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1.  A Homolog of the CtrA Cell Cycle Regulator Is Present and Essential in Sinorhizobium meliloti 
Journal of Bacteriology  2001;183(10):3204-3210.
During development of the symbiotic soil bacterium Sinorhizobium meliloti into nitrogen-fixing bacteroids, DNA replication and cell division cease and the cells undergo profound metabolic and morphological changes. Regulatory genes controlling the early stages of this process have not been identified. As a first step in the search for regulators of these events, we report the isolation and characterization of a ctrA gene from S. meliloti. We show that the S. meliloti CtrA belongs to the CtrA-like family of response regulators found in several α-proteobacteria. In Caulobacter crescentus, CtrA is essential and is a global regulator of multiple cell cycle functions. ctrA is also an essential gene in S. meliloti, and it is expressed similarly to the autoregulated C. crescentus ctrA in that both genes have complex promoter regions which bind phosphorylated CtrA.
doi:10.1128/JB.183.10.3204-3210.2001
PMCID: PMC95222  PMID: 11325950
2.  The diversity and evolution of cell cycle regulation in alpha-proteobacteria: a comparative genomic analysis 
BMC Systems Biology  2010;4:52.
Background
In the bacterium Caulobacter crescentus, CtrA coordinates DNA replication, cell division, and polar morphogenesis and is considered the cell cycle master regulator. CtrA activity varies during cell cycle progression and is modulated by phosphorylation, proteolysis and transcriptional control. In a phosphorylated state, CtrA binds specific DNA sequences, regulates the expression of genes involved in cell cycle progression and silences the origin of replication. Although the circuitry regulating CtrA is known in molecular detail in Caulobacter, its conservation and functionality in the other alpha-proteobacteria are still poorly understood.
Results
Orthologs of Caulobacter factors involved in the regulation of CtrA were systematically scanned in genomes of alpha-proteobacteria. In particular, orthologous genes of the divL-cckA-chpT-ctrA phosphorelay, the divJ-pleC-divK two-component system, the cpdR-rcdA-clpPX proteolysis system, the methyltransferase ccrM and transcriptional regulators dnaA and gcrA were identified in representative genomes of alpha-proteobacteria. CtrA, DnaA and GcrA binding sites and CcrM putative methylation sites were predicted in promoter regions of all these factors and functions controlled by CtrA in all alphas were predicted.
Conclusions
The regulatory cell cycle architecture was identified in all representative alpha-proteobacteria, revealing a high diversification of circuits but also a conservation of logical features. An evolutionary model was proposed where ancient alphas already possessed all modules found in Caulobacter arranged in a variety of connections. Two schemes appeared to evolve: a complex circuit in Caulobacterales and Rhizobiales and a simpler one found in Rhodobacterales.
doi:10.1186/1752-0509-4-52
PMCID: PMC2877005  PMID: 20426835
3.  Phase Resetting Reveals Network Dynamics Underlying a Bacterial Cell Cycle 
PLoS Computational Biology  2012;8(11):e1002778.
Genomic and proteomic methods yield networks of biological regulatory interactions but do not provide direct insight into how those interactions are organized into functional modules, or how information flows from one module to another. In this work we introduce an approach that provides this complementary information and apply it to the bacterium Caulobacter crescentus, a paradigm for cell-cycle control. Operationally, we use an inducible promoter to express the essential transcriptional regulatory gene ctrA in a periodic, pulsed fashion. This chemical perturbation causes the population of cells to divide synchronously, and we use the resulting advance or delay of the division times of single cells to construct a phase resetting curve. We find that delay is strongly favored over advance. This finding is surprising since it does not follow from the temporal expression profile of CtrA and, in turn, simulations of existing network models. We propose a phenomenological model that suggests that the cell-cycle network comprises two distinct functional modules that oscillate autonomously and couple in a highly asymmetric fashion. These features collectively provide a new mechanism for tight temporal control of the cell cycle in C. crescentus. We discuss how the procedure can serve as the basis for a general approach for probing network dynamics, which we term chemical perturbation spectroscopy (CPS).
