View details for DOI 10.1073/pnas.1114444108. The flaS transcript initiation site was identified, and an apparently unique promoter sequence was found to be highly conserved among the genes at the same level of the hierarchy. One determinant is present in the last 15 amino acid residues of CtrA, particularly the terminal Ala-Ala residues, and another is located within the first 56 residues of the CtrA receiver domain. The flagellum is ejected from the swarmer cell and then synthesized de novo later in the cell cycle. Critically, many of these functions occur at defined locations within the cell, and therefore regulators of each module must communicate to remain coordinated in space. In bacteria, the mechanisms that underlie the coordination of chromosome replication and segregation are poorly understood. The probe correlation analysis approach reported here is of general use for large-scale sRNA identification for any sequenced microbial genome. Nucleoid morphology was also abnormal. The transduction of the chemotaxis signal is initiated by a chemoreceptor-CheW-CheA ternary complex at the inner membrane. Alison Lui, SURF Scholar 2014 PhD at UC Berkeley Caltech All use the paradigm of regulatory protein localization as a way of translating genetic information into three-dimensional space. In particular, little is known about the replication of multipartite genomes in bacteria. By combining these data with previous global analysis of cell cycle transcription patterns and gene expression profiles of mutant ctrA strains, we have determined that CtrA directly regulates at least 95 genes. In this study, we uncovered a mechanism by which daughter cell fate, which is specified by the DivJ-DivK-PleC system and effectively encoded in the phosphorylation state of the single-domain RR DivK, is communicated to the CckA-ChpT-CtrA signaling pathway that regulates more than 100 genes for polar differentiation, replication initiation and cell division. phiCbK DNA cosediments with Escherichia coli phage T2 DNA and has therefore been assigned an S(20,w) value of 63.5S. The subcellular localization of the chemotaxis proteins may reflect a general mechanism by which the bacterial cell sequesters different regions of the cell for specialized functions. 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. The researchers, https://doi.org/10.1103/PhysRevLett.130.145001, For questions or comments, contact the SLAC Office of Communications at, A day in the life of an experiment controls engineer, Researchers can see crystals perform their dance moves, GEM Fellowship aims to increase representation in STEM, Researchers resolve decades-long debate about shock-compressed silicon with unprecedented detail, Vintage SLAC accelerator software spreads its wings, Researchers model accelerator magnets' history using machine learning approach, Capturing Molecular Motion using Artificial Intelligence. Most machine learning models don't directly include any notion of particle beam dynamics to speed up learning and reduce the amount of data required, SLAC accelerator scientist and co-author Auralee Edelen said. Here we demonstrate live-cell 3D superresolution imaging of Crescentin-eYFP, a cytoskeletal fluorescent protein fusion, colocalized with the surface of the bacterium Caulobacter crescentus using a double-helix point spread function microscope. Currently: Assistant Professor of Biomedical Sciences DipM localized to the division site in an FtsZ-dependent manner via its PG-binding LysM domains. Caltech-IBS Global Pioneer Fellow 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. This result establishes the editing site as a bona fide target for aminoacyl-tRNA synthetase inhibitors. View details for PubMedCentralID PMC4702786. We focus on mRNA processing, RNA modifications and their roles in development and disease. 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. The kinetic complexity of Caulobacter deoxyribonucleic acid, however, is no greater than that of other bacteria. Analysis of the cloned and sequenced dnaK gene has shown that the deduced amino acid sequence could encode a protein of 67.6 kilodaltons that is 68% identical to the DnaK protein of Escherichia coli and 49% identical to the Drosophila and human hsp70 protein family. 