SpoT regulates DnaA stability and initiation of DNA replication in carbon-starved Caulobacter crescentus

Abstract

Cell cycle progression and polar differentiation are temporally coordinated in Caulobacter crescentus. This oligotrophic bacterium divides asymmetrically to produce a motile swarmer cell that represses DNA replication and a sessile stalked cell that replicates its DNA. The initiation of DNA replication coincides with the proteolysis of the CtrA replication inhibitor and the accumulation of DnaA, the replication initiator, upon differentiation of the swarmer cell into a stalked cell. We analyzed the adaptive response of C. crescentus swarmer cells to carbon starvation and found that there was a block in both the swarmer-to-stalked cell polar differentiation program and the initiation of DNA replication. SpoT is a bifunctional synthase/hydrolase that controls the steady-state level of the stress-signaling nucleotide (p)ppGpp, and carbon starvation caused a SpoT-dependent increase in (p)ppGpp concentration. Carbon starvation activates DnaA proteolysis (B. Gorbatyuk and G. T. Marczynski, Mol. Microbiol. 55:1233-1245, 2005). We observed that SpoT is required for this phenomenon in swarmer cells, and in the absence of SpoT, carbon-starved swarmer cells inappropriately initiated DNA replication. Since SpoT controls (p)ppGpp abundance, we propose that this nucleotide relays carbon starvation signals to the cellular factors responsible for activating DnaA proteolysis, thereby inhibiting the initiation of DNA replication. SpoT, however, was not required for the carbon starvation block of the swarmer-to-stalked cell polar differentiation program. Thus, swarmer cells utilize at least two independent signaling pathways to relay carbon starvation signals: a SpoT-dependent pathway mediating the inhibition of DNA replication initiation, and a SpoT-independent pathway(s) that blocks morphological differentiation.

FIG. 1.

Carbon starvation blocks swarmer-to-stalked cell polar differentiation. (A) Schematic depicting polar changes that occur over the course of the cell cycle. Complexes of the McpA chemoreceptor (green), the FliF flagellar basal body protein (red), and the PilA pilus subunit (blue) are shown. (B) DIC and DAPI fluorescence images of wild-type swarmer cells inoculated into M2 medium possessing or lacking a carbon source (0.2% glucose). Arrowheads point to stalks on synchronized cells grown in the presence of glucose. The percentage of stalked cells was determined by counting at least 100 cells. (C) Immunoblots of cell extracts from a synchronized wild-type culture incubated in minimal medium in the presence (M2G) or absence (M2) of glucose. McpA, FliF, and PilA antibodies were used to probe each protein's accumulation at the indicated time points. (D) Immunoblots of cell extracts from a synchronized ΔspoT strain grown in either the presence or absence of glucose. McpA, FliF, and PilA antibodies were used to probe each protein's accumulation at the indicated time points. (E) DIC and DAPI fluorescence images of ΔspoT swarmer cells inoculated into M2 medium possessing or lacking a carbon source (0.2% glucose). Arrowheads point to stalks on synchronized ΔspoT cells grown in the presence of glucose. The percentage of stalked cells was determined by counting at least 100 cells.

FIG. 2.

Carbon starvation inhibits the initiation of DNA replication and promotes a reduction in the steady-state level of DnaA in swarmer cells. (A) FROS images of the chromosomal replication origin in synchronized swarmer cells grown on minimal medium agarose pads that either possessed (+carbon) or lacked (-carbon) 0.2% glucose. The origin is shown in red, and white arrows point to examples of cells with either a single or a duplicated origin. (B) Quantification of origin duplication as a function of incubation time. The origin duplication status of at least 150 cells was determined at each time point, and the number that possessed a duplicated origin was expressed as a percentage of the total number of cells counted. The experiment to determine the origin duplication status of carbon-starved swarmer cells was performed two times, and the plotted values are the means of the two experiments. (C) FACS analysis of wild-type swarmer cells grown in minimal medium either with (+carbon) or without (-carbon) 0.2% glucose. At the indicated times, aliquots were incubated at 28°C for 3 h with rifampin (15 μg/ml), which allowed cells that had initiated DNA replication prior to rifampin addition to finish chromosome duplication but blocked new rounds of replication initiation. Cells were fixed, stained with Vybrant DyeCycle Orange, and analyzed by flow cytometry. (D) Immunoblots of cell extracts from a synchronized wild-type culture incubated in medium that either possessed (+carbon) or lacked (-carbon) 0.2% glucose. DnaA and CtrA antibodies were used to probe each protein's steady-state levels at the indicated time points. The DnaA antibody cross-reacts with a heat shock protein (Hsp) thought to be GroEL (14) and which runs at a slightly larger apparent molecular weight during SDS-PAGE.

