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. 2015 Oct 19;198(1):187-200.
doi: 10.1128/JB.00658-15. Print 2016 Jan 1.

Identification of the PhoB Regulon and Role of PhoU in the Phosphate Starvation Response of Caulobacter crescentus

Emma A Lubin  1 , Jonathan T Henry  2 , Aretha Fiebig  2 , Sean Crosson  2 , Michael T Laub  3
Affiliations

Identification of the PhoB Regulon and Role of PhoU in the Phosphate Starvation Response of Caulobacter crescentus

Emma A Lubin et al. J Bacteriol. .

Abstract

An ability to sense and respond to changes in extracellular phosphate is critical for the survival of most bacteria. For Caulobacter crescentus, which typically lives in phosphate-limited environments, this process is especially crucial. Like many bacteria, Caulobacter responds to phosphate limitation through a conserved two-component signaling pathway called PhoR-PhoB, but the direct regulon of PhoB in this organism is unknown. Here we used chromatin immunoprecipitation-DNA sequencing (ChIP-Seq) to map the global binding patterns of the phosphate-responsive transcriptional regulator PhoB under phosphate-limited and -replete conditions. Combined with genome-wide expression profiling, our work demonstrates that PhoB is induced to regulate nearly 50 genes under phosphate-starved conditions. The PhoB regulon is comprised primarily of genes known or predicted to help Caulobacter scavenge for and import inorganic phosphate, including 15 different membrane transporters. We also investigated the regulatory role of PhoU, a widely conserved protein proposed to coordinate phosphate import with expression of the PhoB regulon by directly modulating the histidine kinase PhoR. However, our studies show that it likely does not play such a role in Caulobacter, as PhoU depletion has no significant effect on PhoB-dependent gene expression. Instead, cells lacking PhoU exhibit striking accumulation of large polyphosphate granules, suggesting that PhoU participates in controlling intracellular phosphate metabolism.

Importance: The transcription factor PhoB is widely conserved throughout the bacterial kingdom, where it helps organisms respond to phosphate limitation by driving the expression of a battery of genes. Most of what is known about PhoB and its target genes is derived from studies of Escherichia coli. Our work documents the PhoB regulon in Caulobacter crescentus, and comparison to the regulon in E. coli reveals significant differences, highlighting the evolutionary plasticity of transcriptional responses driven by highly conserved transcription factors. We also demonstrated that the conserved protein PhoU, which is implicated in bacterial persistence, does not regulate PhoB activity, as previously suggested. Instead, our results favor a model in which PhoU affects intracellular phosphate accumulation, possibly through the high-affinity phosphate transporter.

