Expression profiling of B.subtils ftsZ depletion cells with ftsZ induced or uninduced for 1 or 2hrs.
ABSTRACT: The past twenty years have seen tremendous advances in our understanding of the mechanisms underlying bacterial cytokinesis, particularly the composition of the division machinery and the factors controlling its assembly. At the same time, however, we understand very little about the relationship between cell division and other cell cycle events in bacteria. Here we report that inhibiting division in Bacillus subtilis and Staphylococcus aureus quickly leads to an arrest in the initiation of new rounds of DNA replication followed by a complete arrest in cell growth. Arrested cells are metabolically active but unable to initiate new rounds of either DNA replication or division when shifted to permissive conditions. Inhibiting DNA replication results in entry into a similar quiescent state, in which cells are unable to resume growth or division when returned to permissive conditions. Our findings suggest the presence of two cell cycle control points: one linking division to the initiation of DNA replication and another linking the initiation of DNA replication to division. Significantly, this evidence contradicts the prevailing view of the bacterial cell cycle as a series of coordinated but uncoupled events. Importantly, the terminal nature of the cell cycle arrest validates the bacterial cell cycle machinery as an effective target for antimicrobial development. Four-condition experiment: ftsZ induced for 1hr, ftsZ depleted for 1hr, ftsZ induced for 2hrs, ftsZ depleted for 2hrs. Biological replicates: 3-4 for each sample. Reference: a mixture of wt RNA from different growth phases and wt backgrounds.
Project description:One of the first steps in bacterial cell division is the polymerization of the tubulin-like protein FtsZ at midcell. The dynamics of FtsZ polymerization is regulated by a set of proteins among which ZapA. A zapA mutation does not result in a clear phenotype in Bacillus subtilis. In this study we used a synthetic-lethal screen to find genes that become essential when ZapA is absent. Three transposon insertions were found in yvcL. Deletion of yvcL in a wild type background had only a mild effect on growth, but a yvcL zapA double mutant is very filamentous and sick. This filamentation is caused by a strong reduction in FtsZ polymerization, suggesting that YvcL is involved in an early stage of cell division. YvcL is 25 % identical and 50 % similar to the Streptomyces coelicolor transcription factor WhiA. WhiA is required for septation of aerial hyphae during sporulation. Using GFP fusions, we show that YvcL localizes at the nucleoid. Surprisingly, transcriptome analyses in combination with a ChIP on chip assay did not provide clear evidence that YvcL functions as a transcription factor. To gain more insight into the function of YvcL, we searched for suppressors of the filamentous phenotype of a ∆yvcL ∆zapA mutant. Transposon insertions in gtaB and pgcA restored normal cell division of the double mutant. The corresponding proteins have been implemented in the metabolic sensing of cell division. We conclude that YvcL (WhiA) is involved in cell division in B. subtilis through an as yet unknown mechanism. Comparing wild tpe Bascillus subtilus (n=3) with Bascillus Subtilis KS400 (n=2) and Bascillus subtilis KS696 (n=2)
Project description:We performed ribosome profiling and RNA-seq on mouse bone marrow derived macrophages (BMDMs) treated with 100ng/ml LPS for 0hr, 1hr, 2hrs, 4hrs, 6hrs to obtain global mRNA translational landscapes during this inflammatory response. Overall design: Examine transcriptomic and translatomic dynamics in mouse BMDMs' response to LPS treatment. Ribo-seq, RNA-Seq, CLIP-seq.
Project description:WhiA is a highly unusual transcriptional regulator related to a family of eukaryotic homing endonucleases. WhiA is required for sporulation in the filamentous bacterium Streptomyces, but WhiA homologues of unknown function are also found throughout the Gram-positive bacteria. To better understand the role of WhiA in Streptomyces development and its function as a transcription factor, we identified the WhiA regulon through a combination of chromatin immunoprecipitation-sequencing (ChIP-seq) and microarray transcriptional profiling, exploiting a new model organism for the genus, Streptomyces venezuelae, which sporulates in liquid culture. The regulon encompasses ~240 transcription units, and WhiA appears to function almost equally as an activator and as a repressor. Bioinformatic analysis of the upstream regions of the complete regulon, combined with DNase I footprinting, identified a short but highly conserved asymmetric sequence, GACAC, associated with the majority of WhiA targets. Construction of a null mutant showed that whiA is required for the initiation of sporulation septation and chromosome segregation in S. venezuelae, and several genes encoding key proteins of the Streptomyces cell division machinery, such as ftsZ, ftsW, and ftsK, were found to be directly activated by WhiA during development. Several other genes encoding proteins with important roles in development were also identified as WhiA targets, including the sporulation-specific sigma factor (WhiG) and the diguanylate cyclase CdgB. Cell division is tightly coordinated with the orderly arrest of apical growth in the sporogenic cell, and filP, encoding a key component of the polarisome that directs apical growth, is a direct target for WhiA-mediated repression during sporulation.
