shapiro lab stanford

Isolation of the full-length ccrM genes from the aquatic bacterium C. crescentus, the soil bacterium R. meliloti, and the intracellular pathogen B. abortus showed that this sequence conservation extends over the entire protein. The M ring, which is at the inner membrane of the cell, has a different structure depending on the method of preparation. Full expression of fliX was found to be dependent on ctrA, and DNase I footprinting analysis demonstrated a direct interaction between CtrA and the fliX promoter. Health Care . steve.nordeen@uchsc.edu 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. 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. First, after entry into S-phase, the newly synthesized origin regions are segregated in an active and directed process, involving the bacterial actin homolog MreB. View details for Web of Science ID 000088048400024, View details for PubMedCentralID PMC16621. Double-stranded side branches between 100 and 600 nucleotide pairs in length were visible in electron micrographs of rapidly reassociating deoxyribonucleic acid isolated by hydroxyapatite chromatography. gyrB and orf-1 are within a newly identified cluster of genes involved in DNA replication and recombination, including dnaN and recF. The resulting plasmid was used as a probe to isolate the flaE region from a wild-type gene bank and to determine the chromosomal location of several deletion and insertion mutations within the flaY/E/F/G cluster. 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. The outcome of these experiences, together with the extraordinary scientists I came to know along the way, was and is an abiding passion to fully understand a simple cell in all its complexity and beauty. We use chromosomal inversions and in vivo time-lapse imaging to show that parS is the Caulobacter site of force exertion, independent of its position in the chromosome. 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. Using in vivo and in vitro analyses of dynamic polar protein complex formation, we show that a polymeric cell polarity protein, SpmX, serves as a direct bridge between the PopZ polymeric network and the cell fate-directing DivJ histidine kinase. The mutant strain, AE6000 , was altered in both of these regulatory functions. The CIR1 and CIR2 motifs exhibit a conserved inverted repeat organization, with a CcrM site in the center of one of the repeats. Although CckA is present throughout the cell cycle, it moves to a cell pole in S phase, and upon cell division it disperses. High temperature and other environmental stresses induce the expression of several heat shock proteins in Caulobacter crescentus, including the molecular chaperones DnaJ, DnaK, GrpE, and GroEL and the Lon protease. Topologically-guided continuous protein crystallization controls bacterial surface layer self-assembly. Signaling hubs at bacterial cell poles establish cell polarity in the absence of membrane-bound compartments. Using structural biology and biochemical findings we proposed a mechanistic basis for TCS pathway coupling in which the DivL pseudokinase is repurposed as a sensor rather than participant in phosphotransduction. Negative control of bacterial DNA replication by a cell cycle regulatory protein that binds at the chromosome origin. Rev. The 0.2 kb fragment contained a homolog of the bacterial gene encoding 4.5 S RNA. Several fla- mutants were also isolated by Tn5-VB32 mutagenesis and shown to confer kanamycin resistance. A developmental mutant of C. crescentus with altered polar surface structures has been isolated. 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. The probe correlation analysis approach reported here is of general use for large-scale sRNA identification for any sequenced microbial genome. The partial IS sequences may represent silent evolutionary remnants or they could modulate the expression of genes carrying these sequences. Bacterial chromosomes are generally approximately 1000 times longer than the cells in which they reside, and concurrent replication, segregation, and transcription/translation of this crowded mass of DNA poses a challenging organizational problem. DNA replication in the dimorphic bacterium Caulobacter crescentus is tightly linked to its developmental cell cycle. The size of the phage and its DNA and the percentage of DNA indicate that the phiCbK phage head is relatively loosely packed. View details for DOI 10.1073/pnas.1000846107, View details for Web of Science ID 000275368400036, View details for PubMedCentralID PMC2842071. (a) In an in vitro reaction, C. crescentus phage phi Cdl major early mRNA synthesized in vitro by host RNA polymerase was processed by RNase III to yield RNA species which co-migrated with phage RNA synthesized in vivo in phi Cdl-infected cells, and (b) an in vitro transcript of a C. crescentus DNA clone containing the entire 16 S gene and part of the 23 S gene was processed by C. crescentus RNase III to yield an RNA product which co-migrated with 16 S RNA. By contrast, our transcriptome analysis shows that >10% of the genes are misexpressed in cells lacking or constitutively over-expressing CcrM. The biogenesis of the bacterial flagellum and chemotaxis apparatus in both Escherichia coli and Caulobacter crescentus requires the ordered expression of over 40 genes whose expression is controlled by a trans-acting regulatory hierarchy. The transduction of the chemotaxis signal is initiated by a chemoreceptor-CheW-CheA ternary complex at the inner membrane. RcdA is required for CtrA polar localization and degradation by ClpXP. CcrM is a DNA adenine methyltransferase found in the alpha subdivision of the proteobacteria. The P1 promoter fires early in S phase and contains a GAnTC sequence that is recognized by the CcrM DNA methyltransferase. The expression of these class II genes initiates assembly of the flagellum just prior to activation of the ccrM promoter in the predivisional cell. 