Regulated DnaA synthesis and Lon-mediated degradation are required to eliminate DnaA upon carbon exhaustion.

<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>_<i>lac</i><i>-dnaA</i> cells from M2G to M2 medium containing 0.02% glucose at t = 0. The culture of <i>P</i>_<i>lac</i><i>-dnaA</i> cells was supplemented with 50 μM IPTG to induce <i>P</i>_<i>lac</i>. 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>_<i>lac</i><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>_<i>lac</i><i>-UTR</i>_<i>dnaA</i><i>-dnaA</i> was grown in the presence of 1 mM IPTG.</p

(p)ppGpp is not required to eliminate DnaA during the entry to stationary phase.

<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 (OD₆₀₀ 0.4) or after growth for 24 hours in stationary phase. The percentage of cells with one chromosome (1N) is indicated.</p

Reduced translation of <i>dnaA</i> accounts for the downregulation of DnaA abundance at the onset of stationary phase.

<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>_{<b><i>lac</i></b>}<i>-UTR</i>_{<b><i>dnaA</i></b>}<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>_{<b><i>dnaA</i></b>}<i>-ΔUTR</i>_{<b><i>dnaA</i></b>}<i>-dnaA</i>) or contain (<i>P</i>_{<b><i>lac</i></b>}<i>-UTR</i>_{<b><i>dnaA</i></b>}<i>-dnaA</i>) the 5'UTR of the <i>dnaA</i> gene. Cells were grown for 12 hours after reaching the maximal OD_<b>600</b> before samples were taken for flow cytometry analysis. The percentage of cells with one chromosome (1N) is indicated.</p

Lon-dependent proteolysis is required to eliminate DnaA and to induce a G1-arrest upon entry to stationary phase.

<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 OD₆₀₀ 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 (OD₆₀₀ 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 OD₆₀₀ 0.4, OD₆₀₀ 1.4 and after growth for 24 hours at the maximum OD₆₀₀ (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 OD₆₀₀ 1.4 and after growth for 24 hours at the maximum OD₆₀₀ (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

Dynamic control of DnaA abundance and DNA replication in response to environmental inputs.

<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