Colocalization experiments with the genomic replicons of A. tumefaciens revealed that the repABC replicons, although preferentially positioned at the cell pole, colocalize only rarely. The centromere-binding protein ParB binds to and destabilizes ParA structures in vitro. View details for Web of Science ID A1984SH73800012. 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 hook structure was purified from nonflagellated mutants and found to be composed of a 70,000-molecular-weight protein component. View details for Web of Science ID A1979HV87000036. 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. To selectively repress and limit chromosome replication, CtrA receives both protein degradation and protein phosphorylation signals. Lateral positions of labeled loci at comparable positions along the length of the cell are strongly correlated when the longitudinal locus positions differ by <0.16 m. View details for Web of Science ID 000181056400008. RNase E formed clusters along the central axis of the cell, while weak clusters of ribosomal protein L1 were deployed throughout the cytoplasm. WebShapiro Lab University of Illinois, Department of Biochemistry. View details for Web of Science ID A1996TQ17000011, View details for Web of Science ID A1996BG26T00006. B.S. Currently: Associate Director of ML/AI Gital, Z., Dye, N. A., Reisenauer, A., Wachi, M., Shapiro, L. A membrane metalloprotease participates in the sequential degradation of a Caulobacter polarity determinant. The penicillin-binding protein PBP2, which is commonly branded as an essential cell elongation-specific transpeptidase, switches its localization from a dispersed, patchy pattern to an accumulation at the FtsZ ring location in response to osmotic upshifts as low as 40 mosmol/kg. The promoters for the flgF operon and the flgH gene use sigma 54 to initiate transcription. Thanks to the researchers from 10 countries who joined us for this inaugural event and to all the Shapiro Lab members who participated, helped organize, and hosted live demos. Caulobacter crescentus wild-type strain CB13 is unable to utilize galactose as the sole carbon source unless derivatives of cyclic AMP are present. Our recent discoveries include findings that ion channels, receptors and signaling proteins are clustered into discrete nanodomains that orchestrate directed signals in ganglia and brain, nanodomains which we have visualized using visible light at 5 nm resolution via STORM nanoscopy. Such nanodomain complexes of proteins direct neuronal excitability in distinct and purposeful ways, guide transcriptional expression, and underlie plasticity, cognition and circuit development in brain. Ph.D. Student, Neurobiology View details for Web of Science ID A1983RA96700072. 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. SURF Scholar 2022- 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. After the equivalent of one generation time, rapid cell death occurred. Cell type determinants in stalked progeny promote entry into S phase, whereas swarmer progeny remain in G1 phase. Individual cells containing enhanced GFP were exposed to a bleaching laser pulse tightly focused at one cell pole. A., Shapiro, L. Spatial complexity and control of a bacterial cell cycle, An antifungal agent inhibits an aminoacyl-tRNA synthetase by trapping tRNA in the editing site. Ph.D. Medical Engineering, Caltech Currently: Interventional Radiology Residency View details for Web of Science ID A1980JX98100009. Our data indicate that differences in the affinity of CtrA approximately P for PfliQ and PccrM regulate, in part, the temporal expression of these genes. Only one of the two chromosomes in the asymmetric Caulobacter predivisional cell initiates replication in the progeny cells. Comerci, C. J., Herrmann, J. n., Yoon, J. n., Jabbarpour, F. n., Zhou, X. n., Nomellini, J. F., Smit, J. n., Shapiro, L. n., Wakatsuki, S. n., Moerner, W. E. Identification of PAmKate as a Red Photoactivatable Fluorescent Protein for Cryogenic Super-Resolution Imaging. Hear Jeffs story. This pathway involves 2 domains serving distinct functions in assembly. Science may have been her second choice, but Shapiro, who joined the Stanford faculty in 1989 after serving as chair of the Microbiology and Immunology Department at Columbia University, has certainly made a rousing success of it: In the past few years, she's garnered an impressive list of national and international awards for her This information, combined with quantitative localization data from epitasis experiments, also identified all previously known proteins affecting such localization. Search results about individual genes are displayed as tables, graphs of time resolved expression profiles, and schematics of protein localization throughout the cell cycle. View details for Web of Science ID A1987G456800007. The Caulobacter crescentus bacteriophage phiCbK was studied with respect to the physical and chemical properties of both the phage and its deoxyribonucleic acid (DNA). 