They also allow an investigation of the regulatory signals that modulate the asymmetric distribution of proteins and their subsequent cell type-specific expression in the initial stages of a developmental program. Their goal is to define these mechanisms using both molecular genetics and biochemistry. The probe carries an altered Tn5 transposon that allows detection of chromosomal promoter regions by virtue of acquired kanamycin resistance. The formation of a flagellum opposite the stalk has been observed by microscope during the differentiation of a stalked cell in preparation for cell division. Postdoc. B., Cohen, M., Delli-Santi, M., Fennell, C., Leinberry, C., Husband, J., Ladd, A., Seitz, W. R., Constanz, B. Blackburn, E., Firtel, R. A., Shapiro, L. GENETIC-ANALYSIS OF A TEMPORALLY TRANSCRIBED CHEMOTAXIS GENE-CLUSTER IN CAULOBACTER-CRESCENTUS. 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. This genetic lesion did not appear to affect directly a fatty acid biosynthetic reaction because fatty acid and phospholipid syntheses were found to continue in the absence of supplement. Because DivL and CckA accumulate at the same cell pole after the initiation of DNA replication and were found to interact in vivo, we propose that DivL recruits CckA to the pole, thereby promoting its autophosphorylation and activity. A localized adaptor protein performs distinct functions at the Caulobacter cell poles. (i) Is the temporal progression of events occurring during bacterial differentiation controlled by regulator gene products? A., McGrath, P. T., Reisenauer, A., McAdams, H. H., Shapiro, L. Single molecules of the bacterial actin MreB undergo directed treadmilling motion in Caulobacter crescentus. Laboratories for Reproductive Biology & Lineberger Comprehensive Cancer Center
7/2016. View details for Web of Science ID 000165870600056. View details for Web of Science ID 000251336000004, View details for PubMedCentralID PMC2175322. The eukaryotic mitotic machinery uses the cytoskeleton to move specific chromosomal regions. Caulobacter crescentus permits detailed analysis of chromosome replication control during a developmental cell cycle. Moerner, W. E., Biteen, J., Conley, N. R., Lee, H., Lord, S. J., Thompson, M. A., Shapiro, L., Liu, N., Samuel, R., Twieg, R. J. DIFFERENTIAL EXPRESSION AND POSITIONING OF CHEMOTAXIS METHYLATION PROTEINS IN CAULOBACTER, CAULOBACTER-CRESCENTUS FATTY ACID-DEPENDENT CELL-CYCLE MUTANT. & Gerardot, C. J. flaNQ promoter expression is localized to the swarmer pole of the predivisional cell, as are other flagellar promoters that possess these regulatory sequences 5' to the start site. Other mutants bearing Tn5 insertions retained cross-reacting MCP activity and were altered only in their methyltransferase and methylesterase activities. Rev. Large structures, such as a flagellum, are anchored at the pole by means of the basal body that traverses the peptidoglycan wall. 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. For example, requirements for DnaA protein and RNA transcription affiliate both origins. Coactivator Studies, Progesterone Receptor, University of North Carolina, Chapel Hill
Although FliL is required for flagellar function, it is not part of the transcriptional hierarchy, supporting the hypothesis that, as is the case for the enterics, the regulatory hierarchy responds to assembly cues rather than directly to the expression of flagellar proteins. In addition, we demonstrated that the fatty acid composition of wild-type C. crescentus can be altered by growing the cells in medium supplemented with any one of a variety of unsaturated fatty acids. Bacterial cells are inherently asymmetric, some more obviously so than others. The L and P rings are connected by a bridge of material at their outer radii. 7/2016. Bowman, G. R., Comolli, L. R., Zhu, J., Eckart, M., Koenig, M., Downing, K. H., Moerner, W. E., Earnest, T., Shapiro, L. Architecture and inherent robustness of a bacterial cell-cycle control system. The bacterium Caulobacter crescentus carries out a distinct differentiation process during its life cycle. article|readcube | press, Researchers Make it Possible for Ultrasound to Reveal Gene Expression in the Body, Vilcek Foundation Prize Awarded to Mikhail Shapiro, CCE Postdoc Receives NIH Pathway to Independence Award, Mikhail Shapiro Wins Roger Tsien Award for Excellence in Chemical Biology, Program Brings Area High School Students, Teachers into Caltech Labs, Switching Brain Circuits On and Off Without Surgery, Mikhail Shapiro Selected as Camille Dreyfus Teacher-Scholar, Taking MRI Technology down to Micrometer Scales, Scientists Design Bacteria to Reflect Sonar Signals for Ultrasound Imaging, Biologists