In an effort to understand this developmental control of replication, we employed pulsed-field gel electrophoresis to localize and to isolate the chromosomal origin of replication. Accurate replication and segregation of the bacterial genome are essential for cell cycle progression. Affiliations: Bioengineering, Biochemistry, Neurobiology Caulobacter crescentus performs chemotaxis by short intermittent reversals of rotation of its single polar flagellum. jkim622@illinois.edu Typically, researchers describe the positions and speeds of particles in a beam in terms of a few summary statistics that provide a rough shape of the beam overall but that approach throws out a lot of potentially useful information. View details for Web of Science ID A1978FP55600049. The biogenesis of the polar flagellum in Caulobacter crescentus is limited to a specific time in the cell cycle and to a specific site on the cell. Caulobacter requires micromolar concentrations of calcium for normal growth and development. Thus, the Fix network is a conserved sensory/signaling module whose transcriptional output has been adapted to the unique physiologies of C. crescentus and the nitrogen-fixing rhizobia. 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. Finally, the C. crescentus and R. meliloti ccrM genes are functionally interchangeable, as the complemented strains are viable and the chromosomes are methylated. Our Research Scientist Two-component signal transduction proteins are known to play a significant role in cell cycle progression. Collaboration: High-throughput Screening, University of Illinois, Department of Biochemistry, Yu Zheng, Molecular and Cellular Biology, Class of 2020, Mara Livezey, PhD, Instructor at the University of Detroit Mercy, Xiaobin Zheng, PhD, Program Director for Health Data Science at Insight Data Science, Lily Mahapatra, MD/PhD, Resident in Anatomic and Clinical Pathology at Washington University School of Medicine in St. Louis, Mathew Cherian, MD/PhD, Resident in Emergency Medicine at the University of New Mexico, Neal D. Andruska, MD/PhD, Resident in Radiation Oncology at Washington University School of Medicine in St. Louis. One of these genes, flbN, is required early in the flagellar assembly process. Caulobacter crescentus undergoes an asymmetric cell division controlled by a genetic circuit that cycles in space and time. Expression of the latter two phenotypes required complex media and both were repressed by glucose. The 29K flagellin was found only in the progeny swarmer cell after cell division. Binding of cyclic GMP is not affected by the addition of cyclic AMP or 5'-GMP, but is inhibited about 50 percent by a 50-fold molar excess of dibutyryl cyclic GMP or cyclic hypoxanthine 3':5'-monophosphate. One of the phosphorylated DNA-binding proteins was identified as the beta' subunit of the host RNA polymerase. C. crescentus is, to our knowledge, the first free-living alpha-class proteobacterium to be sequenced and will serve as a foundation for exploring the biology of this group of bacteria, which includes the obligate endosymbiont and human pathogen Rickettsia prowazekii, the plant pathogen Agrobacterium tumefaciens, and the bovine and human pathogen Brucella abortus. Shapiro, L., Rosen, O. M., AGABIANK, N., Hirsch, A. BACTERIAL DIFFERENTIATION AND PHAGE INFECTION. Here we demonstrate the applicability of one such biosensor, the fluorescent protein roGFP2, for cryo-CLEM experiments. To explore the contribution of translational control, RNA-seq and ribosome profiling were used to assay global transcription and translation levels of individual genes at six times over the cell cycle. We further show that ClpXP localization is required for CtrA proteolysis. Known flagellar genes encode structural and regulatory components that are required for flagellar assembly and function. A novel promoter motif for Caulobacter cell cycle-controlled DNA replication genes, The control of temporal and spatial organization during the Caulobacter cell cycle, Bacterial pathogenesis: Delivering the payload, Caulobacter Lon protease has a critical role in cell-cycle control of DNA methylation. For this study, the team tried a new approach: They built a machine learning model that uses our understanding of beam dynamics to predict the distribution of particles positions and speeds within the beam, collectively known as the beam's phase space distribution. A nitrogen regulatory circuit can be regulated by the intracellular level of tryptophan, which mimics the allosteric role of glutamine in this feedback loop. These plasmids were mated into a wild-type strain and into strains containing various Tn5 insertion and deletion mutations and a temperature-sensitive mutation in the flaYE region. A developmental mutant of C. crescentus with altered polar surface structures has been isolated. One of these mutants was analyzed and shown to map in the Z region of the lactose operon. Dye, N. A., Pincus, Z., Theriot, J. SsrA, or tmRNA, is a small RNA that interacts with selected translating ribosomes to target the nascent polypeptides for degradation. B.S. Caulobacter crescentus carries a flagellum and is motile only during a limited time in its cell cycle. 