Author Summary
During the cell cycle, the cell progresses through a series of stages that are associated with various cell cycle events such as replication of genetic materials. Genetic and molecular dissections have revealed that the cell cycle is regulated by a network of interacting molecules that produces oscillatory dynamics. The major cell cycle regulators have been identified previously in different species and the activity of these regulators oscillates. However, the question of how cell cycle regulators coordinate different cell cycle events during the cell cycle remains controversial. Here, we investigate this question in a model bacterial system for cell cycle, Caulobacter crescentus. We perturb the expression of the master cell cycle regulator ctrA in a pulsatile fashion and quantify the response of the cell cycle to such perturbations. The measured response is contradictory to the existing mechanism of Caulobacter cell cycle control, which views the cell cycle progression as a sequential activation/inhibition process. We propose a new model that involves coupling of multiple oscillators and show the quantitative agreement between this new model and our measurements. We expect this procedure to be generalized and applied to a broad range of systems to obtain information that complements that obtained from other methods.
doi:10.1371/journal.pcbi.1002778
PMCID: PMC3510036  PMID: 23209388
4.  Polarity and cell fate asymmetry in Caulobacter crescentus 
Current opinion in microbiology  2012;15(6):744-750.
The production of asymmetric daughter cells is a hallmark of metazoan development and critical to the life cycle of many microbes, including the α-proteobacterium Caulobacter crescentus. For Caulobacter, every cell division is asymmetric, yielding daughter cells with different morphologies and replicative potentials. This asymmetry in daughter cell fate is governed by the response regulator CtrA, a transcription factor that can also bind and silence the origin of replication. CtrA activity is controlled by a complex regulatory circuit that includes several polarly localized histidine kinases. This circuit ensures differential activation of CtrA in daughter cells, leading to their asymmetric replicative potentials. Here, we review progress in elucidating the molecular mechanisms regulating CtrA and the role of cellular polarity in this process.
doi:10.1016/j.mib.2012.10.011
PMCID: PMC3587792  PMID: 23146566
5.  Loss of the Response Regulator CtrA Causes Pleiotropic Effects on Gene Expression but Does Not Affect Growth Phase Regulation in Rhodobacter capsulatus▿ †  
Journal of Bacteriology  2010;192(11):2701-2710.
The purple nonsulfur photosynthetic bacterium Rhodobacter capsulatus has been extensively studied for its metabolic versatility as well as for production of a gene transfer agent called RcGTA. Production of RcGTA is highest in the stationary phase of growth and requires the response regulator protein CtrA. The CtrA protein in Caulobacter crescentus has been thoroughly studied for its role as an essential, master regulator of the cell cycle. Although the CtrA protein in R. capsulatus shares a high degree of sequence similarity with the C. crescentus protein, it is nonessential and clearly plays a different role in this bacterium. We have used transcriptomic and proteomic analyses of wild-type and ctrA mutant cultures to identify the genes dysregulated by the loss of CtrA in R. capsulatus. We have also characterized gene expression differences between the logarithmic and stationary phases of growth. Loss of CtrA has pleiotropic effects, with dysregulation of expression of ∼6% of genes in the R. capsulatus genome. This includes all flagellar motility genes and a number of other putative regulatory proteins but does not appear to include any genes involved in the cell cycle. Quantitative proteomic data supported 88% of the CtrA transcriptome results. Phylogenetic analysis of CtrA sequences supports the hypothesis of an ancestral ctrA gene within the alphaproteobacteria, with subsequent diversification of function in the major alphaproteobacterial lineages.
doi:10.1128/JB.00160-10
PMCID: PMC2876497  PMID: 20363938
6.  Dynamics of Two Phosphorelays Controlling Cell Cycle Progression in Caulobacter crescentus▿ †  
Journal of Bacteriology  2009;191(24):7417-7429.
In Caulobacter crescentus, progression through the cell cycle is governed by the periodic activation and inactivation of the master regulator CtrA. Two phosphorelays, each initiating with the histidine kinase CckA, promote CtrA activation by driving its phosphorylation and by inactivating its proteolysis. Here, we examined whether the CckA phosphorelays also influence the downregulation of CtrA. We demonstrate that CckA is bifunctional, capable of acting as either a kinase or phosphatase to drive the activation or inactivation, respectively, of CtrA. By identifying mutations that uncouple these two activities, we show that CckA's phosphatase activity is important for downregulating CtrA prior to DNA replication initiation in vivo but that other phosphatases may exist. Our results demonstrate that cell cycle transitions in Caulobacter require and are likely driven by the toggling of CckA between its kinase and phosphatase states. More generally, our results emphasize how the bifunctional nature of histidine kinases can help switch cells between mutually exclusive states.
doi:10.1128/JB.00992-09
PMCID: PMC2786585  PMID: 19783630
7.  Temporal Controls of the Asymmetric Cell Division Cycle in Caulobacter crescentus 
PLoS Computational Biology  2009;5(8):e1000463.