Give Bacteria Thermostat Controls, Designing Ultrasound Tools with Lego-Like Proteins, Newly Named Pew Scholar to Image Gut Bacteria with Sound Waves, Partnership with Heritage Medical Research Institute Will Augment Translational Medicine Research, Abedi Receives Fellowship for New Americans, Caltech Researchers Receive NIH BRAIN Funding, New Method Could Improve Ultrasound Imaging, x@caltech.edu; x=mikhail 1996-2001. Caulobacter has a single circular chromosome whose origin of replication is positioned at one cell pole. View details for Web of Science ID A1996UD48400009, View details for PubMedCentralID PMC177876. The basal body consisted of five rings mounted on a rod. Cell division, essential for the viability of the organism, is dependent on the irreversible differentiation of a flagellated swarmer cell to a mature stalked cell. Biophysical analysis of purified wild type and assembly defective mutant proteins indicates that PopZ self-associates into an elongated trimer, which readily forms a dimer of trimers through lateral contact. Reno, PL, CY McLean, JE Hines, TD Capellini, G Bejerano, and DM Kingsley (2013). The sequential activation of these three subgroups of structural genes reflects the order of assembly of their gene products into the flagellum. 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. Epistasis experiments placed fliX within class II of the flagellar regulatory hierarchy, suggesting that FliX functions at an early stage in flagellar assembly. Congratulations to Sangjin and collaborators on this detailed biophysical study. 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. These controls include temporally regulated transcription and phosphorylation and spatially restricted proteolysis. The Caulobacter life cycle, defined in synchronously growing cultures, includes a sequential series of morphological changes that occur at specific times in the cycle and at specific locations in the cell. This point mutation allows normal flagellin synthesis, stalk formation, equatorial cell division, and rate of growth. The cytoplasmic methylesterase was also found to be differentially synthesized coincident with the period of flagellar biogenesis. A., Shapiro, L., Gitai, Z. DnaA coordinates replication initiation and cell cycle transcription in Caulobacter crescentus. The ctrA gene is preferentially transcribed from a hemimethylated promoter. We identified a domain within the C-terminal 76 amino acids that is necessary and sufficient for accumulation as a single subcellular focus, a domain within the N-terminal 23 amino acids that is necessary for bipolar targeting, and a linker domain between these localization determinants that tolerates large variation. 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. (Credit: Paul Mueller) The scientists developed the platform at the Advanced Light Source, which produces light in the X-ray region of the electromagnetic spectrum. The circuit diagram of the bacteriophage lambda lysislysogeny decision circuit represents connectivity in signal paths of the biochemical components. Attempts to encode large numbers of polymeric, metallic or glass beads in random arrays or in fluid suspension have used a variety of entities to provide coded elements (bits)--fluorescent molecules, molecules with specific vibrational signatures, quantum dots, or discrete metallic layers. Using plasmid complementation, we have mapped the extent of the flaY and flaE genes. However, many of the mechanistic details underlying these functions are unknown. For large aggregates, such as the clusters of MCP, CheA, and CheW complexes, perhaps the size of the aggregate alone prevents displacement. Caulobacter crescentus assembles many of its cellular machines at distinct times and locations during the cell cycle. My career goal is to improve outcomes and experiences of patients and caregivers living with and beyond cancer, and my research and scholarship have contributed to understanding and meeting the needs of the growing population of cancer survivors. Nathan, P., Gomes, S. L., Hahnenberger, K., Newton, A., Shapiro, L. FATTY-ACID DEGRADATION IN CAULOBACTER-CRESCENTUS, TRANSCRIPTION INITIATION INVITRO AND INVIVO AT A HIGHLY CONSERVED PROMOTER WITHIN A 16 S-RIBOSOMAL RNA GENE. The molecular weights of the enzyme subunits were 165,000, 155,000, 101,000, and 44,000, respectively. The transcript synthesized in vitro was shown to be cleaved by C. crescentus RNase III and to release the transfer RNA genes from the downstream 16 S/23 S intergenic spacer region. Shapiro, L., Grossman, L. I., Marmur, J., KLEISCHM, A. K. SYMPOSIUM ON REPLICATION OF VIRAL NUCLEIC ACIDS .2. 