Nutritional Control of DNA Replication Initiation through the Proteolysis and Regulated Translation of DnaA

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.National Institutes of Health (U.S.) (Grant 5R01GM082899

Tomato TFT1 Is Required for PAMP-Triggered Immunity and Mutations that Prevent T3S Effector XopN from Binding to TFT1 Attenuate Xanthomonas Virulence

XopN is a type III effector protein from Xanthomonas campestris pathovar vesicatoria that suppresses PAMP-triggered immunity (PTI) in tomato. Previous work reported that XopN interacts with the tomato 14-3-3 isoform TFT1; however, TFT1's role in PTI and/or XopN virulence was not determined. Here we show that TFT1 functions in PTI and is a XopN virulence target. Virus-induced gene silencing of TFT1 mRNA in tomato leaves resulted in increased growth of Xcv ΔxopN and Xcv ΔhrpF demonstrating that TFT1 is required to inhibit Xcv multiplication. TFT1 expression was required for Xcv-induced accumulation of PTI5, GRAS4, WRKY28, and LRR22 mRNAs, four PTI marker genes in tomato. Deletion analysis revealed that the XopN C-terminal domain (amino acids 344–733) is sufficient to bind TFT1. Removal of amino acids 605–733 disrupts XopN binding to TFT1 in plant extracts and inhibits XopN-dependent virulence in tomato, demonstrating that these residues are necessary for the XopN/TFT1 interaction. Phos-tag gel analysis and mass spectrometry showed that XopN is phosphorylated in plant extracts at serine 688 in a putative 14-3-3 recognition motif. Mutation of S688 reduced XopN's phosphorylation state but was not sufficient to inhibit binding to TFT1 or reduce XopN virulence. Mutation of S688 and two leucines (L64,L65) in XopN, however, eliminated XopN binding to TFT1 in plant extracts and XopN virulence. L64 and L65 are required for XopN to bind TARK1, a tomato atypical receptor kinase required for PTI. This suggested that TFT1 binding to XopN's C-terminal domain might be stabilized via TARK1/XopN interaction. Pull-down and BiFC analyses show that XopN promotes TARK1/TFT1 complex formation in vitro and in planta by functioning as a molecular scaffold. This is the first report showing that a type III effector targets a host 14-3-3 involved in PTI to promote bacterial pathogenesis

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

Reduced <i>TFT1</i> mRNA expression in VIGS tomato leaves correlates with reduced PTI marker mRNA abundance in response to Xcv infection.

<p>Relative mRNA levels for four PTI marker genes (<i>PTI5</i>, <i>GRAS4</i>, <i>WRKY28</i>, and <i>LRR22</i>) in 4 control (TRV2) and 4 <i>TFT1</i>-silenced (TRV2-TFT1) tomato lines. Leaflets on the same branch were inoculated with 1×10⁵ CFU/mL of Xcv or Xcv <i>ΔhrpF</i>. Total RNA isolated from inoculated leaves at 6 HPI was used for Q-PCR. <i>Actin</i> mRNA expression was used to normalize the expression value in each sample. Error bars indicate SD for four plants. Asterisk indicates significant difference (<i>t</i> test, P<0.05) in the infected TRV2-TFT1 lines compared to the similarly infected TRV2 lines.</p

Serine 688 in XopN is required for TFT1 binding in yeast but not <i>in planta</i>.

<p>(<b>A</b>) Schematic of putative 14-3-3 motifs in XopN protein. Black boxes represent regions for putative Mode I and II 14-3-3 binding motifs. Mode II site contains S688. PEST domain is underlined. N-terminal leucines (L64, L65) required for TARK1-binding are labeled. (<b>B</b>) XopN(S688A) mutant does not interact with TFT1 in yeast. Serine 688 in XopN was mutated to alanine. Yeast strain AH109 carrying pXDGATcy86(GAL4-DNA binding domain) containing XopN, and XopN(S688A) was transformed with the following PREY constructs: pGADT7(GAL4 activation domain) alone (Vector) or pGADT7 containing TFT1. Strains were spotted on nonselective (SD-LT) and selective (SD-LTH) medium and then incubated at 30°C for 3d. (<b>C</b>) XopN(S688A) and two phosphomimetic mutants, XopN(S688D) and XopN(S688E), interact with TFT1 in <i>N. benthamiana</i>. Leaves were hand-infiltrated with a suspension (8×10⁸ CFU/mL total) of two <i>A. tumefaciens</i> strains expressing TFT1-HA and XopN-6His or XopN(S688A)-6His or XopN(688D)-6His or XopN(688E)-6His. After 48 h, protein was extracted, purified by Ni⁺ affinity chromatography, and analyzed by protein gel blot analysis using anti-His and anti-HA sera. Expected protein MW: XopN-6His, S688A-6xHis, S688D-6His, and S688E-6His = 78.7 kDa; TFT1-HA = 29.3 kDa. +, protein expressed; −, vector control. (<b>D</b>) Growth of Xcv Δ<i>xopN</i> (vector), Xcv <i>ΔxopN</i> (XopN-HA), Xcv <i>ΔxopN</i> (XopN(S688A)-HA), Xcv <i>ΔxopN</i> (XopN(S688D)-HA, or Xcv <i>ΔxopN</i> (XopN(S688E)-HA in susceptible tomato VF36 leaves. Leaves were inoculated with a 1×10⁵ CFU/mL suspension of bacteria. Number of bacteria in each leaf was quantified at 0 and 10 DPI. Data points represent mean CFU/cm² ± SD of four plants. Different letters at day 10 indicate statistically significant (one-way analysis of variance and Tukey's HSD test, P<0.05) differences between the samples. Vector = pVSP61. (<b>E</b>) Phenotype of tomato leaves inoculated with the strains described in (<b>D</b>). Leaves were photographed at 12 DPI. Analysis was repeated two times.</p