Integration of cell wall synthesis and chromosome segregation during cell division in Caulobacter.

To divide, bacteria must synthesize their peptidoglycan (PG) cell wall, a protective meshwork that maintains cell shape. FtsZ, a tubulin homolog, dynamically assembles into a midcell band, recruiting division proteins, including the PG synthases FtsW and FtsI. FtsWI are activated to synthesize PG and drive constriction at the appropriate time and place. However, their activation pathway remains unresolved. In Caulobacter crescentus, FtsWI activity requires FzlA, an essential FtsZ-binding protein. Through time-lapse imaging and single-molecule tracking of Caulobacter FtsW and FzlA, we demonstrate that FzlA is a limiting constriction activation factor that signals to promote conversion of inactive FtsW to an active, slow-moving state. We find that FzlA interacts with the DNA translocase FtsK and place FtsK genetically in a pathway with FzlA and FtsWI. Misregulation of the FzlA-FtsK-FtsWI pathway leads to heightened DNA damage and cell death. We propose that FzlA integrates the FtsZ ring, chromosome segregation, and PG synthesis to ensure robust and timely constriction during Caulobacter division.

Analysis and remedy of negativity problem in hybrid stochastic simulation algorithm and its application.

BACKGROUND: The hybrid stochastic simulation algorithm, proposed by Haseltine and Rawlings (HR), is a combination of differential equations for traditional deterministic models and Gillespie's algorithm (SSA) for stochastic models. The HR hybrid method can significantly improve the efficiency of stochastic simulations for multiscale biochemical networks. Previous studies on the accuracy analysis for a linear chain reaction system showed that the HR hybrid method is accurate if the scale difference between fast and slow reactions is above a certain threshold, regardless of population scales. However, the population of some reactant species might be driven negative if they are involved in both deterministic and stochastic systems. RESULTS: This work investigates the negativity problem of the HR hybrid method, analyzes and tests it with several models including a linear chain system, a nonlinear reaction system, and a realistic biological cell cycle system. As a benchmark, the second slow reaction firing time is used to measure the effect of negative populations on the accuracy of the HR hybrid method. Our analysis demonstrates that usually the error caused by negative populations is negligible compared with approximation errors of the HR hybrid method itself, and sometimes negativity phenomena may even improve the accuracy. But for systems where negative species are involved in nonlinear reactions or some species are highly sensitive to negative species, the system stability will be influenced and may lead to system failure when using the HR hybrid method. In those circumstances, three remedies are studied for the negativity problem. CONCLUSION: The results of different models and examples suggest that the Zero-Reaction rule is a good remedy for nonlinear and sensitive systems considering its efficiency and simplicity.

Regulated Proteolysis in Bacteria: Caulobacter.

Protein degradation is essential for all living things. Bacteria use energy-dependent proteases to control protein destruction in a highly specific manner. Recognition of substrates is determined by the inherent specificity of the proteases and through adaptor proteins that alter the spectrum of substrates. In the α-proteobacterium Caulobacter crescentus, regulated protein degradation is required for stress responses, developmental transitions, and cell cycle progression. In this review, we describe recent progress in our understanding of the regulated and stress-responsive protein degradation pathways in Caulobacter. We discuss how organization of highly specific adaptors into functional hierarchies drives destruction of proteins during the bacterial cell cycle. Because all cells must balance the need for degradation of many true substrates with the toxic consequences of nonspecific protein destruction, principles found in one system likely generalize to others.

Topological control of the Caulobacter cell cycle circuitry by a polarized single-domain PAS protein.

Despite the myriad of different sensory domains encoded in bacteria, only a few types are known to control the cell cycle. Here we use a forward genetic screen for Caulobacter crescentus motility mutants to identify a conserved single-domain PAS (Per-Arnt-Sim) protein (MopJ) with pleiotropic regulatory functions. MopJ promotes re-accumulation of the master cell cycle regulator CtrA after its proteolytic destruction is triggered by the DivJ kinase at the G1-S transition. MopJ and CtrA syntheses are coordinately induced in S-phase, followed by the sequestration of MopJ to cell poles in Caulobacter. Polarization requires Caulobacter DivJ and the PopZ polar organizer. MopJ interacts with DivJ and influences the localization and activity of downstream cell cycle effectors. Because MopJ abundance is upregulated in stationary phase and by the alarmone (p)ppGpp, conserved systemic signals acting on the cell cycle and growth phase control are genetically integrated through this conserved single PAS-domain protein.

