View details for DOI 10.1073/pnas.0402567101, View details for Web of Science ID 000222278600017, View details for PubMedCentralID PMC438962. Marczynski, G. T., Dingwall, A., Shapiro, L. A CAULOBACTER GENE INVOLVED IN POLAR MORPHOGENESIS. Small noncoding regulatory RNAs (sRNAs) play a key role in the posttranscriptional regulation of many bacterial genes. Mann, T. H., Seth Childers, W., Blair, J. Dingwall, A., Zhuang, W. Y., Quon, K., Shapiro, L. The control of timing and spatial organization during Caulobacter cell differentiation. Unlike Escherichia coli, the intracellular and extracellular concentrations of cyclic AMP in C. crescentus did not vary under several growth conditions, including catabolite repression. 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. Electron micrographs reveal the phage to be among the largest DNA bacteriophages reported, with head dimensions of 64 by 195 nm and a flexible tail 275 nm in length. View details for Web of Science ID A1992JM38600007. The expression of NPTase II in these mutants is read from a chimeric mRNA that initiates in a chromosomal fla promoter and continues through the inserted NPTase II gene. Here, we combine single-molecule tracking and super-resolution microscopy, light-induced subcellular localization, reaction-diffusion modelling and a spatially resolved promoter activation assay to study signal exchange in and out of the 200nm cytoplasmic pole-organizing protein popZ (PopZ) microdomain at the cell pole of the asymmetrically dividing bacterium Caulobacter crescentus4-8. In the bacterium Caulobacter crescentus, many cellular processes are temporally regulated with respect to the cell cycle, and the genes required for these processes are expressed immediately before the products are needed. View details for Web of Science ID A1986E866400004. Bennett Shapiro is a provider established in Stanford, California and his medical specialization is Orthopaedic Surgery. Following the shift to the restrictive temperature protein synthesis continued, but at a reduced rate. 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. B.S. Aris Taychameekiatchai, SURF Scholar 2016-19 PhD at UCSF The GroEL homolog, however, was distributed equally to both the stalked cell and the swarmer cell. We are interested in candidates who will establish a vigorous and innovative research program studying fundamental biological processes in any experimental system. View details for Web of Science ID A1987G981000008. We can now use particle beam data in a more comprehensive, powerful way to improve our scientific goals at accelerators everywhere, he said. When ccrM gene expression is placed under control of a constitutive promoter, these chromosomal sites are fully methylated throughout the cell cycle. View details for Web of Science ID A1976CE95700078. 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. Dynamic flexibility between the 2 domains rationalizes efficient S-layer crystal nucleation on the curved cellular surface. A partial open reading frame 165 base pairs 3' to the end of dnaK encodes a peptide of 190 amino acids that is 59% identical to DnaJ of E. coli. 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. Gonzalez, D., Kozdon, J. View details for DOI 10.1146/annurev.biochem.72.121801.161824. A fliX null mutant is nonmotile, lacks a flagellum, and exhibits a marked cell division defect. Small Molecule Biomodulators for Studying and Treating Diseases Our focus is on the discovery and use S1 mapping and primer extension experiments showed that transcription initiated at two sites 5' to the dnaK coding sequence. Amemiya, K., Bellofatto, V., Shapiro, L., Feingold, J. MIT, Dr. David Mittelstein M.D. Small membrane vesicles from swarmer cells contained [methyl-3H]MCPs both in the flagellated and non-flagellated vesicles, which indicates that the region immediately surrounding the flagellum, as well as the rest of the surface of the swarmer cell, contains [methyl-3H]MCP. Ph.D. Student, Medical Engineering, Defended 2022 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. The recognition of polar bacterial organization is just emerging. x@caltech.edu, x=thamza, Bella Hurvitz It has been shown that DNA replication serves as a checkpoint for flagellar biosynthesis and cell division and that this checkpoint is mediated by the availability of active CtrA. During development of the symbiotic soil bacterium Sinorhizobium meliloti into nitrogen-fixing bacteroids, DNA replication and cell division cease and the cells undergo profound metabolic and morphological changes. 