The cell cycle is important for growth, genome replication, and development in all cells. View details for Web of Science ID A1977DU20100033, View details for Web of Science ID A1976CH91600017. liqunyu2@illinois.edu For questions or comments, contact the SLAC Office of Communications at communications@slac.stanford.edu. The S ring has a triangular cross section, the sides of the triangle abutting the E ring, the rod and the M ring. B.S. Transposition of the Tn5-VB32 promoter probe into the Caulobacter crescentus chromosome generated auxotrophic and motility mutants and Southern blot analysis of DNA from these mutants showed Tn5-VB32 sequences in random-sized chromosomal restriction fragments. This redundant control of gcrA transcription by DnaA (activation) and CtrA (repression) forms a robust switch controlling the decision to proceed through the cell cycle or to remain in the G1 stage. 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. The gene encoding CtrA, a key cell cycle regulatory protein, is transcribed from two promoters. B.S. We identified all essential promoter elements for the cell cycle-regulated genes. Several fla- mutants were also isolated by Tn5-VB32 mutagenesis and shown to confer kanamycin resistance. The actin homolog MreB contributes to bacterial cell shape. CcrM homologs are widespread in the alpha subdivision of gram-negative bacteria. 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. Similarly, hooks with attached rods were shed from nonflagellate mutants, and these structures also lacked the basal rings. Chemical and Biomolecular Engineering, Johns Hopkins University article | press | video. We propose that DnaA serves to coordinate bacterial DNA replication with the onset of chromosome segregation. A high proportion of morphologically aberrant cells, and cells that have undergone an additional chromosome replication initiation, are found in this population. How this is brought about remains one of the most fundamental questions of developmental biology. 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. The ffs36 phenotype results from a single base change in one of the non-conserved stems of the mature RNA, and is completely rescued by a compensating mutation in the opposite strand, providing confirmation of the predicted secondary structure of the 4.5 S RNA. Pharmacological Chemistry, UC San Diego Assays of the differential placement of the promoter-less drug resistance proteins (encoded within the interrupted fla genes) allow us to determine whether the positioning of the fla gene products is controlled by signal sequences in their proteins, by specific mRNA-targeting sequences in the 5'-regulatory regions of these genes, or by specific transcription from only one of the two newly replicated chromosomes in the predivisional cell. By deciphering the underlying design principles, we hope to generate pure populations of these cell-types from embryonic and induced pluripotent stem cells for regenerative medicine. 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. Caulobacter cell division is inherently asymmetric, yielding progeny with different fates: stalked cells and swarmer cells. 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. In this study, we report the use of a highly photostable fluoromodule, dL5, to genetically label proteins in the Gram-negative bacterium Caulobacter crescentus, enabling long-time-scale protein tracking and super-resolution microscopy. It has been shown that DNA replication serves as a checkpoint for flagellar biosynthesis and cell division and that this checkpoint is mediated by the availability of active CtrA. Upon initiation of DNA replication, one copy of the duplicated origin sequence rapidly appears at the opposite cell pole. M.S. A previously uncharacterized essential protein, MipZ, forms a complex with the partitioning protein ParB near the origin of replication and localizes with the duplicated origin regions to the cell poles. Global transcription analysis of synchronized Caulobacter crescentus cells was used to identify 553 genes (19% of the genome) whose messenger RNA levels varied as a function of the cell cycle. One of these sites was located within the 16S gene near its 3' end, and the other two were found at the 5' end of the 23S gene. LOEWY, B., Marczynski, G. T., Dingwall, A., Shapiro, L. INTEGRATION HOST FACTOR IS REQUIRED FOR THE ACTIVATION OF DEVELOPMENTALLY REGULATED GENES IN CAULOBACTER, EXPRESSION OF THE CAULOBACTER HEAT-SHOCK GENE DNAK IS DEVELOPMENTALLY CONTROLLED DURING GROWTH AT NORMAL TEMPERATURES, PLASMID AND CHROMOSOMAL DNA-REPLICATION AND PARTITIONING DURING THE CAULOBACTER-CRESCENTUS CELL-CYCLE. This conclusion is based on the observations that (i) methionine auxotrophs starved of methionine can swim only in the forward direction (comparable to smooth swimming in the enteric bacteria), (ii) a specific set of membrane proteins was found to be methylated in vivo and the incorporated [3H]methyl groups were alkali sensitive, (iii) this same set of membrane proteins incorporated methyl groups from S-adenosylmethionine in vitro, and (iv) out of a total of eight generally nonchemotactic mutants, two were found to swim only in a forward direction and one of these lacked methyltransferase activity. We show that gas vesicles are exceptionally good acoustic antennae, experiencing strong acoustic forces relative to their nm size. Our results identify cell cycle changes in gene expression in response to carbon starvation that involve the prominent role of the FixK FNR/CAP family transcription factor and the CtrA cell cycle regulator. 