Phosphate starvation decouples cell differentiation from DNA replication control in the dimorphic bacterium Caulobacter crescentus.

Upon nutrient depletion, bacteria stop proliferating and undergo physiological and morphological changes to ensure their survival. Yet, how these processes are coordinated in response to distinct starvation conditions is poorly understood. Here we compare the cellular responses of Caulobacter crescentus to carbon (C), nitrogen (N) and phosphorus (P) starvation conditions. We find that DNA replication initiation and abundance of the replication initiator DnaA are, under all three starvation conditions, regulated by a common mechanism involving the inhibition of DnaA translation. By contrast, cell differentiation from a motile swarmer cell to a sessile stalked cell is regulated differently under the three starvation conditions. During C and N starvation, production of the signaling molecules (p)ppGpp is required to arrest cell development in the motile swarmer stage. By contrast, our data suggest that low (p)ppGpp levels under P starvation allow P-starved swarmer cells to differentiate into sessile stalked cells. Further, we show that limited DnaA availability, and consequently absence of DNA replication initiation, is the main reason that prevents P-starved stalked cells from completing the cell cycle. Together, our findings demonstrate that C. crescentus decouples cell differentiation from DNA replication initiation under certain starvation conditions, two otherwise intimately coupled processes. We hypothesize that arresting the developmental program either as motile swarmer cells or as sessile stalked cells improves the chances of survival of C. crescentus during the different starvation conditions

Growth and cell morphology after extended incubation in P-limited media.

(A) Growth curves of wild-type cells in nutrient-replete M2G medium (black), and media limited for C (gray), N (blue), or P (orange). (B) Micrographs of cells in nutrient-replete M2G medium or after incubation in M5G 1 μM phosphate for the indicated amount of time. Scale bars: 5 μm. (PDF)</p

Lon-mediated DnaA proteolysis.

(A) DnaA in vivo stability assay of wild-type and lon::Ω cells in mid-exponential phase in M2G and while experiencing N or P starvation. (B) Quantification of DnaA in vivo stability assays done as shown in (A), from either five (M2G mid-exponential phase), two (N-limited), or three (P-limited) independent replicates for the lon::Ω strain, shown alongside wild-type data from Fig 3B, with error bars showing standard deviations. (C–E) Immunoblots of DnaA after shifting cells to nutrient-limited media, comparing lon::Ω to wild-type cells. For (C) and (D), the wild-type half of the blot was separated from the lon::Ω half, washed, and redeveloped to prevent the high signal of the lon::Ω bands from drowning out the wild-type signal. (F) DNA content as determined by flow cytometry of wild-type and lon::Ω cells after being shifted to C- or P-limited media. Each separate histogram represents 30 000 cells. (G) DNA content presented essentially as in (F), of wild-type and lon::Ω cells in mid-exponential phase in M2G before being shifted to N-limited medium (t = 0 h), and after being shifted to N-limited medium. (PDF)</p

DNA replication initiation is arrested under C, N, and P starvation.

(A) DNA content histograms of cells in mid-exponential phase in M2G before being shifted to nutrient-limited media (t = 0 h) and of cells incubated in C-limited (gray), N-limited (blue), or P-limited (orange) media over time, as measured by flow cytometry. Each separate histogram represents 30 000 cells. 1n and 2n denote one and two chromosome equivalents respectively. (B) Immunoblots of DnaA from nutrient-replete cultures in steady state (t = 0 h) and after shifting cells to nutrient-limited media, or after a mock-shift into nutrient-replete minimal medium (M2G). (C) Quantification of DnaA immunoblots done as in (B), alongside growth curves reproduced from Fig 1A. Means of relative band intensities (normalized to t = 0 h) from three independent replicates are shown with standard deviations; in case of the mock-shifted M2G culture, data from one replicate are shown. (D) Immunoblot of CtrA from nutrient-replete cultures in steady state (t = 0 h) and after shifting cells to nutrient-limited media. (E) Quantification of CtrA immunoblots done as in (D), from either four (C-limited) or three (N-/P-limited) independent replicates, with standard deviations.</p

Induction of <i>dnaA</i> expression from P_<i>lac</i> under starvation conditions.

(A) DNA content as determined by flow cytometry of wild-type cells and Plac-dnaA cells in mid-exponential phase in M2G containing 50 μM IPTG (t = 0 h) and after being shifted to nutrient-limited media containing 1 mM IPTG to induce dnaA overexpression. P starvation data is also shown in Fig 5G. Each separate histogram represents 30 000 cells. (B) DNA content presented as in (A), of a Plac-dnaA culture being split after 24 hours in P-limited medium containing 50 μM IPTG (basal dnaA expression), with one culture half being supplemented with IPTG to a final concentration of 1 mM to boost dnaA expression. (C) Stacked bar chart showing the proportions of swarmer cells (SW), stalked cells (ST), and predivisional (PD) cells in Plac-dnaA cultures treated as in (B). Each bar represents the average of two biological replicates with error bars showing standard deviations. For each biological replicate, 586–1141 cells were counted (average: 801 cells). (D) Immunoblot showing DnaA levels of Plac-dnaA cells in mid-exponential phase in M2G containing 50 μM IPTG (0 h in P-limited medium) and after being treated as in (B). (PDF)</p

(p)ppGpp do not orchestrate the P starvation response.

