Nutritional Control of DNA Replication Initiation through the Proteolysis and Regulated Translation of DnaA
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{"title"=>"Nutritional Control of DNA Replication Initiation through the Proteolysis and Regulated Translation of DnaA", "type"=>"journal", "authors"=>[{"first_name"=>"David J.", "last_name"=>"Leslie", "scopus_author_id"=>"56566124600"}, {"first_name"=>"Christian", "last_name"=>"Heinen", "scopus_author_id"=>"56767842300"}, {"first_name"=>"Frederic D.", "last_name"=>"Schramm", "scopus_author_id"=>"55487187600"}, {"first_name"=>"Marietta", "last_name"=>"Thüring", "scopus_author_id"=>"55749130200"}, {"first_name"=>"Christopher D.", "last_name"=>"Aakre", "scopus_author_id"=>"26533785800"}, {"first_name"=>"Sean M.", "last_name"=>"Murray", "scopus_author_id"=>"56009766200"}, {"first_name"=>"Michael T.", "last_name"=>"Laub", "scopus_author_id"=>"7006013323"}, {"first_name"=>"Kristina", "last_name"=>"Jonas", "scopus_author_id"=>"12799630000"}], "year"=>2015, "source"=>"PLoS Genetics", "identifiers"=>{"scopus"=>"2-s2.0-84938786366", "doi"=>"10.1371/journal.pgen.1005342", "sgr"=>"84938786366", "isbn"=>"1553-7404", "pmid"=>"26134530", "issn"=>"15537404", "pui"=>"605567968"}, "id"=>"cfa16157-7411-3b69-a63a-2dce4e55904b", "abstract"=>"Bacteria can arrest their own growth and proliferation upon nutrient depletion and under various stressful conditions to ensure their survival. However, the molecular mechanisms responsible for suppressing growth and arresting the cell cycle under such conditions remain incompletely understood. Here, we identify post-transcriptional mechanisms that help enforce a cell-cycle arrest in Caulobacter crescentus following nutrient limitation and during entry into stationary phase by limiting the accumulation of DnaA, the conserved replication initiator protein. DnaA is rapidly degraded by the Lon protease following nutrient limitation. However, the rate of DnaA degradation is not significantly altered by changes in nutrient availability. Instead, we demonstrate that decreased nutrient availability downregulates dnaA translation by a mechanism involving the 5' untranslated leader region of the dnaA transcript; Lon-dependent proteolysis of DnaA then outpaces synthesis, leading to the elimination of DnaA and the arrest of DNA replication. Our results demonstrate how regulated translation and constitutive degradation provide cells a means of precisely and rapidly modulating the concentration of key regulatory proteins in response to environmental inputs.", "link"=>"http://www.mendeley.com/research/nutritional-control-dna-replication-initiation-through-proteolysis-regulated-translation-dnaa", "reader_count"=>51, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>3, "Researcher"=>8, "Student > Doctoral Student"=>3, "Student > Ph. D. Student"=>19, "Student > Postgraduate"=>2, "Student > Master"=>4, "Other"=>5, "Student > Bachelor"=>7}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>3, "Researcher"=>8, "Student > Doctoral Student"=>3, "Student > Ph. D. Student"=>19, "Student > Postgraduate"=>2, "Student > Master"=>4, "Other"=>5, "Student > Bachelor"=>7}, "reader_count_by_subject_area"=>{"Engineering"=>1, "Biochemistry, Genetics and Molecular Biology"=>23, "Agricultural and Biological Sciences"=>25, "Chemistry"=>1, "Immunology and Microbiology"=>1}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>1}, "Chemistry"=>{"Chemistry"=>1}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>25}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>23}}, "reader_count_by_country"=>{"United States"=>1}, "group_count"=>0}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/2160111"], "description"=>"<p>(A) Growth curves of wild type (WT), <i>ΔspoT</i> and <i>ΔspoTΔppk1</i> cells grown in PYE. (B) Western Blots showing DnaA protein levels at the indicated optical densities in the three strains. The graphs show quantifications of band intensities. Averages of at least two independent replicates are shown with standard deviations. See also <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005342#pgen.1005342.s001\" target=\"_blank\">S1 Fig</a> for DnaA stability in <i>ΔspoT</i> cells. (C) Western Blots as in (B), but probed with an antibody specific for CtrA. (D) Flow cytometry profiles of WT, <i>ΔspoT</i> and <i>ΔspoTΔppk1</i> cells in exponential phase (OD<sub>600</sub> 0.4) or after growth for 24 hours in stationary phase. The percentage of cells with one chromosome (1N) is indicated.