Rice University, Prof. Jerzy Szablowski Mechanisms that control timing of cell cycle and developmental events include transcriptional cascades, regulated phosphorylation and proteolysis of signal transduction proteins, transient genetic asymmetry, and intercellular communication. The flaD mutant, however, was found to contain a partially assembled basal body consisting of the rod and three hook-distal rings. "New" poles generated at the cell division plane differ from old poles from the previous round of cell division. WebSafety First is designed to be implemented in high school classrooms by health teachers. 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. The depth and diversity of data sets collected by the Caulobacter community makes CauloBrowser a unique and valuable systems biology resource. Therefore, genome-wide codon bias in eubacteria and archaea may be predicted from intergenic sequences that are not translated. This reduction, which was not observed in flagellar hook mutants, was due to a decreased stability of the L-ring protein. The flbN gene was cloned and sequenced, and the time of transcription activation was determined. Three-dimensional colocalization of intracellular protein structures and the cell surface with superresolution optical microscopy opens the door for the analysis of protein interactions in living cells with excellent precision (20-40nm in 3D) over a large field of view (1212m). Lets congratulate Isamar on this awesome achievement, and wish her luck! However, both cell division and stalk formation, which is analogous to a polar division event, require SecA function. We are interested in how Arctic species and populations responded to environmental and habitat change throughout the Pleistocene, and what role ecology, natural history, climate and community-level dynamics played in the The degree of curvature induced by FzlA depended on the nucleotide bound to FtsZ. The CcrM DNA methyltransferase of the alpha-proteobacteria catalyzes the methylation of the adenine in the sequence GAnTC. As bacteria continue to develop resistance toward current antibiotics, we find ourselves in a continual battle to identify new antibacterial agents and targets. The Caulobacter cell cycle is driven by a cascade of transient regulators, starting with the expression of DnaA in G(1) and ending with the expression of the essential CcrM DNA methyltransferase at the completion of DNA replication. Mathematics, Georgia Tech Cedars-Sinai Medical Center, Dr. Mohamad Abedi Moreover, we show that GapR is maintained as a tetramer upon its dissociation from DNA and that tetrameric GapR is capable of binding DNA molecules in vitro Analysis of protein chimeras revealed that two helices of GapR are functionally conserved in H-NS, demonstrating that two evolutionarily distant NAPs with distinct mechanisms of action utilize conserved structural elements to oligomerize and bind DNA.IMPORTANCE Bacteria organize their genetic material in a structure called the nucleoid, which needs to be compact to fit inside the cell and, at the same time, dynamic to allow high rates of replication and transcription. Z., Dill, D. L., McAdams, H. H., Shapiro, L. A pseudokinase couples signaling pathways to enable asymmetric cell division in a bacterium. Temporal control of DNA methylation has been shown to be critical for normal development in the dimorphic Caulobacter life cycle. B.S. x@caltech.edu, x=nnystrom, Claire Rabut, PhD In addition, we found that a rifampicin-resistant RNA polymerase activity dependent on de novo protein synthesis is required for late transcription. Research Technician, 2017-18; Amgen Fellow, 2016 View details for DOI 10.1016/j.ceb.2010.10.013, View details for Web of Science ID 000288349000010. x@caltech.edu, x=rohitk, Rohan Kolhe The essential transcriptional circuitry for growth on rich media includes 10 transcription factors, 2 RNA polymerase sigma factors and 1 anti-sigma factor. Shapiros lab has identified a pathway conserved from insects to humans, and between steroid hormones and peptide hormones in which mitogenic hormones, such as estrogen, and epidermal growth factor (EGF), elicit extremely rapid anticipatory activation of the stress sensor, the unfolded protein response (UPR). The cellular concentration of DnaA is cell cycle-controlled, peaking at the time of replication initiation and gcrA induction. Ph.D Biophysics (Imaging), University of Western Ontario All of the mutants in this cluster exhibited pleiotropic effects on the expression of other flagellar and chemotaxis functions, including the level of synthesis of flagellins, the hook protein and hook protein precursor, and the level of chemotaxis methylation. View details for DOI 10.1128/JB.185.11.3384-3391.2003, View details for Web of Science ID 000183100900016, View details for PubMedCentralID PMC155372. 