Caulobacter Cell Cycle Research Articles

Phosphatase to kinase switch of a critical enzyme contributes to timing of cell differentiation.

The process of cell differentiation is highly regulated in both prokaryotic and eukaryotic organisms. The aquatic bacterium, Caulobacter crescentus, undergoes programmed cell differentiation from a motile swarmer cell to a stationary stalked cell with each cell cycle. This critical event is regulated at multiple levels. Kinase activity of the bifunctional enzyme, PleC, is limited to a brief period when it initiates the molecular signaling cascade that results in cell differentiation. Conversely, PleC phosphatase activity is required for pili formation and flagellar rotation. We show that PleC is localized to the flagellar pole by the scaffold protein, PodJ, which is known to suppress PleC kinase activity in vitro. PleC mutants that are unable to bind PodJ have increased kinase activity in vivo, resulting in premature differentiation. We propose a model in which PodJ regulation of PleC's enzymatic activity contributes to the robust timing of cell differentiation during the Caulobacter cell cycle.

The Underrated Salivary Virome of Men Who Have Sex With Men Infected With HIV.

Salivary virome is important for oral ecosystem, but there are few reports on people living with HIV. We performed metagenomic sequencing to compare composition and functional genes of salivary virobiota between one HIV-negative and four HIV-positive groups in which participants were all men who have sex with men (MSM) with different immunosuppression statuses (five samples per group) to find the evidence that salivary virobiota plays a role in the pathogenesis of oral disease. Acute-stage subjects achieved a positive result of HIV RNA, but HIV antibody negative or indeterminate, whereas individuals with mild, moderate, and severe immunosuppression exhibited CD4+ T-lymphocyte counts of at least 500, 200–499, and less than 200 cells/μL or opportunistic infection, respectively. The results showed the composition of salivary virus genera in subjects with mild immunosuppression was the most similar to that in healthy people, followed by that in the acute stage; under severe immunosuppression, virus genera were suppressed and more similar to that under moderate immunosuppression. Furthermore, abnormally high abundance of Lymphocryptovirus was particularly obvious in MSM with HIV infection. Analysis of KEGG Pathway revealed that Caulobacter cell cycle, which affects cell duplication, became shorter in HIV-positive subjects. It is worth noting that in acute-stage participants, protein digestion and absorption related to the anti-HIV-1 activity of secretory leukocyte protease inhibitor was increased. Moreover, in the severely immunosuppressed subjects, glutathione metabolism, which is associated with the activation of lymphocytes, was enhanced. Nevertheless, the ecological dysbiosis in HIV-positive salivary virobiota possibly depended on the changes in blood viral load, and salivary dysfunction of MSM infected with HIV may be related to CD4 counts. Ribonucleoside diphosphate reductase subunit M1 in purine metabolism was negatively correlated, though weakly, to CD4 counts, which may be related to the promotion of HIV-1 DNA synthesis in peripheral blood lymphocytes. 7-Cyano-7-deazaguanine synthase in folate biosynthesis was weakly positively correlated with HIV viral load, suggesting that this compound was produced excessively to correct oral dysfunction for maintaining normal cell development. Despite the limited number of samples, the present study provided insight into the potential role of salivary virome in the oral function of HIV infected MSM.

Computational modeling of unphosphorylated CtrA:Cori binding in the Caulobacter cell cycle

SummaryIn the alphaproteobacterium, Caulobacter crescentus, phosphorylated CtrA (CtrA∼P), a master regulatory protein, binds directly to the chromosome origin (Cori) to inhibit DNA replication. Using a mathematical model of CtrA binding at Cori site [d], we provide computational evidence that CtrAU can displace CtrA∼P from Cori at the G1-S transition. Investigation of this interaction within a detailed model of the C. crescentus cell cycle suggests that CckA phosphatase may clear Cori of CtrA∼P by altering the [CtrAU]/[CtrA∼P] ratio rather than by completely depleting CtrA∼P. Model analysis reveals that the mechanism allows for a speedier transition into S phase, stabilizes the timing of chromosome replication under fluctuating rates of CtrA proteolysis, and may contribute to the viability of numerous mutant strains. Overall, these results suggest that CtrAU enhances the robustness of chromosome replication. More generally, our proposed regulation of CtrA:Cori dynamics may represent a novel motif for molecular signaling in cell physiology.

