Global regulation of gene expression and cell differentiation in Caulobacter crescentus in response to nutrient availability

Abstract

In a developmental strategy designed to efficiently exploit and colonize sparse oligotrophic environments, Caulobacter crescentus cells divide asymmetrically, yielding a motile swarmer cell and a sessile stalked cell. After a relatively fixed time period under typical culture conditions, the swarmer cell differentiates into a replicative stalked cell. Since differentiation into the stalked cell type is irreversible, it is likely that environmental factors such as the availability of essential nutrients would influence the timing of the decision to abandon motility and adopt a sessile lifestyle. We measured two different parameters in nutrient-limited chemostat cultures, biomass concentration and the ratio of nonstalked to stalked cells, over a range of flow rates and found that nitrogen limitation significantly extended the swarmer cell life span. The transcriptional profiling experiments described here generate the first comprehensive picture of the global regulatory strategies used by an oligotroph when confronted with an environment where key macronutrients are sparse. The pattern of regulated gene expression in nitrogen- and carbon-limited cells shares some features in common with most copiotrophic organisms, but critical differences suggest that Caulobacter, and perhaps other oligotrophs, have evolved regulatory strategies to deal distinctly with their natural environments. We hypothesize that nitrogen limitation extends the swarmer cell lifetime by delaying the onset of a sequence of differentiation events, which when initiated by the correct combination of external environmental cues, sets the swarmer cell on a path to differentiate into a stalked cell within a fixed time period.

FIG. 1.

The Caulobacter cell cycle. Depicted is a schematic diagram of the C. crescentus cell cycle (reviewed in references to 3). Motile swarmer cells possess a polar flagellum and pili. These cells differentiate into stalked cells by shedding the flagellum and initiating DNA replication and stalk biogenesis. As the cell cycle progresses, a flagellum is assembled at the pole opposite the stalk, and subsequent cell division generates two different cell types. CtrA synthesis, phosphorylation, and subsequent degradation are crucial landmark events in the progression of the cell cycle.

FIG. 2.

Isolated swarmer cells fail to differentiate when suspended in medium lacking either a nitrogen or a carbon source. Pure populations of swarmer cells were isolated from M2-glucose cultures, washed, and suspended in media with or without a nitrogen source (9.3 mM NH₄Cl) (A) or a carbon source (0.2% glucose) (B). At the times indicated, a portion of the culture was removed, the cells pelleted, and the lysates were analyzed for flagellin and CtrA via immunoblot.

FIG. 3.

Growth dynamics of Caulobacter cells cultured in a continuous culture under glucose limitation. (A) Comparison of the differing cell cycles of bacterial cells undergoing symmetric or asymmetric division. Maintenance of a steady-state biomass concentration in a chemostat is dependent on the assumption that each progeny cell arising from a given cell division event possesses the capacity to complete a subsequent cell division within the same period of time (T). Therefore, the mean generation time will be constant for all progeny at a fixed dilution rate. This, however, would not be the case for organisms such as Caulobacter that undergo an obligatory asymmetric cell division event that generates progeny with differing generation times. Newly formed swarmer cells possess an extended G₁ period compared to the progeny stalked cell, since they cannot reinitiate the cell cycle immediately following division. As shown, the progeny swarmer cells have a doubling time or reproductive rate (T) that is 1.3 three times longer, under typical laboratory culture conditions, than that of progeny stalked cells. (B) Affect of glucose limitation on growth of Caulobacter cells in a chemostat. The graph shows the relationship between steady-state biomass concentration (OD₆₀₀) versus flow rate (or dilution rate [D]) that is equal to the specific growth rate (μ) of wild-type Caulobacter cells in continuous culture under glucose limitation. Shown below the flow rate scale on the x axis are the calculated doubling times (i.e., D [μ] = ln2/t_D).

FIG. 4.

