B.S. B.S. The P1 promoter fires early in S phase and contains a GAnTC sequence that is recognized by the CcrM DNA methyltransferase. The chemoreceptor-CheW binary complex is polarly localized in the absence of CheA, whereas the chemoreceptor-CheA binary complex is not confined to the cell poles in strains lacking CheW. Also among the genes in this notably large regulon are 14 that encode regulatory proteins, including 10 two-component signal transduction regulatory proteins. Following the shift to the restrictive temperature protein synthesis continued, but at a reduced rate. 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. Regions of homology between the IS elements and ribosomal RNA were observed. Similarly, hooks with attached rods were shed from nonflagellate mutants, and these structures also lacked the basal rings. The inactivation of GlnA promotes the deprivation of glutamine in the cell, which triggers a stringent response. View details for Web of Science ID A1994PM98800011. To explain the phenotype of both the secA and ffs36 strains, we propose that a cell-cycle checkpoint prevents further progression through the cell-cycle in response to increased intracellular levels of heat shock and misfolded proteins. The flbO locus is near the top of the hierarchy, and consequently strains with mutations in this locus are nonmotile and lack the flagellar basal body complex. The examples of polar localization given here are from a variety of bacterial species and concern a disparate array of cellular functions. McGrath, P. T., Lee, H., Zhang, L., Iniesta, A. 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 region of the chromosome defined by flaE mutations contains at least one flagellin structural gene and appears to regulate flagellin synthesis and flagellar assembly. We demonstrate the direct binding between these three proteins and show that a polar microdomain spontaneously assembles when the three proteins are coexpressed heterologously in an Escherichia coli test system. Upon transfer of a mixed population of cells to medium containing lactose as the sole carbon source, these changes were blocked for about 20 hr until beta-galactosidase activity became apparent. The University of Texas Health Science Center at San Antonio, also called UT Health San Antonio, is a leading academic health center with a mission to make lives better through excellence in advanced academics, life-saving research and comprehensive clinical care including health, dental and cancer services. 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. The cellular localization of MipZ thus serves the dual function of positioning the FtsZ ring and delaying formation of the cell division apparatus until chromosome segregation has initiated. We propose that SMC spuriously links the duplicated chromosome immediately after passage of the replication fork. Using structural biology and biochemical findings we proposed a mechanistic basis for TCS pathway coupling in which the DivL pseudokinase is repurposed as a sensor rather than participant in phosphotransduction. View details for Web of Science ID A1976BU75500037. As developmental biologists, we aspire to understand how pluripotent cells become diversified into lineages ranging from brain to blood to bone. 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. Additionally, we investigated the genetic dependence of localization among divisome proteins and the cell cycle regulation of their transcript and protein levels to gain insight into the control mechanisms underlying their assembly. During the normal course of the C. crescentus cell cycle, the polar flagellum with hook and rod was shed into the culture medium without the basal rings. Chemical Engineering, Imperial College London Lee, S. F., Thompson, M. A., Schwartz, M. A., Shapiro, L., Moerner, W. E. Bowman, G. R., Lyuksyutova, A. I., Shapiro, L. Exploring protein superstructures and dynamics in live bacterial cells using single-molecule and superresolution imaging. This study reports the identification and functional characterization of a vanillate-regulated promoter (P(van)) which meets all requirements for application as a multi-purpose expression system in Caulobacter, thus complementing the established xylose-inducible system (P(xyl)). To understand how polar organizing centres are established by PopZ, we investigated a set of mutated PopZ proteins for defects in sub-cellular localization and recruitment activity. Emilio received his B.S. The FtsK C terminus is essential in Caulobacter and is involved in maintaining accurate chromosome partitioning. A null mutation in the Caulobacter crescentus smc gene is conditionally lethal and causes a cell cycle arrest at the predivisional cell stage. By contrast, our transcriptome analysis shows that >10% of the genes are misexpressed in cells lacking or constitutively over-expressing CcrM. Currently: Assistant Professor of Bioengineering The transcription of gyrB and orf1 occurs from the replication-competent chromosome in stalked and predivisional cells and is silenced in swarmer cells. Ricci, D. P., Melfi, M. D., Lasker, K., Dill, D. L., McAdams, H. H., Shapiro, L. An intracellular compass spatially coordinates cell cycle modules in Caulobacter crescentus. 