</p>", "links"=>[], "tags"=>["Lon protease", "Caulobacter crescentus", "DNA replication initiation", "constitutive degradation", "mechanism", "dnaA transcript", "Nutritional Control", "replication initiator protein", "availability downregulates dnaA translation", "DnaA degradation", "cell cycle", "Regulated Translation", "DnaA Bacteria", "DNA replication"], "article_id"=>1471870, "categories"=>["Biological Sciences"], "users"=>["David J. Leslie", "Christian Heinen", "Frederic D. Schramm", "Marietta Thüring", "Christopher D. Aakre", "Seán M. Murray", "Michael T. Laub", "Kristina Jonas"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1005342.g002", "stats"=>{"downloads"=>0, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_p_ppGpp_is_not_required_to_eliminate_DnaA_during_the_entry_to_stationary_phase_/1471870", "title"=>"(p)ppGpp is not required to eliminate DnaA during the entry to stationary phase.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-07-02 02:53:51"}
  • {"files"=>["https://ndownloader.figshare.com/files/2160156", "https://ndownloader.figshare.com/files/2160157", "https://ndownloader.figshare.com/files/2160158", "https://ndownloader.figshare.com/files/2160159", "https://ndownloader.figshare.com/files/2160160", "https://ndownloader.figshare.com/files/2160161", "https://ndownloader.figshare.com/files/2160162", "https://ndownloader.figshare.com/files/2160163", "https://ndownloader.figshare.com/files/2160164", "https://ndownloader.figshare.com/files/2160165", "https://ndownloader.figshare.com/files/2160166", "https://ndownloader.figshare.com/files/2160167", "https://ndownloader.figshare.com/files/2160168"], "description"=>"<div><p>Bacteria can arrest their own growth and proliferation upon nutrient depletion and under various stressful conditions to ensure their survival. However, the molecular mechanisms responsible for suppressing growth and arresting the cell cycle under such conditions remain incompletely understood. Here, we identify post-transcriptional mechanisms that help enforce a cell-cycle arrest in <i>Caulobacter crescentus</i> following nutrient limitation and during entry into stationary phase by limiting the accumulation of DnaA, the conserved replication initiator protein. DnaA is rapidly degraded by the Lon protease following nutrient limitation. However, the rate of DnaA degradation is not significantly altered by changes in nutrient availability. Instead, we demonstrate that decreased nutrient availability downregulates <i>dnaA</i> translation by a mechanism involving the 5' untranslated leader region of the <i>dnaA</i> transcript; Lon-dependent proteolysis of DnaA then outpaces synthesis, leading to the elimination of DnaA and the arrest of DNA replication. Our results demonstrate how regulated translation and constitutive degradation provide cells a means of precisely and rapidly modulating the concentration of key regulatory proteins in response to environmental inputs.</p></div>", "links"=>[], "tags"=>["Lon protease", "Caulobacter crescentus", "DNA replication initiation", "constitutive degradation", "mechanism", "dnaA transcript", "Nutritional Control", "replication initiator protein", "availability downregulates dnaA translation", "DnaA degradation", "cell cycle", "Regulated Translation", "DnaA Bacteria", "DNA replication"], "article_id"=>1471913, "categories"=>["Biological Sciences"], "users"=>["David J. Leslie", "Christian Heinen", "Frederic D. Schramm", "Marietta Thüring", "Christopher D. Aakre", "Seán M. Murray", "Michael T. Laub", "Kristina