Meisenzahl, A. C., Shapiro, L., Jenal, U. Understanding the control logic in the bacterium Caulobacter crescentus has progressed to the point where we now have an integrated systems view of the operation of its entire cell cycle functioning as a state machine. The crystal structure of TadZ from Eubacterium rectale (ErTadZ), in complex with ATP and Mg(2+) , was determined to 2.1 resolution. We mapped transcript units at single base-pair resolution using RNA-seq together with global 5'-RACE. Mutational analysis of two M.Ccr II methylation sites located 3' to the ccrM promoter suggests that methylation might influence the temporally controlled inactivation of ccrM transcription. M.S. In Caulobacter crescentus, the origin of DNA replication is located at the cell pole. B.S. PURIFICATION AND CHARACTERIZATION OF GUANYLATE CYCLASE FROM CAULOBACTER-CRESCENTUS, PLEIOTROPIC MUTATION AFFECTING EXPRESSION OF POLAR DEVELOPMENT EVENTS IN CAULOBACTER-CRESCENTUS, SYNTHESIS AND STRUCTURE OF CAULOBACTER-CRESCENTUS FLAGELLA, DEOXYRIBONUCLEIC ACID-DEPENDENT RIBONUCLEIC-ACID POLYMERASE OF CAULOBACTER-CRESCENTUS. 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. Quon, K. C., Yang, B., Domian, I. J., Shapiro, L., Marczynski, G. T. Transcriptional analysis of the Caulobacter 4.5 S RNA ffs gene and the physiological basis of an ffs mutant with a Ts phenotype, The CcrM DNA methyltransferase is widespread in the alpha subdivision of proteobacteria, and its essential functions are conserved in Rhizobium meliloti and Caulobacter crescentus, Identification of the fliI and fliJ components of the Caulobacter flagellar type III protein secretion system. Flagellar and chemotaxis genes are transcribed at a discrete time in the Caulobacter cell cycle. The rest of the filament (region V) is made up predominantly, if not completely, of the 25 x 10(3) Mr flagellin. Membrane phospholipid synthesis was inhibited in Caulobacter crescentus by growth of a glycerol auxotroph in the absence of glycerol or by treatment with the antibiotic cerulenin. 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. A shift of cells from restrictive to permissive temperature results in rapid degradation of CtrA, initiation of DNA replication, and the resumption of cell cycle progression, including the ordered expression of genes involved in chromosome replication and polar organelle biogenesis. Alternatively, beam scientists can take many measurements of the beam itself and try to reconstruct, sometimes using machine learning, what the beam would look like under different experimental circumstances but those methods require a lot of data and a lot of computational power. The cell cycle-regulated methylation state of Caulobacter DNA mediates the temporal control of transcriptional activation of several key regulatory proteins. Four metrics characterizing the observed localization patterns of each of the three labeled proteins were extracted for hundreds of cell images from each of 854 mapped mutant strains. SURF Scholar 2019- Currently: Assistant Professor of Bioengineering x@caltech.edu, x=wcoleman, George Daghlian To study the relationship between phospholipid synthesis and organelle biogenesis in the dimorphic bacterium Caulobacter crescentus, auxotrophs have been isolated which require exogenous glycerol or glycerol 3-phosphate for growth when glucose is used as the carbon source. Caulobacter crescentus is widely used as a powerful model system for the study of prokaryotic cell biology and development. Upon initiation of DNA replication, one copy of the duplicated origin sequence rapidly appears at the opposite cell pole. B.A. A fusion of the receiver domain and last 15 residues of CtrA to YFP is properly degraded in living cells. 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. Independent mutations in the conserved sequence that lies between the -10 and -35 regions increased transcription, suggesting that a repressor may bind at this site. WebJoy Wu graduated from Stanford University (B.S., Chemistry). beta-Galactosidase-constitutive mutants did not exhibit a cell cycle arrest upon transfer of cultures from glucose to lactose. Currently: Senior Scientist at Shape Therapeutics, Prof. David Maresca The M ring, which is at the inner membrane of the cell, has a different structure depending on the method of preparation. Sirisha Gudavalli, SURF Scholar 2018 BS Bioengineering, Caltech 2021 Our results revealed a picture of divisome assembly with unprecedented temporal resolution. 