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. This heading includes several more specific ancient DNA research projects currently underway in the Shapiro lab. The maintenance of cell shape in Caulobacter crescentus requires the essential gene mreB, which encodes a member of the actin superfamily and the target of the antibiotic, A22. 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. The signal mediators, proteases, response regulators, and kinases, as well as Cori DNA and the replisome, all show distinct patterns of temporal and spatial organization during cell cycle progression. B.S. Affiliations: Bioengineering, Biochemistry, Neurobiology Society for Neuroscience 1991-present. 2010 Wuhan University of Technology Professor, Department of Chemistry Bar-Zion A, Nourmahnad A, Mittelstein DR, Shivaei S, Yoo S, Buss MT, Hurt RC, Malounda D, Abedi MH, Lee-Gosselin A, Swift MB, Maresca D, Shapiro MG*. Iniesta, A. x@caltech.edu, x=mbuss, Yuen Man Kathy Cheung Two GEM Fellows reflect on their summer internships at SLAC and share their thoughts on representation and mentorship. We have carefully mapped out the multiple domains of KCNQ ion channels involved in interactions with phosphatidylinositol 4,5-bisphosphate (PIP2), a lipid signaling molecule of critical importance. In addition, it is becoming increasingly clear that yet another level of information is encoded by the bacterial chromosome - the three-dimensional packaging of the chromosomal DNA molecule itself and its positioning relative to the cell. Suchita Nety, SURF Scholar 2013-17 (Housner Prize) MD-PhD at Harvard-MIT When the swarmer cell differentiated into a stalked cell, the chemoreceptor was specifically degraded by virtue of an amino acid sequence located at its carboxyl terminus. Caltech-MIT-IBS Global Pioneer Fellow We demonstrate here that CcrM is an important target of the Lon protease pathway in C. crescentus. CtrA binds to and silences the origin of replication in swarmer cells. Two phospho-signalling proteins, the transmembrane histidine kinase CckA and the cytoplasmic phosphotransferase ChpT, provide the only phosphate source for the cell fate-determining transcription factor CtrA9-18. 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. Haverford College: Computer Science: For large aggregates, such as the clusters of MCP, CheA, and CheW complexes, perhaps the size of the aggregate alone prevents displacement. Furthermore, the four heat-shock proteins synthesized in the predivisional cell were partitioned in a specific manner upon cell division. Regulated timing of these cellular modules stems from global genetic circuits that allow precise temporal activation with respect to cell cycle progression and cell differentiation. We have examined 35 mutants that have defects in general chemotaxis. Stanford University; Joe Shapiro, University of California Berkeley; Deadline for paper submission. Purified SMC-E1076Q was deficient in ATP hydrolysis and exhibited abnormally stable binding to DNA. B.S. We are interested in the ion currents and signaling pathways regulating excitability of dopaminergic neurons in midbrain, so as to understand the pathophysiology of drug addiction, as well as ion channels in lymphocytes critical to lymphomas. 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. Large structures, such as a flagellum, are anchored at the pole by means of the basal body that traverses the peptidoglycan wall. 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. We are probing the mechanisms of epileptogenesis, and how to prevent its development, focusing on the temporal lobe, particularly circuits in the hippocampus. M.S., Chemical Engineering, 2015 x@caltech.edu, x=ltorress, Alumni We leveraged the ability to isolate synchronous populations of Caulobacter crescentus cells to investigate assembly of the divisome and place the arrival of each component into functional context. These data suggest a more prevalent use of the Shine-Dalgarno sequence for ribosome pausing rather than translation initiation in C. crescentus. To address this need, we have generated CauloBrowser (www.caulobrowser.org), an online resource for Caulobacter studies. Given single-molecule localization precisions of 20-40 nm, a flexible locally weighted image registration algorithm is critical to accurately combine the super-resolution data with <10 nm error. We've shown that we can infer very complicated high-dimensional beam shapes from astonishingly small amounts of data.". These inverted repeat sequences are analogous to the RNase III-processing sites in the E. coli rRNA precursor. The flaD BC motC region was cloned and the genes were localized by subcloning and complementation analysis. Automated image acquisition and analysis allowed us to identify genes that affect the localization of two polar cell cycle histidine kinases, PleC and DivJ, and the pole-specific pili protein CpaE, each tagged with a different fluorescent marker in a single strain. Chromosome segregation in Caulobacter cannot occur unless a dedicated parS guiding mechanism initiates movement. 