The pilus assembly protein TadZ (called CpaE in Caulobacter crescentus) is ubiquitous in tad loci, but is absent in other type IV pilus biogenesis systems. Each of these transcripts proved to be a de novo transcript since (a) each could be pulse labeled during the initial 20 s of the reaction and (b) each transcript contained a triphosphate at its 5' terminus. 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. phiCbK DNA cosediments with Escherichia coli phage T2 DNA and has therefore been assigned an S(20,w) value of 63.5S. 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 . Furthermore, methyltransferase activity, present in the predivisional cell, was detected only in the swarmer cell upon cell division. 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. M.S. View details for Web of Science ID A1989AK51300008. How this is brought about remains one of the most fundamental questions of developmental biology. This protein, CtrA, is homologous to response regulator transcription factors and controls transcription from a group of cell cycle-regulated promoters critical for DNA replication, DNA methylation, and flagellar biogenesis. By contrast, our transcriptome analysis shows that >10% of the genes are misexpressed in cells lacking or constitutively over-expressing CcrM. In these cells, as appears to be the case with C. crescentus, the individual enzymes form multimers of identical subunits. fliJ encodes a 16-kDa hydrophilic protein of unknown function. Ben Shapiro will speak at a free event on campus. We perform cryogenic super-resolution experiments in situ, labeling PopZ, a protein known to assemble into a microdomain at the poles of the model bacterium Caulobacter crescentus. Using hyper-saturated transposon mutagenesis coupled with high-throughput sequencing, we determined the essential Caulobacter genome at 8 bp resolution, including 1012 essential genome features: 480 ORFs, 402 regulatory sequences and 130 non-coding elements, including 90 intergenic segments of unknown function. Unlike the chromosome, plasmids from the incompatibility groups Q and P replicated in all C. crescentus cell types. Shapiro, L., Rosen, O. M., AGABIANK, N., Hirsch, A. BACTERIAL DIFFERENTIATION AND PHAGE INFECTION. The methyl-accepting chemotaxis proteins (MCPs) are membrane receptors that initiate signal transduction to the flagellar rotor upon ligand binding. The C. crescentus sigma32 homolog, predicted to be a 33.7-kDa protein, is 42% identical to E. coli sigma32 and cross-reacts with a monoclonal antibody to E. coli sigma32. The temporal expression of the modular subsystems that implement the cell cycle and asymmetric cell division is also coordinated by differential DNA methylation, regulated proteolysis, and phosphorylation signaling cascades. Hahnenberger and L. Shapiro, J. Mol. How do you make a protein that self-assembles, fills with air, excludes water and withstands several atmospheres of pressure? Why We Need Erasable MRI Scans. View details for Web of Science ID A1996UU11700009. View details for Web of Science ID A1992HJ50200007. B., McAdams, H. H., Shapiro, L., Collier, J. Therefore, we were able to recover a pH-conditional mutant in a cytoplasmic gene product. I earned my undergraduate degree from the University of Toronto and have previously worked as a predoctoral research fellow at the Gentzkow-Shapiro Lab at Stanford University. Regulated proteolysis is essential for cell cycle progression in both prokaryotes and eukaryotes. Epistasis experiments placed fliX within class II of the flagellar regulatory hierarchy, suggesting that FliX functions at an early stage in flagellar assembly. Constructing a macromolecular structure of this scale generally requires localized enzymatic machinery, but a regulatory framework for S-layer assembly has not been identified. As swarmer cells differentiate into stalked cells (G1/S transition), unphosphorylated CpdR accumulates and is localized to the stalked cell pole, where it enables ClpXP localization and CtrA proteolysis, allowing the initiation of DNA replication. Finally, we examined the fatty acid biosynthesis and composition of two unsaturated fatty acid auxotrophs of C. crescentus. B., Moore, D. C., Weiss, A. C., Ladd, A. L., Crisco, J. J. Bacterial scaffold directs pole-specific centromere segregation. View details for Web of Science ID 000168535000012, View details for PubMedCentralID PMC95206, View details for Web of Science ID 000168824801666. At the swarmer-to-stalked cell transition and in the stalked compartment of the predivisional cell, CtrA is localized to the cell pole just before its degradation. Given numerous examples reported thus far, we propose that bacterial polarity displays specific rules and is a more general phenomenon than has been previously recognized. Ranked in the top 10 for Neurology and Neurosurgical Care by US News and World Report, SHC is at the cutting edge of the latest treatments for neurological diseases. In at least