View details for DOI 10.1073/pnas.0604554103, View details for Web of Science ID 000239327200022, View details for PubMedCentralID PMC1544152. However, an active ParE subunit is required for ParC localization to the replisome as it moves from the cell pole to the division plane during chromosome replication. Thanbichler, M., Viollier, P. H., Shapiro, L. MreB actin-mediated segregation of a specific region of a bacterial chromosome. C. crescentus DNA carrying the Tn5-flaE region and adjacent sequences was cloned into pBR325 and selected by transposon-encoded kanamycin resistance. Understanding of the cell cycle control logic in Caulobacter has progressed to the point where we now have an integrated view of the operation of an entire bacterial cell cycle system functioning as a state machine. We propose that the P1 promoter is activated after the initiation of DNA replication in the early predivisional cell. The gliding motility of this bacterium is propelled by a nozzle-like structure that squirts a polysaccharide-containing slime from the pole of the cell (5). Previous microarray experiments revealed that several Caulobacter genes are significantly upregulated in response to uranium but not in response to other heavy metals. A., Deacon, A. M., Shapiro, L. Cell fate regulation governed by a repurposed bacterial histidine kinase. We have performed in vivo genomic binding site analysis of the CtrA protein to identify which of these genes have regulatory regions bound directly by CtrA. Home; Bio; Papers; Data; Press; Main content start Our primary focus is on elucidating the events required for the orderly segregation of homologous chromosomes during meiosis, the crucial process by which diploid germ cells generate haploid gametes. We hypothesize that selective silencing of groups of genes in the chromosomes at the swarmer and stalked poles of the predivisional cell results in the different developmental programs and the difference in replicative ability of the two progeny cells. Thus, it is the signal transduction pathway mediated by CckA that culminates in CtrA activation, which is temporally regulated and essential for cell cycle progression. While recent advances in cryogenic electron microscopy (cryo-EM) allow for the visualization and identification of structures within cells at the nanometer scale, information regarding the cellular environment, such as pH, membrane potential, ionic strength etc. The synthesis of the product of flgJ, the 29K flagellin, occurs prior to the synthesis of the other flagellin proteins. We generated strains that carry ccrM either on a low-copy-number plasmid (strain GR131) or on a moderate-copy-number plasmid (strain GR132). Goley, E. D., Yeh, Y., Hong, S., Fero, M. J., Abeliuk, E., McAdams, H. H., Shapiro, L. The Architecture and Conservation Pattern of Whole-Cell Control Circuitry, Regulatory Response to Carbon Starvation in Caulobacter crescentus. The specific defects responsible for the lack of a chemotactic response have not been determined for the other identified che genes. A consensus sequence for a sigma 54 promoter was found at the appropriate distance 5' to one of two identified transcription start sites. Such rotations did not lead to large-scale changes in gene expression, indicating that genome folding does not strongly affect gene regulation. Only the unphosphorylated form of CpdR localizes and activates ClpXP. Temporal control of DNA methylation has been shown to be critical for normal development in the dimorphic Caulobacter life cycle. Goley, E. D., Iniesta, A. We identify mutations in PopZ that allow scaffold assembly but specifically abrogate interactions with ParA and demonstrate that PopZ/ParA interactions are required for proper chromosome segregation in vivo. It was found that the Tsr protein appeared at the same point in the cell cycle as an endogenous C. crescentus methyl-accepting chemotaxis protein. Transcription activation of flbN in C. crescentus involves the combination of several elements: the NifA-like site is required for full activation, and other sequence elements 5' to the promoter and 3' to the transcription start site are necessary for the correct time of transcription initiation. B.S. The membrane methyl-accepting chemotaxis proteins were shown to be synthesized before cell division, coincident with the synthesis of the components of the flagellum, and to be specifically localized in the membrane of the incipient swarmer cell portion of the predivisional cell. View details for Web of Science ID A1990DB07400012. By analyzing mutations in the dnaX promoter, we identified a motif between the -10 and -35 regions that is required for proper timing of gene expression. Graduate Student (joined @ 06/2017) Bioengineering. The expression of the Caulobacter crescentus homolog of dnaX, which in Escherichia coli encodes both the gamma and tau subunits of the DNA