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. Thus, two transcriptional feedback loops coupled to cell cycle-regulated proteolysis and phosphorylation of the CtrA protein result in the pattern of CtrA activity required for the temporal and spatial control of multiple cell-cycle events. Keiler, K. C., Shapiro, L., Williams, K. P. Identification and cell cycle control of a novel pilus system in Caulobacter crescentus, The Brucella abortus CcrM DNA methyltransferase is essential for viability, and its overexpression attenuates intracellular replication in murine macrophages. David Shapiro | Cancer Center at Illinois Replisome assembly occurs at the chromosomal origin located at the stalked cell pole, coincident with the initiation of DNA replication. 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. View details for Web of Science ID A1983RA96700072. In addition, mutations in either fliQ or fliR exhibit defects in cell division and thus may participate directly or indirectly in the division process. The first parameter correlates with genome GC content, and the second parameter correlates with context-dependent nucleotide bias. Brett SHAPIRO The total group of CtrA-regulated genes includes those involved in polar morphogenesis, DNA replication initiation, DNA methylation, cell division, and cell wall metabolism. WebBrett Shapiro | LIGO Group Group Affiliate Brett Shapiro Group Affiliate, Senior Professional Staff at Johns Hopkins Applied Physics Laboratory Contact brett.shapiro@jhuapl.edu 1200 E. California Blvd, MC210-41 Therefore, this structurally dynamic S-layer responds to environmental conditions as an ion sensor and protects Caulobacter from calcium deficiency stress, a unique mechanism of bacterial adaptation. 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. View details for DOI 10.1073/pnas.1220824110, View details for Web of Science ID 000314558100027, View details for PubMedCentralID PMC3562846. The released flagellum is composed of a filament, hook, and rod. An additional level of control was revealed when it was found that an interruption of DNA replication caused the inhibition of flaS transcription. Here we provide a detailed protocol for the rapid synchronization of Caulobacter NA1000 cells. Monitoring of a fluorescent marker for CtrA showed that the differential degradation of CtrA in the nascent stalk cell compartment occurs only after the cytoplasm is compartmentalized. Chen, S. L., Lee, W., Hottes, A. K., Shapiro, L., McAdams, H. H. A bacterial cell-cycle regulatory network operating in time and space, Identification of long intergenic repeat sequences associated with DNA methylation sites in Caulobacter crescentus and other alpha-proteobacteria, Fluorescence bleaching reveals asymmetric compartment formation prior to cell division in Caulobacter. View details for Web of Science ID A1988P905300045. 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. In contrast to the protein components of the hook and filament, the protein encoded by the flaD gene contains a hydrophobic leader peptide. Mechanical Engineering, Yonsei University During his time with us, he searched for a "first principle project" that defines life by We identified a major promoter transcribing both hrcA and grpE and a minor promoter located within the hrcA coding sequence just upstream of grpE. Ultrasound-controllable engineered bacteria for cancer immunotherapy. Assistant Professor, Department of Molecular and Integrative Physiology Biol. For questions or comments, contact the SLAC Office of Communications at communications@slac.stanford.edu. Flagellar biogenesis and release are developmental events tightly coupled to the cell cycle of Caulobacter crescentus. 38:164-198, 1974; Wolfner et al., J. Mol. Lucy Bernholz This reduction, which was not observed in flagellar hook mutants, was due to a decreased stability of the L-ring protein. View details for Web of Science ID A1986E866400004. Analysis of in vivo and in vitro methylation in synchronized cultures showed that the methylation reaction is lost when the flagellated swarmer cell differentiates into a stalked cell. An analysis of double mutants containing the fatB503 allele and other mutations in membrane biogenesis demonstrated that the cell cycle of AE6001 blocked at a homeostatic state. Class II genes are the earliest to be expressed and are activated at a specific time in the cell cycle by the CtrA response regulator. This movement requires the highly conserved ParABS locus that is essential in Caulobacter. We propose that flagellated stalks arise as a consequence of a failure to eject the flagellum at the correct time in the cell cycle and that the extra stalk lobe is due to a second site for the initiation of stalk biogenesis. View details for DOI 10.1073/pnas.0805258105, View details for Web of Science ID 000258560700056, View details for PubMedCentralID PMC2516238. Schmidt Academy Scholar An impressively diverse array of mechanisms underlies bacterial polarity, including oscillatory systems, phospho-signaling pathways, the sensing of membrane curvature, and the integration of cell cycle regulators with polar maturation. We observed that all plasmids replicated during the C. crescentus cell cycle with comparable kinetics of DNA synthesis, even though we tested plasmids that encode very different known (and putative) replication proteins. The N-terminal atypical receiver domain resembles the canonical receiver domain of response regulators, but has a degenerate, stripped-down 'active site'. In many cases, a large number of upstream signals are funneled to a master regulator of cellular behavior, but it remains unclear how individual proteins can rapidly integrate a complex array of signals within the appropriate spatial niche within the cell. However, live-cell superresolution