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.
The Caulobacter cell cycle is regulated by a network of two-component signal transduction proteins. Phosphorylation and stability of the master transcriptional regulator CtrA are controlled by the CckA-ChpT phosphorelay, and CckA activity is modulated by another response regulator, DivK. In a screen to identify suppressors of the cold-sensitive divK341 mutant, we found point mutations in the essential gene divL. DivL is similar to histidine kinases but has a tyrosine instead of a histidine at the conserved phosphorylation site (Y550). Surprisingly, we found that the ATPase domain of DivL is not essential for Caulobacter viability. We show that DivL selectively affects CtrA phosphorylation but not CtrA proteolysis, indicating that DivL acts in a pathway independent of the CckA-ChpT phosphorelay. divL can be deleted in a strain overproducing the phosphomimetic protein CtrAD51E, but unlike ΔctrA cells expressing CtrAD51E, this strain is profoundly impaired in the control of chromosome replication and cell division. Thus, DivL performs a second function in addition to promoting CtrA phosphorylation. DivL is required for bipolar DivK localization and positively regulates DivK phosphorylation. Our results show that DivL controls two key cell cycle regulators, CtrA and DivK, and that phosphoryl transfer is not DivL's essential cellular activity.
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.
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.
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.
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.
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.
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.
Cell cycle regulated stalk biogenesis in Caulobacter crescentus is controlled by a multi-step phosphorelay system consisting of the hybrid histidine kinase ShkA, the histidine-phosphotransfer protein ShpA and the response regulator TacA. ShpA shuttles phosphoryl groups between ShkA and TacA. When phosphorylated, TacA triggers a downstream transcription cascade for stalk synthesis in an RpoN-dependent manner. The crystal structure of ShpA was determined to 1.52 Å resolution. ShpA belongs to a family of monomeric histidine phosphotransfer (HPt) proteins, which feature a highly conserved four-helix bundle. The phosphorylatable histidine, His56, is located on the surface of the helix bundle and is fully solvent exposed. One end of the four-helix bundle in ShpA is shorter compared to other characterized histidine phosphotransfer proteins, whereas the face that potentially interacts with the response regulators is structurally conserved. Similarities of the interaction surface around the phosphorylation site suggest that ShpA is likely to share a common mechanism for molecular recognition and phosphotransfer with yeast phosphotransfer protein YPD1 despite low overall sequence similarity.
Stalk biogenesis; phosphorelay; two-component signal transduction; Histidine phosphotransfer protein (HPt)
In our previous study, we found wheat TaCHP confers salt tolerance through regulating salt responsive signaling pathways. TaCHP possesses three divergent C1 domains that can specifically bind to phospholipid signaling molecule diacylglycerol (DAG) in animal cells, and most of proteins with this domain have kinase activity. Here, we found that TaCHP localizes both at cytoplasmatic membrane and in nuclei; it has no kinase activity but transcriptional activation activity, and the latter owes to C-terminal two C1 domains. TaCHP transcription was reduced by H2O2 application, but its ectopic expression in Arabidopsis improved both ROS production and scavenging capacity, and enhanced tolerance to H2O2 application. We suggest that TaCHP serve as both a transcription factor and a putative DAG binding protein to confer salt tolerance in part through improving ROS scavenging capacity, and that it is a component of the cross-talk machinery in the phospholipids–ROS–salt responsive signaling pathways.
abiotic stress tolerance; C1 domain; diacylglycerol; phospholipids; ROS; TaCHP; transcription factor
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.
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.
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.
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.
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.
Histidine kinases DivJ and PleC initiate signal transduction pathways that regulate an early cell division cycle step and the gain of motility later in the Caulobacter crescentus cell cycle, respectively. The essential single-domain response regulator DivK functions downstream of these kinases to catalyze phosphotransfer from DivJ and PleC. We have used a yeast two-hybrid screen to investigate the molecular basis of DivJ and PleC interaction with DivK and to identify other His-Asp signal transduction proteins that interact with DivK. The only His-Asp proteins identified in the two-hybrid screen were five members of the histidine kinase superfamily. The finding that most of the kinase clones isolated correspond to either DivJ or PleC supports the previous conclusion that DivJ and PleC are cognate DivK kinases. A 66-amino-acid sequence common to all cloned DivJ and PleC fragments contains the conserved helix 1, helix 2 sequence that forms a four-helix bundle in histidine kinases required for dimerization, autophosphorylation and phosphotransfer. We present results that indicate that the four-helix bundle subdomain is not only necessary for binding of the response regulator but also sufficient for in vivo recognition specificity between DivK and its cognate histidine kinases. The other three kinases identified in this study correspond to DivL, an essential tyrosine kinase belonging to the same kinase subfamily as DivJ and PleC, and the two previously uncharacterized, soluble histidine kinases CckN and CckO. We discuss the significance of these results as they relate to kinase response regulator recognition specificity and the fidelity of phosphotransfer in signal transduction pathways.