(A) The proportions of stalked cells to swarmer cells in Δrel (ΔspoT) cultures subjected to nutrient limitation (blue) as done for the wild type (WT) in Fig 1F. Data for wild-type cultures (gray) are included for comparison. For Δrel, 460–3069 cells were counted per biological replicate (average: 1425 cells). Dashed lines emphasize the mean cell type ratio ± SD of Δrel cultures during mid-exponential phase in M2G medium (t = 0 h). Circles in the top panel depict significant differences determined by an ANOVA test, followed by Bonferroni correction of P values from pairwise post hoc comparisons, of cell type ratios at each time point to the initial ratio (t = 0 h), for each strain separately. (B) Motility of Δrel cells as shown for the wild type in Fig 1G. Wild-type measurements are overlaid for comparison. “ns” highlights nutrient concentrations where Δrel cells did not exhibit a significant motility difference in comparison to nutrient-replete conditions, but where wild-type cells did. (C) Violin plot of cell length measurements, essentially as shown in Fig 1D comparing the Δrel mutant to wild-type. Asterisks at the top depict significant differences comparing Δrel to the wild type at each condition. The bottom panel shows the percent change in average cell length, and depicts significant differences, comparing each starvation condition to nutrient-replete conditions (M2G), for each strain separately. Statistical significance was determined by ANOVA tests followed by Bonferroni correction of P values from pairwise post hoc comparisons; ‘ns’: non-significant; *: P (D) Histograms of the DNA content of 30 000 cells as measured by flow cytometry, comparing the Δrel mutant (blue) to wild-type (black lines) after 24 h in nutrient-limited media. The outlines of the wild-type histograms shown in Fig 2A are overlaid for comparison. 1n and 2n denote one and two chromosome equivalents respectively.</p

The synthesis inhibition conferred by Nt_DnaA is independent on the 5′UTR.

(A) Schematic depiction of dnaA expression reporter constructs from Felletti et al. [34]. (B–C) Growth curves (gray, blue, and orange) and OD600-normalized eGFP fluorescence (green) of cells harboring the dnaA expression reporter constructs depicted in (A), after shift to C-, N-, or P-limited media. Light hues: NtdnaA-eGFP constructs; dark hues: constructs with eGFP alone. eGFP-coding sequences are preceded by 5′ untranslated regions (5′UTRs) either of the artificial 6/13 UTR type (B), or from the lac operon of E. coli (C). (PDF)</p

Noisy fluorescence measurements at the onset of P starvation can be circumvented by using higher initial P concentrations.

Triplicate growth curves and eGFP fluorescence measurements of the dnaA expression reporter construct “UTR-Nt” after shift to M5G with various initial phosphate concentrations. OD600-normalized eGFP fluorescence values were divided by the lowest observed OD600-normalized fluorescence measurement for each “phosphate concentration data set” (resulting in relative OD600-normalized fluorescence), to allow fluorescence patterns to be compared side-by-side. (PDF)</p

Motility assay example pictures.

(A) Representative motility zone (soft agar colony) pictures in grayscale and the spectrum color lookup table of ImageJ (ver 2.1.0) after 72 hours incubation. All pictures are in scale (scale bar: 10 mm). (B) Micrographs of cells withdrawn from motility agar colony edges after 72 hours incubation, exhibiting clear phosphate starvation phenotypes in M5G prepared without phosphate. Scale bars: 5 μm. (PDF)</p

The P starvation-induced cell cycle arrest can be bypassed by <i>dnaA</i> overexpression.

(A) Cell morphology and DNA content of a strain with inducible dnaA expession (Plac-dnaA) grown in M2G under non-depleting conditions (grown with 50 μM IPTG) and under depleting conditions (4 h without IPTG). Quantification of stalked cell fractions was done by manually assigning 1000 cells as either stalked or non-stalked cells, for each condition. The histograms show DNA content of 30 000 cells as measured by flow cytometry. Scale bars: 5 μm. (B) Immunoblots showing DnaA levels of wild-type (WT) and Plac-dnaA cells in mid-exponential phase in M2G containing 50 μM IPTG (t = 0 h) and after being shifted to nutrient-limited media containing 1 mM IPTG to induce dnaA overexpression. (C) Micrographs of cells in nutrient-replete cultures with 50 μM IPTG, and after 24 hours in nutrient-limited media containing 1 mM IPTG to induce dnaA overexpression. (D–F) Stacked bar chart showing the proportions of swarmer cells (SW), stalked cells (ST), and predivisional (PD) cells in wild-type or Plac-dnaA cultures in mid-exponential phase in M2G containing 50 μM IPTG (t = 0 h) and after shifting cells to nutrient-limited media containing 1 mM IPTG to induce dnaA overexpression. Each bar represents the average with error bars showing standard deviations (t = 0 h: six and nine biological replicates for wild-type and Plac-dnaA respectively; t > 0 h: two and three biological replicates for wild-type and Plac-dnaA respectively). For each biological replicate, 917–11 020 cells were counted (average: 2911 cells). (G) DNA content as determined by flow cytometry of wild-type cells and Plac-dnaA cells in mid-exponential phase in M2G containing 50 μM IPTG (t = 0 h) and after being shifted to P-limited medium containing 1 mM IPTG to induce dnaA overexpression. Each separate histogram represents 30 000 cells. 1n and 2n denote one and two chromosome equivalents respectively.</p