polymerase III holoenzyme, is subject to cell cycle control. Here, we show that DnaA, a protein required for the initiation of DNA replication, also functions as a transcriptional activator of gcrA, which in turn activates multiple genes, notably those involved in chromosome replication and segregation. Avedissian, M., Lessing, D., Gober, J. W., Shapiro, L., Gomes, S. L. THE CONTROL OF ASYMMETRIC GENE-EXPRESSION DURING CAULOBACTER CELL-DIFFERENTIATION, IDENTIFICATION OF AN ESSENTIAL SIGMA-32 HOMOLOG IN COULOBACTER-CRESCENIUS, COORDINATE CELL-CYCLE CONTROL OF A CAULOBACTER DNA METHYLTRANSFERASE AND THE FLAGELLAR GENETIC HIERARCHY. Functional homology was demonstrated by complementing the temperature-sensitive growth defect of an E. coli rpoH deletion mutant with the C. crescentus rpoH gene. In Escherichia coli, the Hda protein inactivates DnaA after replication initiation. S-layer proteins (SLPs) regulate their extracellular self-assembly by crystallizing when exposed to an environmental trigger. These changes in DNA methylation could signal differential binding of regulatory proteins to activate or repress transcription. We show that DNA methylation by CcrM is not required for the control of the initiation of chromosome replication or for DNA mismatch repair. Deletions in the 5' region have also revealed that all cis-acting sites required for temporal control of transcription reside within 50 bases of the P2 start site. Shen, X., Collier, J., Dill, D., Shapiro, L., Horowitz, M., McAdams, H. H. Small non-coding RNAs in Caulobacter crescentus. Congratulations to Sangjin and collaborators on this detailed biophysical study. A., Eckart, M. R., Shapiro, L. Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle. Stanford Molecular Pathology and Clinical Genomics groups perform a wide range of diagnostic nucleic acid-based tests for hereditary disorders, cancer diagnosis and management and other conditions. We welcome Emily Maclary, who joined the lab as a postdoctoral fellow. Read More. Time-lapse microscopy of the location of the chromosomal origin and 10 selected loci in the origin-proximal half of the chromosome showed that during DNA replication, as the replisome sequentially copies each locus, the newly replicated DNA segments are moved in chronological order to their final subcellular destination in the nascent half of the predivisional cell. We welcome Raquel Maynez, who joined the lab as an undergraduate researcher. The B, C, and D transcripts were present in equivalent molar ratios, were all smaller than transcript A, and were found to yield RNase III digestion products that were subsets of each other as well as of transcript A. Four transcripts, A, B, C, and D, which ranged in size from 2.9 X 10(6) to 0.53 X 10(6) daltons, were synthesized in vitro by the holoenzyme. The cellular positions of other chromosomal loci were in the wild-type order relative to the parS centromere, but chromosomes remained unsegregated and appeared to be stacked upon one another. View details for Web of Science ID A1993ME00800030. In this paper we report the isolation, characterization and genetic analysis of several C. crescentus mutants altered in membrane lipid synthesis. CtrA activity must be removed from cells at the onset of DNA replication, because phosphorylated CtrA binds to and silences the origin of replication. Because of the many parallels in the function of these biochemically based genetic circuits and electrical circuits, a hybrid modeling approach is proposed that integrates conventional biochemical kinetic modeling within the framework of a circuit simulation. Expression of perP is regulated by a signal transduction system that activates cell type-specific transcription programs and conversion of PodJ(L) to PodJ(S) in response to the completion of cytokinesis. 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. The transient accumulation of DivL at the new cell pole, but not its kinase activity, is required for the localization and activation of CckA. We take advantage of the best feature of both model and non-model organisms, including laboratory mice, wild stickleback fish, and pluripotent stem cells from humans and non-human primates. Carbon starvation activates DnaA proteolysis (B. Gorbatyuk and G. T. Marczynski, Mol. View details for DOI 10.1073/pnas.1220824110, View details for Web of Science ID 000314558100027, View details for PubMedCentralID PMC3562846. We found that MmpA facilitates the degradation of PodJS. Caulobacter crescentus is a model organism for the integrated circuitry that runs a bacterial 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. An adjacent and divergent promoter also has an IHF consensus sequence that binds IHF. We have found that the abundance of SsrA RNA in Caulobacter crescentus is regulated with respect to the cell cycle. 