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. View details for Web of Science ID 000079843900013. Landt, S. G., Abeliuk, E., McGrath, P. T., Lesley, J. Search results about individual genes are displayed as tables, graphs of time resolved expression profiles, and schematics of protein localization throughout the cell cycle. Neuroscience and Behavioral Biology, Emory University GapR target loci are especially enriched in binding sites for the transcription factors GcrA and CtrA and overlap with nearly all of the binding sites for MucR1, a regulator that controls the establishment of swarmer cell fate. Androgen Receptor, University of Illinois Thus, CpdR function is regulated by a feedback loop that incorporates its differential phosphorylation, the transient polar localization and activity of the ClpXP protease, and the clearance of the CpdR by polar ClpXP that, in turn, releases ClpXP from the pole relieving the degradation of CtrA. 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. Mutants in flbN, flaB, flaC and flbO were unable to assemble any basal body structure and are likely to be involved in the early steps of basal body formation. Thus, fatty acid degradation by the beta-oxidation pathway is constitutive in C. crescentus and is only mildly affected by growth in the presence of glucose. Mapping of the transcriptional start site revealed a conserved binding motif for the global response regulator CtrA at the -35 position; this motif was footprinted by purified Caulobacter crescentus CtrA protein in its phosphorylated state. To the best of our knowledge, this is the first demonstration of the use of fluorogen activating proteins for super-resolution imaging in live bacterial cells. B.S. Saurabh, S. n., Perez, A. M., Comerci, C. J., Shapiro, L. n., Moerner, W. E. Dynamic translation regulation in Caulobacter cell cycle control. x@caltech.edu, x=dwwu, Jee Won Yang Even in Escherichia coli, which is generally thought to be symmetrical, old poles are more static than new poles with respect to cell wall assembly (1), and they differ in the deposition of phospholipid domains (2). View details for DOI 10.1101/sqb.2009.74.005, View details for Web of Science ID 000285712600011. In addition to stationary localization factors, dynamic cytoskeletal structures also have a fundamental role in many of these processes. Marczynski, G. T., LENTINE, K., Shapiro, L. REGULATION OF THE CAULOBACTER-CRESCENTUS DNAKJ OPERON. This uncoupling of initiation of replication from CtrA degradation indicates that there is an SsrA-dependent pathway required for correct timing of DNA replication. 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. Quon, K. C., Marczynski, G. T., Shapiro, L. USE OF FLOW-CYTOMETRY TO IDENTIFY A CAULOBACTER 4.5 S RNA TEMPERATURE-SENSITIVE MUTANT DEFECTIVE IN THE CELL-CYCLE, A DEVELOPMENTALLY-REGULATED CHROMOSOMAL ORIGIN OF REPLICATION USES ESSENTIAL TRANSCRIPTION ELEMENTS. One of these sites was located within the 16S gene near its 3' end, and the other two were found at the 5' end of the 23S gene. In the absence of glycerol, the optical density of the culture continued to increase for the equivalent of one generation, although the cells did not divide. View details for DOI 10.1111/j.1365-2958.2011.07954.x, View details for Web of Science ID 000299779200005, View details for PubMedCentralID PMC3272108. The first generation of acoustic reporter genes proved a concept but were insensitive, burdensome and impossible to image continuously. Furthermore, methyltransferase activity, present in the predivisional cell, was detected only in the swarmer cell upon cell division. The Caulobacter DNA methyltransferase CcrM is one of five master cell-cycle regulators. ZS Associates, Undergraduate and Visiting Student Alumni, Mei Yi You, SURF Scholar 2018-2022 PhD at MIT As a proof-of-principle demonstration, we investigate the oxidation/reduction state within vitrified Caulobacter crescentus cells. Collections of genes associated with central cell cycle functional modules (e.g., biosynthesis of stalk, flagellum, or chemotaxis machinery) have consistent but different TE temporal patterns, independent of their operon organization. Dividing cells must coordinate cell cycle events to ensure genetic stability. 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. Little is known about the structure and function of most nucleoid-associated proteins (NAPs) in bacteria. Phage phiCb5 was studied with respect to the physical and chemical properties of both the phage and its RNA. We demonstrate here that two flagellar genes, flaE and flaY, whose products function in trans to modulate the level of transcription of other flagellar genes, are themselves temporally controlled. Further, GapR does not silence the expression of H-NS target genes when expressed in E. coli, suggesting that GapR and H-NS have distinct functions. 