Effects of (p)ppGpp on the progression of the cell cycle of Caulobacter crescentus

Abstract

Bacteria must control the progression of their cell cycle in response to nutrient availability. This regulation can be mediated by guanosine tetra- or pentaphosphate [(p)ppGpp], which are synthesized by enzymes of the RelA/SpoT homologue (Rsh) family, particularly under starvation conditions. Here, we study the effects of (p)ppGpp on the cell cycle of Caulobacter crescentus, an oligotrophic bacterium with a dimorphic life cycle. C. crescentus divides asymmetrically, producing a motile swarmer cell that cannot replicate its chromosome and a sessile stalked cell that is replication competent. The swarmer cell rapidly differentiates into a stalked cell in appropriate conditions. An artificial increase in the levels of (p)ppGpp in nonstarved C. crescentus cells was achieved by expressing a truncated relA gene from Escherichia coli, encoding a constitutively active (p)ppGpp synthetase. By combining single-cell microscopy, flow cytometry approaches, and swarming assays, we show that an increase in the intracellular concentration of (p)ppGpp is sufficient to slow down the swarmer-to-stalked cell differentiation process and to delay the initiation of chromosome replication. We also present evidence that the intracellular levels of two master regulators of the cell cycle of C. crescentus, DnaA and CtrA, are modulated in response to (p)ppGpp accumulation, even in the absence of actual starvation. CtrA proteolysis and DnaA synthesis seem indirectly inhibited by (p)ppGpp accumulation. By extending the life span of the motile nonreproductive swarmer cell and thus promoting dispersal and foraging functions over multiplication under starvation conditions, (p)ppGpp may play a central role in the ecological adaptation of C. crescentus to nutritional stresses.

FIG 1

C. crescentus cells expressing RelA′-FLAG accumulate high levels of (p)ppGpp. (A) Schematics showing the organization of the different domains of the E. coli RelA protein and of the truncated RelA′ and RelA′(E335Q) proteins used in the present study. The TGS/CC/ACT domains form the regulatory region of the protein (3, 7). The star indicates the E335Q mutation. (B) Genetic constructs created to express RelA′-FLAG and RelA′(E335Q)-FLAG in C. crescentus. Plasmid pXTCYC-4-relA′-FLAG or pXTCYC-4-relA′(E335Q)-FLAG was integrated at the native xylX promoter in the C. crescentus chromosome, giving strains JC820 and JC1198, respectively. (C) RelA′-FLAG and RelA′(Q335A)-FLAG proteins are expressed in the JC820 and JC1198 strains, respectively, upon xylose addition in rich medium. JC820, JC1198 and JC835 (control strain) strains were cultivated in exponential phase in PYEG medium, and 0.3% xylose was added (PYEGX) at time zero. Cell extracts collected at time zero and 180 min after xylose addition were used to perform immunoblotting experiments using anti-FLAG antibodies. (D) Intracellular levels of (p)ppGpp in RelA′-FLAG-expressing cells, compared to starved wild-type cells. Strains NA1000 (wild-type strain), JC835 and JC820 were cultivated for 2.5 h in M5GG medium with or without 0.3% xylose. When indicated, NA1000 cells were starved in M5 medium for 90 min. The TLC autoradiograph image shown in the upper part of the figure was used to calculate the pppGpp/(GTP+ppGpp+pppGpp) and ppGpp/(GTP+ppGpp+pppGpp) ratios shown in the lower panel.

FIG 2

The accumulation of (p)ppGpp slows down the growth of C. crescentus cells. (A) Growth of a population of cells upon accumulation of (p)ppGpp. Strains JC820 (expressing relA′-FLAG from the xylX promoter) and JC835 (control strain) were cultivated in exponential phase in PYEG. 0.3% xylose was added (PYEGX) at time 105 min in half of each culture. The absorbance at 660 nm was used as an estimate of cellular mass. (B) The size of cells accumulating (p)ppGpp decreases sharply. Strains JC820 and JC835 were cultivated in exponential phase in PYEG. Xylose at 0.3% was added (PXEGX) at time zero in half of each culture. The forward scattering (FSC) of 20,000 individual cells for each population was measured using a flow cytometer. The FSC is proportional to the size of the cell. Plotted values are the average median FSC value for each strain and condition over three biological replicates normalized by the average median FSC value of JC835 in PYEG at the time zero; error bars refer to the standard deviations. (C) The decrease in size equally affects cells containing one (1N) or two (2N) complete chromosomes. The median FSC value of cells clustering in the neighborhood of the 1N and 2N peak maximum was calculated for three biological replicates after 6 h in PYEG or PYEGX. The plotted values are averages over triplicates for each strain and condition normalized by the average of JC835, PYEG 1N cells; error bars indicate the standard deviations.

FIG 3

Swarming motility of cells expressing different levels of RelA′-FLAG compared to control cells. Strains JC835 (control strain), JC820 (expressing RelA′-FLAG) and JC1198 [expressing RelA′(E335Q)-FLAG] cultivated into PYEG were stabbed into 0.3% PYEG agar containing 0, 0.0024, 0.012, 0.06, or 0.3% xylose and incubated for at room temperature for 4 days. (A) Images of plates with two swarming halos per strain. (B) Relative quantification of the diameters of swarming halos from strains JC835 and JC820 using images shown in panel A. Error bars indicate the standard deviations; stars indicate significance (Student t test, P < 0.05).