imaging has been challenged by the need to image three-dimensional (3D) structures relative to their biological context, such as the cellular membrane. The insertion sequence (IS) elements, IS1 and IS2, present in multiple copies in the Escherichia coli chromosome, are transposable genetic elements of known nucleotide sequence. Here we demonstrate live-cell 3D superresolution imaging of Crescentin-eYFP, a cytoskeletal fluorescent protein fusion, colocalized with the surface of the bacterium Caulobacter crescentus using a double-helix point spread function microscope. View details for DOI 10.1016/j.mib.2016.06.007, View details for PubMedCentralID PMC5069156. Epistasis experiments demonstrated that the fliIJ operon is located in class II of the C. crescentus flagellar regulatory hierarchy, suggesting that the gene products act at an early stage in flagellar assembly. The sequential activation of these three subgroups of structural genes reflects the order of assembly of their gene products into the flagellum. Wright, R., Stephens, C., Zweiger, G., Shapiro, L., Alley, M. R. Cell cycle-controlled proteolysis of a flagellar motor protein that is asymmetrically distributed in the Caulobacter predivisional cell, Identification of a Caulobacter crescentus operon encoding hrcA, involved in negatively regulating heat-inducible transcription, and the chaperone gene grpE. We found that the extent to which MreB localization is perturbed is linearly correlated with the development of pointed cell poles and variable cell widths. Although ribonucleic acid and protein syntheses continued at a reduced rate for the equivalent of one generation in mutant strains, a substantial decrease in the rate of deoxyribonucleic acid synthesis occurred immediately upon glycerol deprivation. Deletion analysis indicated that a 55 bp DNA fragment was sufficient for normal, temporally regulated promoter activity. Goley, E. D., Yeh, Y., Hong, S., Fero, M. J., Abeliuk, E., McAdams, H. H., Shapiro, L. The Architecture and Conservation Pattern of Whole-Cell Control Circuitry, Regulatory Response to Carbon Starvation in Caulobacter crescentus. University of California, Santa Barbara, Dr. Raymond Bourdeau Caltech Examination of the intracellular location of SMC showed that in swarmer cells, which do not replicate DNA, the protein forms two or three foci. Defects in the cheB gene resulted in greatly reduced chemotaxis-associated methylation in vivo and a loss of methylesterase activity in vitro. Society for Neuroscience 1991-present. The diameters of the two upper rings differed, being 32 and 21 nm, respectively. View details for DOI 10.1073/pnas.0307827100, View details for Web of Science ID 000220163800029, View details for PubMedCentralID PMC373487. Because regulatory proteins are among those that reside at specific cellular sites, it is now necessary to consider three-dimensional organization when describing the genetic networks that control bacterial cells. Abedi MH#, Yao M#, Mittelstein DR, Bar-Zion A, Swift MB, Lee-Gosselin A, Barturen-Larrea P, Buss MT, Shapiro MG*. Delft University of Technology, Dr. Pradeep Ramesh This heading includes several more specific ancient DNA research projects currently underway in the Shapiro lab. The transcript start site in front of flaE was determined and the -10 region conforms to the E. coli sigma 28 promoter consensus sequence. 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. Antigen-antibody complex formation occurring within a vector-phage plaque can be used to detect the production of a specific protein from an amplified gene. Three-dimensional colocalization of intracellular protein structures and the cell surface with superresolution optical microscopy opens the door for the analysis of protein interactions in living cells with excellent precision (20-40nm in 3D) over a large field of view (1212m). Human Frontier Science Program Cross-Disciplinary Fellow Rev. Animal Biology, UC Davis SciP is cell cycle-controlled and co-conserved with the global cell cycle regulator CtrA in the -proteobacteria. A revertant regains the normal structures simultaneously. View details for Web of Science ID A1995QP81000003, View details for PubMedCentralID PMC176791, View details for Web of Science ID A1995QJ60200003, View details for Web of Science ID A1995QQ99700450, View details for Web of Science ID A1995QQ99701144. View details for DOI 10.1126/sciadv.abm6570. Neuroscience and Behavioral Biology, Emory University The SMC foci appear randomly distributed in the cell. 2012 University of Texas at Austin, Graduate Student, Biochemistry Full discovery of its essential genome, including non-coding, regulatory and coding elements, is a prerequisite for understanding the complete regulatory network of a bacterial cell. 2017 Indian Institute of Technology, University of Wisconsin-Madison We demonstrate here that each of these genes is also required for the expression of chemotaxis methylation genes known to map elsewhere on the chromosome. By analogy with RNA polymerase from other bacterial sources, they are considered to be components of the C. crescentus holoenzyme, beta', beta, sigma, and alpha, respectively. Chemical Engineering We find that all three proteins exhibit restricted rates of entry into and escape from the microdomain as well as enhanced phospho-signalling within, leading to a submicron gradient of activated CtrA-P19 that is stable and sublinear. View details for Web of Science ID A1989AK51300008. University of California, San Francisco, Gabrielle