Bacterial cell cycle and growth phase switch by the essential transcriptional regulator CtrA

Abstract

Many bacteria acquire dissemination and virulence traits in G1-phase. CtrA, an essential and conserved cell cycle transcriptional regulator identified in the dimorphic alpha-proteobacterium Caulobacter crescentus, first activates promoters in late S-phase and then mysteriously switches to different target promoters in G1-phase. We uncovered a highly conserved determinant in the DNA-binding domain (DBD) of CtrA uncoupling this promoter switch. We also show that it reprograms CtrA occupancy in stationary cells inducing a (p)ppGpp alarmone signal perceived by the RNA polymerase beta subunit. A simple side chain modification in a critical residue within the core DBD imposes opposing developmental phenotypes and transcriptional activities of CtrA and a proximal residue can direct CtrA towards activation of the dispersal (G1-phase) program. Hence, we propose that this conserved determinant in the CtrA primary structure dictates promoter reprogramming during the growth transition in other alpha-proteobacteria that differentiate from replicative cells into dispersal cells.

Figure 1.

ctrA401 as gain of function mutation. (A) Schematic of Caulobacter capsulation (blue) along cell cycle and regulatory interactions that controls Caulobacter transcriptional switch between late S-phase and G1-phase. CtrA controls activity of late S- and G1-phase promoters. SciP which is expressed in G1-phase under the control of CtrA negatively regulates S-phase genes. During cell cycle, capsulation is regulated through expression of hvyA that prevents capsulation in SW cell and under the control of the transcriptional regulators CtrA. (B) Immunoblots showing steady-state levels of HfsJ and SpmX in WT, ΔspoT, ΔpleC, ctrA401 and derivatives in exponential and stationary phase. CCNA_00163 serves as a loading control. (C) Genome wide occupancies of CtrA on the Caulobacter WT, ctrA401 and ctrA401-SS genome as determined by ChIP-Seq. The x-axis represents the nucleotide position on the genome (bp), whereas the y-axis shows the normalized ChIP profiles in read per million (rpm). (D) ChIP-Seq traces of CtrA, CtrA401 (T170I) and CtrA401-SS (T168I/T170I) on different CtrA target promoters. Genes encoded are represented as boxes on the upper part of the graph, gene names and CCNA numbers gene annotation are indicated in the boxes or above. (E, F) Schemes showing the regulatory interactions happening at the late S- and G-phase promoters based on C, D and Table 1.

Figure 2.

Capsulation of the ctrA401 mutant affects the buoyancy switch, bacteriophage sensitivity and motility. (A) Promoter-probe assays of transcriptional reporters carrying a fljL, fliQ (class IV and class II genes, respectively) promoters in WT, ctrA401 and derivatives. Values are expressed as percentages (activity in WT set at 100%). (B) Motility plates (0,3% agar) inoculated with WT, ctrA401, ctrA401motility suppressors 1 and 2 (ctrA401-MS1 and ctrA401-MS2) strains and immunoblot showing the steady state levels of the alpha-flagellin FljK in WT, ΔspoT and ctrA401 cells. (C) Swarm (0.3%) agar inoculated with WT, ctrA401, ctrA401-MS, ΔMGE and Δ03998 (ΔCCNA_03998) cells. Note that motility is also improved when the ctrA401 mutation is transduced into ΔMGE cells (Supplementary Figure S3E), but not restored to the level seen for WT or ctrA401-MS, presumably because of yet unknown mechanism of motility inhibition in ctrA401 or because of unknown contributions conferred by the suppressor mutations on the MGE. (D) Schematic and pictures of cell buoyancy upon centrifugation on density gradient and sensitivity to bacteriophages ϕCr30 and ϕCbK for WT Caulobacter and ctrA401 mutants and derivatives. (E) Promoter-probe assays of hvyA-lacZ transcriptional (left graph) and translational reporters (right graph) in WT, ctrA401 and derivatives. Values are expressed as percentages (activity in WT set at 100%). Data from four independent experiments, error bars are standard deviation. (F) Swarming motility and buoyancy assays in cells overexpressing hvyA-tap fusion under control of P_van on pMT335 plasmid restores the ‘heavy’ cell buoyancy to the ctrA401 cells.

Figure 3.