1973-1974 Stanford University, Senior Researcher SciP is cell cycle-controlled and co-conserved with the global cell cycle regulator CtrA in the -proteobacteria. An additional layer of complexity was recently uncovered, showing that each segment of the chromosome is located at a specific cellular position both during and after the completion of DNA replication, raising the possibility that this positioning contributes to temporal and spatial control of gene expression. 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. Analysis of genome-wide codon bias shows that only two parameters effectively differentiate the genome-wide codon bias of 100 eubacterial and archaeal organisms. SURF Scholar 2022- By combining photoinduced activation of single EYFP fusions and time-lapse imaging, we obtained sub-40 nm resolution images of the filamentous superstructure of the bacterial actin protein MreB in live Caulobacter crescentus cells. Spatial organization and dynamics of RNase E and ribosomes in Caulobacter crescentus. In this report we identify a cluster of genes encoding basal body components and describe their transcriptional regulation. Transcription from the flgF promoter initiates prior to initiation at the internal flgH promoter. Inverted repeat DNA sequences of Caulobacter crescentus have been isolated, characterized, and cloned in a bacteriophage lambda vector. Hahnenberger and L. Shapiro, J. Mol. Postdoctoral Scholar (co-advised with Richard Andersen) With the annotated genome sequence, a full description of the genetic network that controls bacterial differentiation, cell growth, and cell cycle progression is within reach. View details for DOI 10.1101/sqb.2009.74.005, View details for Web of Science ID 000285712600011. The initiation of replication depends on the proteolysis of CtrA. Our correlative approach reveals that SpmX localizes along one side of the cell pole and its extent closely matches that of the PopZ region. Our observations suggest that the processivity of C. crescentus replication requires concomitant phospholipid synthesis and that cell death results from incomplete replication of the chromosome. View details for DOI 10.1016/j.mib.2016.06.007, View details for PubMedCentralID PMC5069156. The algorithm pairs machine-learning techniques with beam physics equations to avoid massive data crunching. Research in the Villeneuve lab is aimed at understanding the molecular and cellular mechanisms underlying the faithful inheritance and function of eukaryotic chromosomes. The E. coli GroEL protein and the related protein from C. crescentus were also induced by treatment with hydrogen peroxide. The availability of temperature-sensitive mutants blocked at various stages of development permits access to both questions. Strains bearing mutations in the pleA gene are pililess and non-flagellated. A mutant of C. crescentus that fails to synthesize flagellin has been isolated. The RF-2 binding region is similar to a NifA binding site normally used in the activation of some sigma 54 promoters involved in nitrogen fixation in other bacteria. Superresolution fluorescence microscopy based on covalent labeling highlights specific proteins and has sufficient sensitivity to observe single fluorescent molecules, but the reconstructions lack detailed cellular context. Here we report a global transcriptional analysis of an oxygen sensory/signaling network in Caulobacter crescentus consisting of the sensor histidine kinase FixL, its cognate response regulator FixJ, the transcriptional regulator FixK, and the kinase inhibitor FixT. Bacterial replication origins move towards opposite ends of the cell during DNA segregation. Constructing a macromolecular structure of this scale generally requires localized enzymatic machinery, but a regulatory framework for S-layer assembly has not been identified. Identification of additional regulatory genes activated by CtrA will serve to directly connect new regulatory modules to the network controlling cell cycle progression. The bacterium Caulobacter crescentus divides asymmetrically to yield two functionally distinct cells: a motile, chemotactic swarmer cell, and a sessile stalked cell with replication and division