The Lon protease temporally restricts polar cell differentiation events during the Caulobacter cell cycle.

The highly conserved protease Lon has important regulatory and protein quality control functions in cells from the three domains of life. Despite many years of research on Lon, only a few specific protein substrates are known in most organisms. Here, we used a quantitative proteomics approach to identify novel substrates of Lon in the dimorphic bacterium Caulobacter crescentus. We focused our study on proteins involved in polar cell differentiation and investigated the developmental regulator StaR and the flagella hook length regulator FliK as specific Lon substrates in detail. We show that Lon recognizes these proteins at their C-termini, and that Lon-dependent degradation ensures their temporally restricted accumulation in the cell cycle phase when their function is needed. Disruption of this precise temporal regulation of StaR and FliK levels in a Δlon mutant contributes to defects in stalk biogenesis and motility, respectively, revealing a critical role of Lon in coordinating developmental processes with cell cycle progression. Our work underscores the importance of Lon in the regulation of complex temporally controlled processes by adjusting the concentrations of critical regulatory proteins. Furthermore, this study includes the first characterization of FliK in C. crescentus and uncovers a dual role of the C-terminal amino acids of FliK in protein function and degradation.

Robust Modulation of a Bacterial Kinase by Protein Phase Separation

The diversity of molecular function and material properties in cytoplasmic biomolecular condensates contributes to the robustness of enzymatic interactions in a crowded cellular environment. However, molecular principles that organize and control chemical reactions in these condensates are poorly understood. We utilized a multi-scale approach combining biochemical reconstitution and single molecule imaging to gain insights into a kinase signaling reaction localized in a phase separated polar microdomain in the Gram-negative bacterium Caulobacter crescentus. Caulobacter divides asymmetrically to yield a sessile stalked cell and a flagellated swarmer cell. Much of this asymmetric division is regulated through different signaling pathways that reside in microdomains at the two cell poles prior to cell division. Specifically, at the stalked pole, two intrinsically disordered proteins, PopZ and SpmX, form a stable complex and sequester the histidine kinase DivJ whose autophosphorylation contributes to the regulation of cell cycle progression. Here, I will discuss the biophysical basis for phase separation of SpmX and PopZ and the implications of this phenomenon in the context of the Caulobacter cell cycle. Using biochemistry and state of the art 3-D single molecule imaging, we have identified molecular features that regulate DivJ diffusion and autophosphorylation in the polar microdomain. We further establish the role of protein topology, crowding, and the extracellular environment on DivJ regulation via phase separation. Our results underscore the relationship between enzyme activity and unstructured biomolecular condensates that govern the physical environment in signaling hubs for robust modulation of biochemical reactions a crowded cytoplasm.

Methodology for Ribosome Profiling of Key Stages of the Caulobacter crescentus Cell Cycle.

Bacterial cell division is the result of a productive round of the cell cycle to yield two daughter cells. The cell cycle is highly coordinated in Caulobacter crescentus where it is driven by a cell cycle gene-regulatory network that coordinates gene expression with the major cell cycle events such as chromosome replication and cell division. Recent ribosomes profiling data showed that 484 genes undergo changes in translation efficiency during the cell cycle, suggesting a broad role for translational control in cell cycle regulation. In this chapter, we focus on how to perform ribosome profiling to measure the translation efficiency across cellular mRNAs at key stages in the Caulobacter cell cycle. This methodology relies on the high-yield ludox gradient synchronization of Caulobacter cells followed by ribosome profiling to measure ribosome density and total RNA-seq to measure mRNA levels.

