Known flagellar genes encode structural and regulatory components that are required for flagellar assembly and function. The research program of the Shapiro lab spans the physiology and modulation of voltage-gated K+ and Ca2+ channels in neurons and non-excitable cells. View details for Web of Science ID 000280561600011, View details for PubMedCentralID PMC3205914. Ultrasound-controllable engineered bacteria for cancer immunotherapy. Flagellated and non-flagellated vesicles were prepared from these cells by immunoaffinity chromatography and the level of MCPs that had been labeled either in vivo or in vitro with methyl-3H was determined. x@caltech.edu, x=mduan, Przemysaw Dutka Transcriptional start sites are identified by analyzing probe signal cross-correlation matrices created from probe pairs tiled every 5 bp upstream of the genes. Wu D, Baresch D, Cook C, Duan M, Malounda D, Maresca D, Abundo MP, Lee J, Shivaei S, Mittelstein DR, Qiu T, Fischer P, Shapiro MG*. We believe in the transformative power of diversity and that great science requires great people with open minds. SLAC is a vibrant multiprogram laboratory that The expression of four of the novel sRNAs changes as the cell cycle progresses. Furthermore, the sequential replication through unmapped Dra I fragments has enabled us to localize their positions on the genome. Disruption of the hrcA gene, homologs of which are also found upstream of grpE in other bacteria, increased transcription of the groESL operon, and this effect was dependent on the presence of an intact CIRCE element. & Aero. Defects in the cheB gene resulted in greatly reduced chemotaxis-associated methylation in vivo and a loss of methylesterase activity in vitro. Although FDA is exercising enforcement discretion of premarket review and other regulations for laboratory-developed tests in the US, certification of the laboratory is required under CLIA to ensure the quality and validity of the tests. Disclosure: Scott Williams has disclosed no relevant financial relationships. The M ring, which is at the inner membrane of the cell, has a different structure depending on the method of preparation. The essential transcriptional circuitry for growth on rich media includes 10 transcription factors, 2 RNA polymerase sigma factors and 1 anti-sigma factor. View details for DOI 10.1371/journal.pgen.1004831, View details for PubMedCentralID PMC4287350. Yeh, Y., Comolli, L. R., Downing, K. H., Shapiro, L., McAdams, H. H. Imaging-Based Identification of a Critical Regulator of FtsZ Protofilament Curvature in Caulobacter. Insightec, Teresa Tran To define the mechanisms that mediate this temporal and spatial control, fla genes whose products are not known were accessed by the insertion of transposon-carried drug resistance markers. We have found that it belongs to an unusual promoter family used by several Caulobacter class II flagellar genes. View details for DOI 10.1111/j.1365-2958.2011.07698.x, View details for Web of Science ID 000292567200009, View details for PubMedCentralID PMC3137890. The rest of the filament (region V) is made up predominantly, if not completely, of the 25 x 10(3) Mr flagellin. Without an S-layer, Caulobacter is even more sensitive to changes in environmental calcium concentration. The genes involved in the biogenesis of the flagellum and the chemotaxis machinery are temporally regulated during the Caulobacter crescentus cell cycle. Here we demonstrate the applicability of one such biosensor, the fluorescent protein roGFP2, for cryo-CLEM experiments. An interesting feature of the differentiation cycle is that the polar organelle may represent a special segregated unit which is operative in the control of the differentiation process. Haverford College: Computer Science: Stanford AI Lab Papers and Talks at ICLR 2023. Postdoctoral Fellow, Stanford University School of Medicine, A.B. Katie Wong, SURF Scholar 2019-21 Associate at ZS Associates Here, we show that the transient midcell localization of ClpXP that precedes cytokinesis requires the FtsZ component of the divisome. Subcellular fractionation showed that FliI is present both in the cytoplasm and in association with the membrane. Tn5 insertion mutants unable to form colonies when oleic acid was the sole carbon source were isolated. Research Scientist 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. A fourth heat-shock protein was detected with antibody to the C. crescentus RNA polymerase. WebShapiroLab Going Deeper We develop technologies to image and control the function of cells deep inside the body. Biophysical Society, 1987-present. Recently, single-molecule imaging and photocontrol have enabled superresolution optical microscopy of cellular structures beyond Abbe's diffraction limit, extending the frontier of noninvasive imaging of structures within living cells. The initiation of DNA replication coincides with the proteolysis of the CtrA replication inhibitor and the accumulation of DnaA, the replication initiator, upon differentiation of the swarmer cell into a stalked cell. The molecular weights of the enzyme subunits were 165,000, 155,000, 101,000, and 