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

Leslie DJ, Heinen C, Schramm FD, Thüring M, Aakre CD, Murray SM, Laub MT, Jonas K - PLoS Genet. (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: LOEWE Center for Synthetic Microbiology, Philipps University Marburg, Marburg, Germany; Max Planck Institute for Terrestrial Microbiology, Marburg, Germany.

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.

No MeSH data available.


Related in: MedlinePlus

The translation of dnaA is modulated by nutrient availability.(A) DnaA protein levels in exponentially grown wild type or Plac 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 S3 Fig). (B) Growth medium-dependent DnaA protein abundance in strains expressing constructs, which either contain or lack the 5'UTR of dnaA (see Fig 4D). The three strains were grown in PYE and M2G and protein abundance was measured by Western blotting. The strain harboring the construct Plac-UTRdnaA-dnaA was grown in the presence of 1 mM IPTG (see also S7 Fig). (C) Protein levels of DnaA and RpoA (loading control) in wild type and ΔspoT 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 OD600 by Western blotting (see also S7 Fig). The growth curve for wild type grown in 1x PYE is shown for comparison and is reproduced from Fig 1A. (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 OD600 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 S7 and S8 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 S7 and S8 Figs).
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pgen.1005342.g005: The translation of dnaA is modulated by nutrient availability.(A) DnaA protein levels in exponentially grown wild type or Plac 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 S3 Fig). (B) Growth medium-dependent DnaA protein abundance in strains expressing constructs, which either contain or lack the 5'UTR of dnaA (see Fig 4D). The three strains were grown in PYE and M2G and protein abundance was measured by Western blotting. The strain harboring the construct Plac-UTRdnaA-dnaA was grown in the presence of 1 mM IPTG (see also S7 Fig). (C) Protein levels of DnaA and RpoA (loading control) in wild type and ΔspoT 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 OD600 by Western blotting (see also S7 Fig). The growth curve for wild type grown in 1x PYE is shown for comparison and is reproduced from Fig 1A. (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 OD600 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 S7 and S8 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 S7 and S8 Figs).

Mentions: A reduction in growth rate during the entry into stationary phase may result from the exhaustion of nutrients or the accumulation of inhibitory waste products, cellular stress, or some combination thereof [40]. We hypothesized that a decrease in nutrient availability might be the signal that ultimately modulates dnaA translation. To test this idea we analyzed DnaA accumulation in growth media containing different amounts of nutrients. M2G, a minimal medium, in which the sole carbon source is glucose, was used as the most nutrient poor medium. We supplemented M2G with increasing amounts of peptone, a pepsin digest consisting of polypeptides and amino acids used in rich media such as PYE. DnaA protein levels during mid-exponential phase were clearly correlated with the complexity of the growth medium (Fig 5A). Increases in the amount of peptone added to M2G were mirrored by increases in DnaA steady-state levels, as measured by Western blotting. In contrast to DnaA, levels of the Lon protease were relatively unaffected by the growth medium (S7 Fig).


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

Leslie DJ, Heinen C, Schramm FD, Thüring M, Aakre CD, Murray SM, Laub MT, Jonas K - PLoS Genet. (2015)

The translation of dnaA is modulated by nutrient availability.(A) DnaA protein levels in exponentially grown wild type or Plac 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 S3 Fig). (B) Growth medium-dependent DnaA protein abundance in strains expressing constructs, which either contain or lack the 5'UTR of dnaA (see Fig 4D). The three strains were grown in PYE and M2G and protein abundance was measured by Western blotting. The strain harboring the construct Plac-UTRdnaA-dnaA was grown in the presence of 1 mM IPTG (see also S7 Fig). (C) Protein levels of DnaA and RpoA (loading control) in wild type and ΔspoT 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 OD600 by Western blotting (see also S7 Fig). The growth curve for wild type grown in 1x PYE is shown for comparison and is reproduced from Fig 1A. (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 OD600 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 S7 and S8 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 S7 and S8 Figs).
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Related In: Results  -  Collection

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pgen.1005342.g005: The translation of dnaA is modulated by nutrient availability.(A) DnaA protein levels in exponentially grown wild type or Plac 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 S3 Fig). (B) Growth medium-dependent DnaA protein abundance in strains expressing constructs, which either contain or lack the 5'UTR of dnaA (see Fig 4D). The three strains were grown in PYE and M2G and protein abundance was measured by Western blotting. The strain harboring the construct Plac-UTRdnaA-dnaA was grown in the presence of 1 mM IPTG (see also S7 Fig). (C) Protein levels of DnaA and RpoA (loading control) in wild type and ΔspoT 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 OD600 by Western blotting (see also S7 Fig). The growth curve for wild type grown in 1x PYE is shown for comparison and is reproduced from Fig 1A. (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 OD600 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 S7 and S8 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 S7 and S8 Figs).
Mentions: A reduction in growth rate during the entry into stationary phase may result from the exhaustion of nutrients or the accumulation of inhibitory waste products, cellular stress, or some combination thereof [40]. We hypothesized that a decrease in nutrient availability might be the signal that ultimately modulates dnaA translation. To test this idea we analyzed DnaA accumulation in growth media containing different amounts of nutrients. M2G, a minimal medium, in which the sole carbon source is glucose, was used as the most nutrient poor medium. We supplemented M2G with increasing amounts of peptone, a pepsin digest consisting of polypeptides and amino acids used in rich media such as PYE. DnaA protein levels during mid-exponential phase were clearly correlated with the complexity of the growth medium (Fig 5A). Increases in the amount of peptone added to M2G were mirrored by increases in DnaA steady-state levels, as measured by Western blotting. In contrast to DnaA, levels of the Lon protease were relatively unaffected by the growth medium (S7 Fig).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: LOEWE Center for Synthetic Microbiology, Philipps University Marburg, Marburg, Germany; Max Planck Institute for Terrestrial Microbiology, Marburg, Germany.

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.

No MeSH data available.


Related in: MedlinePlus