Cell cycle constraints on capsulation and bacteriophage susceptibility.

Despite the crucial role of bacterial capsules in pathogenesis, it is still unknown if systemic cues such as the cell cycle can control capsule biogenesis. In this study, we show that the capsule of the synchronizable model bacterium Caulobacter crescentus is cell cycle regulated and we unearth a bacterial transglutaminase homolog, HvyA, as restriction factor that prevents capsulation in G1-phase cells. This capsule protects cells from infection by a generalized transducing Caulobacter phage (φCr30), and the loss of HvyA confers insensitivity towards φCr30. Control of capsulation during the cell cycle could serve as a simple means to prevent steric hindrance of flagellar motility or to ensure that phage-mediated genetic exchange happens before the onset of DNA replication. Moreover, the multi-layered regulatory circuitry directing HvyA expression to G1-phase is conserved during evolution, and HvyA orthologues from related Sinorhizobia can prevent capsulation in Caulobacter, indicating that alpha-proteobacteria have retained HvyA activity.

ClpXP and ClpAP proteolytic activity on divisome substrates is differentially regulated following the Caulobacter asymmetric cell division.

Proteolytic control of Caulobacter cell cycle proteins is primarily executed by ClpXP, a dynamically localized protease implicated in turnover of several factors critical for faithful cell cycle progression. Here, we show that the transient midcell localization of ClpXP that precedes cytokinesis requires the FtsZ component of the divisome. Although ClpAP does not exhibit subcellular localization, FtsZ is a substrate of both ClpXP and ClpAP in vivo and in vitro. A peptide containing the C-terminal portion of the FtsA divisome protein is a substrate of both ClpXP and ClpAP in vitro but is primarily degraded by ClpAP in vivo. Caulobacter carries out an asymmetric division in which FtsZ and FtsA are stable in stalked cells but degraded in the non-replicative swarmer cell where ClpAP alone degrades FtsA and both ClpAP and ClpXP degrade FtsZ. While asymmetric division in Caulobacter normally yields larger stalked and smaller swarmer daughters, we observe a loss of asymmetric size distribution among daughter cells when clpA is depleted from a strain in which FtsZ is constitutively produced. Taken together, these results suggest that the activity of both ClpXP and ClpAP on divisome substrates is differentially regulated in daughter cells.

DNA methylation in Caulobacter and other Alphaproteobacteria during cell cycle progression.

In Caulobacter crescentus, methylation of DNA by CcrM plays an important part in the regulation of cell cycle progression. Thanks to this methyltransferase, the activity of which is cell cycle regulated, the chromosome transitions between a hemimethylated state in the S-phase to a fully methylated condition in the G1 and G2 phases. Any perturbation in CcrM expression, such as depletion or constitutive expression, causes severe developmental defects. Several studies suggest that the role of CcrM is conserved across the Alphaproteobacteria. In the past few years, the importance of methylation on the expression of cell cycle regulated genes has emerged, suggesting that CcrM-dependent methylation can direct the binding of transcription factors to specific methylated sequences and affect the expression of genes depending on the methylation state of their promoters. CcrM activity has recently been linked to GcrA, a cell cycle master regulator that controls the expression of several genes during S-phase. Here, we review recent findings that establish the global role of methylation in cell cycle progression, and also explore the significance of a CcrM-GcrA epigenetic module that has co-evolved in Alphaproteobacteria, including Caulobacter, in controlling several genes involved in cell division, polarity, and motility.

Caulobacter chromosome in vivo configuration matches model predictions for a supercoiled polymer in a cell-like confinement.