44,000, respectively. Both proteins contain multiple PAS domains, a multifunctional class of sensory domains present across the kingdoms of life. This DNA contains sequence motifs that are common to other bacterial origins, such as five DnaA boxes, an E. coli-like 13-mer, and an exceptional A + T-rich region. Of the 26 genes required for flagellum production, at least 4 of them-flaY, E, F, and G-map together in a single cluster. The genes involved in these processes are widely separated on the chromosome. View details for DOI 10.1073/pnas.0805258105, View details for Web of Science ID 000258560700056, View details for PubMedCentralID PMC2516238. We report herein a class of boron-containing compounds termed borinic esters that have broad spectrum antibacterial activity with minimum inhibitory concentrations (MIC) in the low microgram/mL range. x@caltech.edu, x=blling, Ann Liu We report the identification of another C. crescentus heat shock operon containing two genes, hrcA (hrc for heat shock regulation at CIRCE elements) and a grpE homolog. Biology, Cayetano Heredia University Importantly, a small set of conserved ChpT residues promotes signaling crosstalk and contributes to the branched signaling that activates the master regulator CtrA while inactivating the CtrA degradation signal, CpdR. Ptacin, J. L., Lee, S. F., Garner, E. C., Toro, E., Eckart, M., Comolli, L. R., Moerner, W., Shapiro, L. Polar Remodeling and Histidine Kinase Activation, Which Is Essential for Caulobacter Cell Cycle Progression, Are Dependent on DNA Replication Initiation. In many cases, a large number of upstream signals are funneled to a master regulator of cellular behavior, but it remains unclear how individual proteins can rapidly integrate a complex array of signals within the appropriate spatial niche within the cell. The molecular weight of purified flagellin (subunit of flagella filament) is 25,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We combine quantitative organism-wide fluorescence imaging ("deep imaging"), functional genomics ("deep sequencing"), and statistical modeling to understand the fundamental rules that control collective cell behaviors to optimize tissue organization, regeneration, adaptation, and evolution. Ph.D. 1972 Purdue University We show that the cell-cycle timing of CcrM is critical for Caulobacter fitness. Temporal control of DNA methylation has been shown to be critical for normal development in the dimorphic Caulobacter life cycle. The process of bacterial DNA replication generates chromosomal topological constraints that are further confounded by simultaneous transcription. The addition of rifampicin early after infection inhibited the production of phage, whereas phiCdl production was not inhibited by the addition of rifampicin at any time after infection of a rifampicin-resistant host. Ph.D Biophysics (Imaging), University of Western Ontario In addition to the presence of the fatA506 mutation, this strain was found to contain two other mutations, one of which caused the production of a water-soluble brown-orange pigment (pigA) and another which caused formation of helical cells (hclA). In a mutant strain that failed to assemble a flagellum, the 29K flagellin still segregated to the presumptive swarmer cell, demonstrating that positioning of the protein is independent of filament assembly. 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. 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. Thus, two transcriptional feedback loops coupled to cell cycle-regulated proteolysis and phosphorylation of the CtrA protein result in the pattern of CtrA activity required for the temporal and spatial control of multiple cell-cycle events. Each kind of bacterium also executes its own strategy to find nutrients in its habitat and to cope with conditions of stress from its environment. Large structures, such as a flagellum, are anchored at the pole by means of the basal body that traverses the peptidoglycan wall. Mechanical Engineering, Yonsei University Constructs for mammalian cells and bacteria available on Addgene. Brian Lue, SURF Scholar 2016-19 MD at UT Southwestern Ph.D. Student, Bioengineering Dingwall, A., Zhuang, W. Y., Quon, K., Shapiro, L. The control of timing and spatial organization during Caulobacter cell differentiation. Here, we report that in Caulobacter, a hipA2-encoded bacterial toxin contributes to bacterial persistence by manipulating intracellular amino acid balance. View details for Web of Science ID A1990DY49600006. 