Dynamic translation regulation in Caulobacter cell cycle control

Progression of the Caulobacter cell cycle requires temporal and spatial control of gene expression, culminating in an asymmetric cell division yielding distinct daughter cells. To explore the contribution of translational control, RNA-seq and ribosome profiling were used to assay global transcription and translation levels of individual genes at six times over the cell cycle. Translational efficiency (TE) was used as a metric for the relative rate of protein production from each mRNA. TE profiles with similar cell cycle patterns were found across multiple clusters of genes, including those in operons or in subsets of operons. Collections of genes associated with central cell cycle functional modules (e.g., biosynthesis of stalk, flagellum, or chemotaxis machinery) have consistent but different TE temporal patterns, independent of their operon organization. Differential translation of operon-encoded genes facilitates precise cell cycle-timing for the dynamic assembly of multiprotein complexes, such as the flagellum and the stalk and the correct positioning of regulatory proteins to specific cell poles. The cell cycle-regulatory pathways that produce specific temporal TE patterns are separate from-but highly coordinated with-the transcriptional cell cycle circuitry, suggesting that the scheduling of translational regulation is organized by the same cyclical regulatory circuit that directs the transcriptional control of the Caulobacter cell cycle.

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.

The Global Regulatory Architecture of Transcription during the Caulobacter Cell Cycle

Each Caulobacter cell cycle involves differentiation and an asymmetric cell division driven by a cyclical regulatory circuit comprised of four transcription factors (TFs) and a DNA methyltransferase. Using a modified global 5′ RACE protocol, we globally mapped transcription start sites (TSSs) at base-pair resolution, measured their transcription levels at multiple times in the cell cycle, and identified their transcription factor binding sites. Out of 2726 TSSs, 586 were shown to be cell cycle-regulated and we identified 529 binding sites for the cell cycle master regulators. Twenty-three percent of the cell cycle-regulated promoters were found to be under the combinatorial control of two or more of the global regulators. Previously unknown features of the core cell cycle circuit were identified, including 107 antisense TSSs which exhibit cell cycle-control, and 241 genes with multiple TSSs whose transcription levels often exhibited different cell cycle timing. Cumulatively, this study uncovered novel new layers of transcriptional regulation mediating the bacterial cell cycle.

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.

Using Optically Reversible Spatial Mutations to Dissect the Asymmetric Developmental Program of a Bacterium

Understanding the fundamental basis of cell fate determination is a critical challenge in biology. The Caulobacter bacterium provides the simplest model system for studying asymmetric cell division. Caulobacter's cell cycle regulatory circuit is controlled by a dynamically localized signaling network and targeted proteolysis. This network has been well characterized both genetically and biochemically. However, little is known about the underlying spatiotemporal mechanisms controlling this circuit. Optogenetics techniques offer an unprecedented way to reversibly alter protein localization in vivo in seconds time resolution. We have adapted a light-inducible dimerization system to Caulobacter and introduced light controlled spatial-mutations for driving a diffuse protein to specific cellular addresses. We then used these mutations to develop an in vivo spatiotempotal protein interaction assay (opto-conn) to determine whether two proteins are in complex in vivo at a specific cellular address and time point of the cell cycle. Using opto-conn, we are dissecting Caulobacter's regulation pathways by altering the localization and interactions of its key regulatory proteins.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Ribosome Profiling of the Caulobacter Cell-Cycle

An important goal in understanding cellular differentiation is to determine how the genetic information encoded in the genome is expressed properly in time and space to yield cell types with distinct functions. The bacterium Caulobacter crescentus has proven to be a valuable model organism for studying cellular differentiation processes that occur as a function of the cell cycle. Each Caulobacter cell division is asymmetric, yielding daughter cells with different cell fates. This process requires rapid and specific changes in gene expression during the cell cycle that are controlled at many levels, including transcriptional regulation, transient DNA methylation, differential proteolysis, and protein phospho-signaling. However, relatively little is known about the cell cycle control of mRNA translation. To understand the role of translational regulation in Caulobacter differentiation and asymmetric cell division, we are employing ribosome profiling to monitor genome-wide changes in translation at multiple times during cell cycle progression. Arrested ribosomes are treated with ribonuclease to partially digest the mRNA, yielding short fragments protected by monoribosomes. These ribosome-protected mRNA fragments are purified and prepared for high throughput sequencing to map the position of cellular ribosomes and quantify levels of translation. By comparing ribosome profiling levels with RNA-seq measurements of mRNA levels we find many new cases of genes whose translation only occurs during the proper phase of the cell cycle. We find that a majority of ORFs have a >2-fold change in translation efficiency during the cell cycle. This suggests that Caulobacter uses cell cycle-specific regulation of translation to ensure proper timing of gene expression.