We measured the distance between fluorescent-labeled DNA loci of various interloci contour lengths in Caulobacter crescentus swarmer cells to determine the in vivo configuration of the chromosome. For DNA segments less than about 300 kb, the mean interloci distances, , scale as n(0.22), where n is the contour length, and cell-to-cell distribution of the interloci distance r is a universal function of r/n(0.22) with broad cell-to-cell variability. For DNA segments greater than about 300 kb, the mean interloci distances scale as n, in agreement with previous observations. The 0.22 value of the scaling exponent for short DNA segments is consistent with theoretical predictions for a branched DNA polymer structure. Predictions from Brownian dynamics simulations of the packing of supercoiled DNA polymers in an elongated cell-like confinement are also consistent with a branched DNA structure, and simulated interloci distance distributions predict that confinement leads to "freezing" of the supercoiled configuration. Lateral positions of labeled loci at comparable positions along the length of the cell are strongly correlated when the longitudinal locus positions differ by <0.16 µm. We conclude that the chromosome structure is supercoiled locally and elongated at large length scales and that substantial cell-to-cell variability in the interloci distances indicates that in vivo crowding prevents the chromosome from reaching an equilibrium arrangement. We suggest that the force causing rapid transport of loci remote from the parS centromere to the distal cell pole may arise from the release at the polar region of potential energy within the supercoiled DNA.

Life in a three-dimensional grid.

There have been two sharp demarcations in my life in science: the transition from fine arts to chemistry, which happened early in my career, and the move from New York to Stanford University, which initiated an ongoing collaboration with the physicist Harley McAdams. Both had a profound effect on the kinds of questions I posed and the means I used to arrive at answers. The outcome of these experiences, together with the extraordinary scientists I came to know along the way, was and is an abiding passion to fully understand a simple cell in all its complexity and beauty.

Bacterial chromosomal loci move subdiffusively through a viscoelastic cytoplasm.

Tracking of fluorescently labeled chromosomal loci in live bacterial cells reveals a robust scaling of the mean square displacement (MSD) as τ(0.39). We propose that the observed motion arises from relaxation of the Rouse modes of the DNA polymer within the viscoelastic environment of the cytoplasm. The time-averaged and ensemble-averaged MSD of chromosomal loci exhibit ergodicity, and the velocity autocorrelation function is negative at short time lags. These observations are most consistent with fractional Langevin motion and rule out a continuous time random walk model as an explanation for anomalous motion in vivo.

Polar remodeling and histidine kinase activation, which is essential for Caulobacter cell cycle progression, are dependent on DNA replication initiation.

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 approximately 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 approximately 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.

Systems biology of Caulobacter.

The dynamic range of a bacterial species' natural environment is reflected in the complexity of its systems that control cell cycle progression and its range of adaptive responses. We discuss the genetic network and integrated three-dimensional sensor/response systems that regulate the cell cycle and asymmetric cell division in the bacterium Caulobacter crescentus. The cell cycle control circuitry is tied closely to chromosome replication and morphogenesis by multiple feedback pathways from the modular functions that implement the cell cycle. The sophistication of the genetic regulatory circuits and the elegant integration of temporally controlled transcription and protein synthesis with spatially dynamic phosphosignaling and proteolysis pathways, and epigenetic regulatory mechanisms, form a remarkably robust living system.

A DNA methylation ratchet governs progression through a bacterial cell cycle.

The Caulobacter cell cycle is driven by a cascade of transient regulators, starting with the expression of DnaA in G(1) and ending with the expression of the essential CcrM DNA methyltransferase at the completion of DNA replication. The timing of DnaA accumulation was found to be regulated by the methylation state of the dnaA promoter, which in turn depends on the chromosomal position of dnaA near the origin of replication and restriction of CcrM synthesis to the end of the cell cycle. The dnaA gene is preferentially transcribed from a fully methylated promoter. DnaA initiates DNA replication and activates the transcription of the next cell-cycle regulator, GcrA. With the passage of the replication fork, the dnaA promoter becomes hemimethylated, and DnaA accumulation drops. GcrA then activates the transcription of the next cell-cycle regulator, CtrA, once the replication fork passes through the ctrA P1 promoter, generating two hemimethylated copies of ctrA. The ctrA gene is preferentially transcribed from a hemimethylated promoter. CtrA then activates the transcription of ccrM, to bring the newly replicated chromosome to the fully methylated state, promoting dnaA transcription and the start of a new cell cycle. We show that the cell-cycle timing of CcrM is critical for Caulobacter fitness. The sequential changes in the chromosomal methylation state serve to couple the progression of DNA replication to cell-cycle events regulated by the master transcriptional regulatory cascade, thus providing a ratchet mechanism for robust cell-cycle control.