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. Mutants in the hook operon or in basal body genes caused overproduction of both hook operon and basal body gene chimeric mRNAs, suggesting that negative regulation is superimposed on the positive trans-acting control for these early events in the flagellar hierarchy. View details for DOI 10.1038/sj.emboj.7600927, View details for Web of Science ID 000234952500008, View details for PubMedCentralID PMC1383511. After phage infection at least 40 proteins are phosphorylated; these include DNA-binding proteins, a membrane-associated protein, and several ribosomal proteins. Biteen, J. S., Goley, E. D., Shapiro, L., Moerner, W. E. Structure of the pilus assembly protein TadZ from Eubacterium rectale: implications for polar localization. Several genes involved in chromosome segregation have been identified, and the analysis of their functions and intracellular localization are beginning to shed light on the mechanisms that ensure efficient chromosome segregation. 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. This motif, named RRF (for repression of replication factors), is conserved in the promoters of other coordinately induced replication factors. CtrA binds to and silences the origin. View details for Web of Science ID 000259631900035, View details for PubMedCentralID PMC2566184. WebBio. The principal analysis method involved identification of sets of adjacent probes with unusually high correlation between the individual intergenic probes within the set, suggesting presence of a sRNA. Acetoacetyl coenzyme A (acetoacetyl-CoA) thiolase, an enzyme required for short-chain fatty acid degradation, has been purified to near homogeneity from Caulobacter crescentus. This movement requires the highly conserved ParABS locus that is essential in Caulobacter. Acad. Ryan Rezvani, Amgen Scholar 2014 PhD at UC Irvine The very C-terminal end of FliF contains a turnover determinant, required for the cell cycle-dependent degradation of the MS-ring. 210-450-9060. When samples containing roGFP2 are rapidly cooled to 77K in a manner compatible with cryo-EM, the ratio of excitation peaks remains a faithful indicator of the redox potential at the time of freezing. The 0.22 value of the scaling exponent for short DNA segments is consistent with theoretical predictions for a branched DNA polymer structure. SLAC is a vibrant multiprogram laboratory that DnaA boxes are present upstream of many genes whose expression requires DnaA, and His6-DnaA binds to the promoters of gcrA, ftsZ and podJ in vitro. View details for Web of Science ID 000177770100004. Through quantification by Ripley's K-test and comparison with Monte Carlo simulations, we find the protein is slightly clustered within a mostly uniform distribution throughout the swarmer and stalked stages of the cell cycle but more highly clustered in predivisional cells. Furthermore, galactose uptake was observed to be regulated independently of the galactose catabolic enzymes. A major challenge involves the integration of these diverse data sets into one comprehensive community resource. The use of this promoter suggests that C. crescentus has a cognate sigma factor and predicts that other C. crescentus genes are expressed from this consensus promoter. The distinct control of available CcrM in progeny swarmer and stalked cells serves to protect the hemimethylated state of DNA during chromosome replication, enabling robustness of cell cycle progression. In addition, two minor but as yet unidentified fatty acids were detected. The start site of the fliLM operon lies 166 bp from the divergently transcribed flaCBD operon that encodes several basal body genes. Collaboration: Estrogen Receptor, University of Illinois The hook structure was purified from nonflagellated mutants and found to be composed of a 70,000-molecular-weight protein component. Two additional genes in the flgF, flgG operon, flaD and flgH, both encode proteins with potentially cleavable signal sequences. A mutant of C. crescentus that fails to synthesize flagellin has been isolated. View details for DOI 10.1002/cphc.201100686, View details for Web of Science ID 000301537300020. Director, High-throughput Screening Facility CtrA activity in the cell cycle is controlled both transcriptionally and by phosphorylation. M.S. 