capabilities. Research Technician, 2014 We then demonstrate that A22 completely blocks the movement of newly replicated loci near the origin of replication but has no qualitative or quantitative effect on the segregation of other loci if added after origin segregation. Neuroscience and Behavioral Biology, Emory University Saurabh, S., Chong, T., Bayas, C., Dahlberg, P. D., Moerner, W. E., Shapiro, L. Selective sequestration of signalling proteins in a membraneless organelle reinforces the spatial regulation of asymmetry in Caulobacter crescentus. However, the lesions were mapped to loci that are separated by a substantial distance. But for proteins and small complexes, whether in the periplasm or the membrane, one must invoke a mechanism that prevents the diffusion of these proteins away from the cell pole. View details for Web of Science ID A1989R820200026. Following the shift to the restrictive temperature protein synthesis continued, but at a reduced rate. CcrM is essential for viability in both of these organisms, and we show here that it is also essential in Brucella abortus. Modeling the cell cycle probably requires a top-down modeling approach and a hybrid control system modeling paradigm to treat its combined discrete and continuous characteristics. This effect could be reversed by exogenous N6,O2'-dibutyryl 3':5'-cyclic AMP, and mutants resistant to repression by cyclic GMP derivatives exhibited a pleiotropic phenotype affecting a cyclic AMP-mediated event. This is an example of controlled proteolysis of a cytoplasmic protein that is associated with its active recruitment to a specific subcellular address. x@caltech.edu, x=sdalfonz, Marama Diaz-Asper The flagellum is composed of a transmembrane basal body, a hook and a filament. View details for Web of Science ID A1987G981000008. The sequential changes in the chromosomal methylation state serve to couple the progression of DNA replication to cell-cycle events regulated by the master transcriptional regulatory cascade, thus providing a ratchet mechanism for robust 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. liqunyu2@illinois.edu An unusual polar organelle that mediates directed motility on solid surfaces is found in the nonpathogenic bacterium Myxococcus xanthus. View details for DOI 10.1371/journal.pgen.1004831, View details for PubMedCentralID PMC4287350. Here we investigate the effect on cell polarity of two cytoskeletal elements previously implicated in cell shape determination. 2015 California Institute of Technology, Graduate Student, Biochemistry No extrachromosomal elements were found in spite of systematic attempts to detect their presence. 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. Comparison of the ffs36 strain to a ts secA strain of Caulobacter, also having cell-cycle and DNA replication phenotypes, showed that both exhibit a permanent induction of a heat shock response at the restrictive temperature. 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. WebMikhail G. Shapiro, PhD Professor of Chemical Engineering & Medical Engineering Investigator, Howard Hughes Medical Institute Physics for Medicine Lab and Sorbonne Meisenzahl, A. C., Shapiro, L., Jenal, U. Nature Biotechnology (2023). A century-old mystery for Gas Vesicles. Cell wall morphogenetic protein RodA and penicillin-binding protein PBP1a also change their spatial distribution by accumulating at the division site in response to external osmotic upshifts. Six distinct cellular characteristics, which are peculiar to these bacteria, have been defined and include (i) the synthesis of a polar organelle which may be membranous (21-23), (ii) a satellite DNA in the stalked cell (26), (iii) pili to which RNA bacteriophage specifically adsorb (16, 33), (iv) a single polar flagellum(17), (v) a lipopolysaccharide phage receptor site (27), and (vi) new cell wall material at the flagellated pole of the cell giving rise to a stalk (19, 20). gyrB and orf-1 are within a newly identified cluster of genes involved in DNA replication and recombination, including dnaN and recF. Lab Phone: 626-395-8955, Division of Chemistry and WebShapiroLab Going Deeper We develop technologies to image and control the function of cells deep inside the body. 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. We have a lot of different ways to manipulate particle beams inside of accelerators, but we dont have a really precise way to describe a beams shape and momentum, SLAC accelerator scientist and lead author Ryan Roussel said. View details for Web of Science ID A1983RA96700072. Research Scientist For DNA segments greater than about 300 kb, the mean interloci distances scale as n, in agreement with previous observations. 