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. We have also identified a new structural feature at the flagellated pole and the tip of the stalk: the 10-nm polar particle. Christen, B., Fero, M. J., Hillson, N. J., Bowman, G., Hong, S., Shapiro, L., McAdams, H. H. Bacterial Chromosome Organization and Segregation, System-level design of bacterial cell cycle control, Feedback Control of DnaA-Mediated Replication Initiation by Replisome-Associated HdaA Protein in Caulobacter, Superresolution imaging of protein superstructures in live Caulobacter crescentus cells with EYFP. [email protected], x=kmcgraw, Rucheng Pan View details for Web of Science ID A1990CW01800056. Here, using Caulobacter crescentus as a model, we exploit genome-wide experimental methods to uncover the functions of CcrM, a DNA methyltransferase conserved in most Alphaproteobacteria. A hallmark of the Caulobacter cell cycle is that the progeny cells that result from each cell division differ from one another with respect to structure and developmental program. Since the dnaK coding region is 1.89 kilobases, dnaK and dnaJ may be transcribed as a polycistronic message. Dibutyryl cyclic adenosine 3',5'-monophosphate (AMP) was shown to stimulate expression of the inducible enzymes and, thus, the initiation of the cell cycle. The mutant phenotype indicates that the assembly of the polar surface structures is coordinately regulated and independent of mechanisms regulating cell polarity and division. During the normal course of the C. crescentus cell cycle, the polar flagellum with hook and rod was shed into the culture medium without the basal rings. B.S. A plasmid, whose replication relies only on DNA from the C. crescentus origin of replication, has a distinct temporal pattern of DNA synthesis that resembles that of the bona fide C. crescentus chromosome. Ph.D. Mech. DNA methylation is involved in a diversity of processes in bacteria, including maintenance of genome integrity and regulation of gene expression. Scientists already knew the atoms in perovskites, a promising class of solar cell materials, react favorably to light. The timing of DnaA accumulation was found to be regulated by the methylation state of the dnaA promoter, which in turn depends on the chromosomal position of dnaA near the origin of replication and restriction of CcrM synthesis to the end of the cell cycle. As a result of the altered genetic structure, these tmRNAs are composed of two distinct RNA molecules. Chemistry, Tsinghua University Surface layers (S-layers) are crystalline protein coats surrounding microbial cells. The molecular weights of the enzyme subunits were 165,000, 155,000, 101,000, and 44,000, respectively. View details for DOI 10.1016/j.ceb.2010.10.013, View details for Web of Science ID 000288349000010. 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. The experimental techniques include patch-clamp electrophysiology, cutting-edge molecular biology, advanced imaging techniques such as total internal reflection fluorescence (TIRF) microscopy, Frster resonance energy transfer (FRET), confocal and super-resolution microscopy, molecular and cellular modeling, biophysical chemical analysis of channel complexes and structural biology. 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. 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. Xu, Q., Christen, B., Chiu, H., Jaroszewski, L., Klock, H. E., Knuth, M. W., Miller, M. D., Elsliger, M., Deacon, A. M., Godzik, A., Lesley, S. A., Figurski, D. H., Shapiro, L., Wilson, I. Because mutations in the RRF motif result in constitutive gene expression throughout the cell cycle, this sequence is likely to be the binding site for a cell cycle-regulated transcriptional repressor. Examination of the 16 S rRNA genes from other bacterial species and chloroplasts and 18 S rRNA genes from Xenopus and yeast revealed that the nucleotide sequence of this internal 16 S rRNA promoter region was highly conserved. Work from several Caulobacter labs has revealed that differentiation