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. At three separate chromosomal sites the CcrM recognition sequence is fully methylated in swarmer cells, becomes hemimethylated upon DNA replication in stalked cells, and does not become remethylated until just prior to cell division. CtrA plays key roles in asymmetric cell division and in the timing of chromosome replication. Further, we find that overexpression of the bridge protein SpmX in Caulobacter disrupts this ordered assembly, generating ectopic cell poles containing both PopZ and DivJ. 1973-1974 Stanford University Chengjian Mao Senior Researcher cmao@illinois.edu Transcription of a flbN-reporter gene fusion in an Escherichia coli background was dependent on the presence of a NifA transcription factor supplied by a plasmid-borne Rhizobium meliloti gene encoding NifA. View details for DOI 10.1128/mBio.03020-20. Chromosome replication is restricted to the stalked cell by a unique chromosome origin of replication that may be regulated by a novel cell-specific transcriptional control system. The gene encoding CtrA, a key cell cycle regulatory protein, is transcribed from two promoters. The initiation of replication depends on the proteolysis of CtrA. Like flagellar biogenesis, stalk formation is an asymmetric polar morphogenesis that occurs once each cell cycle in response to internal cell cycle signals. The transcription of gyrB and orf1 occurs from the replication-competent chromosome in stalked and predivisional cells and is silenced in swarmer cells. The Shapiro Family Laboratory of Viral Oncology and Aging Research is shared by four PIs (Drs. Schrader, J. M., Li, G., Childers, W. S., Perez, A. M., Weissman, J. S., Shapiro, L., McAdams, H. H. Cell cycle progression in Caulobacter requires a nucleoid-associated protein with high AT sequence recognition. The Shapiro Lab is part of an extremely collaborative group of scientists and clinician-scientists focusing on the biology and therapeutic targeting of pediatric diseases as outlined above. Proteins involved in chemotaxis methylation reactions have been identified in Caulobacter crescentus and their activities, times of synthesis and cellular positions have been determined. During the replication process, the ParC subunit colocalizes with the replisome, whereas the ParE subunit is dispersed throughout the cell. Brett Shapiro Group Affiliate, Senior Professional Staff at Johns Hopkins Applied Physics Laboratory Contact brett.shapiro@jhuapl.edu Stanford LIGO Group Web Login Stanford University Stanford Home(link is external) Maps & Directions(link is external) Search Stanford(link is external) Emergency Info(link is external) Terms of Use(link is external) The dimorphic bacterium Caulobacter crescentus provides a simple model for cellular differentiation. Chromatographic mobilities suggested that these fatty acids may be a cyclopropane and a branched-chain fatty acid. (i) Is the temporal progression of events occurring during bacterial differentiation controlled by regulator gene products? Nature Methods18, 945-952 (2021). In its role as a global response regulator, CtrA controls the transcription of a diverse group of genes at different times in the Caulobacter crescentus cell cycle. Additional homologous sequences in phi X174 and a leader region of a ribosomal protein gene cluster were also detected. Caulobacter crescentus goes through a series of morphological changes during its life cycle, including the coincident expression of synthesis of flagella, pili, and receptor sites for DNA bacteriophage. A newly identified cell-cycle master regulator protein, GcrA, together with the CtrA master regulator, are key components of a genetic circuit that drives cell-cycle progression and asymmetric polar morphogenesis in Caulobacter crescentus. Regulated timing of these cellular modules stems from global genetic circuits that allow precise temporal activation with respect to cell cycle progression and cell differentiation. Here we demonstrate the applicability of one such biosensor, the fluorescent protein roGFP2, for cryo-CLEM experiments. The specific features of the Caulobacter system which make it a system of choice for studies of the control of sequential events resulting in cellular differentiation can be summarized as follows. The sequence upstream of the translational start site shows little homology to the canonical Shine-Dalgarno ribosome recognition sequence, but the region downstream of the start codon is complementary to a region of 16S rRNA implicated in downstream box recognition. We predict that with any phenotype independent of energy production, however, pH-sensitive mutants