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. The analysis of small predivisional vesicles showed that the MCP content is higher in the flagellated vesicles, and analysis of large flagellated vesicles showed that the MCPs are positioned preferentially in the swarmer cell portion of the predivisional cell. In the absence of DnaA, the CtrA master regulator is cleared by proteolysis during the swarmer-to-stalked cell transition as usual, but DNA replication initiation is blocked. The polar organizing protein Z (PopZ) localizes to the polar regions of C. crescentus where it is known to form a distinct microdomain. A cyclical control circuit composed of four master regulators drives the Caulobacter cell cycle. However, the dynamics of the bacterial actin homolog MreB have yet to be examined in vivo. We determined the cellular positions of the origins of the replicons in the alpha proteobacteria Agrobacterium tumefaciens and Sinorhizobium meliloti and found that they are located at the poles of the cells. View details for DOI 10.1126/science.1175685, View details for Web of Science ID 000272117900037. These results are consistent with a model in which unreplicated DNA is pulled into the replication factory and newly replicated DNA is bidirectionally extruded from the complex, perhaps contributing to chromosome segregation. These changes in DNA methylation could signal differential binding of regulatory proteins to activate or repress transcription. Work in Caulobacter crescentus shows that essential and nonessential proteins localize to discrete positions in the cell as a function of cell-cycle progression. 2) A specific technique has been developed whereby the progress of the differentiation cycle can be accurately measured by adsorption of labeled RNA phage or penetration of labeled phage DNA into specific cell forms. The molecular weight of purified flagellin (subunit of flagella filament) is 25,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. View details for DOI 10.1371/journal.pgen.1004831, View details for PubMedCentralID PMC4287350. Duplication of the chromosome and partitioning of the newly generated daughter strands are interwoven processes driven by the dynamic interplay between the synthesis, segregation and condensation of DNA. The Caulobacter cell cycle is driven by a cascade of transient regulators, starting with the expression of DnaA in G(1) and ending with the expression of the essential CcrM DNA methyltransferase at the completion of DNA replication. Mutations that alter cell curvature and mislocalize the intermediate filament crescentin cluster on the back surface of MreB's structure. They have found a single molecular event present in all cancers studied to date that protects them from macrophages of the innate immune system. Zhou, B., Schrader, J., Christen, B., McAdams, H., Shapiro, L. Osmolality-Dependent Relocation of Penicillin-Binding Protein PBP2 to the Division Site in Caulobacter crescentus. The effect of cyclic AMP on growth on sugars metabolized by inducible enzymes, as well as on sugars metabolized by constitutive enzymes, may represent a regulatory system common to both types of sugar utilization, since they share features that differ from glucose utilization, namely, temperature-sensitive growth and low intracellular concentrations of cyclic guanosine 3',5'-monophosphate. View details for Web of Science ID A1990CW01800056. View details for Web of Science ID 000077377300004, View details for Web of Science ID 000077110800030. The polar accumulation of PopZ occurs by a diffusion/capture mechanism that requires the MreB cytoskeleton. Our goal is to identify and understand the pathways that govern organogenesis of the pancreas, a vital organ with endocrine and exocrine functions. 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. View details for Web of Science ID 000304978400010, View details for PubMedCentralID PMC3370875. Our high-throughput screening methodology can be adapted readily to any sequenced bacterial species, opening the potential for databases of localization regulatory networks across species, and investigation of localization network phylogenies. The cellular localization of MipZ thus serves the dual function of positioning the FtsZ ring and delaying formation of the cell division apparatus until chromosome segregation has initiated. We show that the PopZ oligomeric network forms polar ribosome exclusion zones that change function during cell cycle progression. Strains bearing mutations in the pleA gene are pililess and non-flagellated. MipZ directly interferes with FtsZ polymerization, thereby restricting FtsZ ring formation to midcell, the region of lowest MipZ concentration. Gregg Shapiro, formerly a leading Department of Justice (DOJ) fraud prosecutor, joined with Jeffrey Newman to form Newman & Shapiro in 2021. View details for Web of Science ID A1985ALK8400022. 