The asymmetric cell division cycle of Caulobacter crescentus is orchestrated by an elaborate gene-protein regulatory network, centered on three major control proteins, DnaA, GcrA and CtrA. The regulatory network is cast into a quantitative computational model to investigate in a systematic fashion how these three proteins control the relevant genetic, biochemical and physiological properties of proliferating bacteria. Different controls for both swarmer and stalked cell cycles are represented in the mathematical scheme. The model is validated against observed phenotypes of wild-type cells and relevant mutants, and it predicts the phenotypes of novel mutants and of known mutants under novel experimental conditions. Because the cell cycle control proteins of Caulobacter are conserved across many species of alpha-proteobacteria, the model we are proposing here may be applicable to other genera of importance to agriculture and medicine (e.g., Rhizobium, Brucella).
Author Summary
Because of its small genome size and the ease by which it can be manipulated genetically and biochemically, Caulobacter crescentus provides unique opportunities to study the molecular circuitry controlling the asymmetric cell division cycle of bacteria. A large amount of experimental data accumulated on this model organism in recent years needs to be quantitatively reconciled and analyzed in order to generate a full description of the process. Here, from these experimental clues, we suggest a mechanism for the principal molecular interactions that control DNA synthesis and asymmetric cell division in Caulobacter and construct a quantitative (mathematical) model of the mechanism in order to analyze the temporal dynamics of the control system. The model is centered around three “master regulator” proteins, whose timing of expression is tightly controlled by the progression of DNA replication. The model has been validated against observed phenotypes of wild-type cells and relevant mutants, and predicts phenotypes of novel mutants and of known mutants under novel experimental conditions. It provides a rigorous account of current intuitive ideas of bacterial cell cycle control and advances our understanding of bacterial cell division.
doi:10.1371/journal.pcbi.1000463
PMCID: PMC2714070  PMID: 19680425
8.  Glutamate at the phosphorylation site of response regulator CtrA provides essential activities without increasing DNA binding 
Nucleic Acids Research  2003;31(6):1775-1779.
The essential response regulator CtrA controls the Caulobacter crescentus cell cycle and phosphorylated CtrA∼P preferentially binds target DNA in vitro. The CtrA aspartate to glutamate (D51E) mutation mimics phosphorylated CtrA∼P in vivo and rescues non-viable C.crescentus cells. However, we observe that the CtrA D51E and the unphosphorylated CtrA wild-type proteins have identical DNA affinities and produce identical DNase I protection footprints inside the C.crescentus replication origin. There fore, D51E promotes essential CtrA activities separate from increased DNA binding. Accordingly, we argue that CtrA protein recruitment to target DNA is not sufficient to regulate cell cycle progression.
PMCID: PMC152873  PMID: 12626719
9.  Coordination of Division and Development Influences Complex Multicellular Behavior in Agrobacterium tumefaciens 
PLoS ONE  2013;8(2):e56682.
The α-Proteobacterium Agrobacterium tumefaciens has proteins homologous to known regulators that govern cell division and development in Caulobacter crescentus, many of which are also conserved among diverse α-Proteobacteria. In light of recent work demonstrating similarity between the division cycle of C. crescentus and that of A. tumefaciens, the functional conservation for this presumptive control pathway was examined. In C. crescentus the CtrA response regulator serves as the master regulator of cell cycle progression and cell division. CtrA activity is controlled by an integrated pair of multi-component phosphorelays: PleC/DivJ-DivK and CckA-ChpT-CtrA. Although several of the conserved orthologues appear to be essential in A. tumefaciens, deletions in pleC or divK were isolated and resulted in cell division defects, diminished swimming motility, and a decrease in biofilm formation. A. tumefaciens also has two additional pleC/divJ homologue sensor kinases called pdhS1 and pdhS2, absent in C. crescentus. Deletion of pdhS1 phenocopied the ΔpleC and ΔdivK mutants. Cells lacking pdhS2 morphologically resembled wild-type bacteria, but were decreased in swimming motility and elevated for biofilm formation, suggesting that pdhS2 may serve to regulate the motile to non-motile switch in A. tumefaciens. Genetic analysis suggests that the PleC/DivJ-DivK and CckA-ChpT-CtrA phosphorelays in A. tumefaciens are vertically-integrated, as in C. crescentus. A gain-of-function mutation in CckA (Y674D) was identified as a spontaneous suppressor of the ΔpleC motility phenotype. Thus, although the core architecture of the A. tumefaciens pathway resembles that of C. crescentus there are specific differences including additional regulators, divergent pathway architecture, and distinct target functions.
doi:10.1371/journal.pone.0056682
PMCID: PMC3577659  PMID: 23437210
10.  Physical constraints on the establishment of intracellular spatial gradients in bacteria 
BMC Biophysics  2012;5:17.