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. The Min proteins that govern division site selection in Escherichia coli may be the first example of a system that generates positional information de novo. We propose that during segregation PopZ sequesters free ParA and induces target-proximal regeneration of ParA DNA binding activity to enforce processive and pole-directed centromere segregation, preventing segregation reversals. The type 1 incoherent feedforward circuit motif enhances the pulse-like expression of the downstream genes, and the negative feedback to ctrA expression reduces peak CtrA accumulation. Dye, N. A., Pincus, Z., Fisher, I. C., Shapiro, L., Theriot, J. SLAC is a vibrant multiprogram laboratory that explores how the universe works at the biggest, smallest and fastest scales and invents powerful tools used by scientists around the globe. After graduating he worked for the NASA Marshall Space Flight Center developing non-destructive evaluation techniques for applications related to the US space program. This structure is absent at the flagellar pole but not in the stalks of flbT mutant predivisional cells. 210-450-9060. All four proteins were synthesized in the predivisional cell, but the progeny showed cell type-specific bias in the level of enhanced synthesis after heat shock. As a proof-of-principle demonstration, we investigate the oxidation/reduction state within vitrified Caulobacter crescentus cells. View details for Web of Science ID A1987G196300016. Moreover, initiation of DNA replication is allowed only once per cell cycle. Goley, E. D., Comolli, L. R., Fero, K. E., Downing, K. H., Shapiro, L. Cell pole-specific activation of a critical bacterial cell cycle kinase, Caulobacter PopZ forms a polar subdomain dictating sequential changes in pole composition and function. Among the validated sRNAs, two are candidate transposase gene antisense RNAs. We demonstrate here that in some of these genes, an AT-rich region containing an integration host factor (IHF) consensus binding site lies between the activator and the promoter, and that this region binds IHF in vitro. Many of them are controlled by promoters methylated by CcrM and co-regulated by other global cell cycle regulators, demonstrating an extensive cross talk between DNA methylation and the complex regulatory network that controls the cell cycle of C. crescentus and, presumably, of many other Alphaproteobacteria. The N-terminal atypical receiver domain resembles the canonical receiver domain of response regulators, but has a degenerate, stripped-down 'active site'. The protein product of the adjacent flaY gene was found to be required to regulate the expression of several flagellin proteins and the assembly of a functional flagellum. Bacterial chromosome segregation: Is there a mitotic apparatus? Two GEM Fellows reflect on their summer internships at SLAC and share their thoughts on representation and mentorship. We show here that genes differentially expressed in a ctrA temperature-sensitive (ts) mutant are similarly affected in a cckA ts mutant, that the phosphorylation of CckA coincides temporally with CtrA phosphorylation during the cell cycle, and that CckA is essential for viability because it is required for CtrA phosphorylation. Fluorescence microscopy can potentially be used to reveal this information when specific labels, known as fluorescent biosensors, are used, but there has been minimal use of such biosensors in cryo-CLEM to date. x@caltech.edu, x=rkolhe, Kyle McGraw Zhou, B., Schrader, J. M., Kalogeraki, V. S., Abeliuk, E., Dinh, C. B., Pham, J. Q., Cui, Z. Caltech Cell type-specific phosphorylation and proteolysis of a transcriptional regulator controls the G1-to-S transition in a bacterial cell cycle, Translation of the leaderless Caulobacter dnaX mRNA, Protein localization and cell fate in bacteria, Transcription of genes encoding DNA replication proteins is coincident with cell cycle control of DNA replication in Caulobacter crescentus. Caulobacter crescentus carries a flagellum and is motile only during a limited time in its cell cycle. We participated in the largest prospective studies to date in molecular diagnostics in oncology (CIRCULATE), womens health (SMART), and organ health (Trifecta). Laboratories for Reproductive Biology & Lineberger Comprehensive Cancer Center The