Jonas"], "doi"=>["https://dx.doi.org/10.1371/journal.pgen.1005342.s001", "https://dx.doi.org/10.1371/journal.pgen.1005342.s002", "https://dx.doi.org/10.1371/journal.pgen.1005342.s003", "https://dx.doi.org/10.1371/journal.pgen.1005342.s004", "https://dx.doi.org/10.1371/journal.pgen.1005342.s005", "https://dx.doi.org/10.1371/journal.pgen.1005342.s006", "https://dx.doi.org/10.1371/journal.pgen.1005342.s007", "https://dx.doi.org/10.1371/journal.pgen.1005342.s008", "https://dx.doi.org/10.1371/journal.pgen.1005342.s009", "https://dx.doi.org/10.1371/journal.pgen.1005342.s010", "https://dx.doi.org/10.1371/journal.pgen.1005342.s011", "https://dx.doi.org/10.1371/journal.pgen.1005342.s012", "https://dx.doi.org/10.1371/journal.pgen.1005342.s013"], "stats"=>{"downloads"=>6, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Nutritional_Control_of_DNA_Replication_Initiation_through_the_Proteolysis_and_Regulated_Translation_of_DnaA_/1471913", "title"=>"Nutritional Control of DNA Replication Initiation through the Proteolysis and Regulated Translation of DnaA", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2015-07-02 02:53:51"}
  • {"files"=>["https://ndownloader.figshare.com/files/2160120"], "description"=>"<p>The synthesis and the degradation of DnaA are both subject to control mechanisms that respond to environmental changes. Changes in nutrient availability modulate the rate of DnaA synthesis by a mechanism involving the 5'UTR. Changes in the global protein folding state impact the rate of DnaA degradation by the protease Lon. During exponential growth high levels of nutrients promote translation of DnaA. Although DnaA is constantly degraded, the rate of synthesis is high enough to allow for the accumulation of DnaA and DNA replication initiation. In starvation and stationary phase conditions lower amounts of nutrients cause the translation rate of DnaA to decrease. Because DnaA degradation continues at the same rate as in exponential phase, DnaA is rapidly cleared leading to a cessation of DNA replication. In proteotoxic stress conditions, for example chaperone depletion or thermal stress, nutrients are still available and drive DnaA synthesis. However, Lon-mediated DnaA degradation is stimulated in these conditions leading to the clearance of DnaA and a G1-arrest [<a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005342#pgen.1005342.ref026\" target=\"_blank\">26</a>].</p>", "links"=>[], "tags"=>["Lon protease", "Caulobacter crescentus", "DNA replication initiation", "constitutive degradation", "mechanism", "dnaA transcript", "Nutritional Control", "replication initiator protein", "availability downregulates dnaA translation", "DnaA degradation", "cell cycle", "Regulated Translation", "DnaA Bacteria", "DNA replication"], "article_id"=>1471879, "categories"=>["Biological Sciences"], "users"=>["David J. Leslie", "Christian Heinen", "Frederic D. Schramm", "Marietta Thüring", "Christopher D. Aakre", "Seán M. Murray", "Michael T. Laub", "Kristina Jonas"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1005342.g007", "stats"=>{"downloads"=>0, "page_views"=>13, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Dynamic_control_of_DnaA_abundance_and_DNA_replication_in_response_to_environmental_inputs_/1471879", "title"=>"Dynamic control of DnaA abundance and DNA replication in response to environmental inputs.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-07-02 02:53:51"}
  • {"files"=>["https://ndownloader.figshare.com/files/2160119"], "description"=>"<p>(A) Growth curves and Western Blots showing changes in DnaA and CtrA levels after shifting wild type, <i>Δlon</i>, <i>ΔspoT</i> and <i>P</i><sub><i>lac</i></sub><i>-dnaA</i> cells from M2G to M2 medium containing 0.02% glucose at t = 0. The culture of <i>P</i><sub><i>lac</i></sub><i>-dnaA</i> cells was supplemented with 50 μM IPTG to induce <i>P</i><sub><i>lac</i></sub>. See also <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005342#pgen.1005342.s009\" target=\"_blank\">S9 Fig</a> for DnaA stability before and after glucose exhaustion. (B) Flow cytometry profiles of wild type, <i>Δlon</i>, <i>ΔspoT</i> and <i>P</i><sub><i>lac</i></sub><i>-dnaA</i> cells 0 or 8 hours after shift from M2G to M2 medium containing 0.02% glucose. The percentage of cells with one chromosome (1N) is indicated. (C) Growth curves and Western Blots showing changes in DnaA levels in strains, which either contain or lack the 5'UTR (see <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005342#pgen.1005342.g004\" target=\"_blank\">Fig 4D</a>), after shift from M2G to M2 medium containing 0.02% glucose at t = 0 (see also <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005342#pgen.1005342.s010\" target=\"_blank\">S10 Fig</a>). All strains were grown in the absence of xylose to shut off <i>dnaA</i> expression from the chromosome. The strain harboring the construct <i>P</i><sub><i>lac</i></sub><i>-UTR</i><sub><i>dnaA</i></sub><i>-dnaA</i> was grown in the presence of 1 mM IPTG.