Growth dynamics of Caulobacter cells cultured in an ammonium-limited chemostat. (A) The graph depicts the relationship between steady-state biomass concentration versus (OD₆₀₀) versus flow rate (or dilution rate [D]) of wild-type Caulobacter cells in continuous culture under ammonium limitation. Shown below the flow rate scale on the x axis are the calculated doubling times (t_D). The concentration of cells exhibited two different steady-state phases, phase I (D ≤ 0.05 h⁻¹) where the biomass remained at a relatively high concentration, and phase II (D ≥ 0.05 h⁻¹) where a lower steady-state biomass concentration was established. (B) The graph depicts the relationship between biomass concentration (OD₆₀₀) and flow rate (D h⁻¹) over a time course experiment in an ammonium-limited chemostat culture.

FIG. 5.

Enumeration of cell types in continuous cultures growing under imposed ammonium limitation. The proportion of swarmer cells (i.e., nonstalked) in continuous culture under ammonium limitation as a function of flow rate is depicted. Swarmer cells were enumerated microscopically, either by staining the entire cell (▪) or by determining that fraction of cells exhibiting polarly foci of CpaE-YFP (░⃞), a pilus assembly protein that is polarly localized in swarmer cells. 1,000 to 2,700 cells for each depicted time point were counted from at least three independent cultures that were at equilibrium. The percentage of cells possessing polar YFP-CpaE foci was determined on captured images by counting 500 to 1,000 cells for each time point from three different cultures.

FIG. 6.

Assay of mRNA levels of chemostat-grown Caulobacter cells over a range of flow rates. RNA from wild-type Caulobacter cells growing in ammonium-limited continuous culture at the indicated flow rates was harvested and used to synthesize cDNA, which was subjected to qRT-PCR. The RNA assayed was ftsZ (cell division gene), glnK (PII paralog), and ntrY (sensor kinase). RNA from the Caulobacter surface array gene, rsaA, served as a standard and normalized control for growth rate changes (see Materials and Methods). The graph depicts the fold difference in the normalized qRT-PCR product from samples at each time point compared to the normalized qRT-PCR product generated from RNA extracted at the fastest flow rate sampled (0.26 h⁻¹), which was arbitrarily set at a value of 1 (not depicted in the graph).

FIG. 7.

Effect of ntrY on chemostat growth dynamics under ammonium limitation. (A) Depicted is the nitrogen regulatory operon of C. crescentus. Above each gene symbol is the corresponding genome locus number (see the text for details). (B) The graph depicts the relationship between steady-state biomass concentration versus (OD₆₀₀) versus flow rate of ΔntrY mutant Caulobacter cells in continuous culture under ammonium limitation. Shown below the flow rate scale on the x axis are the calculated doubling times (t_D).

FIG. 8.

Role of RNA polymerase containing σ⁵⁴ (rpoN) on the expression of nitrogen-regulated genes. The activity of glnK-, gltD-, and glnB-lacZ transcriptional reporter fusions was assayed in wild-type and rpoN::Tn5 strains following growth to late logarithmic phase in either complex (PYE) or minimal (M2) medium. The levels of reporter gene expression are shown in units of β-galactosidase activity. The error bars represent the standard deviation of assays performed in triplicate on three independently grown cultures.

FIG. 9.

Effect of rpoN on the expression of genes encoding translation and transcription machinery. RNA was harvested from wild-type and rpoN::Tn5 cells grown under either ammonium or glucose limitation in chemostats operated at a flow rate of 0.034 h⁻¹. The heat map represents expression ratios of ammonium- versus glucose-limited cells.

FIG. 10.

Comparison of transcriptional profiles of wild-type and rpoN::Tn5 cells grown in continuous culture under glucose limitation. (A) Forty genes predicted to be involved in carbon acquisition that exhibited the highest differential expression between wild-type glucose- versus ammonium-limited cultures. The heat map represents expression ratios of glucose- versus ammonium-limited cells. (B) Diagram showing genes induced at least twofold in wild-type and rpoN::Tn5 cells under carbon limitation. Of the 153 genes induced in wild-type cells under carbon limitation, only 41 were also induced in rpoN::Tn5 cells. Conversely, rpoN::Tn5 cells induced 55 genes that were not significantly increased in expression in wild-type cells.