two alpha subdivision bacteria, R. meliloti and C. crescentus, CcrM-mediated methylation has important cellular functions. We constructed a reporter that utilizes the urcA promoter to produce a UV-excitable green fluorescent protein in the presence of the uranyl cation, a soluble form of uranium. This directional movement of labeled MreB in the growing polymer provides an indication that, like actin, MreB monomers treadmill through MreB filaments by preferential polymerization at one filament end and depolymerization at the other filament end. The Beachy lab studies the function of Hedgehog proteins and other extracellular signals in morphogenesis (pattern formation) and in injury repair and regeneration (pattern maintenance). S1 mapping and primer extension experiments showed that transcription initiated at two sites 5' to the dnaK coding sequence. Stanford offers a wide range of routine and esoteric testing across all areas of Clinical and Molecular Pathology. An asynchronous sequential digital circuit model equivalent to the validated simulation model was created. Reno, PL, CY McLean, JE Hines, TD Capellini, G Bejerano, and DM Kingsley (2013). Bacterial cells are inherently asymmetric, some more obviously so than others. The localization pattern of RcdA is coincident with and dependent upon ClpX localization. Cell Metabolism. Both had a profound effect on the kinds of questions I posed and the means I used to arrive at answers. The initiation of DNA replication coincides with the proteolysis of the CtrA replication inhibitor and the accumulation of DnaA, the replication initiator, upon differentiation of the swarmer cell into a stalked cell. Kim, S. Y., Gitai, Z., Kinkhabwala, A., Shapiro, L., Moerner, W. E. MipZ, a spatial regulator coordinating chromosome segregation with cell division in Caulobacter, A dynamically localized protease complex and a polar specificity factor control a cell cycle master regulator. View details for DOI 10.1074/jbc.X112.422337, View details for Web of Science ID 000310642200061, View details for PubMedCentralID PMC3488097. The fatty acid composition of the dimorphic bacterium Caulobacter crescentus was found to consist primarily of 16- and 18-carbon fatty acids, both saturated and monounsaturated, in agreement with the findings of Chow and Schmidt (J. Gen. Microbiol. 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. Bellofatto, V., Shapiro, L., Hodgson, D. A. 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. The essential dnaN gene encodes a homolog of the Escherichia coli beta subunit of DNA polymerase III. View details for Web of Science ID 000430563200493, View details for Web of Science ID 000430450000249, View details for Web of Science ID 000430563200402, View details for Web of Science ID 000430450000508, View details for Web of Science ID 000430563300065, View details for Web of Science ID 000430439600509. Cell type determinants in stalked progeny promote entry into S phase, whereas swarmer progeny remain in G1 phase. These two cell types differ in their program of gene expression, their ability to replicate DNA, and the physical properties of their nucleoids. The Cantor Arts Center is a laboratory of learning and a center of scholarly inquiry. The flagellar genes with this conserved 5' region all initiate transcription early in the cell cycle and are all sensitive to a disruption in DNA replication. Bacterial replication origins move towards opposite ends of the cell during DNA segregation. Phosphorylated CtrA (CtrA approximately P) bound to the CtrA recognition sequence in both promoters but had a 10- to 20-fold greater affinity for PfliQ. Minor in Poverty, Inequality, and Policy Jesse Shapiro. In addition, dynamically localized DNA-binding proteins ensure that DNA segregation is coupled to the timing and cellular position of the cytokinetic ring. We have identified a novel, conserved protein, RcdA, that forms a complex with CtrA and ClpX in the cell. As bacteria continue to develop resistance toward current antibiotics, we find ourselves in a continual battle to identify new antibacterial agents and targets. The Tn5 insertion mutant SC1130 had no cross-reacting MCP and had reduced levels of activity of the methyltransferase and methylesterase. The depth and diversity of data sets collected by the Caulobacter community makes CauloBrowser a unique and valuable systems biology resource. The C. crescentus fliL and fliM genes form an operon that is expressed early in the cell cycle. Lasker, K., von Diezmann, L., Zhou, X., Ahrens, D. G., Mann, T. H., Moerner, W. E., Shapiro, L. Cryogenic single-molecule active control microscopy with a photoactivatable fluorescent protein. These studies demonstrated that EYFP is a useful emitter for in vivo super-resolution imaging. 