Comparison of quantitative methods for cell-shape analysis.

Morphology is an important large-scale manifestation of the global organizational and physiological state of cells, and is commonly used as a qualitative or quantitative measure of the outcome of various assays. Here we evaluate several different basic representations of cell shape - binary masks, distance maps and polygonal outlines - and different subsequent encodings of those representations - Fourier and Zernike decompositions, and the principal and independent components analyses - to determine which are best at capturing biologically important shape variation. We find that principal components analysis of two-dimensional shapes represented as outlines provide measures of morphology which are quantitative, biologically meaningful, human interpretable and work well across a range of cell types and parameter settings.

Spatial complexity and control of a bacterial cell cycle.

A major breakthrough in understanding the bacterial cell cycle is the discovery that bacteria exhibit a high degree of intracellular organization. Chromosomal loci and many protein complexes are positioned at particular subcellular sites. In this review, we examine recently discovered control mechanisms that make use of dynamically localized protein complexes to orchestrate the Caulobacter crescentus cell cycle. Protein localization, notably of signal transduction proteins, chromosome partition proteins, and proteases, serves to coordinate cell division with chromosome replication and cell differentiation. The developmental fate of daughter cells is decided before completion of cytokinesis, via the early establishment of cell polarity by the distribution of activated signaling proteins, bacterial cytoskeleton, and landmark proteins.

[A psychrotolerant Caulobacter sp. from Russian polar tundra soil].

Strain Z-0024, a psychrotolerant aerobic heterotrophic representative of the prosthecate bacteria of the genus Caulobacter, was isolated from a methanotrophic enrichment obtained from Russian polar tundra soil. The cells of the new isolate are vibrios (0.5-0.6 x 1.3-1.8 microm) with a polar stalk. The organism grows in a temperature range from 5 to 36 degrees C, with an optimum at 20 degrees C. The pH range for growth is from 4.5 to 7.0 with an optimum at pH 6.0. Strain Z-0024 utilizes a wide range of organic compounds: sugars, amino acids, volatile fatty acids, and primary alcohols. It tolerates a NaCl concentration in the medium of up to 15 g/l. The G + C content of DNA is 66.6 mol %. The 16S rRNA gene sequence analysis revealed that strain Z-0024 belongs to the cluster of Caulobacter species, showing a 98.8-99.2% sequence similarity to them. DNA-DNA hybridization revealed a low level of homology (24%) between strain Z-0024 and C. vibrioides ATCC 15252. The new isolate is described as Caulobacter sp. Z-0024.

Cell cycle control in bacteria and yeast: a case of convergent evolution?

Superficially similar traits in phylogenetically unrelated species often result from adaptation to common selection pressures. Examples of convergent evolution are known at the levels of whole organisms, organ systems, gene networks and specific proteins. The phenotypic properties of living things, on the other hand, are determined in large part by complex networks of interacting proteins. Here we present a mathematical model of the network of proteins that controls DNA synthesis and cell division in the alpha-proteobacterium, Caulobacter crescentus. By comparing the protein regulatory circuits for cell reproduction in Caulobacter with that in budding yeast (Saccharomyces cerevisiae), we suggest that convergent evolution may have created similar molecular reaction networks in order to accomplish the same purpose of coordinating DNA synthesis to cell division. Although the genes and proteins involved in cell cycle regulation in prokaryotes and eukaryotes are very different and (apparently) phylogenetically unrelated, they seem to be wired together in similar regulatory networks, which coordinate cell cycle events by identical dynamical principles.