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. When ccrM gene expression is placed under control of a constitutive promoter, these chromosomal sites are fully methylated throughout the cell cycle. Signaling hubs at bacterial cell poles establish cell polarity in the absence of membrane-bound compartments. View details for Web of Science ID A1984TT35100004. We have also identified a new structural feature at the flagellated pole and the tip of the stalk: the 10-nm polar particle. Although ClpAP does not exhibit subcellular localization, FtsZ is a substrate of both ClpXP and ClpAPin vivo and in vitro. In the absence of glycerol 3-phosphate, DNA replication was initiated in the stalked cell at the correct time in the cell cycle and at the correct site on the chromosome. One of the phosphorylated DNA-binding proteins was identified as the beta' subunit of the host RNA polymerase. Here, we show that CckA uses its PAS domains to integrate information from DivL and its own oligomerization state to control the balance of its kinase and phosphatase activities. Two rings were in the hook-proximal upper set, and three rings (two narrow and one wide) were in the lower set. This type of gene overlap is also observed in bacterial genes involved in cell division. Except for the hook, there are no morphological features that would otherwise distinguish these regions. Herrmann, J., Smit, J., Shapiro, L., Wakatsuki, S. Two-Color Sted Microscopy to Visualize S-Layer Biogenesis in Caulobacter Crescentus. The ctrA gene is preferentially transcribed from a hemimethylated promoter. Herrmann, J., Li, P., Jabbarpour, F., Chan, A. C., Rajkovic, I., Matsui, T., Shapiro, L., Smit, J., Weiss, T. M., Murphy, M. E., Wakatsuki, S. Asymmetric division yields progeny cells with distinct modes of regulating cell cycle-dependent chromosome methylation. Christen, B., Fero, M. J., Hillson, N. J., Bowman, G., Hong, S., Shapiro, L., McAdams, H. H. Bacterial Chromosome Organization and Segregation, System-level design of bacterial cell cycle control, Feedback Control of DnaA-Mediated Replication Initiation by Replisome-Associated HdaA Protein in Caulobacter, Superresolution imaging of protein superstructures in live Caulobacter crescentus cells with EYFP. Although transcription from the smc promoter is induced during early S phase, a cell cycle transcription pattern previously observed with multiple DNA replication and repair genes, the SMC protein is present throughout the entire cell cycle. B.S. Cell division in Caulobacter crescentus yields a swarmer and a stalked cell. Genetic networks with tens to hundreds of genes are difficult to analyze with currently available techniques. This control circuitry monitors the environment and the internal state of the cell, including the cell topology, as it orchestrates orderly activation of cell cycle subsystems and Caulobacter's asymmetric cell division. To identify factors contributing to the asymmetric biogenesis of polar pili, cytological studies of pilus assembly components were performed. Biology, Caltech The relative copy numbers of these proteins are essential for complex formation, as overexpression of SpmX in Caulobacter reorganizes the polarity of the cell, generating ectopic cell poles containing PopZ and DivJ. SsrA, or tmRNA, is a small RNA found in all bacteria that intervenes in selected translation reactions to target the nascent polypeptide for rapid proteolysis. View details for DOI 10.1016/j.cell.2008.07.015, View details for Web of Science ID 000259318100015, View details for PubMedCentralID PMC2745220. The activity of the enzyme shows an unusual sensitivity to salt levels, apparently dissociating more rapidly from methylated DNA product as the salt level is decreased. Collaboration: View details for Web of Science ID 000168535000012, View details for PubMedCentralID PMC95206, View details for Web of Science ID 000168824801666. 7703 Floyd Curl Drive, MC 7843 Cell cycle progression and polar differentiation are temporally coordinated in Caulobacter crescentus. In the recent years, considerable advances have been made towards understanding the structure and function of the bacterial chromosome. Our current understanding of this system has been limited by the fact that purified protein products are available for only about one-fifth of these genes. Thus, chromosome compaction likely involves dynamic aggregates of SMC bound to DNA. Awards: NIH Pioneer, Mark, IEEE Hertz, Vilcek, Saville, Tsien, Dreyfus, Van Ness, Packard, Sontag, Pew, DARPA YFA, BWF-CASI, Miller, Hertz, Soros, LSRF, TR35 We use a variety of innovative approaches including genomics, computation, biochemistry, and advanced imaging. Each cell division produces two distinct cell types: a swarmer cell and a stalked cell. A crucial function for eukaryotic cytoskeletal filaments is to organize the intracellular space: facilitate communication across the cell and enable the active transport of cellular components. This is an example of controlled proteolysis of a cytoplasmic protein that is associated with its active recruitment to a specific subcellular address. CcrM, an adenine DNA methyltransferase, is essential for viability in Caulobacter crescentus. Beyond direct protein coding, genomes encode regulatory information required to orchestrate the proper timing and levels of gene expression and protein synthesis, and contain binding sites and regulatory sequences to co-ordinate the activities of proteins involved in chromosome repair and maintenance. Analysis of bacterial genome organization and replication using pulsed field gel electrophoresis, THE MOLECULAR-GENETICS OF DIFFERENTIATION, NEGATIVE TRANSCRIPTIONAL REGULATION IN THE CAULOBACTER FLAGELLAR HIERARCHY, AN ESCHERICHIA-COLI CHEMORECEPTOR GENE IS TEMPORALLY CONTROLLED IN CAULOBACTER, THE ORGANIZATION OF THE CAULOBACTER-CRESCENTUS FLAGELLAR FILAMENT. Androgen Receptor, University of Illinois Currently: Assistant Professor of Biomedical Sciences This technique can be used to select for mutants blocked in the various stages of morphogenesis. View details for Web of Science ID A1977EH42100096. Animal Biotechnology, Konkuk University 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. 