Background
Bacteria dynamically regulate their intricate intracellular organization involving proteins that facilitate cell division, motility, and numerous other processes. Consistent with this sophisticated organization, bacteria are able to create asymmetries and spatial gradients of proteins by localizing signaling pathway components. We use mathematical modeling to investigate the biochemical and physical constraints on the generation of intracellular gradients by the asymmetric localization of a source and a sink.
Results
We present a systematic computational analysis of the effects of other regulatory mechanisms, such as synthesis, degradation, saturation, and cell growth. We also demonstrate that gradients can be established in a variety of bacterial morphologies such as rods, crescents, spheres, branched and constricted cells.
Conclusions
Taken together, these results suggest that gradients are a robust and potentially common mechanism for providing intracellular spatial cues.
doi:10.1186/2046-1682-5-17
PMCID: PMC3496868  PMID: 22931750
11.  Regulated proteolysis of a transcription factor complex is critical to cell cycle progression in Caulobacter crescentus 
Molecular microbiology  2013;87(6):1277-1289.
Cell cycle transitions are often triggered by the proteolysis of key regulatory proteins. In Caulobacter crescentus, the G1-S transition involves the degradation of an essential DNA-binding response regulator, CtrA, by the ClpXP protease. Here, we show that another critical cell cycle regulator, SciP, is also degraded during the G1-S transition, but by the Lon protease. SciP is a small protein that binds directly to CtrA and prevents it from activating target genes during G1. We demonstrate that SciP must be degraded during the G1-S transition so that cells can properly activate CtrA-dependent genes following DNA replication initiation and the reaccumulation of CtrA. These results indicate that like CtrA, SciP levels are tightly regulated during the Caulobacter cell cycle. In addition, we show that formation of a complex between CtrA and SciP at target promoters protects both proteins from their respective proteases. Degradation of either protein thus helps trigger the destruction of the other, facilitating a cooperative disassembly of the complex. Collectively, our results indicate that ClpXP and Lon each degrade a critical cell cycle regulator, helping to trigger the onset of S phase and prepare cells for the subsequent programs of gene expression critical to polar morphogenesis and cell division.
doi:10.1111/mmi.12166
PMCID: PMC3596498  PMID: 23368090
12.  Polar Remodeling and Histidine Kinase Activation, Which Is Essential for Caulobacter Cell Cycle Progression, Are Dependent on DNA Replication Initiation▿ †  
Journal of Bacteriology  2010;192(15):3893-3902.
Caulobacter crescentus initiates a single round of DNA replication during each cell cycle. Following the initiation of DNA replication, the essential CckA histidine kinase is activated by phosphorylation, which (via the ChpT phosphotransferase) enables the phosphorylation and activation of the CtrA global regulator. CtrA∼P then blocks the reinitiation of replication while regulating the transcription of a large number of cell cycle-controlled genes. It has been shown that DNA replication serves as a checkpoint for flagellar biosynthesis and cell division and that this checkpoint is mediated by the availability of active CtrA. Because CckA∼P promotes the activation of CtrA, we addressed the question of what controls the temporal activation of CckA. We found that the initiation of DNA replication is a prerequisite for remodeling the new cell pole, which includes the localization of the DivL protein kinase to that pole and, consequently, the localization, autophosphorylation, and activation of CckA at that pole. Thus, CckA activation is dependent on polar remodeling and a DNA replication initiation checkpoint that is tightly integrated with the polar phospho-signaling cascade governing cell cycle progression.
doi:10.1128/JB.00468-10
PMCID: PMC2916389  PMID: 20525830
13.  Microchannel-Nanopore Device for Bacterial Chemotaxis Assays 
Analytical chemistry  2010;82(22):9357-9364.