FIG 4

The accumulation of (p)ppGpp delays the swarmer-to-stalked cell transition. (A) Schematic showing the subcellular localization of CpaE, PleC, and DivJ as a function of the cell cycle when C. crescentus is grown in PYE medium. (B) Quantification of fluorescence microscopy experiments using isolated swarmer cells from strain JC845 (NA1000 pleC::pleC-YFP divJ::divJ-RFP cpaE::cpaE-CFP pXTCYC-4-relA′-FLAG) grown in PYEG. (C) Quantification of fluorescence microscopy experiments using isolated swarmer cells from strain JC845 grown in PYEGX after the synchronization procedure. The graphs shown in panels B and C correspond to the frequency of cells with detectable PleC-YFP or CpaE-CFP foci but no detectable DivJ focus (○), with visible DivJ-RFP foci (□) or with no visible focus (●). The time corresponds to the time after resuspension of the freshly isolated swarmer cells into PYEG or PYEGX. Typical images acquired at 0 and 60 min and used for this quantification are shown in Fig. S5 in the supplemental material. Plotted values are the averages of observations in three microscope fields; error bars indicate the standard deviations.

FIG 5

The accumulation of (p)ppGpp increases the proportion of cells with one chromosome. (A) Histograms of the DNA content of cells accumulating (p)ppGpp. Representative profiles were obtained by flow cytometry analysis of unsynchronized cells from strain JC820 expressing RelA′-FLAG upon xylose addition. Cells were grown to exponential phase in PYEG before 0.3% xylose (PYEGX) was added at time zero. Cells were fixed at the indicated times and stained prior to flow cytometry analysis. The horizontal axis indicates the fluorescence intensity of individual cells and the number (N) of complete chromosomes. The vertical axis indicates the number of cells. (B) The proportion of cells with one chromosome increases when (p)ppGpp accumulates. Quantification of the results of flow cytometry experiments using strain JC820 and the control strain JC835. Cells were grown to exponential phase in PYEG before 0.3% xylose (PYEGX) was added to half of the culture at time zero. The y axis corresponds to the average proportion of cells containing 1N chromosome per cell at the indicated times after xylose addition. Plotted values are the averages of three independent populations; the standard deviations are also indicated.

FIG 6

The accumulation of (p)ppGpp delays the initiation of chromosome replication. (A) Schematics showing the status of chromosome replication as a function of time when swarmer cells are grown in rich medium. The upper schematic shows that DnaN-RFP localizes as a moving focus only when DNA replication is ongoing. The lower schematic shows the replicating circular chromosome of C. crescentus as a function of the cell cycle. The small white and black circles on the lower schematic indicate the chromosomal origin and terminus, respectively. (B) (p)ppGpp accumulation delays the initiation of chromosome replication in swarmer cells. Quantification of flow cytometry experiments using isolated swarmer cells from strain JC861 (NA1000 dnaN::dnaN-RFP pXTCYC-4-relA′-FLAG) grown in PYEG or PYEGX. Cells were sampled at the indicated times, treated with rifampin, and then fixed and stained prior to flow cytometry analysis. The graph shows the percentage of cells with 1N chromosome, corresponding to cells that had not initiated chromosome replication at the indicated times of the cell cycle. (C) (p)ppGpp accumulation delays the assembly of the replisome in swarmer cells. Quantification of fluorescence microscopy experiments using isolated swarmer cells from strain JC861 grown in PYEG or PYEGX. Typical images acquired at different times under each condition and used for this quantification are shown in Fig. S8 in the supplemental material. The graph shows the percentage of cells with visible DnaN-RFP fluorescent foci, corresponding to cells that had initiated DNA replication at the indicated times of the cell cycle. The plotted values are the average percentages over three microscope fields; error bars indicate the standard deviations.

FIG 7

Effects of the accumulation of (p)ppGpp on the intracellular levels and the proteolysis of the DnaA and CtrA master regulators of the C. crescentus cell cycle. (A) The levels of DnaA are decreased upon (p)ppGpp accumulation. Strain JC820 (NA1000 pXTCYC-4-relA′-FLAG) was grown to exponential phase in PYEG and 0.3% xylose was added (PYEGX) to half of the culture at time zero. Cells were collected at the indicated times for immunoblot analysis using antibodies raised against DnaA. (B) The proteolysis of DnaA is not conspicuously faster upon (p)ppGpp accumulation. Strains JC835 (NA1000 pXTCYC-4) and JC820 were grown to exponential phase in PYEG and 0.3% xylose was added to the medium 150 min before the addition of rifampin (10 μg/ml). The addition of rifampin blocked further dnaA transcription and DnaA synthesis. DnaA decay in strains JC820 and JC835 were compared by immunoblot analysis using antibodies raised against DnaA; samples were collected 0, 15, 30, 45, 60, 75, and 90 min after rifampin addition. (C) The levels of CtrA are increased upon (p)ppGpp accumulation. Strain JC820 and JC835 (NA1000 pXTCYC-4) were grown to exponential phase in PYEG and 0.3% xylose was added (PYEGX) to the cultures at time zero. Cells were collected at the indicated times for immunoblot analysis using antibodies raised against CtrA. (D) CtrA proteolysis is inhibited upon (p)ppGpp accumulation. Strains JC835 and JC820 were grown as indicated in panel B, and the stability of CtrA was estimated by immunoblot analysis with antibodies raised against CtrA as in panel B. (E) DnaA and CtrA levels as a function of time after rifampin addition were estimated from the immunoblots shown in panels B and D.