FIG. 3.

Construction of a C. crescentus ΔspoT strain and a complemented ΔspoT strain. (A) Schematic representation of the spoT locus and the ΔspoT strain used to characterize SpoT function. (B) Construction of the ΔspoT-complementing vector pJL38 and the negative control vector, pJL37. (C) Schematic representation of the complemented ΔspoT strain, ΔspoT/pJL38, and the control strains harboring the empty vector ΔspoT/pJL37 and wild-type/pJL37.

FIG. 4.

SpoT is required for rapid adaptation to nutrient downshift from rich medium (PYE) to minimal medium (M2G) and for (p)ppGpp accumulation during carbon starvation. (A) Growth curves of wild-type, ΔspoT, and hisB::Tn5 strains grown in PYE medium, washed in M2G, and then either resuspended in PYE (left panel) or M2G (right panel). (B) One-dimensional thin-layer chromatography analysis of total intracellular nucleotides extracted from wild-type/pJL37 grown in minimal medium with 0.2% glucose (+carbon) as well as total intracellular nucleotides extracted from wild-type/pJL37, ΔspoT/pJL37 (uncomplemented), and ΔspoT/pJL38 (complemented) grown in minimal medium lacking 0.2% glucose (-carbon).

FIG. 5.

Carbon starvation-induced reduction in the steady-state level of DnaA requires a SpoT-dependent activation of DnaA proteolysis. (A) Immunoblots of cell extracts from synchronized swarmer cells of the wild type or a ΔspoT strain which were grown in medium lacking glucose (-carbon). DnaA antibody was used to probe the DnaA steady-state level at the indicated time points. (B) Immunoblots of cell extracts from synchronized swarmer cells of wild-type/pJL37, ΔspoT/pJL37 (uncomplemented), and ΔspoT/pJL38 (complemented) which were grown in medium lacking glucose (-carbon). DnaA antibody was used to probe the protein's steady-state level at the indicated time points. (C) SpoT promotes DnaA proteolysis during carbon starvation. The half-life of DnaA was measured by Western blot analysis of samples taken at the indicated times after the addition of rifampin (10 μg/ml) to block further dnaA transcription and ultimately DnaA synthesis. Samples were taken from synchronized swarmer cells of wild-type and ΔspoT strains that were starved for carbon for 10 min prior to the addition of rifampin. In order to adjust for the nonlinearity of chemiluminescent signal versus protein quantity in Western blots analyzed by chemiluminescent detection, a standard curve was generated to determine the relationship between chemiluminescent DnaA signal and actual DnaA abundance. DnaA levels quantified from the Western blot assay were fit to a single exponential decay curve in order to calculate the half-life.

FIG. 6.

SpoT is required for the carbon starvation-induced block in the initiation of DNA replication. (A) FROS images of the chromosomal origin of replication in swarmer cells of the wild type and a ΔspoT strain incubated on M2 minimal medium agarose pads in the absence of a carbon source. The origin region is shown in red, and white arrows point to examples of cells that have undergone origin duplication. (B) Quantification of the origin duplication status of carbon-starved swarmer cells from wild-type and ΔspoT strains is presented as a function of time. At each time point, at least 150 cells were counted, and the plotted values are the means of two independent experiments. Bars represent 1 standard deviation from the mean. (C) FACS analysis of synchronized ΔspoT swarmer cells grown in M2 minimal medium without (-carbon) 0.2% glucose. At the indicated times, aliquots were incubated at 28°C for 3 h with rifampin (15 μg/ml), which allowed cells that had initiated replication prior to rifampin addition to finish chromosome duplication but blocked new rounds of replication initiation. Cells were fixed, stained with Vybrant DyeCycle Orange, and analyzed by flow cytometry.

FIG. 7.

SpoT is required for swarmer cells to survive carbon starvation. Synchronized wild-type/pJL37, ΔspoT/pJL37 (uncomplemented), and ΔspoT/pJL38 (complemented) swarmer cells were washed in minimal medium lacking a carbon source, suspended in the same medium, and incubated at 28°C. Cell viability was measured as CFU/ml of culture medium and was determined at the indicated time points and normalized to viability measured at time zero. (Insert) A blown-up depiction of carbon starvation survival data from the first 30 h of the experiment. The cell viability values are the means from three independent experiments. Standard deviations were less than 25% of the means.

FIG. 8.

Model for SpoT-dependent control of DnaA proteolysis and the inhibition of DNA replication initiation in carbon-starved swarmer cells. A detailed description of the figure is given in the Discussion section.