Convergence of alarmone and cell cycle signaling from trans-encoded sensory domains

Abstract

Despite the myriad of different sensory domains encoded in bacterial genomes, only a few are known to control the cell cycle. Here, suppressor genetics was used to unveil the regulatory interplay between the PAS (Per-Arnt-Sim) domain protein MopJ and the uncharacterized GAF (cyclic GMP-phosphodiesterase-adenylyl cyclase-FhlA) domain protein PtsP, which resembles an alternative component of the phosphoenolpyruvate (PEP) transferase system. Both of these systems indirectly target the Caulobacter crescentus cell cycle master regulator CtrA, but in different ways. While MopJ acts on CtrA via the cell cycle kinases DivJ and DivL, which control the removal of CtrA at the G1-S transition, our data show that PtsP signals through the conserved alarmone (p)ppGpp, which prevents CtrA cycling under nutritional stress and in stationary phase. We found that PtsP interacts genetically and physically with the (p)ppGpp synthase/hydrolase SpoT and that it modulates several promoters that are directly activated by the cell cycle transcriptional regulator GcrA. Thus, parallel systems integrate nutritional and systemic signals within the cell cycle transcriptional network, converging on the essential alphaproteobacterial regulator CtrA while also affecting global cell cycle transcription in other ways.

Importance: Many alphaproteobacteria divide asymmetrically, and their cell cycle progression is carefully regulated. How these bacteria control the cell cycle in response to nutrient limitation is not well understood. Here, we identify a multicomponent signaling pathway that acts on the cell cycle when nutrients become scarce in stationary phase. We show that efficient accumulation of the master cell cycle regulator CtrA in stationary-phase Caulobacter crescentus cells requires the previously identified stationary-phase/cell cycle regulator MopJ as well as the phosphoenolpyruvate protein phosphotransferase PtsP, which acts via the conserved (p)ppGpp synthase SpoT. We identify cell cycle-regulated promoters that are affected by this pathway, providing an explanation of how (p)ppGpp-signaling might couple starvation to control cell cycle progression in Caulobacter spp. and likely other Alphaproteobacteria. This pathway has the potential to integrate carbon fluctuation into cell cycle control, since in phosphotransferase systems it is the glycolytic product phosphenolpyruvate (PEP) rather than ATP that is used as the phosphor donor for phosphorylation.

FIG 1

MopJ and PtsP are pleiotropic regulators that control motility and cell cycle progression in Caulobacter crescentus. (A) Model showing the C. crescentus cell cycle and the relevant cell cycle transcriptional regulators CtrA and GcrA, as well as the recently described single PAS domain protein MopJ (23). The thin black vertical line represents the flagellar filament (composed of FljK, FljM, and other flagellins), before it rotates (wavy line). The thick vertical black line represents the stalk, and the white oval represents the chromosome, whose replication is initiated at the C. crescentus origin of replication (Cori). The thin slanted black lines represent the polar pili (composed of the PilA pilin). The expression of MopJ and CtrA is transcriptionally activated by GcrA (blue arrows), while CtrA activates expression of the methylase CcrM, the flagellin FljM, and the pilin PilA. Expression of the flagellin FljK by CtrA is indirect (7). Shown underneath is a model of the (p)ppGpp-dependent signaling pathways in stationary-phase C. crescentus cells described in the text. Dashed arrows indicate connections that are poorly defined. (B, top) Motility assay on swarm (0.3%) agar for WT, mopJ::himar, ΔmopJ, and ΔptsP single mutants, the ΔmopJ ΔptsP double mutant, and two spontaneously isolated ΔmopJ motility suppressors, ΔmopJ ptsP(S104P) and ΔmopJ ptsP(Q153P). (Bottom) Complementation of the ΔptsP motility defect with pMT335-ptsP (p335-ptsP), but not with empty pMT335 (p335). WT cells harboring empty pMT335 (p335) are also shown. (C) Domain organization of PtsP from the N to C terminus, indicating the total length in amino acids (aa) of the protein. Asterisks inidcate the position of the suppressive mutation in the PtsP GAF domain. (D) Motility assay on soft (0.3%) agar with WT, ΔmopJ, ΔptsP, ΔmopJ ptsP(S104P), ptsPΔGAF, and ΔmopJ ptsPΔGAF strains. (E) The ptsP(S104P) or ptsP(Q153P) suppressor mutations in ΔmopJ (top) and the deletion of the GAF domain of ptsP in the WT or in the ΔmopJ background (bottom) increased the doubling time of cells. Growth curves are shown for the WT, ΔmopJ ptsP(S104P), ΔmopJ ptsP(Q153P), ptsPΔGAF, and ΔmopJ ptsPΔGAF cells in PYE. Error bars in the graph indicate standard deviations. (F) Immunoblot showing the steady-state levels of PtsP, CtrA, GcrA, and CcrM during the cell cycle of WT cells (top) or ΔmopJ ptsP(S104P) cells (bottom). The time (in minutes) after synchronization is indicated above the blots. (G) Fluorescence and DIC images show the localization pattern of PtsP-GFP (C-terminal fusion of PtsP to GFP) expressed under the control of P_xyl (xylose inducible) at the xylX locus in WT cells.