Ho 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. Collaboration: In-vivo Drug Evaluation, University of Colorado and Health Sciences Center B.Sc. Postdoctoral Fellowship, Radiology, Mayo Clinic Overall, the algorithm is a major paradigm shift in the way we analyze experimental accelerator data at facilities today, Roussel said. Until recently, a dedicated mitotic apparatus that segregates newly replicated chromosomes into daughter cells was believed to be unique to eukaryotic cells. CtrA binds to and regulates the promoters of two genes critical to its temporally controlled proteolysis, divK and clpP, providing a transcriptional feedback loop for the control of cell cycle progression. The S ring has a triangular cross section, the sides of the triangle abutting the E ring, the rod and the M ring. fliI encodes a 50-kDa polypeptide whose sequence is closely related to that of the Salmonella typhimurium FliI protein, an ATPase thought to energize the export of flagellar subunits across the cytoplasmic membrane through a type III protein secretion system. The region of early phiCdl was mapped by hybridizing labeled RNA extracted from phiCdl-infected cells grown in the presence or absence of chloramphenicol to HindIII and HpaI restriction fragments of the phiCdl genome. Superresolution imaging techniques based on sequential imaging of sparse subsets of single molecules require fluorophores whose emission can be photoactivated or photoswitched. View details for Web of Science ID 000280561600011, View details for PubMedCentralID PMC3205914. Models for regulation of Caulobacter early flagellar promoters are discussed in which RNA polymerase containing a novel sigma subunit interacts with an activation factor bound to the central region of the promoter. In this report we describe the isolation and characterization of a flagellar gene, fliX. Dibutyryl cyclic adenosine 3',5'-monophosphate (AMP) was shown to stimulate expression of the inducible enzymes and, thus, the initiation of the cell cycle. A deletion of the ssrA gene, or of the gene encoding SmpB, a protein required for SsrA activity, results in a specific delay in the cell cycle during the G(1)-to-S transition. B.A., Physics, University of Chicago, 1984. WebBrett Shapiro Gravitational waves are predicted to exist by Einstein's Theory of General Relativity. The ccrM gene was cloned, and DNA sequence analysis revealed that the predicted amino acid sequence has 49% identity with the Haemophilus influenzae methyltransferase HinfM. View details for DOI 10.1016/j.molcel.2011.09.010, View details for Web of Science ID 000296212100011, View details for Web of Science ID 000299378306327. x@caltech.edu, x=li.richard, Bill Ling This binding activity was missing from strains containing mutations in flaO and flaW, two genes near the top of the flagellar hierarchy known to be required for hook operon transcription. 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. 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. RcdA is required for CtrA polar localization and degradation by ClpXP. View details for Web of Science ID 000077377300004, View details for Web of Science ID 000077110800030. To help to define the order of assembly of the flagellar components and to identify the genes involved in the early steps of basal body construction, mutants defective in basal body formation have been analyzed. We develop technologies to image and control the function of cells deep inside the body. Using chromosome conformation capture carbon copy (5C), we derive ~13 kb resolution 3D models of the Caulobacter genome. View details for Web of Science ID 000279782000006, View details for PubMedCentralID PMC2916389. Maria Paulene Abundo 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. Shapiro, L., MANSOUR, J., Shaw, P., Henry, S. SYNTHESIS AND UTILIZATION OF FATTY-ACIDS BY WILD-TYPE AND FATTY-ACID AUXOTROPHS OF CAULOBACTER-CRESCENTUS. Ph.D. Biomedical Engineering, University of Minnesota The work at Caltech was led by Norman, former Shapiro lab postdoctoral fellow David Maresca (now assistant professor at Delft University of Technology), and Christopoulos. 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. Ph.D. Neuroscience, Institute of Neuroscience, Shanghai Thus, CpdR function is regulated by a feedback loop that incorporates its differential phosphorylation, the transient polar localization and activity of the ClpXP protease, and the clearance of the CpdR by polar ClpXP that, in turn, releases ClpXP from the pole relieving the degradation of CtrA. Upon asymmetric cell division, swarmer and stalked progeny cells employ distinct mechanisms to control active CcrM. View details for DOI 10.1073/pnas.182411999, View details for Web of Science ID 000178635700088, View details for PubMedCentralID PMC129783. These results demonstrate that the chemotaxis methylation machinery is positionally biased toward one portion of the predivisional cell, and that the time of expression of a set of fla and che genes is correlated with the positioning of their gene products within the cell. View details for DOI 10.1128/mBio.03020-20. Thus, the direct coupling of chromosome replication with the cell cycle is mediated by the ubiquitous two-component signaling proteins. 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. The initiation of chromosomal replication occurs concomitantly with the transition of the motile swarmer cell to the sessile stalked cell.
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