Britos, L., Abeliuk, E., Taverner, T., Lipton, M., McAdams, H., Shapiro, L. Super-Resolution Imaging of the Nucleoid-Associated Protein HU in Caulobacter crescentus. Recent advances in cell-imaging technology with subdiffraction resolution have revealed that the bacterial nucleoid is reliably oriented and highly organized within the cell. American volume -Ryaby, J. T., Sheller, M. R., Levine, B. P., Bramlet, D. G., Ladd, A. L., Carney, D. H.2006;88: 132-139, JOURNAL OF SHOULDER AND ELBOW SURGERY -Srivastava, S., Youngblood, P. L., Rawn, C., Hariri, S., Heinrichs, W. L., Ladd, A. L.2004;13 (2): 196-205, journal of bone and joint surgery. A deletion or base changes in the RF-2 binding region eliminated expression of the flbN gene in E. coli even when a NifA protein was provided in trans, suggesting that a sigma 54 promoter with an upstream activator element is used by the C. crescentus flbN gene. Structure of Anabaena flos-aquae gas vesicles revealed by cryo-ET. A newly discovered NAP in Caulobacter crescentus, GapR, is thought to facilitate the movement of the replication and transcription machines along the chromosome by stimulating type II topoisomerases to remove positive supercoiling. The bacterial cell has less internal structure and genetic complexity than cells of eukaryotic organisms, yet it is a highly organized system that uses both temporal and spatial cues to drive its cell cycle. We determined that a chromosomal DNA-based platform stimulates CcrM degradation by Lon and that the CcrM C terminus both binds to its DNA substrate and is recognized by the Lon protease. View details for Web of Science ID A1995RC46100016, View details for PubMedCentralID PMC177052. Here, we explore the role of the coexpressed MreC protein in Caulobacter and show that it forms a periplasmic spiral that is out of phase with the cytoplasmic MreB spiral. Saurabh, S., Chong, T. N., Bayas, C., Dahlberg, P. D., Cartwright, H. N., Moerner, W. E., Shapiro, L. Delivering the message: How a novel technology enabled the rapid development of effective vaccines. The bacterium Caulobacter crescentus uses a ParA-based partitioning system to segregate newly replicated chromosomal centromeres to opposite cell poles. . The cellular concentration of DnaA is cell cycle-controlled, peaking at the time of replication initiation and gcrA induction. She was a psychologist, did some work at in Materials Science & Engineering, Stanford . A Bacterial Toxin Perturbs Intracellular Amino Acid Balance To Induce Persistence. Biophysical analysis of purified wild type and assembly defective mutant proteins indicates that PopZ self-associates into an elongated trimer, which readily forms a dimer of trimers through lateral contact. Shapiro, L., Quon, K., Marczynski, G., Stephens, C. CAULOBACTER FLIQ AND FLIR MEMBRANE-PROTEINS, REQUIRED FOR FLAGELLAR BIOGENESIS AND CELL-DIVISION, BELONG TO A FAMILY OF VIRULENCE FACTOR EXPORT PROTEINS, Regulation of asymmetry and polarity during the Caulobacter cell cycle, CAULOBACTER FLAGELLAR FUNCTION, BUT NOT ASSEMBLY, REQUIRES FLIL, A NON-POLARLY LOCALIZED MEMBRANE-PROTEIN PRESENT IN ALL CELL-TYPES, CELL-CYCLE ARREST OF A CAULOBACTER-CRESCENTUS SECA MUTANT, BACTERIAL SPORULATION - AN ATP/ADP SWITCH, A CAULOBACTER DNA METHYLTRANSFERASE THAT FUNCTIONS ONLY IN THE PREDIVISIONAL CELL, EXPRESSION OF CAULOBACTER-DNAA AS A FUNCTION OF THE CELL-CYCLE, THE EXPRESSION OF ASYMMETRY DURING CAULOBACTER CELL-DIFFERENTIATION, CHECKPOINTS THAT COUPLE GENE-EXPRESSION TO MORPHOGENESIS. 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 Shapiro lab uses small molecule screening approaches to identify new regulatory pathways important in cancer and other diseases and to identify small molecules with therapeutic potential. The bacterial chromosome encodes information at multiple levels. View details for DOI 10.1073/pnas.1909798116. The Stanford Open Policing Project a unique partnership between the Stanford Computational Journalism Lab and the Stanford Computational Policy Lab is changing that. Delighted to host the first International Symposium on Biomolecular Ultrasound and Sonogenetics. 