Motile bacteria bias the random walk of their motion in response to chemical gradients by the process termed chemotaxis, which allows cells to accumulate in favorable environments and disperse from less favorable ones. In this work, we describe a simple microchannel-nanopore device that establishes a stable chemical gradient for chemotaxis assays in ≤ 1 min. Chemoattractant is dispensed by diffusion through 10 nm diameter pores at the intersection of two microchannels. This design requires no external pump and minimizes the effect of transmembrane pressure, resulting in a stable, reproducible gradient. The microfluidic platform facilitates microscopic observation of individual cell trajectories, and chemotaxis is quantified by monitoring changes in cell swimming behavior in the vicinity of the intersection. We validate this system by measuring the chemotactic response of an aquatic bacterium, Caulobacter crescentus, to xylose concentrations from 1.3 μM to 1.3 M. Additionally, we make an unanticipated observation of increased turn frequency in a chemotaxis-impaired mutant which provides new insight into the chemotaxis pathway in C. crescentus.
doi:10.1021/ac101977f
PMCID: PMC2992552  PMID: 20961116
14.  Conserved Response Regulator CtrA and IHF Binding Sites in the α-Proteobacteria Caulobacter crescentus and Rickettsia prowazekii Chromosomal Replication Origins 
Journal of Bacteriology  2002;184(20):5789-5799.
CzcR is the Rickettsia prowazekii homolog of the Caulobacter crescentus global response regulator CtrA. CzcR expression partially compensates for developmental defects in ctrA mutant C. crescentus cells, and CzcR binds to all five CtrA binding sites in the C. crescentus replication origin. Conversely, CtrA binds to five similar sites in the putative R. prowazekii replication origin (oriRp). Also, Escherichia coli IHF protein binds over a central CtrA binding site in oriRp. Therefore, CtrA and IHF regulatory proteins have similar binding patterns in both replication origins, and we propose that CzcR is a global cell cycle regulator in R. prowazekii.
doi:10.1128/JB.184.20.5789-5799.2002
PMCID: PMC139603  PMID: 12270838
15.  Sinorhizobium meliloti CpdR1 is critical for coordinating cell-cycle progression and the symbiotic chronic infection 
Molecular microbiology  2009;73(4):586-600.
ATP-driven proteolysis plays a major role in regulating the bacterial cell cycle, development and stress responses. In the nitrogen-fixing symbiosis with host plants, Sinorhizobium meliloti undergoes a profound cellular differentiation including endoreduplication of the genome. The regulatory mechanisms governing the alterations of the S. meliloti cell cycle in planta are largely unknown. Here, we report the characterization of two cpdR homologs, cpdR1 and cpdR2, of S. meliloti that encode single-domain response regulators. In Caulobacter crescentus, CpdR controls the polar localization of the ClpXP protease, thereby mediating the regulated proteolysis of key protein(s), such as CtrA, involved in cell-cycle progression. The S. meliloti cpdR1-null mutant can invade the host cytoplasm, however, the intracellular bacteria are unable to differentiate into bacteroids. We show that S. meliloti CpdR1 has a polar localization pattern and a role in ClpX positioning similar to C. crescentus CpdR, suggesting a conserved function of CpdR proteins among f¿-proteobacteria. However, in S. meliloti, free-living cells of the cpdR1-null mutant show a striking morphology of irregular coccoids and aberrant DNA replication. Thus, we demonstrate that CpdR1 mediates the coordination of cell-cycle events, which are critical for both the free-living cell division and the differentiation required for the chronic intracellular infection.
doi:10.1111/j.1365-2958.2009.06794.x
PMCID: PMC2756024  PMID: 19602145
16.  Regulation of podJ Expression during the Caulobacter crescentus Cell Cycle 
Journal of Bacteriology  1999;181(13):3967-3973.
The polar organelle development gene, podJ, is expressed during the swarmer-to-stalked cell transition of the Caulobacter crescentus cell cycle. Mutants with insertions that inactivate the podJ gene are nonchemotactic, deficient in rosette formation, and resistant to polar bacteriophage, but they divide normally. In contrast, hyperexpression of podJ results in a lethal cell division defect. Nucleotide sequence analysis of the podJ promoter region revealed a binding site for the global response regulator, CtrA. Deletion of this site results in increased overall promoter activity, suggesting that CtrA is a negative regulator of the podJ promoter. Furthermore, synchronization studies have indicated that temporal regulation is not dependent on the presence of the CtrA binding site. Thus, although the level of podJ promoter activity is dependent on the CtrA binding site, the temporal control of podJ promoter expression is dependent on other factors.
PMCID: PMC93886  PMID: 10383964
17.  The CtrA Response Regulator Mediates Temporal Control of Gene Expression during the Caulobacter Cell Cycle 
Journal of Bacteriology  1999;181(8):2430-2439.
