Iron chelation increases the tolerance of Escherichia coli to hyper-replication stress.
ABSTRACT: In Escherichia coli, an increase in the frequency of chromosome replication is lethal. In order to identify compounds that affect chromosome replication, we screened for molecules capable of restoring the viability of hyper-replicating cells. We made use of two E. coli strains that over-initiate DNA replication by keeping the DnaA initiator protein in its active ATP bound state. While viable under anaerobic growth or when grown on poor media, these strains become inviable when grown in rich media. Extracts from actinomycetes strains were screened, leading to the identification of deferoxamine (DFO) as the active compound in one of them. We show that DFO does not affect chromosomal replication initiation and suggest that it was identified due to its ability to chelate cellular iron. This limits the formation of reactive oxygen species, reduce oxidative DNA damage and promote processivity of DNA replication. We argue that the benzazepine derivate (±)-6-Chloro-PB hydrobromide acts in a similar manner.
Project description:Yeast cell cycle transcript dynamics in three S. cerevisiae strains grown at 30 degrees Celsius: cdc20 GALL-CDC20 (persistent mitotic CDK activity; CDK on), cdc8-ts (DNA replication checkpoint), GAL-cse4-353 (spindle assembly checkpoint), cdc8-ts cdc20 (DNA replication checkpoint, CDK on), and cdc8-ts cdc20, rad53-1 (DNA replication checkpoint without Rad53 activity, CDK on) in a BF264-15DU background. We compared transcript levels of genes previously shown to be periodically expressed in wild-type cells and in cells lacking all mitotic cyclins (clb1,2,3,4,5,6; CDK off). Two replicate time courses for each of the strains studied are included
Project description:Escherichia coli strains from sewage sample were screened for the presence and expression of heavy metal-translocating zntA gene by PCR and RT-PCR analysis with type culture of K-12 as standard strain. The strain which showed high level of gene expression (SBVP1) was chosen to further study the growth and heavy metal translocation. This superior strain was grown in the presence of ZnSO4, Pb (CH3COO)2 and mixture of ZnSO4, Pb(CH3COO)2 metal salts and the growth was observed at different time points. The cell pellet fraction was found to have more of zinc than lead as determined by atomic absorption spectroscopy indicating the translocation of these metals from media to the cells. However, the intracellular translocation of zinc is affected by the presence of lead in the media. Expression of the zntA gene in bacteria grown in the presence of ZnSO4 was also studied and the molecular analysis results correlate with spectroscopic observations.
Project description:Replication of bacterial chromosomes increases the copy numbers of genes located near the origin of replication relative to genes located near the terminus. Such differential gene dosage is variable and depends on several factors including replication rate, generation time and chromosome size. For vibrios, a diverse family of fast growing marine living gammaproteobacteria, gene dosage effects may be particularly important as they harbour their genome within two differently sized chromosomes. In this series of experiments, gene dosage at the genomic level and its influence on the expression level has been examined in V. parahaemolyticus. For the genomic analyses, gene copy numbers for bacteria in mid-logarithmic phase grown in rich media (Luria-Bertani containing 3% (w/v) NaCl) at two temperatures (37Â°C or 20Â°C) or in minimal media (M9 containing 3% (w/v) NaCl and 0.4% (w/v) glucose) were compared against gene copy numbers for non-replicating cells. For expression analyses, cDNA derived from bacteria in mid-logarithmic phase grown in rich broth at two temperatures (37Â°C or 20Â°C) or in minimal media were compared against gDNA from non-replicating cells. Also, cDNA derived from early (OD600=0.2) or mid-logarithmic phase bacteria (OD600=0.5) in rich broth at 37Â°C, were compared against cDNA derived from mid-logarithmic cells in minimal media (OD600=0.5) or cDNA from early stationary phase rich broth cells (OD600=3.0). Keywords: Comparative genomic and/or cDNA hybridization Samples of genomic DNA and/or cDNA were labeled with Cy3 and Cy5 and cohybridized on six single arrays.