Two-component signal transduction pathways consisting of a histidine kinase and a response regulator are used by prokaryotes to respond to diverse environmental and intracellular stimuli. Most species encode numerous paralogous histidine kinases that exhibit significant structural similarity. Yet in almost all known examples, histidine kinases are thought to function as homodimers. We investigated the molecular basis of dimerization specificity, focusing on the model histidine kinase EnvZ and RstB, its closest paralog in Escherichia coli. Direct binding studies showed that the cytoplasmic domains of these proteins each form specific homodimers in vitro. Using a series of chimeric proteins, we identified specificity determinants at the base of the four-helix bundle in the dimerization and histidine phosphotransfer domain. Guided by molecular coevolution predictions and EnvZ structural information, we identified sets of residues in this region that are sufficient to establish homospecificity. Mutating these residues in EnvZ to the corresponding residues in RstB produced a functional kinase that preferentially homodimerized over interacting with EnvZ. EnvZ and RstB likely diverged following gene duplication to yield two homodimers that cannot heterodimerize, and the mutants we identified represent possible evolutionary intermediates in this process.
The onset of motility late in the Caulobacter crescentus cell cycle depends on a signal transduction pathway mediated by the histidine kinase PleC and response regulator DivK. We now show that pleD, whose function is required for the subsequent loss of motility and stalk formation by the motile swarmer cell, encodes a 454-residue protein with tandem N-terminal response regulator domains D1 and D2 and a novel C-terminal GGDEF domain. The identification of pleD301, a semidominant suppressor of the pleC Mot phenotype, as a mutation predicted to result in a D-53-->G change in the D1 domain supports a role for phosphorylation in the PleD regulator. Disruptions constructed in the pleD open reading frame demonstrated that the gene is not essential and that the pleC phenotype can also be suppressed by a recessive, loss-of-function mutation. These results suggest that PleD is part of a signal transduction pathway controlling stalked-cell differentiation early in the C. crescentus cell cycle.
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.
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.
The virulence of the opportunistic pathogen Pseudomonas aeruginosa involves the coordinate expression of many virulence factors, including type IV pili, which are required for colonization of host tissues and for twitching motility. Type IV pilus function is controlled in part by the Chp chemosensory system, which includes a histidine kinase, ChpA, and two CheY-like response regulators, PilG and PilH. How the Chp components interface with the type IV pilus motor proteins PilB, PilT, and PilU is unknown. We present genetic evidence confirming the role of ChpA, PilG, and PilB in the regulation of pilus extension and the role of PilH and PilT in regulating pilus retraction. Using informative double and triple mutants, we show that (i) ChpA, PilG, and PilB function upstream of PilH, PilT, and PilU; (ii) that PilH enhances PilT function; and (iii) that PilT and PilB retain some activity in the absence of signaling input from components of the Chp system. By site-directed mutagenesis, we demonstrate that the histidine kinase domain of ChpA and the phosphoacceptor sites of both PilG and PilH are required for type IV pilus function, suggesting that they form a phosphorelay system important in the regulation of pilus extension and retraction. Finally, we present evidence suggesting that pilA transcription is regulated by intracellular PilA levels. We show that PilA is a negative regulator of pilA transcription in P. aeruginosa and that the Chp system functionally regulates pilA transcription by controlling PilA import and export.
Progression through the Caulobacter cell cycle is driven by the master regulator CtrA, an essential two-component signaling protein that regulates the expression of nearly 100 genes. CtrA is abundant throughout the cell cycle except immediately prior to DNA replication. However, the expression of CtrA-activated genes is generally restricted to S phase. We identify the conserved protein SciP (small CtrA inhibitory protein) and show that it accumulates during G1, where it inhibits CtrA from activating target genes. The depletion of SciP from G1 cells leads to the inappropriate induction of CtrA-activated genes and, consequently, a disruption of the cell cycle. Conversely, the ectopic synthesis of SciP is sufficient to inhibit CtrA-dependent transcription, also disrupting the cell cycle. SciP binds directly to CtrA without affecting stability or phosphorylation; instead, SciP likely prevents CtrA from recruiting RNA polymerase. CtrA is thus tightly regulated by a protein-protein interaction which is critical to cell-cycle progression.
