We have isolated the sigma 54 gene (rpoN) and describe here a novel role for this alternative sigma-factor in cell differentiation: It is required for the biogenesis of both polar structures, and the disruption of the rpoN gene results in aberrant cell division. CtrA-mediated repression at the origin thus restricts replication to the stalked cell type. Attempts to encode large numbers of polymeric, metallic or glass beads in random arrays or in fluid suspension have used a variety of entities to provide coded elements (bits)--fluorescent molecules, molecules with specific vibrational signatures, quantum dots, or discrete metallic layers. 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. View details for Web of Science ID 000168535000012, View details for PubMedCentralID PMC95206, View details for Web of Science ID 000168824801666. In compartmentalized cells, fluorescence disappears only in the compartment receiving the bleaching beam; in noncompartmentalized cells, fluorescence disappears from the entire cell. American volume -Guitton, T. G., Ring, D.2011;93 (21): 2015-2021, GAIT & POSTURE -Butler, E. E., Ladd, A. L., Louie, S. A., Lamont, L. E., Wong, W., Rose, J. of Mechanical Engineering, 2007. 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. View details for DOI 10.1371/journal.pbio.1001979, View details for Web of Science ID 000344461700017, View details for PubMedCentralID PMC4211667. With the other method, the M ring is similar to that of S. typhimurium; that is, it contacts the S ring only at an outer radius and lacks the button. In a mutant strain that failed to assemble a flagellum, the 29K flagellin still segregated to the presumptive swarmer cell, demonstrating that positioning of the protein is independent of filament assembly. Low levels of the L-ring protein were detected exclusively in the cell envelope of cells lacking the P-ring, suggesting that, in the absence of P-ring assembly, L-ring monomers are unable to form multimeric rings and are thus subject to proteolysis in the periplasm. 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. The promoter sequence of the fliQR operon differs from most known bacterial promoter sequences but is similar to other Caulobacter class II flagellar gene promoter sequences. View details for Web of Science ID 000084010000013. These results suggest that subcellular localization of a prokaryotic protein involves interaction of specific regions of the protein with unique cell sites that contain either localized binding proteins or a specific secretory apparatus. In addition, we demonstrate that borinic esters inhibit menaquinone methyltransferase in gram positive bacteria using a new biochemical assay for MenH from Bacillus subtilis. Sorter, A. M., Dahlberg, P. D., Wang, J., Shapiro, L., Moerner, W. E. Environmental Calcium Controls Alternate Physical States of the Caulobacter Surface Layer. The phenotype of pH-conditional mutants was defined on medium with lactose as the sole carbon source. The Lon protease thus exhibits pleiotropic effects in C. crescentus growth and development. The ensuing accumulation of CtrA results in the activation of the P2 promoter and the repression of the P1 promoter late in the cell cycle. 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. Analysis of the fliX-flgI intergenic region revealed an arrangement of cis-acting elements similar to that of another set of Caulobacter class II and class III flagellar genes, fliL-flgF, that is also divergently transcribed. 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. Faithful cell cycle progression in the dimorphic bacterium Caulobacter crescentus requires spatiotemporal regulation of gene expression and cell pole differentiation. Caulobacter crescentus is a model organism for the integrated circuitry that runs a bacterial cell cycle. A fatty acid auxotroph of Caulobacter crescentus, AE6001, which displays a strict requirement for unsaturated fatty acids to grow on glucose as the carbon source has been isolated. Subscribe: Histocompatibility and Immunogenetics Lab (HLA), COVID-19 Specimen Collection & Handling, Case Consult Service and Reference Lab Testing, PATHOLOGY CONSULTATIONS & REFERENCE LAB TESTING, Stanford Health Care. When these two parameters are calculated for genes from nonmammalian eukaryotic organisms, genes from the same organism again have similar values, and genome-wide codon bias may also be predicted from intergenic sequences. Kim, S., Gitai, Z., Kinkhabwala, A., Shapiro, L., Moerner, W. E. A phospho-signaling pathway controls the localization and activity of a protease complex critical for bacterial cell cycle progression. We determined the cellular positions of the origins of the replicons in the alpha proteobacteria Agrobacterium tumefaciens and Sinorhizobium meliloti and found that they are located at the poles of the cells. 