Project description:Precise chromosome transmission in cell division cycle is maintained by a number of genes. The attempt made in the present study was to isolate temperature-sensitive (ts) fission yeast mutants that display high loss rates of minichromosomes at permissive or semipermissive temperature (designated mis). By colony color assay of 539 ts strains that contain a minichromosome, we have identified 12 genetic loci (mis1-mis12) and determined their phenotypes at restrictive temperature. Seven of them are related to cell cycle block phenotype at restrictive temperature, three of them in mitosis. Unequal distribution of regular chromosomes in the daughters is extensive in mis6 and mis12. Cells become inviable after rounds of cell division due to missegregation. The phenotype of mis5 is DNA replication defect and hypersensitivity to UV ray and hydroxyurea. mis5+ encodes a novel member of the ubiquitous MCM family required for the onset of replication. The mis5+ gene is essential for viability and functionally distinct from other previously identified members in fission yeast, cdc21+, nda1+, and nda4+. The mis11 mutant phenotype was the cell division block with reduced cell size. Progression of the G1 and G2 phases is blocked in mis11. The cloned mis11+ gene is identical to prp2+, which is essential for RNA splicing and similar to a mammalian splicing factor U2AF65.
Project description:Transcriptome profile of S. cerevisiae strains grown in rich ethanol/glycerol media, where the endogenous mitochondrial gene ATP9 has been deleted and relocated to the nucleus by expressing synthetic, codon-optimized versions of the Podospora anserina genes Atp9-5 and Atp9-7. The strains AMY7, AMY8, AMY10, and AMY11 each express one of these genes either from a centromeric or 2-micron plasmid. These strains are derived from RKY26, an atp9 deletion mutant which was not included in this experiment since it does not grow on respiratory media. The isogenic wild-type strain (MR6) was included as a control. Strains were produced for this study, and the arrays used were yeast tiling microarrays from Affymetrix. Note that normalization was performed using genomic DNA hybridizations, the raw data for which can be found in ArrayExpress Accession # E-TABM-1176.
Project description:Many studies are performed on chromosome replication and segregation in Escherichia coli and other bacteria capable of complex replication with C phases spanning several generations. For such investigations an understanding of the replication patterns, including copy numbers of origins and replication forks, is crucial for correct interpretation of the results.Flow cytometry is an important tool for generation of experimental DNA distributions of cell populations. Here, a Visual Basic based simulation program was written for the computation of theoretical DNA distributions for different choices of cell cycle parameters (C and D phase durations, doubling time etc). These cell cycle parameters can be iterated until the best fit between the experimental and theoretical DNA histograms is obtained. The Excel file containing the simulation software is attached as supporting information.Cultures of Escherichia coli were grown at twelve different media and temperature conditions, with following measurements by flow cytometry and simulation of the DNA distributions. A good fit was found for each growth condition by use of our simulation program. The resulting cell cycle parameters displayed clear inter-media differences in replication patterns, but indicated a high degree of temperature independence for each medium. The exception was the poorest medium (acetate), where the cells grew with overlapping replication cycles at 42 °C, but without at the lower temperatures.We have developed an easy-to-use tool for determination of bacteria's cell cycle parameters, and consequently the cells' chromosome configurations. The procedure only requires DNA distribution measurements by flow cytometry. Use of this simulation program for E. coli cultures shows that even cells growing quite slowly can have overlapping replication cycles. It is therefore always important not only to assume cells' replication patterns, but to actually determine the cell cycle parameters when changing growth conditions.
Project description:We compared multiple strains of lab trophozoites to recent clinical isolates. Clinical isolates were grown in xenic media, and maintained many characteristics of the cyst stage of devlopment Keywords: Stage conversion E. histolytica trophozoites of three different strains (HM1:IMSS, Rahman, and 200:NIH) and grown under multiple conditions (Long term axenic growth in TYI, Growth in low glucose media, Mouse adapted parasites and in vivo growth in a mouse model) were compared to recent clinical isolates that had grown in xenic culture for 1-8 weeks.