2001. We have isolated the dnaA gene in order to determine whether this essential and ubiquitous replication initiation protein also contributes to differential replication control in C. crescentus. Our goal is to identify and understand the pathways that govern organogenesis of the pancreas, a vital organ with endocrine and exocrine functions. This DNA is double-stranded as indicated by (i) the sharp increase in extinction coefficient over a narrow range of temperature increase, (ii) an increase in density in CsCl upon thermal denaturation, and (iii) the equivalence of guanine and cytosine as well as adenine and thymine, determined by chemical analysis. Thus, CckA activation is dependent on polar remodeling and a DNA replication initiation checkpoint that is tightly integrated with the polar phospho-signaling cascade governing cell cycle progression. Thus, the temporal control of this methyltransferase appears to contribute to the accurate cell-cycle control of DNA replication and cellular morphology. (ii) Is the differentiation cycle like a biosynthetic pathway where one event must follow another? At least 58 genes, including many flagellar and chemotaxis genes, are regulated by a type 1 incoherent feedforward motif in which CtrA activates sciP, followed by SciP repression of ctrA and CtrA target genes. When active PleC is delocalized in a DeltapodJ mutant, the accumulation of PilA, the downstream target of PleC signaling, is impaired, providing evidence that the polar localization of this histidine kinase stimulates the response signaled by a two-component system. Dynamic flexibility between the 2 domains rationalizes efficient S-layer crystal nucleation on the curved cellular surface. Some inverted repeat DNA sequences were observed to hybridize to different regions of the chromosomal DNA isolated from the morphologically and biochemically distinct swarmer cell and stalked cell populations. This information, combined with quantitative localization data from epitasis experiments, also identified all previously known proteins affecting such localization. Mikhail Shapiro Wins Roger Tsien Award for Excellence in Chemical Biology. Thus, the order of flagellar gene transcription reflects the order of assembly of the protein components. View details for DOI 10.1016/j.cell.2008.07.015, View details for Web of Science ID 000259318100015, View details for PubMedCentralID PMC2745220. Using purified protein in different oxidizing/reducing environments, we generate a calibration curve which can be used to analyze in situ measurements. We show that DNA methylation by CcrM is not required for the control of the initiation of chromosome replication or for DNA mismatch repair. A previously uncharacterized essential protein, MipZ, forms a complex with the partitioning protein ParB near the origin of replication and localizes with the duplicated origin regions to the cell poles. Immunoassays of colonies lysed in situ either by lambda prophage induction or by biochemical means afford a much higher level of sensitivity than the plaque assay probably adequate to detect the production of a few molecules of protein per cell. The mild glucose repression of the acyl-CoA synthase was reversed by exogenous dibutyryl cyclic AMP. Thus, the sequential degradation of PodJ appears to involve regulated intramembrane proteolysis (Rip) by MmpA. Inverted repeat DNA sequences of Caulobacter crescentus have been isolated, characterized, and cloned in a bacteriophage lambda vector. Although transcription from the smc promoter is induced during early S phase, a cell cycle transcription pattern previously observed with multiple DNA replication and repair genes, the SMC protein is present throughout the entire cell cycle. The examples of polar localization given here are from a variety of bacterial species and concern a disparate array of cellular functions. 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 CtrA protein footprints conserved binding sites within the P1 and P2 promoters. In recent years, the subcellular organization of prokaryotic cells has become a focal point of interest in microbiology. FzlA altered FtsZ structure both in vivo and in vitro, forming stable higher-order structures that were resistant to depolymerization by MipZ, a spatial determinant of FtsZ assembly. We show here that two genes, gyrB (encoding the gyrase B subunit) and orf-1, are specifically transcribed from the chromosome in the portion of the predivisional cell destined for the progeny stalked cell. Click here to join the member exclusive portion of my show: https://utm.io/ueSEj. The presence of a plasmid containing the flaYE region allowed the mutant strains to swim and to exhibit chemotaxis, to synthesize increased amounts of the flagellins, to methylate their "methyl-accepting chemotaxis proteins" (MCPs), and to regain wild-type levels of methyltransferase activity. The genes encoding the structural components of the Caulobacter