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. View details for Web of Science ID 000259631900035, View details for PubMedCentralID PMC2566184. Bacteria exhibit a high degree of intracellular organization, both in the timing of essential processes and in the placement of the chromosome, the division site, and individual structural and regulatory proteins. We found most start codons lacked corresponding Shine-Dalgarno sites although ribosomes were observed to pause at internal Shine-Dalgarno sites within the coding DNA sequence (CDS). Purified SMC-E1076Q was deficient in ATP hydrolysis and exhibited abnormally stable binding to DNA. We present evidence that a bacterial signal transduction cascade that couples morphogenesis with cell cycle progression is regulated by dynamic localization of its components. A closed-loop control system drives progression of the coupled stalked and swarmer cell cycles of the bacterium Caulobacter crescentus in a near-mechanical step-like fashion. View details for DOI 10.1128/AEM.01566-07, View details for Web of Science ID 000251474400017, View details for PubMedCentralID PMC2168040. Organization View details for DOI 10.1126/science.1142189, View details for Web of Science ID 000247400500051. The 29K flagellin was found only in the progeny swarmer cell after cell division. We have identified a conserved sequence motif that is present in the promoter of dnaX and several other genes involved in the replication of DNA, all of which show an induction of transcription at the onset of chromosome replication. We propose that changes in the cellular concentration of CtrA approximately P and its interaction with accessory proteins influence the temporal expression of fliQ, ccrM, and other key cell cycle genes and ultimately the regulation of the cell cycle. At specific times in the cell cycle, the bacterium Caulobacter crescentus assembles two major polar organelles, the flagellum and the stalk. In addition, two minor but as yet unidentified fatty acids were detected. These developmental decisions require global changes in genomic readout, and bacteria typically employ intricate (yet poorly understood) signaling networks that enable changes in cell function. Fasten your seatbelt: Developmental biologist Lucy Shapiro, PhD, is driving, and we're zooming through her achievement-packed 40-year career in less than an hour. In this perspective, we highlight recent discoveries in Caulobacter crescentus asymmetric cell division to illuminate diverse mechanisms by which a cellular compass, composed of scaffolding and signaling proteins, directs cell cycle modules to their exact cellular addresses. The Caulobacter chromosome changes progressively from the fully methylated to the hemimethylated state during DNA replication. beta-Galactosidase-constitutive mutants did not exhibit a cell cycle arrest upon transfer of cultures from glucose to lactose. Based on these techniques, the diffusion coefficient and dynamics of the histidine protein kinase PleC, the localization behavior of the polar protein PopZ, and the treadmilling behavior and protein superstructure of the structural protein MreB are investigated with sub-40-nm spatial resolution, all in live cells. "Im telling you this saga because its an example of just one person at Stanford doing curiosity-driven research which can open up the world and affect the world," Shapiro said. Caltech From left: David Shapiro, senior staff scientist at Berkeley Labs Advanced Light Source; William Chueh, associate professor of materials science and engineering at Stanford. It is shown here that C. crescentus catabolizes galactose by the Entner-Duodoroff pathway. Regions of homology between the IS elements and ribosomal RNA were observed. View details for Web of Science ID A1970G593000016, View details for Web of Science ID A1970H419900033, View details for Web of Science ID A1970G466200017. The entire promoter region and an upstream consensus sequence that might be a regulatory element for the flaY gene lies within the carboxyl-terminal coding sequence of the flaE gene. View details for Web of Science ID 000087307700023, View details for PubMedCentralID PMC101938, View details for Web of Science ID 000084722600008. These data suggest a more prevalent use of the Shine-Dalgarno sequence for ribosome pausing rather than translation initiation in C. crescentus. B.S. The derived amino acid sequence indicates that fibN encodes a 25-kilodalton protein with a cleavable leader peptide. Positional information during Caulobacter cell differentiation. A peptide containing the C-terminal portion of the FtsA divisome protein is a substrate of both ClpXP and ClpAPin vitro but is primarily degraded by ClpAPin vivo. We take advantage of the best feature of both model and non-model organisms, including laboratory mice, wild stickleback fish, and pluripotent stem cells from humans and non . 