In addition, we demonstrate that borinic esters inhibit menaquinone methyltransferase in gram positive bacteria using a new biochemical assay for MenH from Bacillus subtilis. The synthesis of the product of flgJ, the 29K flagellin, occurs prior to the synthesis of the other flagellin proteins. The sophistication of the genetic regulatory circuits and the elegant integration of temporally controlled transcription and protein synthesis with spatially dynamic phosphosignaling and proteolysis pathways, and epigenetic regulatory mechanisms, form a remarkably robust living system. A., Eckart, M. R., Shapiro, L. Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle. Caulobacter crescentus divides asymmetrically generating two distinct cell types at each cell division: a stalked cell competent for DNA replication, and a swarmer cell that is unable to initiate DNA replication until it differentiates into a stalked cell later in the cell cycle. The proteins of the Caulobacter cell cycle control system and its internal organization are co-conserved across many alphaproteobacteria species, but there are great differences in the regulatory apparatus' functionality and peripheral connectivity to other cellular subsystems from species to species. The system architecture of Caulobacter cell-cycle control involves top-down control of modular functions by a small number of master regulatory proteins with cross-module signaling coordinating the overall process. Symposium on Problem Fractures of the Hand and Wrist. This DNA contains sequence motifs that are common to other bacterial origins, such as five DnaA boxes, an E. coli-like 13-mer, and an exceptional A + T-rich region. Cell cycle progression in Caulobacter is driven by the master transcriptional regulators CtrA and GcrA. View details for Web of Science ID 000088048400024, View details for PubMedCentralID PMC16621. The B, C, and D transcripts were present in equivalent molar ratios, were all smaller than transcript A, and were found to yield RNase III digestion products that were subsets of each other as well as of transcript A. Lew, M. D., Lee, S. F., Ptacin, J. L., Lee, M. K., Twieg, R. J., Shapiro, L., Moerner, W. E. The Three-Dimensional Architecture of a Bacterial Genome and Its Alteration by Genetic Perturbation. Transcription initiation from this region was also detected in vivo, when the cloned rRNA gene cluster was present on a multi-copy plasmid. A key feature of the lambda genetic circuit is that operons function as active integrated logic components and introduce signal time delays essential for the in vivo behavior of phage lambda. Hodgson, D., Shaw, P., Letts, V., Henry, S., Shapiro, L. ISOLATION AND GENETIC-ANALYSIS OF CAULOBACTER MUTANTS DEFECTIVE IN CELL-SHAPE AND MEMBRANE LIPID-SYNTHESIS, GENERATION OF A TN5 PROMOTER PROBE AND ITS USE IN THE STUDY OF GENE-EXPRESSION IN CAULOBACTER-CRESCENTUS. 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. 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. Lab Objectives. 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. 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. A number of different factors appear to cooperate in condensing DNA into a highly dynamic assembly of supercoiled loops. Strategies to encode or label small particles or beads for use in high-throughput screening and bioassay applications focus on either spatially differentiated, on-chip arrays or random distributions of encoded beads. 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. We propose that the maintenance of DNA topology throughout the cell cycle contributes to the dynamic positioning of the origin sequence within the cell. Rate enhancement of protein crystallization by a discrete nucleation domain may enable engineering of kinetically controllable self-assembling 2D macromolecular nanomaterials. ErTadZ contains an atypical ATPase domain with a variant of a deviant Walker-A motif that retains ATP binding capacity while displaying only low intrinsic ATPase activity. The phospholipid composition of Caulobacter crescentus CB13 and CB15 was determined. The predicted amino acid sequence of the leader peptide of flaD is very similar to the leader peptide of the flagellar basal body P ring of Salmonella typhimurium (M. Homma, Y. Komeda, T. Iino, and R.M. The mutant strain, AE6000 , was altered in both of these regulatory functions. View details for DOI 10.1016/j.tim.2005.03.006, View details for Web of Science ID 000229467100008. The flgH gene, encoding the L-ring protein, is also transcribed from an internal promoter. We report a hybrid simulation of the coupled cell-cycle control system, including asymmetric cell division and responses to external starvation signals, that replicates mRNA and protein concentration patterns and is consistent with observed mutant phenotypes. The distribution of MCPs was examined in flagellated and non-flagellated vesicles isolated from predivisional cells. These inverted repeat sequences are analogous to the RNase III-processing sites in the E. coli rRNA precursor. Mutants of Escherichia coli have been isolated that are able to grow on lactose at pH 7.0 but not at pH 8.1. Because the ultimate expression of cell changes are dependent on selective protein synthesis, specific messenger RNA production-either from DNA present in an organelle or from the chromosome-may prove to be a controlling factor in cell differentiation. We study how the distribution of such signals is regulated in tissues, how cells perceive and respond to distinct concentrations of signals, and how such signaling pathways arose in evolution. The two-component signaling protein CtrA activates or represses the expression of one-quarter of the cell-cycle-regulated genes in Caulobacter crescentus, integrating DNA replication, morphogenesis, and cell division. A fliX null mutant is nonmotile, lacks a flagellum, and exhibits a marked cell division defect. 