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. Small Molecule Biomodulators for Studying and Treating Diseases Our focus is on the discovery and use Research in our laboratory is focused on understanding how regulatory information encoded by the genome is integrated with the transcriptional machinery and chromatin context to allow for emergence of form and function during human embryogenesis and evolution, and how perturbations in this process lead to disease. Perez, D., Dahlberg, P. D., Wang, J., Sartor, A. M., Borden, J. S., Shapiro, L., Moerner, W. E. ATP-responsive biomolecular condensates tune bacterial kinase signaling. These technologies take advantage of biomolecules with Explore SLAC events and learn how to participate. This gene cluster encodes a novel group of pilus assembly proteins. "New" poles generated at the cell division plane differ from old poles from the previous round of cell division. Because CcrM is essential in B. abortus and increased ccrM copy number attenuates survival in host cells, we propose that CcrM is an appropriate target for new antibiotics. Postdoctoral Fellowship, Chemical Engineering, Caltech San Antonio, TX 78229 The asymmetric targeting of proteins to the Caulobacter predivisional cell poles yields dissimilar progeny. Plasmid and phage deoxyribonucleic acid (DNA) harboring bacterial insertion sequence (IS) elements IS1, IS2, and IS5 were characterized and used as probes to detect homologous sequences in various procaryotic and eucaryotic genomes. Thus, the Fix network is a conserved sensory/signaling module whose transcriptional output has been adapted to the unique physiologies of C. crescentus and the nitrogen-fixing rhizobia. x@caltech.edu, x=hsp, Erik Schrunk View details for Web of Science ID A1996VB22500010. View details for Web of Science ID A1997XV69900030, View details for PubMedCentralID PMC179479. We generate multilayered porous films in crystalline silicon using a periodic electrochemical etch. In Caulobacter crescentus, the expression of the dnaKJ operon is regulated both temporally during the normal cell cycle and by heat shock. Learn more about the places where science happens at SLAC: our major facilities, institutes and centers. 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 cellular levels of CtrA and GcrA are temporally and spatially out-of-phase during the cell cycle, with CtrA repressing gcrA transcription and GcrA activating ctrA transcription. However, Cori is distinguished by several features, and especially by five binding sites for the CtrA response regulator protein. WebSafety First is designed to be implemented in high school classrooms by health teachers. Postdoctoral Scholar In addition, dynamically localized DNA-binding proteins ensure that DNA segregation is coupled to the timing and cellular position of the cytokinetic ring. 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. The parS sites, a pair of short contiguous sequence elements known to be involved in chromosome segregation, are positioned at one pole, where they anchor the chromosome to the cell and contribute to the formation of a compact chromatin conformation. Delighted to host the first International Symposium on Biomolecular Ultrasound and Sonogenetics. Thus, PopZ undergoes multiple orders of self-assembly, and the formation of an interconnected superstructure is a key feature of polar organization in Caulobacter. Two-component signal transduction proteins are known to play a significant role in cell cycle progression. View details for Web of Science ID A1997YB26700002. Upon glycerol deprivation, net phospholipid synthesis ceased immediately in a glycerol 3-phosphate auxotroph which was shown to have levels of biosynthetic sn-glycerol 3-phosphate dehydrogenase (E.C. Researchers develop clever algorithm to improve our understanding of particle beams in accelerators, Now, researchers at the Department of Energys SLAC National Accelerator Laboratory, the DOEs Argonne National Laboratory and the University of Chicago have developed an algorithm that more precisely predicts a beams distribution of particle positions and velocities as it zips through an accelerator. The dynamic flow of molecules into and out of these compartments occurs on faster timescales than for membrane-enclosed organelles, presenting a possible mechanism to control spatial patterning within cells. A. To selectively repress and limit chromosome replication, CtrA receives both protein degradation and protein phosphorylation signals. During the normal cell cycle of Caulobacter crescentus, flagella are released into the culture fluid as swarmer cells differentiate into stalked cells. View details for DOI 10.1128/mBio.00448-20. Driks, A., Bryan, R., Shapiro, L., DeRosier, D. J. IMAGE-RECONSTRUCTION OF THE FLAGELLAR BASAL BODY OF CAULOBACTER-CRESCENTUS. Biology, Sichuan University All cells must integrate sensory information to coordinate developmental events in space and time. Primer extension analysis revealed a single transcript originating from a sigma 70-type promoter. We propose that translation of leaderless mRNAs may provide a mechanism by which the ribosome can distinguish between productive and nonproductive templates. 