crescentus flagellum are temporally controlled and their order of expression reflects the sequence of assembly. Despite their small size and lack of obvious intracellular structures, bacteria have a complex and dynamic intracellular organization. Postdoctoral research on super-resolution imaging of bacterial protein assemblies in the Moerner Lab. View details for Web of Science ID A1996UD48400020, View details for PubMedCentralID PMC177887. In particular, little is known about the replication of multipartite genomes in bacteria. The acidic phospholipids, phosphatidylglycerol and cardiolipin, comprise approximately 87% of the total phospholipids. Gilbert Building 371 Jane Stanford Way Stanford, CA 94305 Phone: 650-723-2413 biologyinfo [at] stanford.edu Campus Map For the prior 16 years, he was an Assistant United States Attorney in the United States Attorneys Office for the District of Massachusetts. The histidine kinase signaling protein, PleC, that controls the temporal accumulation of the PilA pilin subunit is asymmetrically localized to the pole at which pili are assembled. The C. crescentus and Escherichia coli MCPs have highly conserved carboxy-terminal domains, and when an E. coli MCP is expressed in C. crescentus, it is targeted to the swarmer cell progeny. Our data demonstrate the potential for further development of borinic esters as antibacterial agents as well as leads to explore more specific inhibitors against two essential bacterial enzymes. View details for DOI 10.1073/pnas.1405188111. An interesting feature of the differentiation cycle is that the polar organelle may represent a special segregated unit which is operative in the control of the differentiation process. We discovered that DivL's catalytic domains have been repurposed as a phosphospecific RR input sensor, thereby reversing the flow of information observed in conventional histidine kinase (HK)-RR systems and coupling a complex network of signaling proteins for cell-fate regulation. 2) A specific technique has been developed whereby the progress of the differentiation cycle can be accurately measured by adsorption of labeled RNA phage or penetration of labeled phage DNA into specific cell forms. View details for Web of Science ID 000279782000006, View details for PubMedCentralID PMC2916389. The DivL pseudo-histidine kinase, positioned at one cell pole, regulates cell-fate by controlling the activation of the global transcription factor CtrA via an interaction with the response regulator (RR) DivK. View details for DOI 10.1016/j.cell.2006.05.038, View details for Web of Science ID 000239224800023. Chromatographic mobilities suggested that these fatty acids may be a cyclopropane and a branched-chain fatty acid. Revertant strains had wild-type levels of glycerol 3-phosphate dehydrogenase activity and normal rates of phospholipid and macromolecular synthesis. (Hons.) Zhou, B., Schrader, J. M., Kalogeraki, V. S., Abeliuk, E., Dinh, C. B., Pham, J. Q., Cui, Z. 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. A major breakthrough in understanding the bacterial cell is the discovery that the cell is highly organized at the level of protein localization. This motif, named RRF (for repression of replication factors), is conserved in the promoters of other coordinately induced replication factors. These discoveries have advanced our understanding of bacterial physiology and provided insight into the evolution of the eukaryotic cytoskeleton. 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. CHARACTERIZATION OF A VIRAL RNA-DEPENDENT RNA POLYMERASE, REPLICATION OF RNA VIRUSES .3. Perez, A. M., Mann, T. H., Lasker, K., Ahrens, D. G., Eckart, M. R., Shapiro, L. Super-Resolution Microscopy and Single-Protein Tracking in Live Bacteria Using a Genetically Encoded, Photostable Fluoromodule. Bayas, C. A., Schrader, J. M., Lee, M. K., Shapiro, L., Moerner, W. E. CauloBrowser: A systems biology resource for Caulobacter crescentus. MreB forms dynamic spirals in MreC-depleted cells, and MreC localizes helically in the presence of the MreB-inhibitor A22, indicating that each protein can form a spiral independently of the other. Particular progress has been made in understanding the structure, replication, and segregation of the bacterial chromosome. View details for PubMedCentralID PMC2937403. These compounds were identified by screening for inhibitors against Caulobacter crescentus CcrM, an essential DNA methyltransferase from gram negative alpha-proteobacteria. Goley, E. D., Dye, N. A., Werner, J. N., Gitai, Z., Shapiro, L. CrfA, a