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. Here, we describe an imaging scheme that correlates cryogenic single-molecule fluorescence localizations with CET reconstructions. In addition, mutations in either fliQ or fliR exhibit defects in cell division and thus may participate directly or indirectly in the division process. Menlo Park, Calif. The Department of Energys SLAC National Accelerator Laboratory and Stanford University today announced the launch of a new joint battery center at SLAC. Stanford Model checking also showed that mechanisms involving methylation-state changes in regulatory promoter regions during DNA replication increase the robustness of the cell-cycle control. Economic Analysis & Policy Mapping of the transcriptional start site revealed a conserved binding motif for the global response regulator CtrA at the -35 position; this motif was footprinted by purified Caulobacter crescentus CtrA protein in its phosphorylated state. Robertson, G. T., Reisenauer, A., Wright, R., Jensen, R. B., Jensen, A., Shapiro, L., Roop, R. M. Dynamic spatial regulation in the bacterial cell, Chromosome segregation during the prokaryotic cell division cycle, Differential localization of two histidine kinases controlling bacterial cell differentiation, The Caulobacter crescentus smc gene is required for cell cycle progression and chromosome segregation. Thanks to all the lab members, collaborators and friends who joined us for the annual Shapiro Lab beach party in Oceanside, CA! She attended the NIH-funded Medical Scientist Training Program at Duke University, where she earned her Ph.D. with Dr. Anthony Means studying calmodulin-dependent kinases in male germ cell development. Saurabh, S., Perez, A. M., Comerci, C. J., Shapiro, L., Moerner, W. E. A cell cycle kinase with tandem sensory PAS domains integrates cell fate cues. The replisome gradually moves to midcell as DNA replication proceeds and disassembles upon completion of DNA replication. 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. This heading includes several more specific ancient DNA research projects currently underway in the Shapiro lab. The first parameter correlates with genome GC content, and the second parameter correlates with context-dependent nucleotide bias. View details for DOI 10.1016/j.mib.2004.10.005, View details for Web of Science ID 000225782400003, View details for Web of Science ID 000224648800052. The dimorphic bacterium Caulobacter crescentus provides a simple model for cellular differentiation. In wild-type cells, ATP hydrolysis opens the SMC dimer, freeing one chromosome to segregate to the opposite pole. When the swarmer cell differentiated into a stalked cell, the chemoreceptor was specifically degraded by virtue of an amino acid sequence located at its carboxyl terminus. An additional level of control was revealed when it was found that an interruption of DNA replication caused the inhibition of flaS transcription. In addition, the C-terminal region of FtsK is required for the localization of the topoisomerase IV ParC subunit to the replisome to facilitate chromosomal decatenation prior to cell division. The most easily recognized asymmetries involve surface structures, e.g., flagella, pili, and stalks that are preferentially assembled at one pole by many bacteria. STALKED BACTERIA - PROPERTIES OF DEOXRIYBONUCLEIC ACID BACTERIOPHAGE PHICBK, SPECIFIC ASSAY FOR DIFFERENTIATION IN STALKED BACTERIUM CAULOBACTER-CRESCENTUS, PHAGE-SPECIFIC AND HOST PROTEINS IN REPLICATION OF BACTERIOPHAGE RNA. Wagenknecht, T., DeRosier, D., Shapiro, L., WEISSBORN, A. PHOSPHOLIPID BIOSYNTHESIS IS REQUIRED FOR STALK ELONGATION IN CAULOBACTER-CRESCENTUS. Many of them are controlled by promoters methylated by CcrM and co-regulated by other global cell cycle regulators, demonstrating an extensive cross talk between DNA methylation and the complex regulatory network that controls the cell cycle of C. crescentus and, presumably, of many other Alphaproteobacteria. This structure is absent at the flagellar pole but not in the stalks of flbT mutant predivisional cells. Since the phage is extremely salt-sensitive, a purification procedure was devised which avoided contact with solutions of high ionic strength. Determination of the DivL crystal structure and biochemical analysis of wild-type and site-specific DivL mutants revealed that the DivL PAS domains regulate binding specificity for DivKP over DivK, which is modulated by an allosteric intramolecular interaction between adjacent domains. Dahlberg, P. D., Sartor, A. M., Wang, J., Saurabh, S., Shapiro, L., Moerner, W. E. Integration of cell cycle signals by multi-PAS domain kinases. This uncoupling of initiation of replication from CtrA degradation indicates that there is an SsrA-dependent pathway required for correct timing of DNA replication. We isolated 35 unique A22-resistant Caulobacter strains with single amino acid substitutions near the nucleotide binding site of MreB. CcrM is transiently present near the end of DNA replication when it rapidly methylates the adenine in hemimethylated GANTC sequences. View details for DOI 10.1073/pnas.1220824110, View details for Web of Science ID 000314558100027, View details for PubMedCentralID PMC3562846. However, molecular mechanisms governing rapid protein crystallization in vivo or in vitro are largely unknown. WebBrett Shapiro. czhang8@illinois.edu The promoter sequence does not resemble that recognized by any known bacterial sigma factor. Expression of fliX is under cell cycle control, with transcription beginning relatively early in the cell cycle and peaking in Caulobacter predivisional cells. The genome contains multiple clusters of genes encoding proteins essential for survival in a nutrient poor habitat. x@caltech.edu, x=jwekselb, Di Wu, PhD We propose that the molecular polarity inherent in an actin-like filament is translated into a mechanism for directing global cell polarity. Collaboration: Estrogen Receptor, University of Illinois Therefore, flagellar genes at or near the top of the hierarchy may be controlled, in part, by a unique transcription factor and may be responsive to the same DNA replication cues that mediate other cell cycle events, such as cell division. E. coli ribosomal RNA contains sequences homologous to insertion sequences IS1 and IS2. Also among the genes in this notably large regulon are 14 that encode regulatory proteins, including 10 two-component signal transduction regulatory proteins. Bennett Shapiro is a provider established in Stanford, California and his medical specialization is Orthopaedic Surgery.
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