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. Among such structures are actin-organizing centers, which mediate the movement of certain pathogenic bacteria within the cytoplasm of an animal host cell; organized arrays of membrane receptors, which govern chemosensory behavior in swimming bacteria; and asymmetrically positioned septa, which generate specialized progeny in differentiating bacteria. Shapiro, Andersen, and Tanter then received an NIH BRAIN Initiative grant to pursue the research. In addition, the site provides a genome viewer that enables customizable visualization of all published high-throughput genomic data. Several fla- mutants were also isolated by Tn5-VB32 mutagenesis and shown to confer kanamycin resistance. Acyl-CoA synthase activity was shown to be the same in oleic acid-grown cells and in cells grown in the presence of succinate, a carbon source not affected by catabolite repression. 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. Postdoctoral Scholar Caulobacter goes to great lengths to control the time and place of the activity of this critical regulatory factor during the cell cycle. In this Review, we summarize the current knowledge on localization mechanisms in bacteria, with an emphasis on the role of polymeric protein assemblies in the directed movement and positioning of macromolecular complexes. These mutants and the wild-type strain were used to determine the pathway of galactose catabolism in this organism. A. B.S. The conserved nucleotides in the promoter region are clustered in the -10, -20 to -30, and -35 regions. The ffs gene is transcribed throughout the cell-cycle and is transcribed at similar rates in mutant (ffs36) and wild-type strains, but in the mutant the 4.5 S RNA is unstable leading to lower 4.5 S RNA levels. View details for Web of Science ID A1989AP19100046. Cut through the jargon while exploring our research. 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. Bayas, C., Wang, J., Lee, M. K., Schrader, J. M., Shapiro, L., Moerner, W. E. Herrmann, J., Jabbarpour, F., Bargar, P. G., Nomellini, J. F., Li, P., Lane, T. J., Weiss, T. M., Smit, J., Shapiro, L., Wakatsuki, S. Ultra-photostable, genetically directed fluoromodule enables STED nanoscopy and long time scale single protein tracks in live bacteria. Cell division in Gram-negative organisms requires coordinated invagination of the multilayered cell envelope such that each daughter receives an intact inner membrane, peptidoglycan (PG) layer and outer membrane (OM). Hodgson, D., Shaw, P., Letts, V., Henry, S., Shapiro, L. ISOLATION AND GENETIC-ANALYSIS OF CAULOBACTER MUTANTS DEFECTIVE IN CELL-SHAPE AND MEMBRANE LIPID-SYNTHESIS, GENERATION OF A TN5 PROMOTER PROBE AND ITS USE IN THE STUDY OF GENE-EXPRESSION IN CAULOBACTER-CRESCENTUS. We describe the identification of 27 novel Caulobacter crescentus sRNAs by analysis of RNA expression levels assayed using a tiled Caulobacter microarray and a protocol optimized for detection of sRNAs. In wild-type cells, the origin is located at the flagellated pole of swarmer cells and, immediately after the initiation of DNA replication in stalked cells, one of the origins moves to the opposite pole, giving a bipolar localization of the origins. How do you make a protein that self-assembles, fills with air, excludes water and withstands several atmospheres of pressure? The IHF and enhancer sites are 3' to the transcription start site in this promoter. 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. Bellofatto, V., Shapiro, L., Hodgson, D. A. Hodgson, D., Shaw, P., OCONNELL, M., Henry, S., Shapiro, L. PURIFICATION AND CHARACTERIZATION OF AN RNA PROCESSING ENZYME FROM CAULOBACTER-CRESCENTUS, DIFFERENTIAL TEMPLATE RECOGNITION BY THE CAULOBACTER-CRESCENTUS AND THE ESCHERICHIA-COLI RNA-POLYMERASES, METHYLATION INVOLVED IN CHEMOTAXIS IS REGULATED DURING CAULOBACTER DIFFERENTIATION. x@caltech.edu, x=pdutka, Abdullah Farooq Although several sequence and structural motifs that are conserved among one-piece tmRNAs have been lost, the alpha-proteobacterium Caulobacter crescentus produces a functional two-piece tmRNA. Health information on this site is not meant to be used to diagnose or treat conditions. Chemical and Biomolecular Engineering, Johns Hopkins University We have identified a proline-rich polar protein, PopZ, required to anchor the separated Caulobacter crescentus chromosome origins at the cell poles, a function that is essential for maintaining chromosome organization and normal cell division. PodJL is synthesized in the early predivisional cell and is later proteolytically converted to PodJS. Professor, Department of Pathology 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. We have found that the trans regulation that modulates the amount of the flagellins and the chemotaxis proteins can be separated from the temporal control of fla and che gene expression. The transient accumulation of DivL at the new cell pole, but not its kinase activity, is required for the localization and activation of CckA.
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