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. Microbiol. 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. Stalked pole assembly, in turn, triggers the initiation of chromosome replication, which signals the formation of a new PopZ zone at the opposite cell pole, where it functions to anchor the newly duplicated centromere that has traversed the long axis of the cell. Single-molecule imaging demonstrated physical anticorrelation between sequestered CcrM and chromosomal DNA, thus preventing DNA remethylation. Therefore, genome-wide codon bias in eubacteria and archaea may be predicted from intergenic sequences that are not translated. B.S. SsrA RNA is stable in G(1)-phase cells and late S-phase cells but is degraded with a half-life of 4 to 5 min at the onset of S phase. 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). Knowing the transcription start site enables targeted searching for regulatory-protein binding motifs in the promoter regions of genes with similar expression patterns. (5) Together, these regulatory proteins create a genetic circuit in which the cellular concentrations of CtrA and GcrA oscillate spatially and temporally to control daughter cell differentiation and cell cycle progression. 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. The fact that the movement of these 10 loci is, like that of the origin, directed and rapid, and occurs at a similar rate, suggests that the same molecular machinery serves to partition and place many, if not most, chromosomal loci at defined subcellular sites. Dr. Shapiro's laboratory question in developmental biology involves the mechanisms used to generate the three-dimensional organization of a cell from a one-dimensional genetic code. View details for DOI 10.1073/pnas.1014395107, View details for Web of Science ID 000283749000046, View details for PubMedCentralID PMC2973855, View details for DOI 10.4161/cc.9.20.13521, View details for Web of Science ID 000283058300001. 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. in Integrative Biology, University of California, Berkeley, Professor, Department of Biology, University of Utah, Adjunct Professor, Department of Human Genetics, University of Utah, Adjunct Associate Professor, Department of Human Genetics, University of Utah, Associate Professor, Department of Biology, University of Utah, Assistant Professor, Department of Biology, University of Utah, Member, Molecular Biology Program, University of Utah, Resident Biology Tutor, Leverett House, Harvard College, Research Assistant, Cardiovascular Research Center, Massachusetts General Hospital and Harvard Medical School, Research Assistant, Museum of Comparative Zoology, Harvard University, Museum Preparator, University of California Museum of Paleontology, James E. Talmage Presidential Endowed Chair, University of Utah, Myriad Genetics Award of Research Excellence, Early Career Development Award, National Science Foundation, Early Career Teaching Award, University of Utah, Career Award in the Biomedical Sciences, Burroughs Wellcome Fund, Best Symposium Presentation by a Postdoctoral Fellow, Society for Developmental Biology, Helen Hay Whitney Postdoctoral Research Fellowship, D. Dwight Davis Award for Best Student Paper, Society for Integrative and Comparative Biology, Stoye Award for Best Student Presentation, American Society of Ichthyologists and Herpetologists, Museum of Comparative Zoology/Harvard Medical School Jeffries Wyman Scholarship in Anatomy, Phi Beta Kappa, Alpha Chapter of California, BIOL 5510: Evolutionary Developmental Biology, HGEN 6091: Evolution and Development (co-taught with N. Elde, G. Kardon, and S. Sakonju), BIOL 7964: Advanced Topics in Ecology and Evolution (Team-taught course), Woods Hole MBL: Lecturer, summer Embryology course, Teaching staff, NIH Stickleback Molecular Genetics Summer Course (multiple times), Program staff, Stanford Summer Research Program, Instructor, Biology and Evolution of the Dinosauria (co-taught with L. Claessens), Teaching Fellow, Evolution of the Vertebrates (multiple times), Teaching Fellow, Structure and Physiology of the Vertebrates (multiple times), Teaching Fellow, Advanced Structure and Physiology of the Vertebrates (multiple times), Teaching Fellow, Functional and Comparative Vertebrate Anatomy (extension school), Biology tutor, Athletic Study Center and Disabled Students Program, Department of Biology, 257 South 1400 East. IEEE Engineering in Medicine and Biology Society. How organismic complexity is generated during embryonic and post-embryonic development. Yoo S, Mittelstein DR, Hurt RC, Lacroix JJ, Shapiro MG*. 