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. View details for Web of Science ID A1996UD48400020, View details for PubMedCentralID PMC177887. This genetic lesion did not appear to affect directly a fatty acid biosynthetic reaction because fatty acid and phospholipid syntheses were found to continue in the absence of supplement. The precise and robust regulation of gene expression is a cornerstone for complex biological life. Biteen, J. S., Thompson, M. A., Tselentis, N. K., Bowman, G. R., Shapiro, L., Moerner, W. E. A bacterial control circuit integrates polar localization and proteolysis of key regulatory proteins with a phospho-signaling cascade, Caulobacter requires a dedicated mechanism to initiate chromosome segregation. In spite of their small size, bacteria have a remarkably complex internal organization and external architecture. SURF Scholar 2021- New molecules and mechanisms for MR imaging and magnetic actuation. Although the phospholipid composition of swarmer and stalked cells was indistinguishable in continuously labeled cultures if the two cell types were pulse-labeled for a short time period, marked differences in the pattern of phospholipid synthesis were detected. Postdoctoral Scholar, 2014-16 To test their ideas, the team used their model to interpret experimental data from the Argonne Wakefield Accelerator at the DOE's Argonne National Laboratory. Therefore, genome-wide codon bias in eubacteria and archaea may be predicted from intergenic sequences that are not translated. Home | Department | Faculty | Education | Labs | Publications | Giving | Contact, Terms of Use | Privacy | Copyright | Trademarks | Non-Discrimination | Accessibility, https://facultypositions.stanford.edu/en-us/job/493432, Heidi Chen in Gill Bejerano & David Kingsley's lab successfully defended her thesis titled Whole-genome comparisons identify enhancers underlying repeated fin evolution in diverse fishes, Mollie Friedlander Qian defended her thesis titled "Discovering new functions of the diabetes gene HNF1A in human pancreatic islets". The Caulobacter Tol-Pal complex is thus a key component of cell envelope structure and function, mediating OM constriction at the final step of cell division as well as the positioning of a protein localization factor. The chemotaxis proteins are synthesized in the predivisional cell and then partition only to the swarmer cell upon division. We found that MmpA facilitates the degradation of PodJS. 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. A mutation in the flaD gene results in the assembly of a partial basal body which is missing the outermost P and L rings as well as the external hook and filament (K.M. Thus, a dynamically localized ClpXP proteolysis complex in concert with a cytoplasmic factor provides temporal and spatial specificity to protein degradation during a bacterial cell cycle. x@caltech.edu, x=zyjin, Whee-Soo Kim, PhD Aero. Until recently, a dedicated mitotic apparatus that segregates newly replicated chromosomes into daughter cells was believed to be unique to eukaryotic cells. B.S. The insertion sequence (IS) elements, IS1 and IS2, present in multiple copies in the Escherichia coli chromosome, are transposable genetic elements of known nucleotide sequence. These genes all share the same 5' cis-regulatory elements: a sigma 54 promoter, a binding site for integration host factor (IHF), and an enhancer sequence, known as the ftr element. The Tn5 insertion mutant SC1130 had no cross-reacting MCP and had reduced levels of activity of the methyltransferase and methylesterase. Several products involved in flagellar biogenesis and the chemotaxis machinery are segregated to the swarmer cell. The M ring, which is at the inner membrane of the cell, has a different structure depending on the method of preparation. We have found that it belongs to an unusual promoter family used by several Caulobacter class II flagellar genes. Electrical and Computer Engineering, University of Rochester The outer and inner membranes of C. crescentus CB13 were separated, and phospholipid analysis revealed heterogeneity with respect to the relative amounts of phosphatidylglycerol and cardiolipin in the two membranes. Several fla- mutants were also isolated by Tn5-VB32 mutagenesis and shown to confer kanamycin resistance. Analysis of deletions and base substitutions in the 5' region of the operon established the presence of two functional promoters: a heat shock-inducible promoter, P1, with characteristics of a sigma 32 promoter, and an adjacent sigma 70-like promoter, P2. These results provide access to the functions of C. crescentus heat-shock proteins under both normal and stress conditions. View details for DOI 10.1016/j.bpj.2011.02.022, View details for Web of Science ID 000289494200001, View details for PubMedCentralID PMC3072666. Proteins are positioned at particular sites in bacteria, including the cell pole, the incipient division plane, and the septum. 