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. In compartmentalized cells, fluorescence disappears only in the compartment receiving the bleaching beam; in noncompartmentalized cells, fluorescence disappears from the entire cell. The second region is adjacent to the hook and is approximately 10 nm in length. Many recent studies have revealed exquisite subcellular localization of proteins, DNA, and other molecules within bacterial cells, giving credence to the concept of prokaryotic anatomy. Homology modelling of the N-terminal atypical receiver domain of CpaE indicates that it has a conserved protein-protein binding surface similar to that of the polar localization module of the social mobility protein FrzS, suggesting a similar function. Pasadena, CA, USA 91125. Class II genes are the earliest to be expressed and are activated at a specific time in the cell cycle by the CtrA response regulator. Here, we review the progress that has been made towards understanding the mechanisms by which bacterial cytoskeletal proteins influence cellular organization. Ellen Min, SURF Scholar 2021 BS Computer Science, Caltech 2024 (exp) Genetic regulatory hierarchy in Caulobacter development. View details for DOI 10.1111/j.1365-2958.2008.06172.x, View details for Web of Science ID 000254641600007, View details for Web of Science ID 000208467800418, View details for Web of Science ID 000255316100052. x@caltech.edu, x=zyjin, Whee-Soo Kim, PhD The polarly localized DivK response regulator promotes CtrA localization and proteolysis, but it does not directly recruit CtrA to the cell pole. Caulobacter crescentus performs chemotaxis by short intermittent reversals of rotation of its single polar flagellum. It will bring together the resources and expertise of the national lab, the university and Silicon Valley to accelerate the deployment of batteries and other energy storage The availability of CcrM is controlled in two ways: (1) the ccrM gene is transcribed only in the predivisional. Global transcription analysis of synchronized Caulobacter crescentus cells was used to identify 553 genes (19% of the genome) whose messenger RNA levels varied as a function of the cell cycle. (Hons) Integrative Biomedical Sciences, University of Edinburgh 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. Defects in the cheR gene resulted in a loss of the ability to methylate the methyl-accepting chemotaxis proteins. View details for DOI 10.1073/pnas.1612579113, View details for Web of Science ID 000384528900022, View details for PubMedCentralID PMC5056096. Hillson, N. J., Hu, P., Andersen, G. L., Shapiro, L. A comprehensive set of plasmids for vanillate- and xylose-inducible gene expression in Caulobacter crescentus. 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. In addition, minor phospholipids were detected in the swarmer cells that were not detected in stalked cells. We estimate that there are approximately 350 inverted repeat regions per Caulobacter genome. Faithful chromosome segregation is an essential component of cell division in all organisms. Clearance of active CtrA at the G1/S transition allows the initiation of DNA replication and cell-cycle progression. The hemE gene also appears to be translated from a leaderless mRNA. View details for Web of Science ID A1987G196300016. Caulobacter crescentus wild-type strain CB13 is unable to utilize galactose as the sole carbon source unless derivatives of cyclic AMP are present. Thus, dynamic changes in cellular location of critical signal proteins provide a novel mechanism for the control of the prokaryote cell cycle. These include the morphological transition of a swarmer cell to a replication-competent stalked cell and the subsequent polarized distribution of specific gene products that results in an asymmetric predivisional cell. Our structural results also suggest that TadZ localizes to the pole through the atypical receiver domain during an early stage of pili biogenesis, and functions as a hub for recruiting other pili components, thus providing insights into the Tad pilus assembly process. Discover our history and vision for the future. Pasadena, CA, USA 91125, Copyright 2023 Mikhail G. Shapiro | Powered by, Scientists, Postdoc Scholars & Graduate Students. Nature Nanotechnology 16, 14031412 (2021). Evidence that there is transcriptional control of flgJ expression includes the following: (1) The initial appearance of flgJ message was coincident with the onset of 29K flagellin protein synthesis, and (2) expression of an NPT II reporter gene driven by the flgJ promoter was temporally correct.
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