Project description:We compared the expression profile of TTHA1939 deletion mutant strain of Thermus thermophils HB8 with that of wild-type. The mutant strain grown in minimum essential medium (CS medium) exhibited growth arrest and cellular aggregation during the logarithm growth phase. In this phase, 99 genes were suppressed and 63 genes were activated for the mutant strain. The suppressed genes included genes of ribosomal proteins, TCA cycle, amino acid biosynthesis, fatty acid biosynthesis, cobalamin biosynthesis, transporters, and cell division including dnaB and ftsZ. The activated genes included genes of nitrogen metabolism, stress proteins, and proteases. We suggested that these transcriptional alterations in the mutant cell were induced by the ppGpp accumulation which was prevented by ppGpp degradation activity of TTHA1939 in WT cell. Keywords: gene deletion, minimum essential medium, logarithm growth phase Overall design: Wild-type and the mutant strains were precultured in rich medium (TR medium) for two times at 70 oC and then subcultured to minimum essential medium (CS medium). These cells were harvested during the logarithm growth phase (800 minutes from the start of culture). Total RNA were extracted from each strain and used for the cDNA synthesis. For the biological replication, above experiments were performed four times independently. The cDNA fragments were labeled with biotin-dideoxy UTP, using terminal transferase according to the manufacturer’s instructions (ENZO Biochem. Inc., Farmingdale, NY). The 3’-terminal-labeled cDNA (2 micro g) was hybridized to a TTHB8401 GeneChip (Affymetrix, Santa Clara, CA). The Probe Array was scanned with a GeneArray Scanner (Affymetrix), and then, the image data was scaled to the target intensity by one-step Tukey’s biweight algorithm using GeneChip Operating software, version 1.0 (Affymetrix, Santa Clara, CA). The data analysis was performed by using GeneSpring GX (Agilent Tech.). The genes which had detection call of “presence” more than 4 times from 8 samples were used for following analysis. The normalized intensities for the mutant strains were calculated by using the intensities for wild-type strains of each data set, and then, the average of these intensities were used. The genes which produced P value < 0.1 in the Student’s t-test were extracted. Among them, the genes whose expression level was different more than 2 fold between two strains were considered as significant.
Project description:Novak1997 - Cell Cycle
Modeling the control of DNA replication in fission yeast.
This model is described in the article:
Modeling the control of DNA replication in fission yeast.
Novak B., Tyson JJ.
Proc. Natl. Acad. Sci. U.S.A. 1997:94(17):9147-52
A central event in the eukaryotic cell cycle is the decision to commence DNA replication (S phase). Strict controls normally operate to prevent repeated rounds of DNA replication without intervening mitoses ("endoreplication") or initiation of mitosis before DNA is fully replicated ("mitotic catastrophe"). Some of the genetic interactions involved in these controls have recently been identified in yeast. From this evidence we propose a molecular mechanism of "Start" control in Schizosaccharomyces pombe. Using established principles of biochemical kinetics, we compare the properties of this model in detail with the observed behavior of various mutant strains of fission yeast: wee1(-) (size control at Start), cdc13Delta and rum1(OP) (endoreplication), and wee1(-) rum1Delta (rapid division cycles of diminishing cell size). We discuss essential features of the mechanism that are responsible for characteristic properties of Start control in fission yeast, to expose our proposal to crucial experimental tests.
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Project description:Oncogene-induced senescence is an anti-proliferative stress response program that acts as a fail-safe mechanism to limit oncogenic transformation and is regulated by the retinoblastoma protein (RB) and p53 tumor suppressor pathways. We identify the atypical E2F family member E2F7 as the only E2F transcription factor potently upregulated during oncogene-induced senescence, a setting where it acts in response to p53 as a direct transcriptional target. Once induced, E2F7 binds and represses a series of E2F target genes and cooperates with RB to efficiently promote cell cycle arrest and limit oncogenic transformation. Disruption of RB triggers a further increase in E2F7, which induces a second cell cycle checkpoint that prevents unconstrained cell division despite aberrant DNA replication. Mechanistically, E2F7 compensates for the loss of RB in repressing mitotic E2F target genes. Overall design: Examination of E2F7 binding in either growing or senescent IMR90 cells with different hairpins.
Project description:Successive division events in the spherically shaped bacterium Staphylococcus aureus are oriented in three alternating perpendicular planes. The mechanisms that underlie this relatively unique pattern of division and coordinate it with chromosome segregation remain largely unknown. Thus far, the only known spatial regulator of division in this organism is the nucleoid occlusion protein Noc that inhibits assembly of the cytokinetic ring over the chromosome. However, Noc is not essential in S. aureus, indicating that additional regulators are likely to exist. To search for these factors, we screened for mutants that are synthetic lethal with Noc inactivation. Our characterization of these mutants led to the discovery that S. aureus Noc also controls the initiation of DNA replication. We show that cells lacking Noc over-initiate and mutations in the initiator gene dnaA suppress this defect. Importantly, these dnaA mutations also partially suppress the division problems associated with ∆noc. Reciprocally, we show that over-expression of DnaA enhances the over-initiation and cell division phenotypes of cells lacking Noc. Thus, a single factor both blocks cell division over chromosomes and helps to ensure that new rounds of DNA replication are not initiated prematurely. This degree of economy in coordinating key cell biological processes has not been observed in rod-shaped bacteria and may reflect the challenges posed by the reduced cell volume and complicated division pattern of this spherical pathogen. Overall design: Tn-seq experiments were performed on wild type and mutant cells of Staphylococcus aureus RN4220 (Nair et al,PMID: 21378186 ) growing in tryptic soy broth (TSB) at 30°C with aeration.