In its role as a global response regulator, CtrA controls the transcription of a diverse group of genes at different times in the Caulobacter crescentus cell cycle. To understand the differential regulation of CtrA-controlled genes, we compared the expression of two of these genes, the fliQ flagellar gene and the ccrM DNA methyltransferase gene. Despite their similar promoter architecture, these genes are transcribed at different times in the cell cycle. PfliQ is activated earlier than PccrM. Phosphorylated CtrA (CtrA∼P) bound to the CtrA recognition sequence in both promoters but had a 10- to 20-fold greater affinity for PfliQ. This difference in affinity correlates with temporal changes in the cellular levels of CtrA. Disrupting a unique inverted repeat element in PccrM significantly reduced promoter activity but not the timing of transcription initiation, suggesting that the inverted repeat does not play a major role in the temporal control of ccrM expression. Our data indicate that differences in the affinity of CtrA∼P for PfliQ and PccrM regulate, in part, the temporal expression of these genes. However, the timing of fliQ transcription but not of ccrM transcription was altered in cells expressing a stable CtrA derivative, indicating that changes in CtrA∼P levels alone cannot govern the cell cycle transcription of these genes. We propose that changes in the cellular concentration of CtrA∼P and its interaction with accessory proteins influence the temporal expression of fliQ, ccrM, and other key cell cycle genes and ultimately the regulation of the cell cycle.
PMCID: PMC93667  PMID: 10198005
18.  New criteria for selecting the origin of DNA replication in Wolbachia and closely related bacteria 
BMC Genomics  2007;8:182.
Background
The annotated genomes of two closely related strains of the intracellular bacterium Wolbachia pipientis have been reported without the identifications of the putative origin of replication (ori). Identifying the ori of these bacteria and related alpha-Proteobacteria as well as their patterns of sequence evolution will aid studies of cell replication and cell density, as well as the potential genetic manipulation of these widespread intracellular bacteria.
Results
Using features that have been previously experimentally verified in the alpha-Proteobacterium Caulobacter crescentus, the origin of DNA replication (ori) regions were identified in silico for Wolbachia strains and eleven other related bacteria belonging to Ehrlichia, Anaplasma, and Rickettsia genera. These features include DnaA-, CtrA- and IHF-binding sites as well as the flanking genes in C. crescentus. The Wolbachia ori boundary genes were found to be hemE and COG1253 protein (CBS domain protein). Comparisons of the putative ori region among related Wolbachia strains showed higher conservation of bases within binding sites.
Conclusion
The sequences of the ori regions described here are only similar among closely related bacteria while fundamental characteristics like presence of DnaA and IHF binding sites as well as the boundary genes are more widely conserved. The relative paucity of CtrA binding sites in the ori regions, as well as the absence of key enzymes associated with DNA replication in the respective genomes, suggest that several of these obligate intracellular bacteria may have altered replication mechanisms. Based on these analyses, criteria are set forth for identifying the ori region in genome sequencing projects.
doi:10.1186/1471-2164-8-182
PMCID: PMC1914354  PMID: 17584494
19.  Modularity of the bacterial cell cycle enables independent spatial and temporal control of DNA replication 
Current biology : CB  2011;21(13):1092-1101.
Summary
Background
Complex regulatory circuits in biology are often built of simpler sub-circuits, or modules. In most cases, the functional consequences and evolutionary origins of modularity remain poorly defined.
Results
Here, by combining single-cell microscopy with genetic approaches, we demonstrate that two separable modules independently govern the temporal and spatial control of DNA replication in the asymmetrically dividing bacterium Caulobacter crescentus. DNA replication control involves DnaA, which promotes initiation, and CtrA, which silences initiation. We show that oscillations in DnaA activity dictate the periodicity of replication while CtrA governs the asymmetric replicative fates of daughter cells. Importantly, we demonstrate that DnaA activity oscillates independently of CtrA.
Conclusions
The genetic separability of spatial and temporal control modules in Caulobacter reflects their evolutionary history. DnaA is the central component of an ancient and phylogenetically widespread circuit that governs replication periodicity in Caulobacter and most other bacteria. By contrast, CtrA, which is found only in the asymmetrically dividing α-proteobacteria, was integrated later in evolution to enforce replicative asymmetry on daughter cells.
doi:10.1016/j.cub.2011.05.040
PMCID: PMC3143580  PMID: 21683595
20.  The CckA-ChpT-CtrA Phosphorelay System Is Regulated by Quorum Sensing and Controls Flagellar Motility in the Marine Sponge Symbiont Ruegeria sp. KLH11 
PLoS ONE  2013;8(6):e66346.