requires concerted regulation by several two-component system (TCS) signaling pathways that are differentially positioned at the poles of the predivisional cell (Figure 1). Here we investigate the effect on cell polarity of two cytoskeletal elements previously implicated in cell shape determination. Cell death also occurred when phospholipid synthesis was inhibited by cerulenin. Enzyme from pure populations of stalked cells, as well as populations enriched in swarmer and predivisional cells, appeared identical in subunit structure and template requirements. The actin homolog MreB contributes to bacterial cell shape. To understand how ChpT interacts with multiple signaling partners, we solved the crystal structure of ChpT at 2.3 resolution. A high proportion of morphologically aberrant cells, and cells that have undergone an additional chromosome replication initiation, are found in this population. Bozdemir, E., Vigil, F.A., Bugay, V., Espinoza, L., Chun, S.H., Hobbs, M., Khoury, S., Holstein, D., Sanchez, I., Cavazos, J., Brenner, R., Carver, C.M., Hastings, S.D., Cook, M.E., and. When an active CtrA protein is present at the wrong time in the cell cycle, owing to expression of a mutant CtrA derivative that is active in the absence of phosphorylation and is not turned over during the cell cycle, the G1-to-S transition is blocked and the cell cycle aborts. The dynamic behavior of these proteins is often intrinsically linked to their function as polarity determinants. Finally, we examined the fatty acid biosynthesis and composition of two unsaturated fatty acid auxotrophs of C. crescentus. WebShapiroLab Going Deeper We develop technologies to image and control the function of cells deep inside the body. The molecular weight of the genome was estimated to be approximately 28.8 X 10(6) on the basis of the relative electrophoretic mobilities of the restriction fragments. Point mutations in one of the DnaA boxes abolish replication in C. crescentus. View details for Web of Science ID 000185536700040. Chemistry, expected 2023 ChpT functions as a histidine-containing phosphotransfer protein (HPt) that shuttles a phosphoryl group from the receiver domain of CckA, the upstream hybrid histidine kinase (HK), to one of two downstream response regulators (CtrA or CpdR) that controls cell-cycle progression. Protein secretion is essential for bacterial cell growth and virulence, so it could be a suitable target for new therapeutic agents. Thus, it is the signal transduction pathway mediated by CckA that culminates in CtrA activation, which is temporally regulated and essential for cell cycle progression. View details for DOI 10.1073/pnas.0807448105, View details for Web of Science ID 000260360500041, View details for PubMedCentralID PMC2563096. Gober, J. W., Xu, H., Dingwall, A. K., Shapiro, L. Temporal and spatial regulation of developmentally expressed genes in Caulobacter, IDENTIFICATION OF A CAULOBACTER BASAL BODY STRUCTURAL GENE AND A CIS-ACTING SITE REQUIRED FOR ACTIVATION OF TRANSCRIPTION, REGULATORY INTERACTIONS BETWEEN PHOSPHOLIPID-SYNTHESIS AND DNA-REPLICATION IN CAULOBACTER-CRESCENTUS. This heading includes several more specific ancient DNA research projects currently underway in the Shapiro lab. Thus, the sequential degradation of PodJ appears to involve regulated intramembrane proteolysis (Rip) by MmpA. Hillson, N. J., Hu, P., Andersen, G. L., Shapiro, L. A comprehensive set of plasmids for vanillate- and xylose-inducible gene expression in Caulobacter crescentus. WebNatera advances molecular diagnostics with integrity and scientific rigor, and supports integration of information provided by our tests into health care decision making. Caulobacter crescentus contains a single chromosome that is replicated once during a defined period in the cell cycle. The high-resolution strategy used here is applicable to high-throughput, full genome essentiality studies and large-scale genetic perturbation experiments in a broad class of bacterial species. Furthermore, the FtsK N terminus is required to either assemble or maintain FtsZ rings at the division plane. Analysis of bacterial genome organization and replication using pulsed field gel electrophoresis, THE MOLECULAR-GENETICS OF DIFFERENTIATION, NEGATIVE TRANSCRIPTIONAL REGULATION IN THE CAULOBACTER FLAGELLAR HIERARCHY, AN ESCHERICHIA-COLI CHEMORECEPTOR GENE IS TEMPORALLY CONTROLLED IN CAULOBACTER, THE ORGANIZATION OF THE CAULOBACTER-CRESCENTUS FLAGELLAR FILAMENT. A systematic search for motifs associated with CcrM