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Figures

FIG 1
FIG 1
A strain producing PhoB with a C-terminal 3×FLAG epitope behaves like the wild type (WT) under phosphate-replete and phosphate-limited conditions. (A) Growth curves of the strains indicated, in minimal medium with 10 mM or 50 μM phosphate. (B) β-Galactosidase assays for a PpstC-lacZ (left) and PpstS-lacZ (right) reporter in minimal medium with 10 mM phosphate (top) or 50 μM phosphate (bottom). The strains indicated were grown in minimal medium with 10 mM extracellular phosphate, washed, resuspended in medium with 10 mM or 50 μM phosphate, and grown for 7 h to an OD600 between 0.3 and 0.4. For panels A and B, data points indicate the averages of results from three independent replicates, with error bars representing the standard deviations (SD). (C) Phase-contrast microscopy of the indicated strains expressing wild-type phoB. Bars, 2 μm.
FIG 2
FIG 2
ChIP-Seq reveals genome-wide binding patterns of PhoB. (A) qPCR of PpstC, a PhoB-activated promoter, and PCC1294, a negative control, using DNA samples after ChIP with an anti-FLAG antibody. Strains expressed either phoB-3×FLAG or wild-type phoB and were grown in minimal medium with 10 mM phosphate or 50 μM phosphate or in rich medium (PYE), with a pstS::Tn5 mutation introduced to constitutively activate PhoB. The y axis indicates fold enrichment of a locus in the ChIP output sample, compared to the input DNA. (B) Numbers of ChIP-Seq reads at three loci. Strains produced either PhoB-3×FLAG or wild-type PhoB and were grown in PYE or minimal medium with 50 μM phosphate; a pstS::Tn5 mutation on the chromosome is indicated when appropriate. Plots are scaled to the maximum read count for each locus. (C) Full-genome ChIP-Seq profiles for the same strains and growth conditions indicated in panel B. Samples from strains producing PhoB-3×FLAG are represented in red; samples from control strains expressing wild-type PhoB are represented in black below the x axes. The positions of genes from panel B are indicated.
FIG 3
FIG 3
PhoB binds to the promoters of Pho regulon genes upon phosphate limitation. (A) Overlap between the sets of genes containing significant PhoB peaks in ChIP-Seq samples from cells grown in minimal medium with 50 μM phosphate or in PYE while harboring a pstS::Tn5 mutation that mimics constitutive phosphate starvation. (B) Genes whose promoters showed >5-fold enrichment by ChIP-Seq analysis of pstS::Tn5 cells grown in rich PYE medium and a >1.7-fold expression change in pstS::Tn5 cells assayed by microarray analysis. ChIP fold enrichment is shown for the same set of genes in PYE and in minimal medium containing 50 μM phosphate. Fold enrichment relative to the input DNA is represented using colors, as indicated, and expression changes assayed by microarray analysis of the pstS::Tn5 and pstS::Tn5 ΔphoB strains are shown. The one-line annotation for each gene or operon whose promoter is bound by PhoB is listed; genes potentially in the same operon are separated by slashes. CC numbers from the original CB15 genome annotation are listed, except for genes newly annotated in the CB15N genome (with CCNA numbers). Note that small RNA genes have CCNAR numbers. (C) MEME was used to identify a motif from sequences of peaks >13-fold enriched in the pstS::Tn5 ChIP-Seq sample. The E value is 1.3 × 10−20. PhoB binding sites (pho boxes) and the putative −35 site are labeled.
FIG 4
FIG 4
Depleting PhoU does not phenocopy a pstS null mutant. (A) Phase-contrast microscopy and flow cytometry analysis of DNA contents for the strains indicated, in the presence and absence of vanillate (van). Microscopic images were taken 8 h after the removal of vanillate. Bar, 2 μm. For flow cytometry, samples were collected 16 h after the removal of vanillate. (B) Numbers of CFU of the indicated strains, grown in PYE. Numbers of CFU reported are the averages of results from two replicates and indicate the number of CFU per 1 ml normalized to an OD600 of 1.
FIG 5
FIG 5
Depleting PhoU does not produce the same gene expression changes seen in a pstS mutant. (A) Expression changes after depletion of PhoU for 2, 5, 7, and 16 h. Expression values are the averages of results from two replicates. Genes upregulated at least 2-fold in the pstS::Tn5 strain are shown. Green, direct PhoB targets, as determined by ChIP-Seq. The genes labeled are those that were ≥2-fold upregulated in the phoU depletion strain at the 7-h time point and found to be direct PhoB targets. (B) Venn diagrams indicating the numbers of genes whose expression changed at least 2-fold, compared to their expression in the wild type, in the pstS::Tn5 strain or in the phoU depletion strain after 7 or 16 h of depletion. (C) Expression changes in a PhoU depletion strain harboring the suppressor mutation pstS::Tn5. Data are for the same genes shown in panel A. Numbers of hours after the removal of vanillate are indicated. (D) β-Galactosidase assay of PpstC-lacZ and PpstS-lacZ reporter expression after PhoU depletion in PYE. wt, wild type. Data points indicate the averages from three independent replicates, with error bars representing the SD.
FIG 6
FIG 6
Mutations in the pst and pho genes suppress phoU depletion lethality, and polyphosphate accumulates upon phoU depletion in Caulobacter crescentus. (A) Locations of Tn5 insertions that suppressed phoU depletion lethality are indicated by arrows. Bars denote directly adjacent but not cooperonic loci. phoU, which was deleted in this background, is shown in black. (B) Light microscopy of the noted strains in the presence and absence of vanillate. Arrows indicate dark granules that may represent polyphosphate (Fig. 7). Bars, 2 μm. (C) Numbers of CFU for the strains indicated, with (left) or without (right) vanillate, are shown, with data points representing the averages of results from two replicates and given as the number of CFU in 1 ml normalized to an OD600 of 1.
FIG 7
FIG 7
Polyphosphate accumulates upon PhoU depletion in Caulobacter crescentus. (A) The strains indicated were grown either with or without vanillate for the times indicated. Cells were imaged by phase-contrast and epifluorescence microscopy using a filter set specific for DAPI-polyphosphate. Phase images were overlaid with the fluorescence image at 100% fluorescence excitation intensity (in green) for the 9-h time points and at a 30% excitation intensity (in magenta) for the 22-h time points. Bars, 2 μm. (B) Numbers of CFU for the strains indicated, with or without vanillate, are shown, with data points representing the averages of results from two replicates and given as the number of CFU in 1 ml normalized to an OD600 of 1.
FIG 8
FIG 8
Model for PhoU function and regulation of the response to extracellular phosphate limitation in Caulobacter. The high-affinity transporter PstSCAB imports inorganic phosphate (red) into the cytoplasm. When flux through the transporter decreases, the histidine kinase PhoR is likely directly activated to autophosphorylate and then phosphotransfer to PhoB. Phosphorylated PhoB can bind to pho boxes (blue) in target genes, including the pst transporter. PhoU likely does not couple the Pst transporter to PhoR and instead is proposed to inhibit phosphate uptake by the Pst system or possibly regulate intracellular phosphate metabolism.
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