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. 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. Reisenauer, A., Kahng, L. S., McCollum, S., Shapiro, L. Changing views on the nature of the bacterial cell: from biochemistry to cytology, Feedback control of a master bacterial cell-cycle regulator. A penile spine/vibrissa enhancer sequence is missing in modern and extinct humans but is retained in multiple primates with penile spines and sensory vibrissae. View details for DOI 10.1111/j.1365-2958.2008.06172.x, View details for Web of Science ID 000254641600007, View details for Web of Science ID 000208467800418, View details for Web of Science ID 000255316100052. The essential dnaN gene encodes a homolog of the Escherichia coli beta subunit of DNA polymerase III. Our work demonstrates the conserved extreme polar localization of circular chromosome origins in these alpha proteobacteria and is also the first to specify the cellular location of origin regions from the repABC family. Under conditions which inhibited DNA synthesis but permitted phospholipid synthesis, i.e., growth of a temperature-sensitive DNA elongation mutant at the restrictive temperature or treatment with hydroxy-urea, stalk elongation occurred normally. Biomolecular enhancers for fUS. 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. We find that the CtrA proteolysis adaptor protein PopA assumes distinct oligomeric states at the two cell poles through asymmetrically distributed c-di-GMP: dimeric at the stalked pole and monomeric at the swarmer pole. This positional bias of MCPs within predivisional cells could reflect either a large compartment or membrane domain within the incipient swarmer cell, or a gradient of MCPs, with the highest concentration in the vicinity of the flagellum. In particular, the distribution of HU, which is the most abundant NAP, has received little attention. In C. crescentus, additional control mechanisms ensure that the transcription of these genes is initiated at the correct time in the cell cycle. Topoisomerases play a key role in ensuring orderly replication and partition of DNA in the face of a continuously changing DNA tertiary structure. Leonard, K. R., Maizel, J. V., AGABIANK, N., Shapiro, L., KLEINSCH, A. K. EFFECT OF DIBUTYRYLADENOSINE 3'-5'-CYCLIC MONOPHOSPHATE ON GROWTH AND DIFFERENTIATION IN CAULOBACTER-CRESCENTUS. 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. Additionally, we investigated the genetic dependence of localization among divisome proteins and the cell cycle regulation of their transcript and protein levels to gain insight into the control mechanisms underlying their assembly. Based on the correlation of the physical and genetic maps derived by Ely and Gerardot [Ely, B. The addition of dibutyryl cyclic AMP to the blocked cultures brought about the resumption of cell differentiation, growth, and the appearance of beta-galactosidase activity within 1 hr. Marczynski, G. T., LENTINE, K., Shapiro, L. REGULATION OF THE CAULOBACTER-CRESCENTUS DNAKJ OPERON. We present evidence that DivK, an essential single-domain response regulator, contributes to the control of the G(1)-S transition by signaling the temporally controlled proteolysis of CtrA. Using a modified global 5' RACE protocol, we globally mapped transcription start sites (TSSs) at base-pair resolution, measured their transcription levels at multiple times in the cell cycle, and identified their transcription factor binding sites. View details for Web of Science ID A1978FP55600049, View details for Web of Science ID A1978FP11300023, View details for Web of Science ID A1977DL60800021. Mohr, C. D., MacKichan, J. K., Shapiro, L. A cell cycle-regulated adenine DNA methyltransferase from Caulobacter crescentus processively methylates GANTC sites on hemimethylated DNA. A simple antibody-based bioassay using fluorescently tagged proteins demonstrates the encoding strategy in biologically relevant media. The methylation state of 4,515 GANTC sites, preferentially positioned in intergenic regions, changed progressively from full to hemimethylation as the replication forks advanced. A genetically engineered transposon promoter probe, Tn5-VB32, containing a promoterless gene encoding neomycin phosphotransferase II (NPTase II) was used to generate a series of non-motile (fla-), kanamycin resistant strains of C. crescentus. To investigate the potential role of the actin-like MreB protein in bacterial chromosome segregation, we first demonstrate that MreB is the direct target of the small molecule A22. A developmental mutant of C. crescentus with altered polar surface structures has been isolated. CtrA binds to and silences the origin. Caulobacter crescentus undergoes an asymmetric cell division controlled by a genetic circuit that cycles in space and time. There have been two sharp demarcations in my life in science: the transition from fine arts to chemistry, which happened early in my career, and the move from New York to Stanford University, which initiated an ongoing collaboration with the physicist Harley McAdams. Studies of the genetic network that controls the Caulobacter cell cycle have identified a response regulator, CtrA, that controls, directly or indirectly, one-quarter of the 553 cell cycle-regulated genes. Interested applicants should visit https://facultypositions.stanford.edu/en-us/job/493432 for our full ad and more information about how to apply. The diameters of the two upper rings differed, being 32 and 21 nm, respectively. Insertions within temporally regulated genes, such as those involved in flagellar