essential dnaN gene encodes a homolog of the Escherichia coli beta subunit of DNA polymerase III. x@caltech.edu, x=mdiazasp, Bulge Gungoren We use a variety of innovative approaches including genomics, computation, biochemistry, and advanced imaging. These results are consistent with a model in which unreplicated DNA is pulled into the replication factory and newly replicated DNA is bidirectionally extruded from the complex, perhaps contributing to chromosome segregation. The NPI number of this provider is 1114219102 and was assigned on May 2011. B.S. The role of these genes in flagellar biogenesis was examined by immunoprecipitation of mutant cell extracts with antiflagellin antibody. To understand the mechanism by which ccrM expression is regulated during the cell cycle, we have identified and characterized the ccrM promoter region. In addition, transcription of phiCdl DNA by the E. coli enzyme produced a subset of transcripts not synthesized by the C. crescentus enzyme. The experimental techniques include patch-clamp electrophysiology, cutting-edge molecular biology, advanced imaging techniques such as total internal reflection fluorescence (TIRF) microscopy, Frster resonance energy transfer (FRET), confocal and super-resolution microscopy, molecular and cellular modeling, biophysical chemical analysis of channel complexes and structural biology. A., Shapiro, L., Gitai, Z. DnaA coordinates replication initiation and cell cycle transcription in Caulobacter crescentus. The CtrA master transcriptional regulator is a central control element in Caulobacter cell cycle progression and polar morphogenesis. View details for Web of Science ID A1994MQ78200018, View details for Web of Science ID A1994NV05900013, View details for Web of Science ID A1993MH32400028. The cell cycle control circuitry is tied closely to chromosome replication and morphogenesis by multiple feedback pathways from the modular functions that implement the cell cycle. Biochemistry, University of Washington, Seattle von Diezmann, A., Lasker, K., Mann, T. H., Ahrens, D. G., Shapiro, L., Moerner, W. E. Probing Asymmetric Behavior of a Cell Cycle Regupatory Protein in Live Caulobacter using Single-Molecule Imaging, A Red Fluorescent Protein for Cryogenic Single-Molecule Superresolution Imaging. B.A., Physics, University of Chicago, 1984. CHAMPER, R., Bryan, R., Gomes, S. L., Purucker, M., Shapiro, L. ANALYSIS OF THE PLEIOTROPIC REGULATION OF FLAGELLAR AND CHEMOTAXIS GENE-EXPRESSION IN CAULOBACTER-CRESCENTUS BY USING PLASMID COMPLEMENTATION. At the moment, Safety First is unavailable as it is being edited and revised by our REACH lab team. We report that CtrA binds five sites within the chromosome replication origin in vitro. SciP overexpression disrupts the balance between activation and repression of the CtrA-SciP coregulated genes yielding filamentous cells and loss of viability. and M.S. Ph.D. Student, Bioengineering Biology, Caltech Millions of possible codes can be prepared this way. Because the Lon protease is present throughout the cell cycle, it is likely that the level of CcrM in the cell is controlled by a dynamic balance between temporally varied transcription and constitutive degradation. x@caltech.edu, x=ltorress, Alumni This technique can be used to select for mutants blocked in the various stages of morphogenesis. In addition, minor phospholipids were detected in the swarmer cells that were not detected in stalked cells. Berdis, A. J., Lee, I., Coward, J. K., Stephens, C., Wright, R., Shapiro, L., Benkovic, S. J. Caulobacter crescentus provides an excellent model system for the bacterial cell cycle whereby cells can be rapidly synchronized in a G0 state by density centrifugation. Surface layers (S-layers) are crystalline protein coats surrounding microbial cells. At the nonpermissive temperature, the cell cycle blocks prior to the de novo synthesis of flagella and chemotaxis proteins that normally occurs in the predivisional cell. These data suggest a more prevalent use of the Shine-Dalgarno sequence for ribosome pausing rather than translation initiation in C. crescentus. B.S. cell, and (2) the CcrM protein is rapidly degraded prior to cell division. Thus, both phosphorylation and proteolysis are critical determinants of bacterial cell cycle control in a manner that is analogous to the control of the eukaryotic cell cycle. Croucher Scholar A series of simple, in situ immunoassays have been developed which can be used in screening for translation products of genes cloned in vitro recombination