</p>", "links"=>[], "tags"=>["Lon protease", "Caulobacter crescentus", "DNA replication initiation", "constitutive degradation", "mechanism", "dnaA transcript", "Nutritional Control", "replication initiator protein", "availability downregulates dnaA translation", "DnaA degradation", "cell cycle", "Regulated Translation", "DnaA Bacteria", "DNA replication"], "article_id"=>1471878, "categories"=>["Biological Sciences"], "users"=>["David J. Leslie", "Christian Heinen", "Frederic D. Schramm", "Marietta Thüring", "Christopher D. Aakre", "Seán M. Murray", "Michael T. Laub", "Kristina Jonas"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1005342.g006", "stats"=>{"downloads"=>2, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Regulated_DnaA_synthesis_and_Lon_mediated_degradation_are_required_to_eliminate_DnaA_upon_carbon_exhaustion_/1471878", "title"=>"Regulated DnaA synthesis and Lon-mediated degradation are required to eliminate DnaA upon carbon exhaustion.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-07-02 02:53:51"}
  • {"files"=>["https://ndownloader.figshare.com/files/2160116"], "description"=>"<p>(A) Modeled DnaA synthesis (blue) and <i>dnaA</i> translation (red) rates over the growth curve in wild type <i>C</i>. <i>crescentus</i>. Synthesis and translation rates were mathematically determined as described in the Materials and Methods. (B) Transcript levels of <i>dnaA</i>, <i>katG</i> and <i>l13p</i> at the indicated optical densities in a wild type culture as determined by qPCR. Average values of relative expression changes of two independent experiments are shown with standard deviations. (C) Transcript levels as determined by microarray analysis of <i>dnaA</i>, <i>katG</i> and <i>l13p</i> as well as selected genes involved in stress responses in wild type grown to late stationary phase. Levels are relative to transcript levels of a culture grown in exponential phase. (D) Schematics of different expression constructs (not to scale), which either contain or lack the 5'UTR of the <i>dnaA</i> gene. The constructs were expressed from a low copy plasmid in a strain background in which the native copy of <i>dnaA</i> is under the control of a xylose inducible promoter (strain GM2471). (E) Changes in DnaA protein over the growth curve in strains expressing either of the three constructs shown in (D). The bottom graphs show the average band intensity of at least two independent experiments with standard deviations. All strains were grown in the absence of xylose to shut off <i>dnaA</i> expression from the chromosome. The strain harboring the construct <i>P</i><sub><b><i>lac</i></b></sub><i>-UTR</i><sub><b><i>dnaA</i></b></sub><i>-dnaA</i> was grown in the presence of 1 mM IPTG (see also <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005342#pgen.1005342.s006\" target=\"_blank\">S6 Fig</a>). (F) Flow cytometry profiles of the strains carrying plasmids that either lack (<i>P</i><sub><b><i>dnaA</i></b></sub><i>-ΔUTR</i><sub><b><i>dnaA</i></b></sub><i>-dnaA</i>) or contain (<i>P</i><sub><b><i>lac</i></b></sub><i>-UTR</i><sub><b><i>dnaA</i></b></sub><i>-dnaA</i>) the 5'UTR of the <i>dnaA</i> gene. Cells were grown for 12 hours after reaching the maximal OD<sub><b>600</b></sub> before samples were taken for flow cytometry analysis. The percentage of cells with one chromosome (1N) is indicated.</p>", "links"=>[], "tags"=>["Lon protease", "Caulobacter crescentus", "DNA replication initiation", "constitutive degradation", "mechanism", "dnaA transcript", "Nutritional Control", "replication initiator protein", "availability downregulates dnaA translation", "DnaA degradation", "cell cycle", "Regulated Translation", "DnaA Bacteria", "DNA replication"], "article_id"=>1471875, "categories"=>["Biological Sciences"], "users"=>["David J. Leslie", "Christian Heinen", "Frederic D. Schramm", "Marietta Thüring", "Christopher D. Aakre", "Seán M. Murray", "Michael T. Laub", "Kristina Jonas"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1005342.g004", "stats"=>{"downloads"=>1, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Reduced_translation_of_dnaA_accounts_for_the_downregulation_of_DnaA_abundance_at_the_onset_of_stationary_phase_/1471875", "title"=>"Reduced translation of <i>dnaA</i> accounts for the downregulation of DnaA abundance at the onset of stationary phase.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-07-02 02:53:51"}