A dynamically localized protease complex and a polar specificity factor control a cell cycle master regulator.

Regulated proteolysis is essential for cell cycle progression in both prokaryotes and eukaryotes. We show here that the ClpXP protease, responsible for the degradation of multiple bacterial proteins, is dynamically localized to specific cellular positions in Caulobacter where it degrades colocalized proteins. The CtrA cell cycle master regulator, that must be cleared from the Caulobacter cell to allow the initiation of chromosome replication, interacts with the ClpXP protease at the cell pole where it is degraded. We have identified a novel, conserved protein, RcdA, that forms a complex with CtrA and ClpX in the cell. RcdA is required for CtrA polar localization and degradation by ClpXP. The localization pattern of RcdA is coincident with and dependent upon ClpX localization. Thus, a dynamically localized ClpXP proteolysis complex in concert with a cytoplasmic factor provides temporal and spatial specificity to protein degradation during a bacterial cell cycle.

Two independent spiral structures control cell shape in Caulobacter.

The actin homolog MreB contributes to bacterial cell shape. Here, we explore the role of the coexpressed MreC protein in Caulobacter and show that it forms a periplasmic spiral that is out of phase with the cytoplasmic MreB spiral. Both mreB and mreC are essential, and depletion of either protein results in a similar cell shape defect. MreB forms dynamic spirals in MreC-depleted cells, and MreC localizes helically in the presence of the MreB-inhibitor A22, indicating that each protein can form a spiral independently of the other. We show that the peptidoglycan transpeptidase Pbp2 also forms a helical pattern that partially colocalizes with MreC but not MreB. Perturbing either MreB (with A22) or MreC (with depletion) causes GFP-Pbp2 to mislocalize to the division plane, indicating that each is necessary but not sufficient to generate a helical Pbp2 pattern. We show that it is the division process that draws Pbp2 to midcell in the absence of MreB's regulation, because cells depleted of the tubulin homolog FtsZ maintain a helical Pbp2 localization in the presence of A22. By developing and employing a previously uncharacterized computational method for quantitating shape variance, we find that a FtsZ depletion can also partially rescue the A22-induced shape deformation. We conclude that MreB and MreC form spatially distinct and independently localized spirals and propose that MreB inhibits division plane localization of Pbp2, whereas MreC promotes lengthwise localization of Pbp2; together these two mechanism ensure a helical localization of Pbp2 and, thereby, the maintenance of proper cell morphology in Caulobacter.

A membrane metalloprotease participates in the sequential degradation of a Caulobacter polarity determinant.

Caulobacter crescentus assembles many of its cellular machines at distinct times and locations during the cell cycle. PodJ provides the spatial cues for the biogenesis of several polar organelles, including the pili, adhesive holdfast and chemotactic apparatus, by recruiting structural and regulatory proteins, such as CpaE and PleC, to a specific cell pole. PodJ is a protein with a single transmembrane domain that exists in two forms, full-length (PodJL) and truncated (PodJS), each appearing during a specific time period of the cell cycle to control different aspects of polar organelle development. PodJL is synthesized in the early predivisional cell and is later proteolytically converted to PodJS. During the swarmer-to-stalked transition, PodJS must be degraded to preserve asymmetry in the next cell cycle. We found that MmpA facilitates the degradation of PodJS. MmpA belongs to the site-2 protease (S2P) family of membrane-embedded zinc metalloproteases, which includes SpoIVFB and YluC of Bacillus subtilis and YaeL of Escherichia coli. MmpA appears to cleave within or near the transmembrane segment of PodJS, releasing it into the cytoplasm for complete proteolysis. While PodJS has a specific temporal and spatial address, MmpA is present throughout the cell cycle; furthermore, periplasmic fusion to mRFP1 suggested that MmpA is uniformly distributed around the cell. We also determined that mmpA and yaeL can complement each other in C. crescentus and E. coli, indicating functional conservation. Thus, the sequential degradation of PodJ appears to involve regulated intramembrane proteolysis (Rip) by MmpA.