The Caulobacter crescentus Homolog of DnaA (HdaA) Also Regulates the Proteolysis of the Replication Initiator Protein DnaA

Abstract

It is not known how diverse bacteria regulate chromosome replication. Based on Escherichia coli studies, DnaA initiates replication and the homolog of DnaA (Hda) inactivates DnaA using the RIDA (regulatory inactivation of DnaA) mechanism that thereby prevents extra chromosome replication cycles. RIDA may be widespread, because the distantly related Caulobacter crescentus homolog HdaA also prevents extra chromosome replication (J. Collier and L. Shapiro, J Bacteriol 191:5706-5715, 2009, http://dx.doi.org/10.1128/JB.00525-09). To further study the HdaA/RIDA mechanism, we created a C. crescentus strain that shuts off hdaA transcription and rapidly clears HdaA protein. We confirm that HdaA prevents extra replication, since cells lacking HdaA accumulate extra chromosome DNA. DnaA binds nucleotides ATP and ADP, and our results are consistent with the established E. coli mechanism whereby Hda converts active DnaA-ATP to inactive DnaA-ADP. However, unlike E. coli DnaA, C. crescentus DnaA is also regulated by selective proteolysis. C. crescentus cells lacking HdaA reduce DnaA proteolysis in logarithmically growing cells, thereby implicating HdaA in this selective DnaA turnover mechanism. Also, wild-type C. crescentus cells remove all DnaA protein when they enter stationary phase. However, cells lacking HdaA retain stable DnaA protein even when they stop growing in nutrient-depleted medium that induces complete DnaA proteolysis in wild-type cells. Additional experiments argue for a distinct HdaA-dependent mechanism that selectively removes DnaA prior to stationary phase. Related freshwater Caulobacter species also remove DnaA during entry to stationary phase, implying a wider role for HdaA as a novel component of programed proteolysis.

Importance: Bacteria must regulate chromosome replication, and yet the mechanisms are not completely understood and not fully exploited for antibiotic development. Based on Escherichia coli studies, DnaA initiates replication, and the homolog of DnaA (Hda) inactivates DnaA to prevent extra replication. The distantly related Caulobacter crescentus homolog HdaA also regulates chromosome replication. Here we unexpectedly discovered that unlike the E. coli Hda, the C. crescentus HdaA also regulates DnaA proteolysis. Furthermore, this HdaA proteolysis acts in logarithmically growing and in stationary-phase cells and therefore in two very different physiological states. We argue that HdaA acts to help time chromosome replications in logarithmically growing cells and that it is an unexpected component of the programed entry into stationary phase.

FIG 1

(A) Locations of CcrM DNA methylation/HinfI target sites (GANTC) at the C. crescentus chromosome origin of replication (Cori). The adenines of these GANTC sites are methylated only within a narrow period, and this cell cycle property is used to detect extra chromosome replication. The restriction endonuclease HinfI only cuts GANTC DNA unmethylated on both strands that are produced by the passage of extra replication forks during the same cell cycle. Cori is located between the hemE and duf299 genes and the two BamHI sites used for Southern blot analysis below. (B) Southern blot analysis. C. crescentus GM3700 cells depleted of HdaA initiate extra chromosome DNA replication that is detected as new unmethylated DNA. GM3700 cells were grown in PYE medium supplemented with xylose and at 0 h, the culture was split into PYE medium with (+) or without (−) xylose. Samples were taken at the indicated times, and total chromosomal DNA was prepared, digested with the restriction endonucleases BamHI and HinfI, and used for Southern blot analysis. Southern blot membranes were hybridized with ³²P-radiolabeled DNA prepared from a 1.6-kb BamHI Cori fragment (shown above in panel A).

FIG 2

The clearing of DnaA from stationary-phase cells requires HdaA. (A) GM3700 and wild-type (NA1000) cells were grown in PYE medium supplemented with xylose, the cultures were split into PYE medium with (+) or without (−) xylose, and samples were taken at 0 h (lanes 1 to 4) and after 20 h (lanes 5 to 8). Sample loadings per lane were adjusted to the same cell mass equivalent to 1.0 ml at an OD₆₆₀ of 0.1. Ponceau S staining confirmed that equal amounts of total cell protein were transferred to the membrane, and immunoblot analysis was performed with anti-DnaA serum. The initial abundances of DnaA during the logarithmic phase were similar in the GM3700 and wild-type cells (lanes 1 to 4). Therefore, the manipulations that shifted the wild-type and GM3700 cells into medium without xylose did not affect the DnaA protein levels (lanes 1 to 2 versus 3 to 4). These cultures restarted logarithmic growth at OD₆₆₀ of 0.4 at 0 h, and their corresponding stationary-phase samples (lanes 5 to 8) were taken at 20 h. Wild-type and GM3700 cells grown in PYE medium with xylose efficiently cleared the DnaA protein (lanes 5 to 7). Only the GM3700 cells grown without xylose retained DnaA protein after 20 h (lane 8). (B) GM3700 cells were grown to the stationary phase in M2G minimal media with xylose (M2GX [lane 1]) and without xylose (M2G [lane 2]). The culture of lane 2 was sampled and incubated for an extra 2 h with 0.2% xylose (lane 3) and with 0.02% xylose (lane 4). Immunoblot analysis was performed with anti-DnaA serum, as shown above (A).