  • {"files"=>["https://ndownloader.figshare.com/files/2160109"], "description"=>"<p>(A) Growth phase-dependent changes in DnaA and CtrA protein levels in wild type (WT) and <i>Δlon</i> cells. The upper graphs show growth curves of WT and <i>Δlon</i> cells grown in rich medium (PYE). Western Blots show DnaA or CtrA protein levels at the indicated OD<sub>600</sub> and after overnight growth in stationary phase (ON). The same set of samples was used in both Western blots. The bottom graphs show quantifications of band intensities. Averages of at least two independent replicates are shown with standard deviations. (B) Flow cytometry profiles and phase contrast microscopy images of wild type (WT) and <i>Δlon</i> cells in exponential phase (OD<sub>600</sub> 0.4) or after growth for 24 hours in stationary phase. The percentage of cells containing one chromosome (1N), two chromosomes (2N) or more than two chromosomes (>2N) are shown in tables. (C) Number and subcellular localization of origins of replication in wild type and <i>Δlon</i> cells at OD<sub>600</sub> 0.4, OD<sub>600</sub> 1.4 and after growth for 24 hours at the maximum OD<sub>600</sub> (stationary phase). Origins were labeled using a strain, which contains a <i>tetO</i> operator array close to the origin and the repressor gene <i>tetR-YFP</i> under the control of an inducible promoter. The number of origins per cell was quantified and graphically displayed. (D) DnaN-YFP foci in wild type and <i>Δlon</i> cells at OD<sub>600</sub> 1.4 and after growth for 24 hours at the maximum OD<sub>600</sub> (stationary phase). <i>dnaN-YFP</i> expression was induced by addition of 40 mM vanillate to the growth medium 1.5–2 hours prior to sampling. The number of foci was counted and graphically displayed.</p>", "links"=>[], "tags"=>["Lon protease", "Caulobacter crescentus", "DNA replication initiation", "constitutive degradation", "mechanism", "dnaA transcript", "Nutritional Control", "replication initiator protein", "availability downregulates dnaA translation", "DnaA degradation", "cell cycle", "Regulated Translation", "DnaA Bacteria", "DNA replication"], "article_id"=>1471868, "categories"=>["Biological Sciences"], "users"=>["David J. Leslie", "Christian Heinen", "Frederic D. Schramm", "Marietta Thüring", "Christopher D. Aakre", "Seán M. Murray", "Michael T. Laub", "Kristina Jonas"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1005342.g001", "stats"=>{"downloads"=>0, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Lon_dependent_proteolysis_is_required_to_eliminate_DnaA_and_to_induce_a_G1_arrest_upon_entry_to_stationary_phase_/1471868", "title"=>"Lon-dependent proteolysis is required to eliminate DnaA and to induce a G1-arrest upon entry to stationary phase.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-07-02 02:53:51"}
  • {"files"=>["https://ndownloader.figshare.com/files/2160117"], "description"=>"<p>(A) DnaA protein levels in exponentially grown wild type or <i>P</i><sub><i>lac</i></sub> cells when cultured in PYE or in M2G (minimal medium) supplemented with the indicated amounts of peptone (P) or peptone and yeast extract (YE). Band intensities were quantified (bottom); data points represent averages of two independent experiments with standard deviations (see also <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005342#pgen.1005342.s003\" target=\"_blank\">S3 Fig</a>). (B) Growth medium-dependent DnaA protein abundance in strains expressing constructs, which either contain or lack the 5'UTR of <i>dnaA</i> (see <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005342#pgen.1005342.g004\" target=\"_blank\">Fig 4D</a>). The three strains were grown in PYE and M2G and protein abundance was measured by Western blotting. The strain