP levels were artificially manipulated, alterations of c-di-GMP levels in the divK strain had minimal impact on phenotypes. These results suggest that DivK performs a critical function in the integration of c-di-GMP signaling into the B. subvibrioides cell cycle. IMPORTANCE Cyclic di-GMP and associated signaling proteins are widespread in bacteria, but their role in physiology is often complex and difficult to predict through genomic level analyses. In C. crescentus, c-di-GMP has been integrated into the developmental cell cycle, but there is increasing evidence that environmental factors can impact this system as well. The research presented here suggests that the integration of these signaling networks could be more complex than previously hypothesized, which could have a bearing on the larger field of c-di-GMP signaling. In addition, this work further reveals similarities and differences in a conserved regulatory network between organisms in the same taxonomic family, and the results show that gene conservation does not necessarily imply close functional conservation in genetic pathways

Characterization of CdbS, a PilZ domain protein involved in chromosome organization and segregation during heat shock stress in Myxococcus xanthus

The second messenger c-di-GMP regulates a wide variety of processes in bacteria that are often related to changes in lifestyle. Unexpectedly, we recently reported a link between c-di-GMP and chromosome organization. Specifically, the DNA-binding protein CdbA binds c-di-GMP, is essential for viability, and important for chromosome organization and segregation in Myxococcus xanthus. CdbA is highly abundant and binds ]500 sites on the chromosome but its depletion causes no or only modest changes in transcription. Based on these findings, we proposed that CdbA is a nucleoid-associated protein whose activity is modulated by c-di-GMP. Most nucleoid-associated proteins are not essential. Therefore, to explore the CdbA essentiality, suppressor mutants that were viable in the absence of CdbA were isolated. Among eight suppressors, seven had mutations in mxan_4328 that encodes a stand-alone PilZ domain protein, henceforth CdbS. The inactivation of cdbS completely suppressed the lethal CdbA depletion phenotype, and cdbS in otherwise wild-type cells was dispensable for viability. Notably, CdbA depletion, without affecting transcription of cdbS, resulted in a four-fold increased CdbS level. Moreover, overexpression of cdbS phenocopied the CdbA depletion phenotype. These observations support that the defects caused by CdbA depletion are the result of CdbS over-accumulation. In vitro, purified CdbS binds c-di-GMP, but the function of CdbS is independent of c-di-GMP binding in vivo. In in vivo pull-down experiments with an active CdbS-FLAG protein, significantly enriched proteins included five chaperones and co-chaperones including two PilZ-Hsp70 proteins, henceforth CsdK1 and CsdK2, a DnaJ homolog and a GrpE homolog. csdK1 as well as csdK2 were transcriptionally upregulated in response to CdbA depletion, and the resulting increased CsdK1 and CsdK2 accumulation lead to an elevated CdbS level. Searching for a physiological function of this system, we found that CdbS accumulation increased in response to high temperature stress at 37°C in a CsdK1- and CsdK2-dependent manner and caused accelerated cell death at this temperature. In total, our data support that increased CdbS accumulation caused by either CdbA depletion or high temperature stress, by an unknown mechanism, results in chromosome segregation and organization defects, thereby causing cell division inhibition and cell death. We speculate that the CdbA/CsdK1/CsdK2/CdbS system could be linked to c-di-GMP signaling and that altered cellular levels of c-di-GMP level modulate DNA binding by CdbA and, ultimately, the cellular level of CdbS. Finally, we speculate that if this system is aberrantly or excessively activated it has detrimental effects on cell viability

Insights into the activation mechanism of PopA, a cyclic di-GMP effector protein involved in cell cycle and development of "Caulobacter Crescentus"

In 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