Comparisons of the proteome of abortifacient Chlamydia psittaci isolates from sheep by two-dimensional gel electrophoresis identified a novel abundant protein with a molecular mass of 61.4 kDa and an isoelectric point of 6.41. C-terminal sequence analysis of this protein yielded a short peptide sequence that had an identical match to the viral coat protein (VP1) of the avian chlamydiaphage Chp1. Electron microscope studies revealed the presence of a 25-nm-diameter bacteriophage (Chp2) with no apparent spike structures. Thin sections of chlamydia-infected cells showed that Chp2 particles were located to membranous structures surrounding reticulate bodies (RBs), suggesting that Chp2 is cytopathic for ovine C. psittaci RBs. Chp2 double-stranded circular replicative-form DNA was purified and used as a template for DNA sequence analysis. The Chp2 genome is 4,567 bp and encodes up to eight open reading frames (ORFs); it is similar in overall organization to the Chp1 genome. Seven of the ORFs (1 to 5, 7, and 8) have sequence homologies with Chp1. However, ORF 6 has a different spatial location and no cognate partner within the Chp1 genome. Chlamydiaphages have three viral structural proteins, VP1, VP2, and VP3, encoded by ORFs 1 to 3, respectively. Amino acid residues in the φX174 procapsid known to mediate interactions between the viral coat protein and internal scaffolding proteins are conserved in the Chp2 VP1 and VP3 proteins. We suggest that VP3 performs a scaffolding-like function but has evolved into a structural protein.
The alternation of eukaryotic life cycles between haploid and diploid phases is crucial for maintaining genetic diversity. In some organisms, the growth and development of haploid and diploid phases are nearly identical, and one might suppose that all genes required for one phase are likely to be critical for the other phase. Here, we show that targeted disruption of the chpA (cysteine- and histidine-rich-domain- [CHORD]-containing protein A) gene in haploid Aspergillus nidulans strains gives rise to chpA knockout haploids and heterozygous diploids but no chpA knockout diploids. A. nidulans chpA heterozygous diploids showed impaired conidiophore development and reduced conidiation. Deletion of chpA from diploid A. nidulans resulted in genome instability and reversion to a haploid state. Thus, our data suggest a vital role for chpA in maintenance of the diploid phase in A. nidulans. Furthermore, the human chpA homolog, Chp-1, was able to complement haploinsufficiency in A. nidulans chpA heterozygotes, suggesting that the function of CHORD-containing proteins is highly conserved in eukaryotes.
RNA interference (RNAi) is critical for the assembly of heterochromatin at fission yeast centromeres. Central to this process is the RNA-induced Initiation of Transcriptional gene Silencing (RITS) complex, which physically anchors small non-coding RNAs to chromatin. RITS includes Ago1, the chromodomain protein Chp1, and Tas3, which bridges between Chp1 and Ago1. Chp1 is a large protein with, apart from its chromodomain, no recognizable domains. Here we describe how the structured C-terminal half of Chp1 binds the Tas3 N-terminal domain, revealing Chp1's tight embrace of Tas3. The structure also reveals a PIN domain at the C-terminal tip of Chp1 that controls subtelomeric transcripts through a post-transcriptional mechanism. We suggest that the Chp1-Tas3 complex provides a solid and versatile platform to recruit both RNAi-dependent and RNAi-independent gene-silencing pathways for locus-specific regulation of heterochromatin.
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.
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.
The bacterium Caulobacter crescentus encodes a two-component signalling protein, LovK, that contains an N-terminal photosensory LOV domain coupled to a C-terminal histidine kinase. LovK binds a flavin cofactor, undergoes a reversible photocycle, and displays regulated ATPase and autophosphorylation activity in response to visible light. Femtosecond to nanosecond visible absorption spectroscopy demonstrates congruence between full-length LovK and isolated LOV domains in the mechanism and kinetics of light-dependent cysteinyl-C4(a) adduct formation and rupture, while steady-state absorption and fluorescence line narrowing (FLN) spectroscopies reveal unique features in the electronic structure of the LovK flavin cofactor. In agreement with other sensor histidine kinases, ATP binds specifically to LovK with micromolar affinity. However, ATP binding to the histidine kinase domain of LovK has no apparent effect on global protein structure as assessed by differential Fourier transform infrared (FTIR) spectroscopy. Cysteinyl adduct formation results in only minor changes in the structure of LovK as determined by differential FTIR. This study provides insight into the structural underpinnings of LOV-mediated signal transduction in the context of a full-length histidine kinase. In particular, the data provide evidence for a model in which small changes in the tertiary/quaternary structure of LovK, as triggered by photon detection in the N-terminal LOV sensory domain, are sufficient to regulate histidine kinase activity.