2012 University of Texas at Austin, Graduate Student, Biochemistry A 0.2 kb fragment of DNA located immediately upstream of the Caulobacter homolog of the Escherichia coli dnaX gene was able to completely rescue the temperature-sensitive phenotype of LS439. 1973-1974 Stanford University. View details for Web of Science ID 000167833700095, View details for PubMedCentralID PMC31192. Shapiro JS, Bakken S, Hyun S, Melton GB, Schlegel C, Johnson SB. We demonstrated that the expression of a gene, flaD, that is involved in the formation of the flagellar basal body is under temporal control and is transcribed relatively early in the cell cycle, before the hook and flagellin genes are transcribed. The commonly used, monomeric EYFP enabled imaging of intracellular protein structures beyond the optical resolution limit ('super-resolution' imaging) in living cells. Caulobacter crescentus cell division is asymmetric and yields distinct swarmer cell and stalked cell progeny. Ph.D. 1993 Shanghai Institute of Biochemistry Driks, A., Bryan, R., Shapiro, L., DeRosier, D. J. IMAGE-RECONSTRUCTION OF THE FLAGELLAR BASAL BODY OF CAULOBACTER-CRESCENTUS. Dingwall, A., Zhuang, W. Y., Quon, K., Shapiro, L. The control of timing and spatial organization during Caulobacter cell differentiation. Recent studies on the dimorphic bacterium Caulobacter crescentus (Caulobacter) highlight mechanisms by which positional information is integrated with temporal modes of cell cycle regulation. We report that CtrA binds five sites within the chromosome replication origin in vitro. Research in the Villeneuve lab is aimed at understanding the molecular and cellular mechanisms underlying the faithful inheritance and function of eukaryotic chromosomes. University of Illinois Postdoc. Here, we report a strategy deployed by the asymmetrically dividing bacterium Caulobacter crescentus where a regulatory protein is programmed to perform distinct functions at the opposing cell poles. Since the phage is extremely salt-sensitive, a purification procedure was devised which avoided contact with solutions of high ionic strength. 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 provide a universal strategy for defining the coding potential of bacterial genomes by applying ribosome profiling, RNA-seq, global 5'-RACE, and liquid chromatography coupled with tandem mass spectrometry (LC-MS) data to the 4-megabase C. crescentus genome. In order to elucidate the basic mechanisms whereby a cell dictates its own defined morphogenic changes, we have found it helpful to study an organism that can be manipulated both biochemically and genetically. for "her pioneering discovery that the bacterial cell is controlled by an integrated genetic circuit functioning in time and space that serves as a systems engineering paradigm underlying cell differentiation and ultimately the generation of diversity in all organisms." We imaged fusions of dL5 to three different proteins in live Caulobacter cells using stimulated emission depletion microscopy, yielding a 4-fold resolution enhancement compared to diffraction-limited imaging. The polar particles appear as a cluster of approximately 1 to 10 stain-excluding rings, visible in electron micrographs of negatively stained wild-type cells. In addition, transcription of phiCdl DNA by the E. coli enzyme produced a subset of transcripts not synthesized by the C. crescentus enzyme. The Rule of Law comprises a number of principles of a formal and procedural character, addressing the way in which a community is governed. dL5-MG complexes emit 2-fold more photons before photobleaching compared to organic dyes such as Cy5 and Alexa 647 in vitro, and 5-fold more photons compared to eYFP in vivo. Chen, S. L., Lee, W., Hottes, A. K., Shapiro, L., McAdams, H. H. A bacterial cell-cycle regulatory network operating in time and space, Identification of long intergenic repeat sequences associated with DNA methylation sites in Caulobacter crescentus and other alpha-proteobacteria, Fluorescence bleaching reveals asymmetric compartment formation prior to cell division in Caulobacter. Imaging and controlling cellular function with ultrasound. Comerci, C. J., Herrmann, J., Yoon, J., Jabbarpour, F., Zhou, X., Nomellini, J. F., Smit, J., Shapiro, L., Wakatsuki, S., Moerner, W. E. Robust Modulation of a Bacterial Kinase by Protein Phase Separation. Accurate replication and segregation of the bacterial genome are essential for cell cycle progression. When parS is moved farther from the origin, the cell waits for parS to be replicated before segregation can begin. View details for DOI 10.1073/pnas.1612579113, View details for Web of Science ID 000384528900022, View details for PubMedCentralID PMC5056096. By deciphering the underlying design principles, we hope to generate pure populations of these cell-types from embryonic and induced pluripotent stem cells for regenerative medicine.
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