small noncoding RNA regulator of adaptation to carbon starvation in Caulobacter crescentus. This reporter is specific for uranium and has little cross specificity for nitrate (<400 microM), lead (<150 microM), cadmium (<48 microM), or chromium (<41.6 microM). FzlA is conserved in -proteobacteria and was found to be functionally critical for cell division in Caulobacter. Out of 2726 TSSs, 586 were shown to be cell cycle-regulated and we identified 529 binding sites for the cell cycle master regulators. The ubiquitous DnaA protein is a major regulator of all three bacterial origins. DEOXYRIBONUCLEIC-ACID SEQUENCE HOMOLOGIES AMONG BACTERIAL INSERTION SEQUENCE ELEMENTS AND GENOMES OF VARIOUS ORGANISMS, CELL-CYCLE-ASSOCIATED REARRANGEMENT OF INVERTED REPEAT DNA-SEQUENCES. shapiro lab stanfordcancer man psychologycancer man psychology Collaboration: The mutant lacks a flagellum and pili, and may have an abnormal DNA phage receptor site. 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. CcrM is a DNA adenine methyltransferase found in the alpha subdivision of the proteobacteria. Fatty acid degradation was investigated in Caulobacter crescentus, a bacterium that exhibits membrane-mediated differentiation events. Here, we report that CckA, the histidine kinase upstream of CtrA, employs a tandem-PAS domain sensor to integrate two distinct spatiotemporal signals. Signals from probes binding the same message are correlated. We report the regulatory response of C. crescentus to carbon starvation, based on combined high-throughput proteome and transcriptome analyses. The Global Regulatory Architecture of Transcription during the Caulobacter Cell Cycle. Growth on mannose, however, is stimulated by exogenous dibutyryl cyclic AMP without a concomitant increase in the specific activity of the mannose catabolic enzymes. We have also identified a new structural feature at the flagellated pole and the tip of the stalk: the 10-nm polar particle. Schrader, J. M., Zhou, B., Li, G., Lasker, K., Childers, W. S., Williams, B., Long, T., Crosson, S., McAdams, H. H., Weissman, J. S., Shapiro, L. The Coding and Noncoding Architecture of the Caulobacter crescentus Genome. 1994-1996. They have found a single molecular event present in all cancers studied to date that protects them from macrophages of the innate immune system. Particularly, hybrid tags that combine a fluorescent or fluorogenic dye with a genetically encoded protein (such as enzymatic labels) have been used successfully in multiple cell types. View details for DOI 10.1101/sqb.2009.74.005, View details for Web of Science ID 000285712600011. We demonstrate quantitative multicolor three-dimensional (3D) subdiffraction imaging of the structural arrangement of fluorescent protein fusions in living Caulobacter crescentus bacteria. View details for Web of Science ID A1997XE30000021, View details for PubMedCentralID PMC179208. 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. We demonstrate that SciP binds to DNA at a motif distinct from the CtrA binding motif that is present in the promoters of genes co-regulated by SciP and CtrA. This oligotrophic bacterium divides asymmetrically to produce a motile swarmer cell that represses DNA replication and a sessile stalked cell that replicates its DNA. Stallmeyer, M. J., Hahnenberger, K. M., Sosinsky, G. E., Shapiro, L., DeRosier, D. J. TEMPORAL REGULATION AND OVERLAP ORGANIZATION OF 2 CAULOBACTER FLAGELLAR GENES. Topoisomerases play a key role in ensuring orderly replication and partition of DNA in the face of a continuously changing DNA tertiary structure. Genetics 197:405-420. In an intensive 4-week period, students learn fundamental skills focused on scientific proposal writing, including writing and criticizing a proposal on the scientific topic of their choice. Although ribonucleic acid and protein syntheses continued at a reduced rate for the equivalent of one generation in mutant strains, a substantial decrease in the rate of deoxyribonucleic acid synthesis occurred immediately upon glycerol deprivation. We generate multilayered porous films in crystalline silicon using a periodic electrochemical etch. Site-directed mutagenesis confirmed that a conserved nucleotide in this sigma 54 promoter consensus sequence was required for transcription. How toxin activation triggers persistence and induces a systemic stress response in the alphaproteobacteria remains unclear. Minor in Poverty, Inequality, and Policy Jesse Shapiro. 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