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". Shapiro (2018). Purucker, M., Bryan, R., Amemiya, K., Ely, B., Shapiro, L. CHARACTERIZATION OF THE PROTEINS OF THE CAULOBACTER-CRESCENTUS FLAGELLAR FILAMENT - PEPTIDE ANALYSIS AND FILAMENT ORGANIZATION. Life Sciences Research Foundation / Amgen Fellow It emerged that multiple mechanisms cooperate to establish a dynamic assembly of supercoiled domains, which are stacked in consecutive order to adopt a defined higher-level organization. Ph.D. Student, Biology 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. After the equivalent of one generation time, rapid cell death occurred. Formal model-checking analysis of the digital circuit showed that the cell-cycle control is robust to intrinsic stochastic variations in reaction rates and nutrient supply, and that it reliably stops and restarts to accommodate nutrient starvation. M.S. This suggests a role for HrcA in negative regulation of heat shock gene expression. 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. Candidate, David Geffen School of Medicine at UCLA Large structures, such as a flagellum, are anchored at the pole by means of the basal body that traverses the peptidoglycan wall. View details for Web of Science ID A1989R914200005. The C. crescentus enzyme appeared similar to the Pseudomonas aeruginosa enzyme and unlike the Escherichia coli enzyme with respect to subunit molecular weights and failure to separate into core and sigma components upon phosphocellulose chromatography. (5) Together, these regulatory proteins create a genetic circuit in which the cellular concentrations of CtrA and GcrA oscillate spatially and temporally to control daughter cell differentiation and cell cycle progression. Bryan, R., Purucker, M., Gomes, S. L., Alexander, W., Shapiro, L. GENETIC-ANALYSIS AND CHARACTERIZATION OF A CAULOBACTER-CRESCENTUS MUTANT DEFECTIVE IN MEMBRANE BIOGENESIS. Rock, F. L., Mao, W., Yaremchuk, A., Tukalo, M., Crepin, T., Zhou, H., Zhang, Y., Hernandez, V., Akama, T., Baker, S. J., Plattner, J. J., Shapiro, L., Martinis, S. A., Benkovic, S. J., Cusack, S., Alley, M. R. High-throughput identification of transcription start sites, conserved promoter motifs and predicted regulons. jkim622@illinois.edu The probe correlation analysis approach reported here is of general use for large-scale sRNA identification for any sequenced microbial genome. CcrM is essential for viability in both of these organisms, and we show here that it is also essential in Brucella abortus. The expression of the Caulobacter ccrM gene and the activity of its product, the M.Ccr II DNA methyltransferase, are limited to a discrete portion of the cell cycle (G. Zweiger, G. Marczynski, and L. Shapiro, J. Mol. Functional homology was demonstrated by complementing the temperature-sensitive growth defect of an E. coli rpoH deletion mutant with the C. crescentus rpoH gene. View details for Web of Science ID 000361534800042, View details for PubMedCentralID PMC4541484. The switch to the second phospholipid profile was observed to occur at the predivisional cell stage. Society of General Physiology, 2002-present. View details for Web of Science ID A1977DU20100033, View details for Web of Science ID A1976CH91600017. & Gerardot, C. J. In addition, two minor but as yet unidentified fatty acids were detected. An SMC ATPase mutant disrupts chromosome segregation in Caulobacter, Three-dimensional superresolution colocalization of intracellular protein superstructures and the cell surface in live Caulobacter crescentus. The enzyme acts processively during methylation of specific DNA sequences, indicating a preferred order of product release in which S-adenosylhomocysteine is released from enzyme before fully methylated DNA. View details for DOI 10.1111/j.1365-2958.2011.07698.x, View details for Web of Science ID 000292567200009, View details for PubMedCentralID PMC3137890. RcdA is required for CtrA polar localization and degradation by ClpXP. At normal growth temperature (30 degrees C), a different start site was identified 3' to the heat shock start site that conformed to the E. coli sigma 70 promoter consensus sequence. View details for Web of Science ID 000088048400024, View details for PubMedCentralID PMC16621. Transcription of Escherichia coli and Caulobacter crescentus phage DNAs by their respective host RNA polymerase was examined to determine their ability to recognize specific transcription signals on the heterologous template. 