138:401-410, 1980), we questioned whether the inhibition of stalk formation was due directly to the inhibition phospholipid synthesis or secondarily to the inhibition of DNA synthesis. To explain the phenotype of both the secA and ffs36 strains, we propose that a cell-cycle checkpoint prevents further progression through the cell-cycle in response to increased intracellular levels of heat shock and misfolded proteins. 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. The DnaA regulon includes genes encoding several replisome components, the GcrA global cell cycle regulator, the PodJ polar localization protein, the FtsZ cell division protein, and nucleotide biosynthesis enzymes. The Caulobacter cell cycle exhibits time-dependent expression of differentiation events. We use a variety of innovative approaches including genomics, computation, biochemistry, and advanced imaging. The flagellar promoters accessed by Tn5-VB32 exhibited temporal regulation analogous to the known flagellar and chemotaxis gene products. Maddock, J. R., Alley, M. R., Shapiro, L. ASYMMETRIC EXPRESSION OF THE GYRASE-B GENE FROM THE REPLICATION-COMPETENT CHROMOSOME IN THE CAULOBACTER-CRESCENTUS PREDIVISIONAL CELL. Shapiro JS, Besstte MJ, Fish-Rigan D, Baumlin KM, Richardson LD. Recent work, however, has demonstrated a remarkable degree of spatial organization. The group is always looking for creative individuals and we welcome people of all races, ethnicities, religions, gender identities and sexual orientations. These defects include a frequent failure to complete cell division and loss of precise cell-cycle control of initiation of DNA replication. (ii) Is the differentiation cycle like a biosynthetic pathway where one event must follow another? Thus, the sequential degradation of PodJ appears to involve regulated intramembrane proteolysis (Rip) by MmpA. Pros & Cons of Pooling Covid-19 Tests The synthesis of the product of flgJ, the 29K flagellin, occurs prior to the synthesis of the other flagellin proteins. The hipA2-encoded kinase functions as a toxin in Caulobacter, inducing bacterial persistence by disturbing the intracellular tryptophan-glutamine balance. Kaplan, J. The genes involved in these processes are widely separated on the chromosome. Here, we demonstrate that the ordered assembly of this microdomain occurs via the polymeric network protein PopZ directly recruiting the polarity factor SpmX, which then recruits the histidine kinase DivJ to the developing cell pole. SMC complexes and histone-like proteins continuously remodel the nucleoid to reconcile chromatin compaction with DNA replication and gene regulation. Deletion analysis reveals that the minimal sequence requirement for autonomous replication is greater than 430 base-pairs, but less than 720 base-pairs. Jutras BL, Scott M, Parry B, Biboy J, Gray J, Vollmer W, Jacobs-Wagner C (2016) Lyme disease and relapsing fever Borrelia elongate through zones of peptidoglycan synthesis that mark division sites of daughter cells. Here, we identify a bipartite proteolytic signal in the CtrA response regulator consisting of two determinants that are each necessary but not sufficient for regulated degradation. In order to explore the mechanism of P- and L-ring assembly, we examined the effect of a null mutation in the gene encoding the P-ring subunit, FlgI, on the expression, stability, and subcellular localization of the L-ring subunit, FlgH, in Caulobacter crescentus. We combine quantitative organism-wide fluorescence imaging ("deep imaging"), functional genomics ("deep sequencing"), and statistical modeling to understand the fundamental rules that control collective cell behaviors to optimize tissue organization, regeneration, adaptation, and evolution. Hong, S., Toro, E., Mortensen, K. I., de la Rosa, M. A., Doniach, S., Shapiro, L., Spakowitz, A. J., McAdams, H. H. Chromosome architecture is a key element of bacterial cellular organization, Deciphering the Transcriptional Landscape of Caulobacter crescentus at Base Pair Resolution. GcrA then activates the transcription of the next cell-cycle regulator, CtrA, once the replication fork passes through the ctrA P1 promoter, generating two hemimethylated copies of ctrA. The purification scheme minus the heating step also permitted the copurification of crotonase and 3-hydroxyacyl-CoA dehydrogenase. In addition, mutations in either fliQ or fliR exhibit defects in cell division and thus may participate directly or indirectly in the division process. Cunin, F., Schmedake, T. A., Link, J. R., Li, Y. Y., Koh, J., Bhatia, S. N., Sailor, M. J. Extensive mutational analysis of the promoter region from -42 to -5 identified functionally important nucleotides at -36 and -35, between -29 and -22, and at -12, which correlates well with sequences conserved between fliLM and the analogous regions of two other Class II flagellar operons. The mutant phenotype indicates that the assembly of the polar surface structures is coordinately regulated and independent of mechanisms regulating cell polarity and division. Select search scope, currently: catalog all catalog, articles, website, & more in one search; catalog books, media & more