2016 University of Illinois at Chicago, Graduate Student, Biochemistry Postdoc. x@caltech.edu, x=msoufi, Margaret Swift View details for Web of Science ID A1994MQ78200018, View details for Web of Science ID A1994NV05900013, View details for Web of Science ID A1993MH32400028. An inducible promoter is a useful tool for the controlled expression of a given gene. The basal body consisted of five rings mounted on a rod. Analysis of mutant alleles of ctrA and point mutations in one of the CtrA binding sites in the origin demonstrate that CtrA represses replication in vivo. Postdoctoral Fellowship, Radiology, Mayo Clinic Chen, J. C., Viollier, P. H., Shapiro, L. Spatial complexity of mechanisms controlling a bacterial cell cycle, An actin-like gene can determine bacterial cell polarity, A genetic oscillator and the regulation of cell cycle progression in Caulobacter crescentus, Rapid and sequential movement of individual chromosomal loci to specific subcellular locations during bacterial DNA replication. The genetic mechanisms that control asymmetric cell divisions--yielding progeny cells that differ from one another--have been conserved among prokaryotes, eukaryotic microbes, and higher organisms. Menlo Park, Calif. The Department of Energys SLAC National Accelerator Laboratory and Stanford University today announced the launch of a new joint battery This vast structural blueprint of specific positional information is manifested in various ways, directing chromosome compaction, accessibility, attachment to the cell envelope, supercoiling, and general architecture and arrangement of the chromosome relative to the cell body. The order of appearance of labeled restriction fragments revealed that the chromosome replicates bidirectionally at a fork movement rate of 21 kilobases per minute. x@caltech.edu, x=crabut, (Amir)Hossein Salahshoor, PhD We have identified a novel, conserved protein, RcdA, that forms a complex with CtrA and ClpX in the cell. We show here that a previously unidentified single domain-response regulator, CpdR, when in the unphosphorylated state, binds to ClpXP and, thereby, causes its localization to the cell pole. We generated strains that carry ccrM either on a low-copy-number plasmid (strain GR131) or on a moderate-copy-number plasmid (strain GR132). View details for Web of Science ID A1997YA84900001. M.D. B.S. CtrA binds to and regulates the promoters of two genes critical to its temporally controlled proteolysis, divK and clpP, providing a transcriptional feedback loop for the control of cell cycle progression. FliF is proteolytically turned over during swarmer-to-stalked cell differentiation, coinciding with the loss of the flagellum, suggesting that its degradation is coupled to flagellar release. View details for Web of Science ID 000250487600069. Spatial control of intracellular signaling relies on signaling proteins sensing their subcellular environment. Caulobacter crescentus is motile by virtue of a polar flagellum assembled during the predivisional stage of the cell cycle. Although FliL is required for flagellar function, it is not part of the transcriptional hierarchy, supporting the hypothesis that, as is the case for the enterics, the regulatory hierarchy responds to assembly cues rather than directly to the expression of flagellar proteins. The achievements of our students and fellows have been recognized by many honors and awards, and many Stanford Developmental Biology alumni have become leaders in biomedical research, teaching, and medicine. The transcript start site in front of flaE was determined and the -10 region conforms to the E. coli sigma 28 promoter consensus sequence. Despite their small size, bacteria have a remarkably intricate internal organization. Full expression of fliX was found to be dependent on ctrA, and DNase I footprinting analysis demonstrated a direct interaction between CtrA and the fliX promoter. The Shapiro laboratory has leveraged its expertise in ERa action in cancer to identify new pathways of hormone action that drive proliferation, metastases, and therapy resistance, and new types of selective anticancer therapeutics. ChpT functions as a histidine-containing phosphotransfer protein (HPt) that shuttles a phosphoryl group from the receiver domain of CckA, the upstream hybrid histidine kinase (HK), to one of two downstream response regulators (CtrA or CpdR) that controls cell-cycle progression. Brain & Bio Engineering, Korea Adv. Bacteria adapt to shifts from rapid to slow growth, and have developed strategies for long-term survival during prolonged starvation and stress conditions. Tome Biosciences, Prof. JrmeLacroix 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. View details for DOI 10.1073/pnas.062065699, View details for Web of Science ID 000174856000089, View details for PubMedCentralID PMC123699. Currently: Associate Director of ML/AI 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. 