Bi-modal Distribution of the Second Messenger c-di-GMP Controls Cell Fate and Asymmetry during the Caulobacter Cell Cycle

Many bacteria mediate important life-style decisions by varying levels of the second messenger c-di-GMP. Behavioral transitions result from the coordination of complex cellular processes such as motility, surface adherence or the production of virulence factors and toxins. While the regulatory mechanisms responsible for these processes have been elucidated in some cases, the global pleiotropic effects of c-di-GMP are poorly understood, primarily because c-di-GMP networks are inherently complex in most bacteria. Moreover, the quantitative relationships between cellular c-di-GMP levels and c-di-GMP dependent phenotypes are largely unknown. Here, we dissect the c-di-GMP network of Caulobacter crescentus to establish a global and quantitative view of c-di-GMP dependent processes in this organism. A genetic approach that gradually reduced the number of diguanylate cyclases identified novel c-di-GMP dependent cellular processes and unraveled c-di-GMP as an essential component of C. crescentus cell polarity and its bimodal life cycle. By varying cellular c-di-GMP concentrations, we determined dose response curves for individual c-di-GMP-dependent processes. Relating these values to c-di-GMP levels modeled for single cells progressing through the cell cycle sets a quantitative frame for the successive activation of c-di-GMP dependent processes during the C. crescentus life cycle. By reconstructing a simplified c-di-GMP network in a strain devoid of c-di-GMP we defined the minimal requirements for the oscillation of c-di-GMP levels during the C. crescentus cell cycle. Finally, we show that although all c-di-GMP dependent cellular processes were qualitatively restored by artificially adjusting c-di-GMP levels with a heterologous diguanylate cyclase, much higher levels of the second messenger are required under these conditions as compared to the contribution of homologous c-di-GMP metabolizing enzymes. These experiments suggest that a common c-di-GMP pool cannot fully explain spatiotemporal regulation by c-di-GMP in C. crescentus and that individual enzymes preferentially regulate specific phenotypes during the cell cycle.

Identification of ClpP substrates in Caulobacter crescentus reveals a role for regulated proteolysis in bacterial development

Energy-dependent proteases ensure the timely removal of unwanted proteins in a highly selective fashion. In Caulobacter crescentus, protein degradation by the ClpXP protease is critical for cell cycle progression; however, only a handful of substrates are currently known. Here, we use a trapping approach to identify putative substrates of the ClpP associated proteases in C. crescentus. Biochemical validation of several of these targets reveals specific protease recognition motifs and suggests a need for ClpXP-specific degradation beyond degradation of known cell cycle regulators. We focus on a particular instance of regulated proteolysis in Caulobacter by exploring the role of ClpXP in degrading the stalk synthesis transcription factor TacA. We show that TacA degradation is controlled during the cell cycle dependent on the ClpXP regulator CpdR and that stabilization of TacA increases degradation of another ClpXP substrate, CtrA, while restoring deficiencies associated with prolific CpdR activity. Together, our work reveals a number of new validated ClpXP substrates, clarifies rules of protease substrate selection, and demonstrates how regulated protein degradation is critical for Caulobacter development and cell cycle progression.

RK2 plasmid dynamics in Caulobacter crescentus cells – two modes of DNA replication initiation

Undisturbed plasmid dynamics is required for the stable maintenance of plasmid DNA in bacterial cells. In this work, we analysed subcellular localization, DNA synthesis and nucleoprotein complex formation of plasmid RK2 during the cell cycle of Caulobacter crescentus. Our microscopic observations showed asymmetrical distribution of plasmid RK2 foci between the two compartments of Caulobacter predivisional cells, resulting in asymmetrical allocation of plasmids to progeny cells. Moreover, using a quantitative PCR (qPCR) method, we estimated that multiple plasmid particles form a single fluorescent focus and that the number of plasmids per focus is approximately equal in both swarmer and predivisional Caulobacter cells. Analysis of the dynamics of TrfA-oriV complex formation during the Caulobacter cell cycle revealed that TrfA binds oriV primarily during the G1 phase, however, plasmid DNA synthesis occurs during the S and G2 phases of the Caulobacter cell cycle. Both in vitro and in vivo analysis of RK2 replication initiation in C. crescentus cells demonstrated that it is independent of the Caulobacter DnaA protein in the presence of the longer version of TrfA protein, TrfA-44. However, in vivo stability tests of plasmid RK2 derivatives suggested that a DnaA-dependent mode of plasmid replication initiation is also possible.

