Comparative analysis of Caulobacter chromosome replication origins

Area of Science:

• Microbiology
• Molecular Biology
• Bacterial Genetics

Background:

• Caulobacter crescentus (CB15) exhibits cell-cycle-specific chromosome replication, initiated only in stalked cells, not swarmers.
• Replication control mechanisms involve integration host factor (IHF), CcrM DNA methylation, DnaA boxes, and CtrA binding sites.
• Previous studies identified IHF and CcrM sites in model Caulobacter, but their conservation varies across species.

Purpose of the Study:

• To investigate the dimorphic control of chromosome replication in Caulobacter species.
• To isolate and characterize replication origins (oris) from freshwater (FWC) and marine (MCS) Caulobacter.
• To determine the roles of DnaA boxes and CtrA binding sites in FWC and MCS oris.

Main Methods:

• Isolation of replication origins from freshwater and marine Caulobacter species.
• Sequence analysis to identify conserved DnaA boxes and CtrA binding sites.
• Site-directed mutagenesis of an MCS10 replication origin to assess the function of CtrA and DnaA binding sites.

Main Results:

• DnaA boxes and CtrA binding sites were identified in FWC and MCS oris, often in proximity, suggesting regulatory interaction.
• Mutagenesis of the MCS10 ori revealed that mutations in the DnaA box decreased replication, while mutations in the overlapping CtrA site increased replication.
• Both FWC and MCS oris utilize CtrA to repress replication, but phylogenetic analysis indicates this CtrA usage evolved independently.

Conclusions:

• CtrA plays a conserved role in repressing DNA replication initiation across diverse Caulobacter species.
• The independent evolution of CtrA usage in replication control demonstrates convergent evolution of bacterial origins.
• Understanding these mechanisms provides insights into bacterial chromosome replication and evolutionary strategies.