Bacteria respond to their environment via signal transduction pathways, often two-component type systems that function through phosphotransfer to control expression of specific genes. Phosphorelays are derived from two-component systems but are comprised of additional components. The essential cckA-chpT-ctrA phosphorelay in Caulobacter crescentus has been well studied and is important in orchestrating the cell cycle, polar development and flagellar biogenesis. Although cckA, chpT and ctrA homologues are widespread among the Alphaproteobacteria, relatively few is known about their function in the large and ecologically significant Roseobacter clade of the Rhodobacterales. In this study the cckA-chpT-ctrA system of the marine sponge symbiont Ruegeria sp. KLH11 was investigated. Our results reveal that the cckA, chpT and ctrA genes positively control flagellar biosynthesis. In contrast to C. crescentus, the cckA, chpT and ctrA genes in Ruegeria sp. KLH11 are non-essential and do not affect bacterial growth. Gene fusion and transcript analyses provide evidence for ctrA autoregulation and the control of motility-related genes. In KLH11, flagellar motility is controlled by the SsaRI system and acylhomoserine lactone (AHL) quorum sensing. SsaR and long chain AHLs are required for cckA, chpT and ctrA gene expression, providing a regulatory link between flagellar locomotion and population density in KLH11.
doi:10.1371/journal.pone.0066346
PMCID: PMC3692519  PMID: 23825536
21.  Polar Localization of the CckA Histidine Kinase and Cell Cycle Periodicity of the Essential Master Regulator CtrA in Caulobacter crescentus▿ †  
Journal of Bacteriology  2009;192(2):539-552.
The phosphorylated form of the response regulator CtrA represses DNA replication initiation and regulates the transcription of about 100 cell cycle-regulated genes in Caulobacter crescentus. CtrA activity fluctuates during the cell cycle, and its periodicity is a key element of the engine that drives cell cycle progression. The histidine kinase CckA controls the phosphorylation not only of CtrA but also of CpdR, whose unphosphorylated form promotes CtrA proteolysis. Thus, CckA has a central role in establishing the cell cycle periodicity of CtrA activity by controlling both its phosphorylation and stability. Evidence suggests that the polar localization of CckA during the cell cycle plays a role in CckA function. However, the exact pattern of CckA localization remains controversial. Here, we describe a thorough, quantitative analysis of the spatiotemporal distribution of a functional and chromosomally produced CckA-monomeric green fluorescent protein fusion that affects current models of cell cycle regulation. We also identify two cis-acting regions in CckA that are important for its proper localization and function. The disruption of a PAS-like motif in the sensor domain affects the stability of CckA accumulation at the poles. This is accompanied by a partial loss in CckA function. Shortening an extended linker between β-sheets within the CckA catalysis-assisting ATP-binding domain has a more severe effect on CckA polar localization and function. This mutant strain exhibits a dramatic cell-to-cell variability in CpdR levels and CtrA cell cycle periodicity, suggesting that the cell cycle-coordinated polar localization of CckA may be important for the robustness of signal transduction and cell cycle progression.
doi:10.1128/JB.00985-09
PMCID: PMC2805319  PMID: 19897656
22.  Bacterial chemotaxis in linear and nonlinear steady microfluidic gradients 
Nano letters  2010;10(9):3379-3385.
Diffusion-based microfluidic devices can generate steady, arbitrarily shaped chemical gradients without requiring fluid flow and are ideal for studying chemotaxis of free-swimming cells such as bacteria. However, if microfluidic gradient generators are to be used to systematically study bacterial chemotaxis, it is critical to evaluate their performance with actual quantitative chemotaxis tests. We characterize and compare three diffusion-based gradient generators by confocal microscopy and numerical simulations, select an optimal design and apply it to chemotaxis experiments with Escherichia coli in both linear and nonlinear gradients. Comparison of the observed cell distribution along the gradients with predictions from an established mathematical model shows very good agreement, providing the first quantification of chemotaxis of free-swimming cells in steady nonlinear microfluidic gradients and opening the door to bacterial chemotaxis studies in gradients of arbitrary shape.