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. Only the unphosphorylated form of CpdR localizes and activates ClpXP. These findings provide a biochemical and physiological basis for RsaA's calcium-binding behavior, which extends far beyond calcium's commonly accepted role in aiding S-layer biogenesis or oligomerization and demonstrates a connection to cellular fitness. Interested applicants should visit https://facultypositions.stanford.edu/en-us/job/493432 for our full ad and more information about how to apply. Using these motifs, we predict coregulated genes. Our lab takes an interdisciplinary approach to understand the systems biology of a living cell. We found that MmpA facilitates the degradation of PodJS. In parallel with the FliL protein, FliX copurifies with the membrane fraction, and although its expression is cell cycle controlled, the protein is present throughout the cell cycle. 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. 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. Our lab takes an interdisciplinary approach to understand the systems biology of a living cell. Stephens, C., Reisenauer, A., Wright, R., Shapiro, L. Flagellar assembly in Caulobacter crescentus: A basal body P-ring null mutation affects stability of the L-ring protein, Cell cycle control by an essential bacterial two-component signal transduction protein. The aggregation pattern changes as a function of the cell cycle both during and upon completion of chromosome replication. Bacteria exhibit a high degree of intracellular organization, both in the timing of essential processes and in the placement of the chromosome, the division site, and individual structural and regulatory proteins. CtrA is a member of the response regulator family of two component signal transduction systems. We show that the cell-cycle timing of CcrM is critical for Caulobacter fitness. The methyl-accepting chemotaxis proteins, the methyl-transferase and the methylesterase were all shown to be active in the flagella-bearing swarmer cell, but all three activities were lost after the swarmer cells shed their flagellum and differentiated into a stalked cell. A promoter probe, Tn5-VB32, was constructed and placed in a P group R plasmid containing bacteriophage Mu sequences, allowing transfer of the transposon to bacteria such as Caulobacter, Rhizobium, and Agrobacterium without retention of the plasmid. Quon, K. C., Yang, B., Domian, I. J., Shapiro, L., Marczynski, G. T. Transcriptional analysis of the Caulobacter 4.5 S RNA ffs gene and the physiological basis of an ffs mutant with a Ts phenotype, The CcrM DNA methyltransferase is widespread in the alpha subdivision of proteobacteria, and its essential functions are conserved in Rhizobium meliloti and Caulobacter crescentus, Identification of the fliI and fliJ components of the Caulobacter flagellar type III protein secretion system. We postulate that IHF mediates the formation of a higher order structure between the divergent promoter regions in a manner analogous to the nucleosome-like structure generated for lambda-Escherichia coli DNA recombination and that this higher order structure modulates transcription. Ultrasound-controllable engineered bacteria for cancer immunotherapy. One of these, a fatty acid bradytroph, AE6002, was shown to be due to a mutation in the fatA gene. Differential translation of operon-encoded genes facilitates precise cell cycle-timing for the dynamic assembly of multiprotein complexes, such as the flagellum and the stalk and the correct positioning of regulatory proteins to specific cell poles. 2007: 506506. The chemotaxis proteins are synthesized in the predivisional cell and then partition only to the swarmer cell upon division. Two questions related to control processes can now readily be approached experimentally. These genes are controlled in a positive trans-acting hierarchy that reflects the order of assembly of the flagellum. Each Caulobacter cell cycle involves differentiation and an asymmetric cell division driven by a cyclical regulatory circuit comprised of four transcription factors (TFs) and a DNA methyltransferase. Two lines of evidence are presented which show that an RNase III activity functions as a processing enzyme in C. crescentus. We are a discovery-driven research group working at the interface between developmental biology, bioengineering, and statistical physics. Here, utilizing genetic, biochemical, and biophysical studies of GapR in light of a recently published DNA-bound crystal structure of GapR, we identified the structural elements involved in oligomerization and DNA binding.
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