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. Postdoctoral Scholar Chromosome replication in Caulobacter crescentus is tightly regulated to ensure that initiation occurs at the right time and only once during the cell cycle. View details for PubMedCentralID PMC2937403. At three separate chromosomal sites the CcrM recognition sequence is fully methylated in swarmer cells, becomes hemimethylated upon DNA replication in stalked cells, and does not become remethylated until just prior to cell division. Importantly, RNase E cluster positions correlated with the subcellular location of chromosomal loci of two highly transcribed rRNA genes, suggesting that RNase E's function in rRNA processing occurs at the site of rRNA synthesis. Mark S. Shapiro, Ph.D. - Cellular and Integrative Physiology Hocking, J., Priyadarshini, R., Takacs, C. N., Costa, T., Dye, N. A., Shapiro, L., Vollmer, W., Jacobs-Wagner, C. Three-Dimensional Super-Resolution Imaging of the Midplane Protein FtsZ in Live Caulobacter crescentus Cells Using Astigmatism. Congratulations to Avinoam and colleagues on showing that cells can be engineered to migrate into tumors and damage them from the inside upon receiving a focused ultrasound stimulus. Inverted repeat DNA sequences of Caulobacter crescentus have been isolated, characterized, and cloned in a bacteriophage lambda vector. View details for Web of Science ID 000259631900035, View details for PubMedCentralID PMC2566184. This year's LaskerDebakey Clinical Research Award honors Katalin Kariko and Drew Weissman for the development of a therapeutic technology based on nucleoside-modification of messenger RNA, enabling the rapid development of the highly effective COVID-19 vaccines. The mature two-piece tmRNAs are predicted to have a tRNA-like domain and an mRNA-like domain similar to those of standard one-piece tmRNAs, with a break located in the loop containing the tag reading frame. In contrast to the protein components of the hook and filament, the protein encoded by the flaD gene contains a hydrophobic leader peptide. LIGO Group. In vitro experiments showed that the lack of in vivo methylation in cheR mutants was due to the absence of methyltransferase activity. Biomedical Sciences, Zhejiang University Join us. 2023 SLAC National Accelerator Laboratory is operated by Stanford University for the U.S. Department of Energy Office of Science. However, their use in bacteria has been limited due to challenges imposed by a complex bacterial cell wall. B.S. During polar maturation, the PopZ-centromere tether is broken, and the PopZ zone at that pole then switches function to act as a recruitment factor for the ordered addition of multiple proteins that promote the transformation of the flagellated pole into a stalked pole. Nature Biotechnology (2023). One-third of these target genes encode putative TonB-dependent receptors, suggesting CrfA plays a role in the surface modification of C. crescentus, facilitating the uptake of nutrients during periods of carbon starvation. Transcription from a strong promoter within the origin occurs uniquely from the replication-competent chromosome at the stalked pole of the predivisional cell. Bayas, C. A., Schrader, J. M., Lee, M. K., Shapiro, L., Moerner, W. E. CauloBrowser: A systems biology resource for Caulobacter crescentus. Our Department is home to about 60 graduate students and 80 postdoctoral fellows who pursue innovative research projects at the leading edge of Developmental Biology. Comerci, C. J., Herrmann, J. n., Yoon, J. n., Jabbarpour, F. n., Zhou, X. n., Nomellini, J. F., Smit, J. n., Shapiro, L. n., Wakatsuki, S. n., Moerner, W. E. Identification of PAmKate as a Red Photoactivatable Fluorescent Protein for Cryogenic Super-Resolution Imaging. The ribonucleic acid (RNA) bacteriophage phiCb5, which specifically infects only one form of the dimorphic stalked bacterium Caulobacter crescentus, has been obtained in high yield. The Caulobacter crescentus bacteriophage phiCbK was studied with respect to the physical and chemical properties of both the phage and its deoxyribonucleic acid (DNA). 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. Accordingly, we identified, cloned, and sequenced a chromosomal locus, xylX, from Caulobacter crescentus which is required for growth on xylose as the sole carbon source and showed that transcription from a single site is dependent on the presence of xylose in the growth medium. A binding protein specific for cyclic guanosine 3':5'-monophosphate (cyclic GMP) has been partially purified from extracts of the eubacterium Caulobacter crescentus and resolved from cyclic adenosine 3':5'-monophosphate (cyclic AMP)-binding activity. View details for Web of Science ID A1995RP99400003. The host RNA polymerase appears to be involved in the early transcription program of the Caulobacter crescentus bacteriophage phiCdl. WebGraduate student, Sarah Cohen laboratory, Romberg Tiburon Center for Environmental Studies, 3152 Paradise Drive, Tiburon, CA 94920, (415) 338-3754 daniellendesmet gmail.com We view Developmental Biology in the broadest sense, encompassing Microbes to Humans and employing a wide variety of molecular and genetic approaches as well as systems level biology, engineering, and computational science. Biology, Sichuan University Milu T. Cherian, PhD, Senior