Intragenic suppressor mutations of ctrA401 alter DNA-binding. (A) DIC microscopy, 8X zoom images and FACS analysis of the WT, ctrA401 and derivatives carrying the pilA::P_pilA-nptII transcriptional reporter. Microscopy pictures and DNA content (FL1-A channel) quantification were performed during exponential growth in PYE. ctrA401 MS2 pilA::P_pilA-nptII cells resistant to 40 μg/ml of kanamycin (kan) were screened by microscopy for the presence of stalk (yellow arrow). DIC images and FACS analysis of the ctrA401 and ctrA401- MS2 show cells that are elongated, accumulating more than 2n chromosomes (red arrow) and stalkless in exponential phase. Selection of ctrA401 MS2 pilA::P_pilA-nptII cells on PYE plates supplemented with 40 μg/ml of kanamycin and screening by DIC microscopy for the presence of stalk (yellow arrow, 8X zoom) lead to the identification of the ctrA401-SS (T170I/T168I) mutation. ctrA401-SS cells do not elongate and do not accumulate extra-chromosome (green arrow). (B) On the left, the alignment of the 28 amino acids around the T170 and the T168 shows the amino acid replacements in ctrA401, ctrA401-SS compared to WT. On the right, the alignment of CtrA protein sequences from several members of the alpha-proteobacteria phylum is shown. (C) Motility (0.3% agar) plates inoculated with WT, ctrA401, ctrA401-SS cells. The T168I amino acid change restores motility to ctrA401 cells. (D) Tn insertion bias in coding sequences (CDS) of ctrA401 (T170I) cells relative to WT cells (orange dots) and ctrA (T170A) cells relative to WT (black dots) cells as determined by Tn-Seq. The abscissa shows position as function of genome position, and the ordinate provides Tn-insertion ratios. Peaks show CDSs with the highest number of Tn insertions. Noncoding sequences are not included.

Figure 4.

CtrA is substantially reprogrammed in stationary phase. (A) Promoter-probe assays of transcriptional reporters carrying a pleA and fliQ promoter in WT, ΔspoT, ctrA401 and derivatives. Transcription from P_pleA-lacZ and P_fliQ-lacZ in WT and ctrA401 is strongly induced in stationary phase in a SpoT-dependent manner. Values are expressed as percentages (activity in WT in exponential phase is set at 100%). (B) Genome-wide occupancies of CtrA on the Caulobacter WT genome in exponential phase compared to CtrA occupancies in WT, ΔspoT and ΔptsP genome in stationary phase as determined by ChIP-Seq using antibodies to CtrA. The x-axis represents the nucleotide position on the genome (bp), whereas the y-axis shows the normalized ChIP profiles in read per million (rpm). (C) ChIP-Seq traces of CtrA on different CtrA-binding promoter regions in WT cells in exponential phase, in WT and ΔspoT in stationary phase. Genes encoded are represented as boxes on the upper part of the graph, gene names and CCNA numbers gene annotation are indicated in the boxes or above. (D) Immunoblot showing steady-state levels of SciP in WT, ΔspoT, ctrA401, ΔspoT ctrA401, ΔsciP and Δlon, in exponential and stationary phase cells. MreB serves as a loading control. (E) Promoter-probe assays of sciP-translational reporters in WT, ΔspoT, ctrA401 and derivatives. Values are expressed as percentages (activity in WT set at 100%). Data from four independent experiments, error bars are standard deviation.

Figure 5.

CtrA reprogramming in stationary phase is mediated by RpoB via (p)ppGpp. (A) Identification of the rpoB^H559P mutation as motility suppressor of the ΔptsP ΔspoT mutant that restores CtrA steady state levels in stationary phase. Swarm (0.3%) agar plates inoculated with WT, ΔptsP ΔspoT, WT rpoB* and ΔptsP ΔspoT rpoB*. rpoB^H559P mutation confers hypermotility to WT and ΔptsP ΔspoT cells. Immunoblot analysis (right) of the steady-state levels of CtrA in exponential and stationary phase of WT, ΔspoT, ΔptsP ΔspoT, WT rpoB* and ΔptsP ΔspoT rpoB* cells. CCNA_00163 serves as a loading control. (B) FACS analysis and DNA content (FL1-A) quantification in exponential and stationary phase of WT, ΔspoT, ctrA401, rpoB* and derivatives. Cells expressing the constitutive active form of E. coli RelA (RelA’) are also shown. rpoB* and ectopic induction of (p)ppGpp lead to G1 block in exponential and stationary phase. In stationary phase, ctrA401 cells are elongated and do not replicate while disruption of spoT improves this defect. (C) Genome-wide occupancies of CtrA on the Caulobacter WT, ΔptsP ΔspoT, WT rpoB* and ΔptsP ΔspoT rpoB* genome in exponential phase. The rpoB* mutation impacts the occupancy of CtrA on chromatin. Stars indicate two highly enriched peaks in rpoB* at the CCNA_03890 and CCNA_03426 loci. (D) ChIP-Seq traces of CtrA on the two CtrA-binding promoter regions highlighted with stars in (C) in WT and rpoB* cells in exponential phase and in WT and ΔspoT cells in stationary phase. Gene names are represented as boxes on the upper part of the graph and CCNA gene annotation are indicated in the boxes or above. (E) Promoter-probe assays of transcriptional reporters carrying the CCNA_03890 promoter in WT, rpoB*, ΔspoT rpoB* and ΔptsP ΔspoT rpoB*. Values are expressed as percentages (activity in WT set at 100%). (F) Immunoblots showing steady-state levels of SciP and CtrA in WT, ΔspoT, ctrA401, ΔspoT ctrA401, rpoB* and rpoB* ctrA401 in exponential and stationary phase.