Project description:Mutations in the tumor suppressor BRCA2 predominantly predispose to breast cancer. Paradoxically, while loss of BRCA2 promotes tumor formation, it also causes cell lethality, although how lethality is triggered is unclear. Here, we generate BRCA2 conditional non-transformed human mammary epithelial cell lines using CRISPR-Cas9. Cells are inviable upon BRCA2 loss, which leads to replication stress associated with under replication, causing mitotic abnormalities, 53BP1 nuclear body formation in the ensuing G1 phase, and G1 arrest. Unexpected from other systems, the role of BRCA2 in homologous recombination, but not in stalled replication fork protection, is primarily associated with supporting human mammary epithelial cell viability, and, moreover, preventing replication stress, a hallmark of pre-cancerous lesions. Thus, we uncover a DNA under replication-53BP1 nuclear body formation-G1 arrest axis as an unanticipated outcome of homologous recombination deficiency, which triggers cell lethality and, we propose, serves as a barrier that must be overcome for tumor formation.BRCA2 mutations promote tumour formation while also paradoxically causing cell lethality. Here the authors generate conditional BRCA2 loss in a non-transformed human mammary cell line and see increased replication stress due to under-replication of DNA.
Project description:Cyclical inactivation of B-type cyclins has been proposed to be required for alternating DNA replication and mitosis. Destruction box-dependent Clb5p degradation is strongly increased in mitotic cells, and constitutive overexpression of Clb5p lacking the destruction box resulted in rapid accumulation of inviable cells, frequently multiply budded, with DNA contents ranging from unreplicated to apparently fully replicated. Loss of viability correlated with retention of nuclear Clb5p at the time of nuclear division. CLB2-Deltadb overexpression that was quantitatively comparable to CLB5-Deltadb overexpression with respect to Clb protein production and Clb-associated kinase activity resulted in a distinct phenotype: reversible mitotic arrest with uniformly replicated DNA. Simultaneous overexpression of CLB2-Deltadb and CLB5-Deltadb overexpressers similarly resulted in a uniform arrest with replicated DNA, and this arrest was significantly more reversible than that observed with CLB5-Deltadb overexpression alone. These results suggest that Clb2p and not Clb5p can efficiently block mitotic completion. We speculate that CLB5-Deltadb overexpression may be lethal, because persistence of high nuclear Clb5p-associated kinase throughout mitosis leads to failure to load origins of replication, thus preventing DNA replication in the succeeding cell cycle.
Project description:5-Aminolevulinic acid (5-ALA) has been approved for clinical photodynamic therapy (PDT) due to its negligible photosensitive toxicity. However, the curative effect of 5-ALA is restricted by intracellular biotransformation inactivation of 5-ALA and potential DNA repair of tumor cells. Inspired by the crucial function of iron ions in 5-ALA transformation and DNA repair, a liposomal nanomedicine (MFLs@5-ALA/DFO) with intracellular iron ion regulation property was developed for boosting the PDT of 5-ALA, which was prepared by co-encapsulating 5-ALA and DFO (deferoxamine, a special iron chelator) into the membrane fusion liposomes (MFLs). MFLs@5-ALA/DFO showed an improved pharmaceutical behavior and rapidly fused with tumor cell membrane for 5-ALA and DFO co-delivery. MFLs@5-ALA/DFO could efficiently reduce iron ion, thus blocking the biotransformation of photosensitive protoporphyrin IX (PpIX) to heme, realizing significant accumulation of photosensitivity. Meanwhile, the activity of DNA repair enzyme was also inhibited with the reduction of iron ion, resulting in the aggravated DNA damage in tumor cells. Our findings showed MFLs@5-ALA/DFO had potential to be applied for enhanced PDT of 5-ALA.