harboring the construct <i>P</i><sub><i>lac</i></sub><i>-UTR</i><sub><i>dnaA</i></sub><i>-dnaA</i> was grown in the presence of 1 mM IPTG (see also <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005342#pgen.1005342.s007\" target=\"_blank\">S7 Fig</a>). (C) Protein levels of DnaA and RpoA (loading control) in wild type and <i>ΔspoT</i> cells in PYE and M2G when grown at 30°C and in wild type cells when grown in PYE and M2G at room temperature (20°C). (D) Growth phase-dependent changes in DnaA protein levels in wild type cells when grown in 2x (red) and 0.5x (green) PYE. Cells were grown in the respective media to stationary phase. DnaA protein abundance was measured at the indicated culture OD<sub>600</sub> by Western blotting (see also <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005342#pgen.1005342.s007\" target=\"_blank\">S7 Fig</a>). The growth curve for wild type grown in 1x PYE is shown for comparison and is reproduced from <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005342#pgen.1005342.g001\" target=\"_blank\">Fig 1A</a>. (E) Changes in growth rate (upper graph) and DnaA protein levels after nutrient addition to a culture grown in stationary phase. A culture was grown for two hours in stationary phase (at OD<sub>600</sub> 1.5) before concentrated PYE was added to a final concentration of 1x (orange), 5x (red) or 10x (dark blue) that of PYE medium. As controls, the culture was either maintained in stationary phase (no addition, green) or backdiluted (1:10) into fresh PYE medium (backdilution, blue). DnaA protein levels were analyzed by Western blotting at the indicated time points (see also <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005342#pgen.1005342.s007\" target=\"_blank\">S7</a> and <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005342#pgen.1005342.s008\" target=\"_blank\">S8</a> Figs). (F) Changes in growth rate (upper graph) and DnaA after glucose addition to a carbon starved culture. A culture grown in M2G was shifted to M2 medium containing 0.02% glucose to induce carbon starvation. Two hours after the resulting growth arrest the culture was split into two subcultures. One of them remained untreated (no addition, green line), the other culture was supplemented with 0.2% glucose (glucose addition, red line). A third culture was grown in M2G medium throughout the experiment (M2G, blue line). DnaA protein levels were analyzed by Western blotting at the indicated time points (see also <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005342#pgen.1005342.s007\" target=\"_blank\">S7</a> and <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005342#pgen.1005342.s008\" target=\"_blank\">S8</a> Figs).</p>", "links"=>[], "tags"=>["Lon protease", "Caulobacter crescentus", "DNA replication initiation", "constitutive degradation", "mechanism", "dnaA transcript", "Nutritional Control", "replication initiator protein", "availability downregulates dnaA translation", "DnaA degradation", "cell cycle", "Regulated Translation", "DnaA Bacteria", "DNA replication"], "article_id"=>1471876, "categories"=>["Biological Sciences"], "users"=>["David J. Leslie", "Christian Heinen", "Frederic D. Schramm", "Marietta Thüring", "Christopher D. Aakre", "Seán M. Murray", "Michael T. Laub", "Kristina Jonas"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1005342.g005", "stats"=>{"downloads"=>1, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_translation_of_dnaA_is_modulated_by_nutrient_availability_/1471876", "title"=>"The translation of <i>dnaA</i> is modulated by nutrient availability.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-07-02 02:53:51"}