FIG 3

HdaA is required for DnaA proteolysis in logarithmically growing C. crescentus cells. (A) GM3700 cells were logarithmically grown in PYE medium supplemented with glucose and xylose (PYEGX), and the culture was split into media with xylose (PYEGX) and without xylose (PYEG) and grown for an extra 3 h to deplete HdaA. Tetracycline (1.5 μg/ml) was next added to halt translation, and samples of equal optical density were taken at 30-min intervals for immunoblot analysis with anti-DnaA serum. (B) The HdaA depletion period was changed to 0, 2, and 5 h. Tetracycline was added, and the cultures were sampled at 0 and 4 h. Otherwise these immunoblot experiments were performed as in Fig. 2A.

FIG 4

C. crescentus cells depleted of HdaA can still degrade DnaA protein in response to sudden and severe nitrogen starvation both during logarithmic growth and during stationary phase. (A) Wild-type C. crescentus (NA1000) and GM3700 cells were grown logarithmically in M2G with xylose (+X), and their cultures were split into M2GX and M2G media. The cultures were grown for an extra 4 h to deplete HdaA from the M2G-grown GM3700 cells, and as before, DnaA abundance was measured by immunoblot analysis. These cells had equally large amounts of DnaA protein (lanes 1 to 4). Next these cells were transferred to the corresponding media lacking ammonium (−N), and therefore without the sole nitrogen source, for an additional 2 h, and DnaA abundance was measured by immunoblot analysis (lanes 5 to 8). DnaA protein was efficiently cleared, even in cells depleted of HdaA (lane 8, GM3700 M2G–N). (B) In lanes 1 and 2, GM3700 cells were grown to stationary phase in M2GX and M2G media. In the next 3 lanes, cells from the M2G culture were transferred to fresh medium lacking ammonium (M2G–N) or to fresh M2GX and fresh M2G media and then incubated for an additional 2 h. DnaA abundance was measured by immunoblot analysis of equal-cell-mass samples. Stable DnaA protein in stationary-phase M2G is efficiently removed by nitrogen starvation (lane 3, M2G–N).

FIG 5

The used-medium (exhausted-medium) shift experiment. The top panel shows the DnaA immunoblot of logarithmically growing cells. The bottom panel shows the DnaA immunoblot of the same cells after they were shifted for 2 h to “used” PYE medium (the remaining liquid separated from a stationary-phase GM3700 PYE batch culture). This 2-h starvation condition completely removed DnaA protein from wild-type cells (lanes 1 and 2), while substantial DnaA protein was retained in HdaA-depleted cells (lane 4). The removal of most of the DnaA protein in the lane 3 control further argues that HdaA is a direct cause of DnaA proteolysis. The retention of some DnaA in lane 3 probably results from experimental manipulations: Specifically, this could result from weaker hdaA expression by the artificial Pxyl fusion and from the metabolism and exhaustion of xylose during the growth and the starvation periods.

FIG 6

The abundance of DnaA decreases with the same kinetics in both wild-type (NA1000) and in ΔspoT cells as they enter stationary phase. (A) Immunoblot analysis to determine the abundance of DnaA in wild-type and ΔspoT cells (LS4427) as they transition from growing cells to stationary-phase cells. Samples were taken at 2-h intervals, and as before, DnaA abundance was measured by immunoblot analysis of equal-cell-mass samples. (B) The quantitation of the DnaA protein bands from the immunoblot in panel A. (C) The graph shows the optical densities of the PYE cultures at the sampling times in panel A. Both cultures entered stationary phase at the same rate, and both reached the same saturating cell densities.

FIG 7

DnaA alleles R357H and DnaA R357A increase DnaA protein stability and chromosome replication. (A) Immunoblot measurements of DnaA protein stability in cells expressing the WT DnaA and ATPase-deficient mutants DnaA R357H and DnaA R357A. Replicating Pxyl::dnaA plasmids pGM3859 (WT), pGM3861 (R357H), and pGM3862 (R357A) were introduced into wild-type C. crescentus strain NA1000 grown logarithmically in PYEG (glucose) medium. Half of each culture was shifted to PYEX (xylose) medium for 2 h. Next, tetracycline (1.5 μg/ml) was added, and these cultures were sampled for immunoblot analysis at 0, 30, and 60 min, as shown. (B) Chromosome DNA fluorescence cytometry of the 0-min cultures in panel A. This table presents the percentage of cells with threshold DNA fluorescence above the 2-chromosome peak. These samples were taken immediately before tetracycline was added and analyzed by fluorescence cytometry as described in Materials and Methods: i.e., rifampin and cephalexin were added to complete chromosome replication without cell division. The inserted picture shows the plot of DNA fluorescence versus cell numbers from the WT DnaA PYEX culture sample, which was used to set the DNA fluorescence threshold for the other culture samples.

FIG 8

DnaA is cleared from wild-type C. crescentus (NA1000) and from related freshwater Caulobacter strains (FWC 14, 21, and 38) during stationary phase. DnaA abundance was measured by immunoblot analysis, as before. Samples were taken from logarithmic-growth-phase (L) PYE cultures and after the same cultures reached stationary phase (S), ∼20 h later. As before, all samples were adjusted to an equal cell mass equivalent to 1.0 ml at an optical density of 0.1. Control lanes 1 and 2 confirm antiserum specificity and show that C. crescentus strain GM2471 (Pxyl::dnaA) produces DnaA in xylose (X) but not glucose (G) PYE medium. Lanes 3 to 10 show that each of the Caulobacter strains decreases DnaA abundance in stationary phase relative to the logarithmic growth phase.