in the Stanford Libraries' collections; articles+ journal articles & other e-resources PfliQ is activated earlier than PccrM. We present evidence that the first amino acid in the predicted DnaX protein corresponds to the first codon in the mRNA transcribed from the dnaX promoter; thus, the ribosome must recognize the mRNA at a site downstream of the start codon in an unusual but not unprecedented fashion. Ph.D. in Biology, Harvard University. Joshua Jelly-Schapiro is the author of Island People: The Caribbean and the World (Knopf, 2016) and the co-editor, with Rebecca Solnit, of Nonstop Metropolis: A New York City Atlas (California, 2016). Ph.D., 2015, University of Utah (Oncological Sciences) NIH Developmental Biology Training Grant graduate trainee. View details for DOI 10.1002/cphc.201100686, View details for Web of Science ID 000301537300020. View details for DOI 10.1016/j.cell.2005.12.033. View details for Web of Science ID A1994PM98800011. 2015 California Institute of Technology, Graduate Student, Biochemistry Here, we show that the cytoplasmic response regulator, DivK, exhibits a dynamic, cyclical localization that culminates in asymmetric distribution of DivK within the two cell types that are characteristic of the Caulobacter cell cycle; DivK is dispersed throughout the cytoplasm of the progeny swarmer cell and is localized to the pole of the stalked cell. The P1 promoter fires early in S phase and contains a GAnTC sequence that is recognized by the CcrM DNA methyltransferase. The phage is composed of 57% DNA. DnaA initiates DNA replication and activates the transcription of the next cell-cycle regulator, GcrA. Control of sequential cell changes at the level of transcription has long been postulated and has recently been substantiated in the case of Bacillus sporulation (6). These controls include temporally regulated transcription and phosphorylation and spatially restricted proteolysis. Using DnaA depletion and induction in synchronized cell populations, we have analysed global transcription patterns to identify the differential regulation of normally co-expressed genes. The contribution of each promoter for genes transcribed from multiple promoters is identified. The uranium reporter construct was effective for discriminating contaminated groundwater samples (4.2 microM uranium) from uncontaminated groundwater samples (<0.1 microM uranium) collected at the Oak Ridge Field Research Center. 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. Previous microarray experiments revealed that several Caulobacter genes are significantly upregulated in response to uranium but not in response to other heavy metals. Nature Communications13, 493 (2022). Flagellated and non-flagellated vesicles were prepared from these cells by immunoaffinity chromatography and the level of MCPs that had been labeled either in vivo or in vitro with methyl-3H was determined. (i) Is the temporal progression of events occurring during bacterial differentiation controlled by regulator gene products? These discoveries have advanced our understanding of bacterial physiology and provided insight into the evolution of the eukaryotic cytoskeleton. A specialized protein secretion pathway is used by some Gram-negative bacterial pathogens for delivery of virulence factors directly into mammalian host cells. Roberts, R. C., Toochinda, C., Avedissian, M., Baldini, R. L., Gomes, S. L., Shapiro, L. Regulation of a heat shock sigma(32) homolog in Caulobacter crescentus, A cell cycle-regulated bacterial DNA methyltransferase is essential for viability. The C-terminal crystallization domain forms the physiological 2-dimensional (2D) crystal lattice, but full-length protein crystallizes multiple orders of magnitude faster due to the N-terminal nucleation domain. View details for Web of Science ID 000233399500043. We describe the identification of 27 novel Caulobacter crescentus sRNAs by analysis of RNA expression levels assayed using a tiled Caulobacter microarray and a protocol optimized for detection of sRNAs. 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. 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. Cell division in Caulobacter crescentus yields progeny cells that differ with respect to cell structure and developmental program. Using site-directed mutagenesis, we provide the first demonstration that natural enhancer sequences and IHF binding elements that reside 3' to the sigma 54 promoter of a bacterial gene, flaNQ, are required for transcription of the operon, in vivo. Yoo S, Mittelstein DR, Hurt RC, Lacroix JJ, Shapiro MG*. This structure is absent at the flagellar pole but not in the stalks of flbT mutant predivisional cells. Here we provide a detailed protocol for the rapid synchronization of Caulobacter NA1000 cells. As a result of the altered genetic structure, these tmRNAs are composed of two distinct RNA molecules. She is the Virginia and D.K. Homologous sequences were then detected by computer analysis of the published IS1 and IS2 nucleotide sequences. We show that the peptidoglycan transpeptidase Pbp2 also forms a helical pattern that partially colocalizes with MreC but not MreB. 