Feedback control of a master bacterial cell-cycle regulator

Abstract

The transcriptional regulator CtrA controls several key cell-cycle events in Caulobacter crescentus, including the initiation of DNA replication, DNA methylation, cell division, and f lagellar biogenesis. CtrA is a member of the response regulator family of two component signal transduction systems. Caulobacter goes to great lengths to control the time and place of the activity of this critical regulatory factor during the cell cycle. These controls include temporally regulated transcription and phosphorylation and spatially restricted proteolysis. We report here that ctrA expression is under the control of two promoters: a promoter (P1) that is active only in the early predivisional cell and a stronger promoter (P2) that is active in the late predivisional cell. Both promoters exhibit CtrA-mediated feedback regulation: the early P1 promoter is negatively controlled by CtrA, and the late P2 promoter is under positive feedback control. The CtrA protein footprints conserved binding sites within the P1 and P2 promoters. We propose that the P1 promoter is activated after the initiation of DNA replication in the early predivisional cell. The ensuing accumulation of CtrA results in the activation of the P2 promoter and the repression of the P1 promoter late in the cell cycle. Thus, two transcriptional feedback loops coupled to cell cycle-regulated proteolysis and phosphorylation of the CtrA protein result in the pattern of CtrA activity required for the temporal and spatial control of multiple cell-cycle events.

The Caulobacter crescentus ctrA P1 promoter is essential for the coordination of cell cycle events that prevent the overinitiation of DNA replication

“…The following primers were used to create wild-type ctrA, ctrA P1 only and ctrA P2 only translational reporters in pJC326C (Chen et al, 2006). The forward primer amplifies the same nucleotides that are found in the 59 end of the ctrA transcriptional reporters created earlier (Domian et al, 1999). To amplify the wild-type and ctrA P1 mutant promoters, the following primers were used: ctrA-TNL-US-Fwd, 59-AAAA-AGATCTAGGCCTCGATTTTCTCGATTTCTT-39; ctrA-TNL-DS-Rev, 59-AAAACTGCAGATCCTCGATCAACAGTACGCGCAT-39.…”

“…To amplify the wild-type and ctrA P1 mutant promoters, the following primers were used: ctrA-TNL-US-Fwd, 59-AAAA-AGATCTAGGCCTCGATTTTCTCGATTTCTT-39; ctrA-TNL-DS-Rev, 59-AAAACTGCAGATCCTCGATCAACAGTACGCGCAT-39. The following primers were used to create a loss-of-function mutation (Domian et al, 1999) (specific mutated nucleotides underlined) in the ctrA P2 promoter by sequential PCR. The upstream (US) and downstream (DS) primer sets were used to respectively amplify the US and DS sequences (relative to the mutation).…”

“…The newly synthesized strand is unmethylated because the CcrM methyltransferase is only active at the end of the cell cycle. After CtrA accumulates, it represses its own transcription from P1 and activates transcription from P2 (Domian et al, 1999). P1 is activated in early predivisional cells, whereas P2 is activated in late predivisional cells.…”

“…To explore the role of P1 in cell cycle progression, we inactivated the P1 promoter at the native ctrA locus by inserting the same 5 bp spacer between the 235 and 210 sites of the P1 promoter that had previously been shown to prevent transcription from P1, but not P2, on a plasmid-based transcriptional reporter (Domian et al, 1999). Here, we characterize the effect of this mutation at the native ctrA locus.…”

“…GcrA notably activates genes involved in DNA replication elongation and chromosome segregation and contributes to the expression of CtrA by activating the first promoter of ctrA, P1, which is regulated by N 6 -adenosine methylation (Reisenauer & Shapiro, 2002). When ctrA P1 becomes hemimethylated during DNA replication (Domian et al, 1999;Stephens et al, 1996), GcrA can activate its transcription (Holtzendorff et al, 2004). The newly synthesized strand is unmethylated because the CcrM methyltransferase is only active at the end of the cell cycle.…”