will be recovered only in surface elements. MmpA appears to cleave within or near the transmembrane segment of PodJS, releasing it into the cytoplasm for complete proteolysis. Located on the first floor of the Shapiro Undergraduate Library, the Lab bosts ownership of four 3D printers, a 3D printer pen, a silhouette vinyl cutter, an 18th century letter press, a heavy duty singer sewing machine, two soldering stations, and a VR station with an Oculus and Vive. A major breakthrough in understanding the bacterial cell cycle is the discovery that bacteria exhibit a high degree of intracellular organization. The assembly of a functional flagellum in the bacterium Caulobacter crescentus requires the protein products of approximately 30 genes expressed in a temporally discrete and spatially distinct manner. 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. View details for DOI 10.1128/JB.185.11.3384-3391.2003, View details for Web of Science ID 000183100900016, View details for PubMedCentralID PMC155372. Millions of possible codes can be prepared this way. View details for Web of Science ID A1995QB30700010, View details for PubMedCentralID PMC176597, View details for Web of Science ID A1995BG35H00001. View details for Web of Science ID 000227028900009. A., Ryan, K. R., Shapiro, L., McAdams, H. H. The bifunctional FtsK protein mediates chromosome partitioning and cell division in Caulobacter, DnaA couples DNA replication and the expression of two cell cycle master regulators, Cytokinesis signals truncation of the PodJ polarity factor by a cell cycle-regulated protease. View details for Web of Science ID A1996TQ17000011, View details for Web of Science ID A1996BG26T00006. When samples containing roGFP2 are rapidly cooled to 77K in a manner compatible with cryo-EM, the ratio of excitation peaks remains a faithful indicator of the redox potential at the time of freezing. Hierarchical formation of higher-order SpmX oligomers nucleates new PopZ microdomain assemblies at the incipient lateral cell poles, driving localized outgrowth. This vast structural blueprint of specific positional information is manifested in various ways, directing chromosome compaction, accessibility, attachment to the cell envelope, supercoiling, and general architecture and arrangement of the chromosome relative to the cell body. Yuan (Soso) Xue, Bioengineering (09/2015-03/2016). American volume -Cassidy, C., Jupiter, J. A., Hottes, A. K., Tan, M. H., Hillson, N. J., Hu, P., Shapiro, L., McAdams, H. H. The push and pull of the bacterial cytoskeleton, Chromosome organization and segregation in bacteria, PHYS 489-Direct observation of MreB treadmilling in Caulobacter by single-molecule fluorescence microscopy. Cell division in Gram-negative organisms requires coordinated invagination of the multilayered cell envelope such that each daughter receives an intact inner membrane, peptidoglycan (PG) layer and outer membrane (OM). We further show that CckA oligomerizes through a multidomain interaction that is critical for stimulation of kinase activity by DivL, while DivL stimulation of CckA phosphatase activity is independent of CckA homooligomerization. Bacteria have evolved several different mechanisms to target protein complexes, membrane vesicles and DNA to specific positions within the cell. A Bacterial Toxin Perturbs Intracellular Amino Acid Balance To Induce Persistence. This result allowed us to deduce that the mechanism of fatty acid desaturation in C. crescentus is anaerobic, as it is in E. coli. View details for DOI 10.1128/mBio.00448-20. 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. Bacterial cells utilize toxin-antitoxin systems to inhibit self-reproduction, while maintaining viability, when faced with environmental challenges. The sequential changes in the chromosomal methylation state serve to couple the progression of DNA replication to cell-cycle events regulated by the master transcriptional regulatory cascade, thus providing a ratchet mechanism for robust cell-cycle control. In this study, we uncovered a mechanism by which daughter cell fate, which is specified by the DivJ-DivK-PleC system and effectively encoded in the phosphorylation state of the single-domain RR DivK, is communicated to the CckA-ChpT-CtrA signaling pathway that regulates more than 100 genes for polar differentiation, replication initiation and cell division. A., Eckart, M. R., Shapiro, L. Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle. Examination of the intracellular location of SMC showed that in swarmer cells, which do not replicate DNA, the protein forms two or three foci. The rapid synchronizability and high cell yields of Caulobacter make this organism a powerful model system for studies of the bacterial cell cycle. Minor in Poverty, Inequality, and Policy Jesse Shapiro. Pubmedcentralid PMC176597, View details for Web of Science ID 000275368400036, View details for PubMedCentralID PMC155372 remnants they... Process, the fluorescent protein roGFP2, for cryo-CLEM experiments and orf-1 are within a identified! Analysis shows that > 10 % of the chemotaxis signal is initiated by a ternary! Tn5-Vb32 mutagenesis and shown to confer kanamycin resistance P1 promoter fires early in S phase and contains a sequence... 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