Analyst at Oncology, SmartAnalyst, Inc. Irene Aninye, PhD, Senior Program Associate at the American Association for the Advancement of Science, Rui Huang, Graduate Student in Biochemistry at Duke University, Jeffrey Trost, Medical Student at University of Virginia School of Medicine, Khin-Khin Soe Wu, Medical Student at Rosalind Franklin School of Medicine, Amanda Etheridge, Medical Student at University of Illinois College of Medicine. Hurt RC#, Buss MT#, Duan M#, Wong K, You MY, Sawyer DP, Swift MB, Dutka P, Barturen-Larrea P, Mittelstein DR, Jin Z, Abedi MH, Farhadi A, Dephande R, Shapiro MG*. This enzyme, like RNase III isolated from Escherichia coli, processes precursor ribosomal RNAs and polycistronic phage mRNAs and has a monomeric Mr of approximately 20,000. Biochemistry, Jagiellonian University Ryan Rezvani, Amgen Scholar 2014 PhD at UC Irvine We generate multilayered porous films in crystalline silicon using a periodic electrochemical etch. The latter two motifs are implicated in essential C. crescentus replication functions, because they are contained within specific deletions that abolish replication. University of California, San Francisco, Gabrielle Ho Of the 26 genes required for flagellum production, at least 4 of them-flaY, E, F, and G-map together in a single cluster. Analysis of a chimeric flgJ-NPT II transcriptional fusion showed that the flgJ regulatory sequences do not control the segregation of the 29K flagellin to the swarmer cell progeny, suggesting that correct segregation depends on the protein product. A conditional genetic screen for CckA mislocalization mutants, using automated fluorescence microscopy and an image processing platform, revealed that the essential DivL protein kinase promotes CckA localization, autophosphorylation, and activity at the new cell pole. View details for Web of Science ID A1996UD48400020, View details for PubMedCentralID PMC177887. These results suggest that the interdependence between chromosome partitioning and cell division in Caulobacter is mediated, in part, by the FtsK protein. They have found a single molecular event present in all cancers studied to date that protects them from macrophages of the innate immune system. Research Technician Upon initiation of replication, an 8-kb region of the chromosome containing both the origin and parS moves rapidly to the opposite pole. Analysis of in vivo and in vitro methylation in synchronized cultures showed that the methylation reaction is lost when the flagellated swarmer cell differentiates into a stalked cell. SMC complexes and histone-like proteins continuously remodel the nucleoid to reconcile chromatin compaction with DNA replication and gene regulation. Using cluster analysis of the resulting set of 12-element vectors for each of these strains, we identified 52 strains with mutations that affected the localization pattern of the three tagged proteins. University of Washington, Dr. Danny Sawyer We demonstrate that sequences 3' to the transcription start site do not contribute to temporal control. Asymmetric cell division in Caulobacter crescentus yields daughter cells that have different cell fates. Chemical Engineering, MIT B.Sc. WebJonathan Schapiro, MD, Adjunct Clinical Professor, Stanford University School of Medicine, Stanford, California . SLAC is operated by Stanford University for the U.S. Department of Energys Office of Science. SciP is cell cycle-controlled and co-conserved with the global cell cycle regulator CtrA in the -proteobacteria. Lasker, K., Abraham, A., Childers, W., Shapiro, L. Global methylation state at base-pair resolution of the Caulobacter genome throughout the cell cycle. The resulting plasmid was used as a probe to isolate the flaE region from a wild-type gene bank and to determine the chromosomal location of several deletion and insertion mutations within the flaY/E/F/G cluster. The gene is located 2 kb from the origin of replication. We show that the peptidoglycan transpeptidase Pbp2 also forms a helical pattern that partially colocalizes with MreC but not MreB. Congratulations to Mohamad, Michael and collaborators on this new paper demonstrating the local delivery of checkpoint inhibitors inside solid by ultrasound-controlled probiotic agents. In C. crescentus, the Fix network is required for normal cellular growth during hypoxia and controls expression of genes encoding four distinct aerobic respiratory terminal oxidases and multiple carbon and nitrogen metabolic enzymes. View details for Web of Science ID 000227028900009. Pasadena, CA, USA 91125. Several temporally controlled flagellar genes in Caulobacter crescentus require a sigma 54 promoter and upstream sites for transcription activation. B.S. View details for DOI 10.1073/pnas.1909798116. Control of sequential cell changes at the level of transcription has long been postulated and