FIG 2

MopJ and PtsP promote the accumulation of G₁-phase cells. (A and B) FACS analysis of ΔmopJ and ΔptsP mutant strains and the ΔmopJ ΔptsP double mutant strain showed a reduction in G₁ phase. Genome content (FL1-A channel) and cell size (FSC-A channel) were analyzed by FACS during exponential (A) and stationary (B) phases in M2G. (C and D) ΔmopJ and ΔptsP single mutants and the ΔmopJ ΔptsP double mutant showed filamentation. DIC images of WT, ΔmopJ and ΔptsP single mutants, and the ΔmopJ ΔptsP double mutant during exponential (C) and stationary (D) growth phases in M2G.

FIG 3

PtsP regulates CtrA synthesis in stationary phase. (A and B) Promoter-probe assays of transcriptional reporters carrying a fljM, sciP, pilA, or fljK promoter fused to a promoterless lacZ gene in WT, ΔmopJ or ΔptsP single mutants, the ΔmopJ ΔptsP double mutant, and suppressor mutants ΔmopJ ptsP(S104P) and ΔmopJ ptsP(Q153P) in exponential (exp.) (A) and stationary (stat.) (B) phases. The graphs show lacZ-encoded β-galactosidase activities, measured in Miller units. Error bars indicate standard deviations (SD). (C and D) Immunoblot showing the steady-state levels of the major flagellin FljK, the SciP negative regulator, and the PilA structural subunit of the pilus filament in WT, ΔmopJ and ΔptsP single mutants, the ΔmopJ ΔptsP double mutant, and suppressor mutants ΔmopJ ptsP(S104P) and ΔmopJ ptsP(Q153P) in exponential (C) and stationary (D) phases. The steady-state levels of the MreB actin are shown as a loading control. (E) Immunoblot showing the steady-state levels of CtrA (or CtrA-M2), PtsP (or PtsPΔGAF) and MreB (loading control) in various mutants in exponential and stationary phases. (F) Promoter-probe assays of transcriptional reporters carrying the ctrA promoter fused to a promoterless lacZ gene in the WT and various mutants in exponential (left) and stationary (right) growth phases. The graphs show lacZ-encoded β-galactosidase activities measured relative to the WT. Error bars show the SD.

FIG 4

Genetic and physical interactions between PtsP and the (p)ppGpp synthase SpoT. (A) Domain organization of Caulobacter SpoT. The asterisk marks the position of the suppressor mutation. The hydrolase and synthase domains are also indicated, along with two conserved regulatory domains in the C-terminal part of SpoT. (B) Motility assay on a swarm agar plate of WT, ΔptsP and ΔspoT single mutants, ΔmopJ ΔptsP, ΔptsP mopJ::himar1, and ΔspoT mopJ::himar1 double mutants, the spontaneous motility suppressor of the ΔptsP mutant, ΔptsP spoT(Δ22), and the ΔptsP spoT(Δ22) mopJ::himar1 triple mutant. (C) Swarm agar assay with WT and ΔmopJ and ΔptsP single mutants upon expression of the constitutive active form of E. coli RelA fused to the FLAG (M2) tag (RelA′-M2) in the presence of xylose. The controls harboring the inactivated form of RelA′ (RelA′-E335Q-M2) and the empty vector are also shown. The arrowhead points to the increase in motility in ΔptsP cells upon (p)ppGpp production by RelA′ induction. (D) Identification of SpoT by tandem mass spectrometry (MS/MS) on a silver stained gel following tandem affinity purification (TAP) from extracts of WT cells expressing PtsP-TAP from pMT335 under the control of the P_van promoter. (E) Coimmunoprecipitation (Co−IP) of PtsP with green fluorescent protein (GFP)-tagged SpoT from a GFP-TRAP affinity matrix (ChromoTek GmbH, Planegg-Martinsried, Germany). Precipitated samples were probed for the presence of PtsP by immunoblotting using antibodies against PtsP. Cell lysates used as input are also shown. (F) Promoter-probe assays of transcriptional reporters carrying the fljM, sciP, pilA, or fljK promoter fused to a promoterless lacZ gene in WT and ΔptsP spoT(Δ22) cells in stationary phase. Error bars show the standard deviations (SD). (G) Promoter-probe assays of transcriptional reporter carrying the mopJ promoter fused to a promoterless lacZ gene in WT, ΔptsP and ΔspoT single mutants, ΔptsP spoT(Δ22), ΔmopJ ptsP(S104P), ΔmopJ ptsP(Q153P) suppressor mutants, and ptsPΔGAF and ΔmopJ ptsPΔGAF mutants cells in stationary (top) and exponential (bottom) phases. Error bars show SD.