An essential transcription factor, SciP, enhances robustness of Caulobacter cell cycle regulation

Abstract

A cyclical control circuit composed of four master regulators drives the Caulobacter cell cycle. We report that SciP, a helix-turn-helix transcription factor, is an essential component of this circuit. SciP is cell cycle-controlled and co-conserved with the global cell cycle regulator CtrA in the α-proteobacteria. SciP is expressed late in the cell cycle and accumulates preferentially in the daughter swarmer cell. At least 58 genes, including many flagellar and chemotaxis genes, are regulated by a type 1 incoherent feedforward motif in which CtrA activates sciP, followed by SciP repression of ctrA and CtrA target genes. We demonstrate that SciP binds to DNA at a motif distinct from the CtrA binding motif that is present in the promoters of genes co-regulated by SciP and CtrA. SciP overexpression disrupts the balance between activation and repression of the CtrA-SciP coregulated genes yielding filamentous cells and loss of viability. The type 1 incoherent feedforward circuit motif enhances the pulse-like expression of the downstream genes, and the negative feedback to ctrA expression reduces peak CtrA accumulation. The presence of SciP in the control network enhances the robustness of the cell cycle to varying growth rates.

Fig. 1.

The expression of sciP is cell cycle-regulated. (A) Upper: The transcript levels of sciP (solid line) and ctrA (dotted line) over the course of the cell cycle, beginning with swarmer cells, as determined by microarray analysis (18). Lower: Northern blot analysis of sciP mRNA as a function of the cell cycle. RNA was extracted from a synchronized population of WT C. crescentus (LS101) at the indicted time intervals. sciP mRNA was detected using a probe against nucleotides 169 to 197 within the sciP coding region. A single transcript of approximately 500 bp peaked at the 120-min time point. (B) Immunoblots of samples from a synchronized population of WT cells (LS101) using antibodies to SciP and to three cell cycle regulators, GcrA, CtrA, and CckA. Upon cell division, swarmer and stalked cells were separated and immunoblot analysis showed that SciP and CtrA appeared specifically in the swarmer cell, whereas GcrA appeared specifically in the stalked cell.

Fig. 2.

CtrA activates the expression of sciP. (A) The nucleotide sequence of the sciP promoter is shown. A CtrA-binding motif, TTAA(n7)TTATT, is underlined, and a DnaA motif with a 7/9 match to the consensus sequence is boxed. Numbers are relative to the +1 transcription start site (24). (B) Left: Immunoblots show that 0.3% xylose-induced expression of a nondegradable phosphomimetic of ctrA, ctrAD51EΔ3Ω, on a high copy plasmid, increased the level of SciP accumulation. Serving as controls, the level of GcrA accumulation decreased, whereas the level of zapA, a gene whose expression is independent of a CtrA-binding site (9), remained unchanged. Right: Immunoblot showing that the SciP protein level decreased with time after LS2195 (ctrA401ts) cells were shifted from 28 °C to 37 °C, compared with no change for WT LS101 cells. As controls, 2 h after the shift to the restrictive temperature, ZapA levels remained unchanged and the level of GcrA, whose expression is inhibited by CtrA (5), increased. (C) Quantitative real-time PCR was used to measure the relative transcript levels of ctrA, sciP, gcrA, and zapA in strain LS4190 upon addition of 0.3% xylose to induce expression of ctrAD51EΔ3Ω. Left: The transcript level of ctrA increased approximately 11-fold, whereas that of sciP increased approximately sixfold. Right: The transcript level of gcrA decreased by approximately sevenfold, whereas that of zapA did not change significantly. In both panels, white bars indicate 0 min, striped bars indicate 15 min, stippled bars indicate 30 min, and gray bars indicate 60 min after addition of 0.3% xylose. The error bars indicate SDs from three technical replicates.

Fig. 3.

Overexpression of sciP represses transcription of the ctrA master regulator. (A) Immunoblots of SciP and three essential cell cycle regulators—CtrA, CcrM, and GcrA—in strain MHT68, in which sciP is under the control of the xylose-inducible promoter on a high copy plasmid, at 0, 30, and 90 min after the addition of 0.3% xylose. The protein levels of CtrA and CcrM, but not GcrA, decreased upon SciP overproduction. (B) Measurements of the relative transcript levels of sciP and ctrA by quantitative real-time PCR in strains bearing an empty plasmid (MHT338) or a plasmid-borne sciP expressed from a vanillate-inducible promoter (MHT171) in a background containing WT sciP. In both cases, cultures were grown in the presence of 0.5 mM vanillate for the times indicated. The transcript levels of ctrA decreased as sciP levels increased upon induction with vanillate. The error bars indicate the SDs from three technical replicates. (C) DNase I protection (footprinting) of the ctrA P1 promoter by purified His₆-SciP. The antisense strand was radiolabeled at the 5′ end and the first four lanes contain the sequencing ladder of the promoter region. His₆-SciP was added in increasing concentrations from 6.4 nM to 512 nM and the last lane contains no purified protein. Black vertical bars on the right indicate regions of His₆-SciP protection against DNase I digestion. Numbers are relative to the +1 transcription start site (15). (D) Sequence of the region upstream of the transcription start site of the P1 promoter of ctrA. The two SciP-binding sites are underlined and the CtrA-binding site is boxed (25).

Fig. 4.

SciP is a global transcriptional regulator that controls the expression of late cell cycle genes. (A) A subset of genes in the SciP regulon. ChIP column: “x” indicates promoters detected. OE (overexpression) column: “x” indicates response detected in our overexpression experiment; “G” indicates response reported in ref. . (B) The consensus SciP binding motif.

Fig. 5.

SciP is an integral component of the core circuit that controls cell cycle progression in Caulobacter. (A) ChIP followed by quantitative real-time PCR showed that SciP bound directly to the promoter regions of genes encoding CtrA, the CcrM DNA methyltransferase, and the PilA pilus subunit, as well as to the promoter regions of a subset of flagellar and chemotaxis genes. The endogenous sciP coding region on the chromosome was replaced by FLAG-sciP to create strain MHT267. Immunoprecipitation was performed on WT cells expressing untagged sciP (strain LS101; white bars) and on cells that expressed FLAG-sciP (strain MHT267; gray bars) using antibodies to the FLAG tag. The error bars indicate SDs from two technical replicates. The promoter of CC2677, which served as a control, does not contain a SciP motif (18). (B) Ribbon diagram depicting a homologous protein of SciP (Protein Data Bank ID code 1PDN) docked to DNA. Structural homology and sequence alignment was established by FFAS (20). Residues mutated in this study are red and residues mutated by Gora et al. (14) are green. All mutations were found to be in the HTH DNA-binding domain of SciP. (C) Topology of the core cell cycle circuit showing how sciP is connected to the other four previously known components, namely dnaA, gcrA, ctrA, and ccrM. CtrA activates the transcription of multiple flagellar and chemotaxis genes. It also activates the production of SciP in the late predivisional cell. SciP then directly represses the transcription of ctrA as well as many late predivisional cell and swarmer cell specific genes that had been activated by CtrA. CtrA represses the ctrA P1 promoter and autoactivates the P2 promoter.