1972-1973 Stanford University Medical School The complete genome sequence of Caulobacter crescentus was determined to be 4,016,942 base pairs in a single circular chromosome encoding 3,767 genes. However, these mutants efficiently transported fatty acids and had beta-oxidation enzyme levels comparable with that of the wild type. View details for DOI 10.1073/pnas.0604503103, View details for Web of Science ID 000239327200021, View details for PubMedCentralID PMC1544151. Both proteins contain multiple PAS domains, a multifunctional class of sensory domains present across the kingdoms of life. Such rotations did not lead to large-scale changes in gene expression, indicating that genome folding does not strongly affect gene regulation. Biochemistry, Jagiellonian University The genes that encode the components and regulatory proteins of the Caulobacter crescentus flagellum are transcribed at specific times in the cell cycle. View details for Web of Science ID A1987J718100012. The RNase III activity isolated from C. crescentus cell extracts has potential use in the analysis of specific RNA species because it was found to be more stringent in the recognition of cleavage sites than the E. coli enzyme. Individual cells containing enhanced GFP were exposed to a bleaching laser pulse tightly focused at one cell pole. An asynchronous sequential digital circuit model equivalent to the validated simulation model was created. Ph.D. 1972 Purdue University Lasker, K., Schrader, J. M., Men, Y., Marshik, T., Dill, D. L., McAdams, H. H., Shapiro, L. A cell cycle kinase with tandem sensory PAS domains integrates cell fate cues. Cell type- and compartment-specific localization of the CtrA global transcriptional regulator is essential for proper cell-cycle progression, and subcellular localization of key chromosome partitioning proteins is correlated with proper nucleoid segregation. Single-molecule super-resolution imaging provides a non-invasive method for nanometer-scale imaging and is ideally suited to investigations of quasi-static structures within live cells. Biophysics of neuromodulation with ultrasound and other forms of energy. Although interactions between the chromosome and the cytoplasmic membrane are believed to be a functional component of the temporal regulation of DNA replication, the ability of this secA mutant to initiate replication at the nonpermissive temperature suggests that SecA-dependent events are not involved in this process. The phenotype of pH-conditional mutants was defined on medium with lactose as the sole carbon source. Lee, M., Schrader, J., Li, G., Weissman, J., McAdams, H., Shapiro, L., Moerner, W. E. DNA Segregation and Partitioning in Caulobacter Crescentus: Super-Resolving Protein Colocalization at the Cell Pole. The PodJ protein was found to exist in two forms, a truncated 90-kDa and a full-length 110-kDa form, each controlling a different aspect of polar development and each localizing to the cell poles at a specific time in the cell cycle. Although not essential for viability, DeltadipM cells exhibited gross morphological defects, including cell widening and filamentation, indicating a role in cell shape maintenance and division that we show requires its LytM domain. Ph.D. Student, Biochemistry and Molecular Biophysics, Defended 2019 Signals from probes binding the same message are correlated. x@vis.caltech.edu, x=rosie, Ruby Zhang This osmolality-dependent relocation to the division apparatus is initiated within less than a minute, while restoration to the patchy localization pattern is dependent on cell growth and takes 1 to 2 generations. This conclusion is based on two observations: the low level of synthesis of flagellins and chemotaxis proteins in flaY and flaE mutant strains occurred at the correct time in the cell cycle, and complementation with plasmids containing intact flaY and flaE genes resulted in the synthesis of normal levels of flagellins and chemotaxis gene products with the maintenance of temporal cell cycle control. A subpopulation of the smc null mutant cells had mislocalized origins or termini, showing that the smc null mutation gives DNA segregation defects. The Nucleoid-Associated Protein GapR Uses Conserved Structural Elements To Oligomerize and Bind DNA. In addition, increasing the cellular content of sigma32 by introducing a plasmid-encoded copy of rpoH induced DnaK expression in C. crescentus cultures grown at 30 degrees C. The C. crescentus rpoH gene was transcribed from either of two heat shock consensus promoters. Rapid clearance of the master regulator, CtrA, by the ClpXP protease is a critical event that enables the initiation of chromosome replication at specific times in the cell cycle. Chemical Engineering, Rensselaer Polytechnic Institute, Dr. Nikita Reznik View details for Web of Science ID 000084010000013. 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. Homologous sequences were then detected by computer analysis of the published IS1 and IS2 nucleotide sequences. View details for DOI 10.1371/journal.pbio.1001979, View details for Web of Science ID 000344461700017, View details for PubMedCentralID PMC4211667. The position of genetic loci on the chromosome is thereby linearly correlated with their position in the cell. A., Xu, Q., Childers, W. S., Mathews, I. I., Kern, J. W., Eckart, M., Deacon, A. M., Shapiro, L. Retrospective. Compartmentalization of the predivisional cell is a critical event in the establishment of the differential distribution of regulatory factors that specify cell fate.
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