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. Dividing cells must coordinate cell cycle events to ensure genetic stability. We investigate the midplane protein FtsZ in Caulobacter crescentus with super-resolution imaging based on fluorescent-protein photoswitching and the natural polymerization/depolymerization dynamics of FtsZ associated with the Z-ring. pilA transcription is regulated by the global two-component response regulator CtrA, which is essential for the expression of multiple cell cycle events, providing a direct link between assembly of the pilus organelle and bacterial cell cycle control. The synthesis of these proteins occurs only in the Caulobacter crescentus predivisional cell coincident with the biosynthesis of the polar flagellum. Since an early effect of inhibiting phospholipid synthesis in C. crescentus is the termination of deoxyribonucleic acid (DNA) replication (I. Contreras, R. Bender, A. Weissborn, K. Amemiya J. D. Mansour, S. Henry, and L. Shapiro, J. Mol. The flagellum is ejected from the swarmer cell and then synthesized de novo later in the cell cycle. Using a cosmid library we isolated a clone that complemented SC1130. Other developmental abnormalities exhibited by the lon null mutant, such as the formation of abnormally long stalks, appear to be unrelated to altered chromosome methylation state. In recent years, the subcellular organization of prokaryotic cells has become a focal point of interest in microbiology. The original point mutation is predicted to disrupt the stem structure in the 4.5 S RNA thus providing a rationale for the genetic basis of the LS439 phenotype. The polar organizing protein Z (PopZ) localizes to the polar regions of C. crescentus where it is known to form a distinct microdomain. Bioengineering (CS minor), Caltech The constraining features for membrane components are not known. The transcription start sites in vitro and in vivo were shown to be identical by S1 nuclease mapping and were found to be located approximately 300 nucleotides upstream from the 3' end of the 16 S rRNA gene. View details for Web of Science ID A1981LG93700035. S1 mapping and primer extension experiments showed that transcription initiated at two sites 5' to the dnaK coding sequence. Postdoctoral Fellowship, U Chicago Nature Communications13, 1585 (2022). Currently: Research Scientist While super-resolution imaging has greatly benefited from highly photostable fluorophores, a shortage of photostable fluorescent labels for bacteria has limited its use in these small but relevant organisms. (1-3) In the alpha-proteobacterium, Caulobacter crescentus, the CtrA global transcriptional regulator exhibits a spatially and temporally dynamic expression pattern across the cell cycle. Achieving proper polarity is essential for cellular function. Among the validated sRNAs, two are candidate transposase gene antisense RNAs. Bacterial chromosomes are generally approximately 1000 times longer than the cells in which they reside, and concurrent replication, segregation, and transcription/translation of this crowded mass of DNA poses a challenging organizational problem. The conserved nucleotides in the promoter region are clustered in the -10, -20 to -30, and -35 regions. The bacterium Caulobacter crescentus uses two-component phospho-signalling to regulate spatially distinct cell cycle events through the master regulator CtrA. Cultures of the bacterium have been synchronized and an assay has been developed for monitoring the course of morphogenesis by the selective adsorption of radioactive RNA bacteriophage. Therefore, flagellar genes at or near the top of the hierarchy may be controlled, in part, by a unique transcription factor and may be responsive to the same DNA replication cues that mediate other cell cycle events, such as cell division. Analysis of the C. crescentus homolog of dnaX, which in Escherichia coli encodes both the gamma and tau subunits of the DNA polymerase III holoenzyme, identified the dnaX transcription start site and showed that activity from the dnaX promoter is stimulated fourfold at the onset of DNA replication. We seek to understand the mechanisms that regulate self-renewal, proliferation and differentiation in adult stem cell linages. View details for DOI 10.1111/j.1365-2958.2005.04912.x, View details for Web of Science ID 000233170700012. It was found that the Tsr protein appeared at the same point in the cell cycle as an endogenous C. crescentus methyl-accepting chemotaxis protein. Our inability to obtain fatty acid degradation mutants after a wide search, coupled with the high constitutive levels of the beta-oxidation enzymes, suggest that fatty acid turnover, as has proven to be the case fatty acid biosynthesis, might play an essential role in membrane biogenesis and cell cycle events in C. crescentus. The sequence upstream of the translational start site shows little homology to the canonical Shine-Dalgarno ribosome recognition sequence, but the region downstream of the start codon is complementary to a region of 16S rRNA implicated in downstream box recognition. Little is known about the structure and function of most nucleoid-associated proteins (NAPs) in bacteria. Mutants of Escherichia coli have been isolated that are able to grow on lactose at pH 7.0 but not at pH 8.1. This redundant control of gcrA transcription by DnaA (activation) and CtrA (repression) forms a robust switch controlling the decision to proceed through the cell cycle or to remain in the G1 stage.
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