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. View details for Web of Science ID A1979HV87000036. While studying for his undergrad economics degree at Stanford, Sam Shapiro got involved in a mentorship program in which he was paired with a student from Stanford Graduate School of Business. 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. Finally, we examined the fatty acid biosynthesis and composition of two unsaturated fatty acid auxotrophs of C. crescentus. Here, we demonstrate that the Caulobacter crescentus SLP readily crystallizes into sheets in vitro via a calcium-triggered multistep assembly pathway. Dividing cells must coordinate cell cycle events to ensure genetic stability. Such organization is transmitted from one generation to the next by progressive segregation of daughter chromosomes and anchoring of DNA to the cell envelope. We examine the temporal and spatial regulation of the Caulobacter cell cycle, bacterial chromosome segregation and cytokinesis, and Bacillus subtilis sporulation. Recent advances in cell-imaging technology with subdiffraction resolution have revealed that the bacterial nucleoid is reliably oriented and highly organized within the cell. Our results broadly demonstrate how signaling factors can leverage information from their subcellular niche to drive spatiotemporal control of cell signaling. View details for DOI 10.1128/JB.188.4.1497-1508.2006, View details for PubMedCentralID PMC1367234. Nierman, W. C., Feldblyum, T. V., Laub, M. T., Paulsen, I. T., Nelson, K. E., Eisen, J., Heidelberg, J. F., Alley, M. R., Ohta, N., Maddock, J. R., Potocka, I., Nelson, W. C., Newton, A., Stephens, C., Phadke, N. D., Ely, B., DeBoy, R. T., Dodson, R. J., Durkin, A. S., Gwinn, M. L., Haft, D. H., Kolonay, J. F., Smit, J., Craven, M. B., Khouri, H., Shetty, J., Berry, K., Utterback, T., Tran, K., Wolf, A., Vamathevan, J., Ermolaeva, M., White, O., Salzberg, S. L., Venter, J. C., Shapiro, L., Fraser, C. M. Dynamic localization of a cytoplasmic signal transduction response regulator controls morphogenesis during the Caulobacter cell cycle, Global analysis of the genetic network controlling a bacterial cell cycle. 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). The flgH gene, encoding the L-ring protein, is also transcribed from an internal promoter. Here, we identify an essential histidine kinase, CckA, that is responsible for CtrA activation by phosphorylation. To explore the global extent of this regulatory mechanism, we determined the methylation state of the entire chromosome at every base pair at five time points in the cell cycle using single-molecule, real-time sequencing. Perhaps the sequential morphogenic changes exhibited by Caulobacter are dependent on the initial synthesis of this organelle. The origins of replication of many different bacteria have been shown to reside at specific subcellular locations, but the mechanisms underlying their positioning and segregation are still being elucidated. Interestingly, M. xanthus, which has nozzles at both poles, can reverse direction by closing one nozzle and opening the other in response to end-to-end interactions between cells. We have also partially purified the Caulobacter homolog of IHF and demonstrate that it can facilitate in vitro integrase-mediated lambda recombination. McGrath, P. T., Iniesta, A. 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. One of these genes, flbN, is required early in the flagellar assembly process. 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. View details for Web of Science ID 000314586600005, View details for PubMedCentralID PMC3660146, View details for Web of Science ID 000342772500028. We show that DnaA coordinates DNA replication initiation with cell cycle progression by acting as a global transcription factor. Using an inactive form of PleC (PleCH610A) that lacks the catalytic histidine residue, we provide evidence that PleC activity is responsible for the asymmetric distribution of CpaE and itself to only one of the two cell poles. The ssrA deletion phenotype is not due to accumulation of stalled ribosomes, because the deletion is not complemented by a mutated version of SsrA that releases ribosomes but does not target proteins for degradation. Doi 10.1128/AEM.01566-07, View details for Web of Science ID 000247400500051 as yet unidentified fatty acids were.! Have also partially purified the Caulobacter crescentus requires spatiotemporal regulation of the coupled stalked and swarmer cycles. Homology between the is elements and ribosomal RNA were observed leverage information from their subcellular to... Protease thus exhibits pleiotropic effects in C. crescentus daughter chromosomes and anchoring of DNA to the stalked type... 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