experiments with either phage or plasmid vectors. At room temperature, the ratio of roGFP2 emission brightness when excited at 425 nm or 488 nm is known to report on the local redox potential. We propose that the maintenance of DNA topology throughout the cell cycle contributes to the dynamic positioning of the origin sequence within the cell. The initiation of chromosomal replication occurs concomitantly with the transition of the motile swarmer cell to the sessile stalked cell. fliQ and fliR are class II flagellar genes residing near the top of the regulatory hierarchy that determines the order of flagellar gene transcription. Two distinct protein complexes, the flagellum and the pilus biogenesis machinery, are asymmetrically assembled at one pole of the Caulobacter predivisional cell. Ann Zhu, SURF Scholar 2021 BS Chemical Engineering, Caltech 2024 (exp) The flagellum is ejected from the swarmer cell and then synthesized de novo later in the cell cycle. Herrmann, J., Comerci, C. J., Jabbarpour, F., Shapiro, L., Moerner, W. E., Wakatsuki, S. Dissection of Protein Function Within a Bacterial Biomolecular Condensate by In Vitro Reconstitution. Three Caulobacter crescentus heat-shock proteins were shown to be immunologically related to the Escherichia coli heat-shock proteins GroEL, Lon and DnaK. Ph.D. Student, Bioengineering, Defended 2020 View details for Web of Science ID A1989T211600005. As a first step in the search for regulators of these events, we report the isolation and characterization of a ctrA gene from S. meliloti. The membrane-bound DivJ and PleC histidine kinases, which are asymmetrically localized at the opposite poles of the predivisional cell, control the temporal and spatial localization of DivK. Cell cycle progression in Caulobacter is driven by the master transcriptional regulators CtrA and GcrA. The researchersdetailed their algorithm and method in April in Physical Review Letters. Previous studies have implicated two histidine kinases, DivJ and PleC, and the response regulator, DivK, in the regulation of morphogenesis in the dimorphic bacterium Caulobacter crescentus. STALKED BACTERIA - PROPERTIES OF DEOXRIYBONUCLEIC ACID BACTERIOPHAGE PHICBK, SPECIFIC ASSAY FOR DIFFERENTIATION IN STALKED BACTERIUM CAULOBACTER-CRESCENTUS, PHAGE-SPECIFIC AND HOST PROTEINS IN REPLICATION OF BACTERIOPHAGE RNA. In ultrasound gradients, GVs and cells expressing them get pushed more strongly and in the opposite direction from other biological materials. Harvard University, Dr. Anupama Lakshmanan Deoxyribonucleic acid-dependent ribonucleic acid (RNA) polymerase (EC 2.7.7.6) was purified from the dimorphic bacterium Caulobacter crescentus at three stages in development. Visualization of dynamic protein structures in live cells is crucial for understanding the mechanisms governing biological processes. enels@illinois.edu Strikingly, cells lacking DipM also showed OM blebbing at the division site, at cell poles and along the cell body. As a result, we can selectively push them, trap them, pattern them and sort them selectively under acoustic remote control. Recent work has uncovered mechanisms that ensure the execution of many events at different times during the cell cycle and at specific places in the cell. 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. The dynamic behavior of these proteins is often intrinsically linked to their function as polarity determinants. When mated into a wild-type strain, plasmids bearing deletions in the flaY region were found to be recessive. WPI, B.S. The map position of another mutation in membrane lipid biogenesis, the glycerol-3-PO4 auxotroph gpsA505, was also determined. Biomedical Engineering, Zhejiang University Our goal is to define these mechanisms using both molecular genetics and biochemistry. We study regulation of M-channels by a variety of receptors coupled to Gq/11 G proteins, via numerous cytoplasmic 2nd-messingers, including lipids, protein kinases and phosphatases, and intracellular Ca2+ ions acting through as calmodulin. View details for Web of Science ID A1989U499000006. The effect of cyclic AMP on growth on sugars metabolized by inducible enzymes, as well as on sugars metabolized by constitutive enzymes, may represent a regulatory system common to both types of sugar utilization, since they share features that differ from glucose utilization, namely, temperature-sensitive growth and low intracellular concentrations of cyclic guanosine 3',5'-monophosphate.

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