SpoT Regulates DnaA Stability and Initiation of DNA Replication in Carbon-StarvedCaulobacter crescentus

Abstract

Cell cycle progression and polar differentiation are temporally coordinated in Caulobacter crescentus. This oligotrophic bacterium divides asymmetrically to produce a motile swarmer cell that represses DNA replication and a sessile stalked cell that replicates its DNA. The initiation of DNA replication coincides with the proteolysis of the CtrA replication inhibitor and the accumulation of DnaA, the replication initiator, upon differentiation of the swarmer cell into a stalked cell. We analyzed the adaptive response of C. crescentus swarmer cells to carbon starvation and found that there was a block in both the swarmer-to-stalked cell polar differentiation program and the initiation of DNA replication. SpoT is a bifunctional synthase/hydrolase that controls the steady-state level of the stress-signaling nucleotide (p)ppGpp, and carbon starvation caused a SpoT-dependent increase in (p)ppGpp concentration. Carbon starvation activates DnaA proteolysis (B. Gorbatyuk and G. T. Marczynski, Mol. Microbiol. 55:1233-1245, 2005). We observed that SpoT is required for this phenomenon in swarmer cells, and in the absence of SpoT, carbon-starved swarmer cells inappropriately initiated DNA replication. Since SpoT controls (p)ppGpp abundance, we propose that this nucleotide relays carbon starvation signals to the cellular factors responsible for activating DnaA proteolysis, thereby inhibiting the initiation of DNA replication. SpoT, however, was not required for the carbon starvation block of the swarmer-to-stalked cell polar differentiation program. Thus, swarmer cells utilize at least two independent signaling pathways to relay carbon starvation signals: a SpoT-dependent pathway mediating the inhibition of DNA replication initiation, and a SpoT-independent pathway(s) that blocks morphological differentiation.

Bacteriostatic versus bactericidal activity of ciprofloxacin in Escherichia coli assessed by flow cytometry using a novel far-red dye

“…As fluorescent stains with the Vybrant DyeCycle series have proven to be effective in staining live eukaryotic cells 18 and fixing prokaryotic cells, such as those of the Gram-negative bacterium Caulobacter crescentus, 19 the present work investigates the ability of DCR to stain bacterial DNA and further evaluates ciprofloxacin activity in the Gram-negative bacterium E. coli.…”

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

“…Samples for flowcytometry were isolated from exponentially growing cells treated with rifampicin for 3 h as described by Winzeler & Shapiro (1995), except that DyeCycle Orange (Invitrogen) was used to stain the DNA as in a recent publication (Lesley & Shapiro, 2008). Data were collected using a FACStar Plus machine (Becton Dickinson) and analysed using FlowJo software (Tree Star).…”

“…The primer sequences were as follows: CtrADp1USFwd, 59-AAAAAAGCTTAACACGGCTCGCGCCTGG-AGATGGGGCCGTT-39; CtrADp1USRev, 59-CGTCGGAGGAAT-GGTTAATCTGATGAAGCTTGCGAATCGGGTGCAAGCCGCGT-39; CtrADp1DSFwd, 59-ACGCGGCTTGCACCCGATTCGCAAGCTTCAT-CAGATTAACCATTCCTCCGACG-39; CtrADp1DSRev, 59-AAAACT-TAAGTTGATGACCGACTGGGCGTGACCCTTCGAA-39. The resulting PCR fragment was cloned into pNPTS138, electroporated into NA1000, and resolved using sucrose selection (Lesley & Shapiro, 2008). DNA sequencing of PCR products using the following primers identified colonies carrying the P1 mutation: CtrA-FL-Fwd, 59-TGGCGTCA-TCGGCAGTCAACTT-39; CtrA-FL-Rev, 59-CTTAACGCTGTAACG-CCGTTCG-39.…”

Replication initiator DnaA binds at the Caulobacter centromere and enables chromosome segregation

“…To determine whether the translocation of the apparently single CFP-ParB focus observed in the presence of limited DnaA levels occurs in the absence of DNA replication, we measured the total chromosomal content in DnaA-limited cells by using fluorescence-activated cell sorting (FACS). The P vanA -dnaA depletion strain was treated with chloramphenicol under conditions in which reinitiation of DNA replication was prevented while allowing the completion of replication (17). In the presence of vanillate (dnaA expression induced), cells contained either one (1n) or two (2n) copies of the chromosome ( Fig.…”