doi:10.1021/nl101204e
PMCID: PMC2935935  PMID: 20669946
Bacterial chemotaxis; Microfluidics; Gradient generator; Hydrogel
23.  A Quantitative Study of the Division Cycle of Caulobacter crescentus Stalked Cells 
Progression of a cell through the division cycle is tightly controlled at different steps to ensure the integrity of genome replication and partitioning to daughter cells. From published experimental evidence, we propose a molecular mechanism for control of the cell division cycle in Caulobacter crescentus. The mechanism, which is based on the synthesis and degradation of three “master regulator” proteins (CtrA, GcrA, and DnaA), is converted into a quantitative model, in order to study the temporal dynamics of these and other cell cycle proteins. The model accounts for important details of the physiology, biochemistry, and genetics of cell cycle control in stalked C. crescentus cell. It reproduces protein time courses in wild-type cells, mimics correctly the phenotypes of many mutant strains, and predicts the phenotypes of currently uncharacterized mutants. Since many of the proteins involved in regulating the cell cycle of C. crescentus are conserved among many genera of α-proteobacteria, the proposed mechanism may be applicable to other species of importance in agriculture and medicine.
Author Summary
The cell cycle is the sequence of events by which a growing cell replicates all its components and divides them more or less evenly between two daughter cells. The timing and spatial organization of these events are controlled by gene–protein interaction networks of great complexity. A challenge for computational biology is to build realistic, accurate, predictive mathematical models of these control systems in a variety of organisms, both eukaryotes and prokaryotes. To this end, we present a model of a portion of the molecular network controlling DNA synthesis, cell cycle–related gene expression, DNA methylation, and cell division in stalked cells of the α-proteobacterium Caulobacter crescentus. The model is formulated in terms of nonlinear ordinary differential equations for the major cell cycle regulatory proteins in Caulobacter: CtrA, GcrA, DnaA, CcrM, and DivK. Kinetic rate constants are estimated, and the model is tested against available experimental observations on wild-type and mutant cells. The model is viewed as a starting point for more comprehensive models of the future that will account, in addition, for the spatial asymmetry of Caulobacter reproduction (swarmer cells as well as stalked cells), the correlation of cell growth and division, and cell cycle checkpoints.
doi:10.1371/journal.pcbi.0040009
PMCID: PMC2217572  PMID: 18225942
24.  The CcrM DNA Methyltransferase of Agrobacterium tumefaciens Is Essential, and Its Activity Is Cell Cycle Regulated 
Journal of Bacteriology  2001;183(10):3065-3075.
DNA methylation is now recognized as a regulator of multiple bacterial cellular processes. CcrM is a DNA adenine methyltransferase found in the alpha subdivision of the proteobacteria. Like the Dam enzyme, which is found primarily in Escherichia coli and other gamma proteobacteria, it does not appear to be part of a DNA restriction-modification system. The CcrM homolog of Agrobacterium tumefaciens was found to be essential for viability. Overexpression of CcrM is associated with significant abnormalities of cell morphology and DNA ploidy. Mapping of the transcriptional start site revealed a conserved binding motif for the global response regulator CtrA at the −35 position; this motif was footprinted by purified Caulobacter crescentus CtrA protein in its phosphorylated state. We have succeeded in isolating synchronized populations of Agrobacterium cells and analyzing their progression through the cell cycle. We demonstrate that DNA replication and cell division can be followed in an orderly manner and that flagellin expression is cyclic, consistent with our observation that motility varies during the cell cycle. Using these synchronized populations, we show that CcrM methylation of the chromosome is restricted to the late S phase of the cell cycle. Thus, within the alpha subdivision, there is a conserved cell cycle dependence and regulatory mechanism controlling ccrM expression.
doi:10.1128/JB.183.10.3065-3075.2001
PMCID: PMC95206  PMID: 11325934
25.  Feedback Control of DnaA-Mediated Replication Initiation by Replisome-Associated HdaA Protein in Caulobacter▿  
Journal of Bacteriology  2009;191(18):5706-5716.
Chromosome replication in Caulobacter crescentus is tightly regulated to ensure that initiation occurs at the right time and only once during the cell cycle. The timing of replication initiation is controlled by both CtrA and DnaA. CtrA binds to and silences the origin. Upon the clearance of CtrA from the cell, the DnaA protein accumulates and allows loading of the replisome at the origin. Here, we identify an additional layer of replication initiation control that is mediated by the HdaA protein. In Escherichia coli, the Hda protein inactivates DnaA after replication initiation. We show that the Caulobacter HdaA homologue is necessary to restrict the initiation of DNA replication to only once per cell cycle and that it dynamically colocalizes with the replisome throughout the cell cycle. Moreover, the transcription of hdaA is directly activated by DnaA, providing a robust feedback regulatory mechanism that adjusts the levels of HdaA to inactivate DnaA.
doi:10.1128/JB.00525-09
PMCID: PMC2737981  PMID: 19633089

Results 1-25 (715852)