Regulated proteolysis of a transcription factor complex is critical to cell cycle progression in Caulobacter crescentus

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 an important cell cycle regulator, helping to trigger the onset of S phase and prepare cells for the subsequent programmes of gene expression critical to polar morphogenesis and cell division.

The Caulobacter crescentus ctrA P1 promoter is essential for the coordination of cell cycle events that prevent the overinitiation of DNA replication

The master regulator CtrA oscillates during the Caulobacter cell cycle due to temporally regulated proteolysis and transcription. It is proteolysed during the G1-S transition and reaccumulates in predivisional cells as a result of transcription from two sequentially activated promoters, P1 and P2. CtrA reinforces its own synthesis by directly mediating the activation of P2 concurrently with repression of P1. To explore the role of P1 in cell cycle control, we engineered a mutation into the native ctrA locus that prevents transcription from P1 but not P2. As expected, the ctrA P1 mutant exhibits striking growth, morphological and DNA replication defects. Unexpectedly, we found CtrA and its antagonist SciP, but not DnaA, GcrA or CcrM accumulation to be dramatically reduced in the ctrA P1 mutant. SciP levels closely paralleled CtrA accumulation, suggesting that CtrA acts as a rheostat to modulate SciP abundance. Furthermore, the reappearance of CtrA and CcrM in predivisional cells was delayed in the P1 mutant by 0.125 cell cycle unit in synchronized cultures. High levels of ccrM transcription despite low levels of CtrA and increased transcription of ctrA P2 in the ctrA P1 mutant are two examples of robustness in the cell cycle. Thus, Caulobacter can adjust regulatory pathways to partially compensate for reduced and delayed CtrA accumulation in the ctrA P1 mutant.

Regulated Proteolysis as a Force to Control the Cell Cycle

In this issue of Structure, Rood and colleagues report that substrate architecture is a key factor in promoting the complete and processive degradation of the Caulobacter cell cycle regulator PdeA by the protease ClpXP. This investigation highlights the important role that the adaptor protein CpdR serves in regulating presentation of PdeA to ClpXP.

A Cell-Type-Specific Protein-Protein Interaction Modulates Transcriptional Activity of a Master Regulator in Caulobacter crescentus

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.

Trade-Off Between Localization and Expression Levels in Flagellar Pole Development of the Bacterium Caulobacter Crescentus

The bacterium Caulobacter crescentus is a model organism for cell cycle regulation and development. Upon division Caulobacter differentiates into two phenotypically distinct cells, a sessile stalked cell and a motile swarmer cell. Throughout the Caulobacter cell cycle, the localization of several key proteins is highly regulated. In this study, we address the importance of spatial localization in signal transduction and development using synthetic redesign of protein localization coupled with mathematical modeling. Development at the flagellar pole is controlled by the response regulator DivK, whose phosphorylation state is controlled by the histidine kinase DivJ and the phosphatase PleC. PleC localizes to the swarmer cell pole, while DivJ localizes at the stalked pole. To address the importance of localization, To address this question, we have constructed strains with a variety of PleC and DivJ localization patterns, including delocalization and mislocalization to the opposite pole. To determine whether phenotypic changes can be explained purely on the basis of altered localization, we have developed a mathematical model that suggests that flagellar pole development does not rely critically on precise localization of DivJ and PleC, and that developmental defects due to complete mislocalization of PleC can be compensated for by overexpression. Our results indicate that localization is not absolutely necessary for some cellular functions, but that localized proteins enhance the robustness of the system to fluctuations.