DNA methylation sites revealed four long (>100-bp) motifs (CIR sequences) present in up to 21 copies in Caulobacter crescentus. However, molecular mechanisms governing rapid protein crystallization in vivo or in vitro are largely unknown. Two genes encoding the major components of the flagellar filament, the 25K and the 27.5K flagellins, are expressed coincident with flagellar assembly. Saurabh, S., Perez, A., Comerci, C., Shapiro, L., Moerner, W. A Localized Complex of Two Protein Oligomers Controls the Orientation of Cell Polarity. The CtrA master transcriptional regulator is a central control element in Caulobacter cell cycle progression and polar morphogenesis. Bowman, G. R., Comolli, L. R., Gaietta, G. M., Fero, M., Hong, S., Jones, Y., Lee, J. H., Downing, K. H., Ellisman, M. H., McAdams, H. H., Shapiro, L. High-throughput identification of protein localization dependency networks. Wheeler, R. T., Gober, J. W., Shapiro, L. DNA replication - Bringing the mountain to mohammed, Microbial asymmetric cell division: Localization of cell fate determinants, A membrane-associated protein, FliX, is required for an early step in Caulobacter flagellar assembly. Milton and Francis Clauser Doctoral Prize Winner (top PhD thesis at Caltech) Here we demonstrate that the Caulobacter PopZ scaffold creates an organizing center at the cell pole that actively regulates polar centromere transport by the ParA partition system. Small Molecule Biomodulators for Studying and Treating Diseases Our focus is on the discovery and use The switch to the second phospholipid profile was observed to occur at the predivisional cell stage. 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. Bacterial chromosome segregation: Is there a mitotic apparatus? Homology between the IS elements and various genomes was determined by hybridizing labeled DNA containing IS1, IS2, and IS5 sequences to Southern blots of chromosomal DNA cleaved with restriction endonucleases. Cumulatively, this study uncovered novel new layers of transcriptional regulation mediating the bacterial cell cycle. The second PAS domain interacts with the asymmetrically partitioned second messenger cyclic-di-GMP, inhibiting kinase activity while stimulating phosphatase activity, consistent with the selective inactivation of CtrA in the incipient stalked cell compartment. Upon the clearance of CtrA from the cell, the DnaA protein accumulates and allows loading of the replisome at the origin. A century-old mystery for Gas Vesicles. In particular, the distribution of HU, which is the most abundant NAP, has received little attention. Our results revealed a picture of divisome assembly with unprecedented temporal resolution. Our work demonstrates how a second messenger provides spatiotemporal cues to change the physical behavior of an effector protein, thereby facilitating asymmetry. The PleA protein contains a region that is similar to a peptidoglycan-hydrolytic active site, and a point mutation at this site in PleA results in the loss of flagellum and pili biogenesis. WebThe Shapiro lab is part of the Department of Molecular and Cellular Biology at the University of Guelph. The flaD BC motC region was cloned and the genes were localized by subcloning and complementation analysis. We show that the S. meliloti CtrA belongs to the CtrA-like family of response regulators found in several alpha-proteobacteria. We discovered an essential DNA-associated protein, GapR, that is required for Caulobacter growth and asymmetric division. These include the MopD and MopE proteins from Erwinia carotovora, the Spa9 and Spa29 proteins from Shigella flexneri, and the YscS protein from Yersinia pestis. By focusing on the biogenesis of the polar flagellum and the proteins of the chemosensory system, several laboratories have now defined an extensive network of genes whose temporal expression is controlled in the predivisional cell. Nuclease S1 analysis also revealed a protected fragment whose size was consistent with a transcript initiating in vivo at a consensus "nif" promoter sequence in front of the flaY gene. View details for Web of Science ID A1997XE30000021, View details for PubMedCentralID PMC179208. By expressing an inducible roGFP2-PopZ fusion we can visualize individual microdomains in the context of their redox environment. Caltech The distinct control of available CcrM in progeny swarmer and stalked cells serves to protect the hemimethylated state of DNA during chromosome replication, enabling robustness of cell cycle progression.