  • {"files"=>["https://ndownloader.figshare.com/files/2160114"], "description"=>"<p>(A) <i>In vivo</i> degradation assays showing DnaA stability in the DnaK/J depletion strain. Cells were grown in PYE with xylose (blue) or PYE with glucose for 4.5 hrs (red) to deplete DnaK/J, before chloramphenicol was added to shut-off protein synthesis. DnaA abundance was monitored by Western Blotting. Band intensities were quantified (bottom); averages of two independent replicates are shown with standard deviations (see also <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005342#pgen.1005342.s002\" target=\"_blank\">S2 Fig</a>). (B) <i>In vivo</i> degradation assays showing DnaA stability in wild type cells during exponential growth (OD<sub><b>600</b></sub> 0.4) and at the onset of stationary phase (OD<sub><b>600</b></sub> 1.0). Band intensities of Western Blots (top) were quantified (bottom); averages of three independent replicates are shown with standard deviations (see also <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005342#pgen.1005342.s002\" target=\"_blank\">S2 Fig</a>). (C) Modeled protein abundance over the growth curve for different DnaA protein half-lives. The blue line shows measured changes in DnaA abundance over time as cells are grown to stationary phase (see also Figs <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005342#pgen.1005342.g001\" target=\"_blank\">1A</a> and <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005342#pgen.1005342.g003\" target=\"_blank\">3E</a>). The solid red line shows predicted changes in protein abundance, assuming that degradation increases in a linear manner, protein synthesis is constant and that the half-life of DnaA is 23 min at OD<sub><b>600</b></sub> 0.4 and 20 min at OD<sub><b>600</b></sub> 1.0 (see <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005342#sec015\" target=\"_blank\">Materials and Methods</a> for the mathematical model). The dashed red line shows predicted protein abundance if the half-life is 23 min at OD<sub><b>600</b></sub> 0.4 and 9.1 min at OD<sub><b>600</b></sub> 1.0, the latter value having been found by best fit to the data. (D) <i>In vivo</i> degradation assays showing DnaA stability in <i>Δlon</i> cells during exponential growth (OD<sub><b>600</b></sub> 0.4) or at the entry into stationary phase (OD<sub><b>600</b></sub> 1.0). Band intensities of Western Blots (top) were quantified (bottom); averages of two independent replicates are shown with standard deviations (see also <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005342#pgen.1005342.s002\" target=\"_blank\">S2 Fig</a>). (E) Growth phase-dependent changes in DnaA protein levels in wild type and a strain in which the native copy of <i>dnaA</i> is under the control of an IPTG inducible promoter (<i>P</i><sub><b><i>lac</i></b></sub>). 1 mM IPTG was added to the culture to induce constitutive <i>dnaA</i> expression (see also <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005342#pgen.1005342.s004\" target=\"_blank\">S4</a> and <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005342#pgen.1005342.s005\" target=\"_blank\">S5</a> Figs). The bottom graphs show the average band intensity of at least three independent experiments with standard deviations. (F) Flow cytometry profiles of wild type (WT) and the <i>P</i><sub><b><i>lac</i></b></sub><b><i>-</i></b><i>dnaA</i> strain in exponential phase (OD<sub><b>600</b></sub> 0.4) or after growth for 24 hours in stationary phase. The percentage of cells with one chromosome (1N) is indicated.</p>", "links"=>[], "tags"=>["Lon protease", "Caulobacter crescentus", "DNA replication initiation", "constitutive degradation", "mechanism", "dnaA transcript", "Nutritional Control", "replication initiator protein", "availability downregulates dnaA translation", "DnaA degradation", "cell cycle", "Regulated Translation", "DnaA Bacteria", "DNA replication"], "article_id"=>1471873, "categories"=>["Biological Sciences"], "users"=>["David J. Leslie", "Christian Heinen", "Frederic D. Schramm", "Marietta Thüring", "Christopher D. Aakre", "Seán M. Murray", "Michael T. Laub", "Kristina Jonas"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1005342.g003", "stats"=>{"downloads"=>1, "page_views"=>16, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Downregulating_DnaA_on_entry_to_stationary_phase_is_not_due_to_faster_proteolysis_but_changes_in_DnaA_synthesis_/1471873", "title"=>"Downregulating DnaA on entry to stationary phase is not due to faster proteolysis, but changes in DnaA synthesis.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-07-02 02:53:51"}

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Relative Metric

{"start_date"=>"2015-01-01T00:00:00Z", "end_date"=>"2015-12-31T00:00:00Z", "subject_areas"=>[]}
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