biosynthesis and chemotaxis functions, can now be used directly to monitor transcriptional regulation from Caulobacter promoter sequences. To define the mechanisms that mediate this temporal and spatial control, fla genes whose products are not known were accessed by the insertion of transposon-carried drug resistance markers. The bacterium Caulobacter crescentus has morphologically and functionally distinct cell poles that undergo sequential changes during the cell cycle. We are interested in candidates who will establish a vigorous and innovative research program studying fundamental biological processes in any experimental system. The 92,000 Mr Lon homolog and the 37,000 Mr RNA polymerase subunit were preferentially synthesized in the stalked cell, whereas the synthesis of the 62,000 Mr GroEL homolog was enhanced in the progeny swarmer cell. The cell-cycle control has a cyclical genetic circuit composed of four regulatory proteins with tight coupling to processive chromosome replication and cell division subsystems. View details for DOI 10.1128/JB.185.6.1825-1830.2003, View details for Web of Science ID 000181448900009, View details for PubMedCentralID PMC150134. We discuss the genetic network and integrated three-dimensional sensor/response systems that regulate the cell cycle and asymmetric cell division in the bacterium Caulobacter crescentus. 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. View details for Web of Science ID A1997YB26700002. Six distinct cellular characteristics, which are peculiar to these bacteria, have been defined and include (i) the synthesis of a polar organelle which may be membranous (21-23), (ii) a satellite DNA in the stalked cell (26), (iii) pili to which RNA bacteriophage specifically adsorb (16, 33), (iv) a single polar flagellum(17), (v) a lipopolysaccharide phage receptor site (27), and (vi) new cell wall material at the flagellated pole of the cell giving rise to a stalk (19, 20). A., Deacon, A. M., Shapiro, L. Using Optically Reversible Spatial Mutations to Dissect the Asymmetric Developmental Program of a Bacterium. View details for DOI 10.1046/j.1365-2958.2003.03576.x, View details for Web of Science ID 000184224700005, View details for DOI 10.1073/pnas.1332806100, View details for Web of Science ID 000183845800003, View details for PubMedCentralID PMC164599. 2015;33 (11): 1639-1645, journal of hand surgery -Park, M. J., Ganjoo, K. N., Ladd, A. L.2015;40 (8): 1620-1624, CLINICAL ORTHOPAEDICS AND RELATED RESEARCH -Ladd, A. L.2015;473 (8): 246063, JOURNAL OF BIOMECHANICS -Halilaj, E., Rainbow, M. J., Moore, D. C., Laidlaw, D. H., Weiss, A. C., Ladd, A. L., Crisco, J. J. Perturbing either MreB (with A22) or MreC (with depletion) causes GFP-Pbp2 to mislocalize to the division plane, indicating that each is necessary but not sufficient to generate a helical Pbp2 pattern. The apparent dissociation constant for the cyclic GMP-binding protein complex is 1.1 X 10(-6) M. View details for Web of Science ID A1975AM69800061, View details for Web of Science ID A1974U579000028. The second thing I discovered is that Stanford has a special hair loss clinic, with the sole doctor at the clinic being Dr. Anthony Oro, after whom the Oro Laboratory is Research in the Department of Developmental Biology at Stanford is aimed at understanding the molecular mechanisms that generate and maintain diverse cell types during development. Here, we report that in Caulobacter, a hipA2-encoded bacterial toxin contributes to bacterial persistence by manipulating intracellular amino acid balance. Here we demonstrate that the bacterium Caulobacter crescentus segregates its chromosome using a partitioning (Par) apparatus that has surprising similarities to eukaryotic spindles. Thus, we propose that the Caulobacter chromosomal origins have specific cellular addresses and that the SMC protein plays important roles in maintaining chromosome structure and in partitioning. View details for DOI 10.1038/sj.emboj.7600927, View details for Web of Science ID 000234952500008, View details for PubMedCentralID PMC1383511. A general mechanism in bacteria to rescue stalled ribosomes and to clear the cell of incomplete polypeptides involves an RNA species, tmRNA (SsrA), which functions as both a tRNA and an mRNA. Regulatory factors that initiate forespore-specific transcription during Bacillus subtilis sporulation respond to adenosine nucleotide ratios. Also, cell pole morphogenesis and initiation of chromosome replication normally occurring at the swarmer-to-stalked cell transition are uncoupled in carbon-starved cells. Phase separation in many eukaryotic condensates has been shown to be responsive to intracellular adenosine triphosphate (ATP) levels, although the consequences of these mechanisms for enzymes sequestered within the condensates are unknown. Expression of the ccrM gene was found to be restricted to the portion of the cell cycle immediately prior to cell division. Imaging and controlling cellular function with ultrasound. Biteen, J. S., Thompson, M. A., Tselentis, N. K., Bowman, G. R., Shapiro, L., Moerner, W. E. A bacterial control circuit integrates polar localization and proteolysis of key regulatory proteins with a phospho-signaling cascade, Caulobacter requires a dedicated mechanism to initiate chromosome segregation. Able to grow on lactose at pH 7.0 but not at pH 7.0 not. By Ely and Gerardot [ Ely, B exhibits a marked cell.... And innovative research program studying fundamental biological processes in any experimental system kinetically controllable self-assembling 2D macromolecular nanomaterials not pH..., View details for DOI 10.1038/sj.emboj.7600927, View details for Web of Science 000234952500008. 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