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. S-layer proteins (SLPs) regulate their extracellular self-assembly by crystallizing when exposed to an environmental trigger. Biol. In this context, we have found that the flgF operon and the distal flgI gene encoding the P-ring, share a sigma 54 activator sequence (class IIA) that differs from the flgH L-ring gene sigma 54 activator site (class IIB) and the hook cluster (class IIC) sigma 54 activator site. Caulobacter crescentus initiates a single round of DNA replication during each cell cycle. We propose that the P1 promoter is activated after the initiation of DNA replication in the early predivisional cell. In order to elucidate the basic mechanisms whereby a cell dictates its own defined morphogenic changes, we have found it helpful to study an organism that can be manipulated both biochemically and genetically. Speaking this week as part of the "What Matters to Me and Why" series hosted by the Stanford Office of Religious Life, Shapiro said the topic prompted her to ponder why she was so passionate about the world of molecules and cells, a world invisible to most people. We have attempted to develop the studies initiated by Poindexter,Stove and Stanier, and Schmidt and Stanier (16, 17, 20) with the Caulobacter genus so that these bacteria can serve as a model system for prokaryotic differentiation. Caulobacter crescentus assembles many of its cellular machines at distinct times and locations during the cell cycle. Analysis of the cloned and sequenced dnaK gene has shown that the deduced amino acid sequence could encode a protein of 67.6 kilodaltons that is 68% identical to the DnaK protein of Escherichia coli and 49% identical to the Drosophila and human hsp70 protein family. Thus, the hook operon upstream region contains a sequence element that responds to a temporally controlled trans-acting factor(s), and in concert with a second sequence element causes the timed activation of transcription. View details for DOI 10.1038/s41467-019-10650-x. The course will serve as a literature-based introductory guide for synthesis of ideas in developmental biology and cancer, with an emphasis on evolutionary analysis and Includes bibliographical references (leaves 124-126). We seek to understand the mechanisms that regulate self-renewal, proliferation and differentiation in adult stem cell linages. Molecular genetics of simple developmental systems. 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. 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. Welcome ARGs 2.0! View details for Web of Science ID A1991EW29800007. A localized adaptor protein performs distinct functions at the Caulobacter cell poles. Vital to bacterial survival is the faithful propagation of cellular signals, and in Caulobacter crescentus, ChpT is an essential mediator within the cell-cycle circuit. 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. Join the team at Stanford Anatomic Pathology and Clinical Labs. Jian Ciao - Postdoc, Matthew Scott Lab. We found that MmpA facilitates the degradation of PodJS. Here we demonstrate that the bacterium Caulobacter crescentus segregates its chromosome using a partitioning (Par) apparatus that has surprising similarities to eukaryotic spindles. The protein components of the flagellar hook and filament are synthesized just prior to their assembly. We imaged fusions of dL5 to three different proteins in live Caulobacter cells using stimulated emission depletion microscopy, yielding a 4-fold resolution enhancement compared to diffraction-limited imaging. Professor of Neuroscience and Biological Engineering Heritage Principal Investigator Director of the Molecular and Cellular Neuroscience Center of the Chen Institute at Caltech Flagellar and chemotaxis genes are transcribed at a discrete time in the Caulobacter cell cycle. A major direction in the lab is to understand how such long-range interactions occur, how they achieve target specificity, and how they may be reprogrammed by alterations to the genome sequence. Driks, A., Bryan, R., Shapiro, L., DeRosier, D. J. IMAGE-RECONSTRUCTION OF THE FLAGELLAR BASAL BODY OF CAULOBACTER-CRESCENTUS. PodJ provides the spatial cues for the biogenesis of several polar organelles, including the pili, adhesive holdfast and chemotactic apparatus, by recruiting structural and regulatory proteins, such as CpaE and PleC, to a specific cell pole. Examined the fatty acid biosynthesis and composition of two distinct RNA molecules H., Shapiro, L., Hodgson D.! And composition of two distinct RNA molecules expressed early in the predivisional cell that... Protein appeared at the Caulobacter cell cycle as an endogenous C. crescentus, CcrM-mediated methylation has important functions! 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