Evidence for Two Different Regulatory Mechanisms Linking Replication and Segregation of Vibrio cholerae Chromosome II

Abstract

Understanding the mechanisms that coordinate replication initiation with subsequent segregation of chromosomes is an important biological problem. Here we report two replication-control mechanisms mediated by a chromosome segregation protein, ParB2, encoded by chromosome II of the model multichromosome bacterium, Vibrio cholerae. We find by the ChIP-chip assay that ParB2, a centromere binding protein, spreads beyond the centromere and covers a replication inhibitory site (a 39-mer). Unexpectedly, without nucleation at the centromere, ParB2 could also bind directly to a related 39-mer. The 39-mers are the strongest inhibitors of chromosome II replication and they mediate inhibition by binding the replication initiator protein. ParB2 thus appears to promote replication by out-competing initiator binding to the 39-mers using two mechanisms: spreading into one and direct binding to the other. We suggest that both these are novel mechanisms to coordinate replication initiation with segregation of chromosomes. Replication and segregation are the two main processes that maintain chromosomes in growing cells. In eukaryotes, the two processes are restricted to distinct phases of the cell cycle. In bacteria, segregation follows replication initiation with a modest lag. Influences of one process on the other have been postulated. The act of replication has been suggested to provide a motive force in chromosome segregation. Moreover, segregation proteins (ParA) have been found to interact with and control the replication initiator, DnaA. Here we show that in V. cholerae chromosome II, which is believed to have originated from a plasmid, a centromere binding protein (ParB) could control replication by two distinct mechanisms: spreading from a centromeric site into the replication-control region, and direct binding to the primary replication-control site, which has limited homology to the centromeric site. These studies establish that Par proteins can influence replication by at least three mechanisms. Homologous Par proteins participate in plasmid segregation but they are not known to influence plasmid replication. The expanded role of Par proteins appears likely to have been warranted to coordinate chromosomal replication and segregation with the cell cycle, which appears less of an issue in plasmid maintenance.