has recently been substantiated in the case of Bacillus sporulation (6). Included are those involved in chemotaxis, outer membrane channel function, degradation of aromatic ring compounds, and the breakdown of plant-derived carbon sources, in addition to many extracytoplasmic function sigma factors, providing the organism with the ability to respond to a wide range of environmental fluctuations. Ph.D. Acoustic Physics, Physics for Medicine Lab and Sorbonne University B.S. Aminoacyl-transfer RNA (tRNA) synthetases, which catalyze the attachment of the correct amino acid to its corresponding tRNA during translation of the genetic code, are proven antimicrobial drug targets. In addition, the C-terminal region of FtsK is required for the localization of the topoisomerase IV ParC subunit to the replisome to facilitate chromosomal decatenation prior to cell division. M.Eng. Caulobacter crescentus carries a flagellum and is motile only during a limited time in its cell cycle. View details for Web of Science ID 000079843900013. Jonathan S. Shapiro Managing Director, The EROS Group, LLC. Thrombin-Activatable Carboxypeptidase B2 (CPB2) in the Crosstalk between Coagulation, Thrombosis, Inflammation and Innate Immunity Procarboxypeptidase B2 circulates as an inactive enzyme and is activated by the thrombin/thrombomodulin complex on endothelial cell surface. A number of well-characterized instances of polar localization of bacterial proteins, including the chemoreceptor complex in both C. crescentus and E. coli, the maltose-binding protein in E. coli, the B. japonicum surface attachment proteins, and the actin tail of L. monocytogenes within a mammalian cell, involve proteins or protein complexes that facilitate bacterial interaction with the environment, either the extracellular milieux or that within a plant or mammalian host. Because DivL and CckA accumulate at the same cell pole after the initiation of DNA replication and were found to interact in vivo, we propose that DivL recruits CckA to the pole, thereby promoting its autophosphorylation and activity. The biogenesis of the bacterial flagellum and chemotaxis apparatus in both Escherichia coli and Caulobacter crescentus requires the ordered expression of over 40 genes whose expression is controlled by a trans-acting regulatory hierarchy. We have developed a technique, termed superresolution by power-dependent active intermittency and points accumulation for imaging in nanoscale topography (SPRAIPAINT) that combines imaging of intracellular enhanced YFP (eYFP) fusions (SPRAI) with stochastic localization of the cell surface (PAINT) to image two different fluorophores sequentially with only one laser. Ph.D. Student, Bioengineering, Defended 2020 Society for Neuroscience 1991-present. In order to study the regulation of these genes, plasmids were constructed that contain either an intact flaYE region or deletions in the region of flaY. 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. In addition, we found that a rifampicin-resistant RNA polymerase activity dependent on de novo protein synthesis is required for late transcription. Epistasis experiments demonstrated that the fliIJ operon is located in class II of the C. crescentus flagellar regulatory hierarchy, suggesting that the gene products act at an early stage in flagellar assembly. The characteristics that differentiate one daughter cell from the other result from differential transcription and subcellular positioning of regulatory and structural proteins. View details for DOI 10.1073/pnas.1220824110, View details for Web of Science ID 000314558100027, View details for PubMedCentralID PMC3562846. Human Frontier Science Program Cross-Disciplinary Fellow Michael Garrett, MBRS-RISE Fellow 2019 PACE Diagnostics View details for Web of Science ID A1996TR39200015, View details for PubMedCentralID PMC177711. Shapiro test is a statistical test used to check whether the considered data is normally distributed data or not. Despite the essential role of the CckA histidine kinase in the control of cell cycle events, the factors that signal its activation at a specific time in the cell cycle have remained elusive. 3) Temperature-sensitive mutants of Caulobacter that are restricted in macromolecular synthesis and development at elevated temperatures have been isolated. Sequence analysis of a complementing subclone revealed that this locus encodes at least two proteins that are homologs of the Salmonella typhimurium and Escherichia coli flagellar proteins FliL and FliM. Quantitative Multicolor Subdiffraction Imaging of Bacterial Protein Ultrastructures in Three Dimensions. We show that the products of transcriptional reporter gene fusions to a flagellin gene and to the flagellar hook operon are segregated to the progeny swarmer cell.
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