Excess SeqA Leads to Replication Arrest and a Cell Division Defect in Vibrio cholerae

Although most bacteria contain a single circular chromosome, some have complex genomes, and allVibrio species studied so far contain both a large and a small chromosome. In recent years, the divided genome ofVibrio cholerae has proven to be an interesting model system with both parallels to and novel features compared with the genome ofEscherichia coli . While factors influencing the replication and segregation of both chromosomes have begun to be elucidated, much remains to be learned about the maintenance of this genome and of complex bacterial genomes generally. An important aspect of replicating any genome is the correct timing of initiation, without which organisms risk aneuploidy. During DNA replication inE. coli , newly replicated origins cannot immediately reinitiate because they undergo sequestration by the SeqA protein, which binds hemimethylated origin DNA. This DNA is already methylated by Dam on the template strand and later becomes fully methylated; aberrant amounts of Dam or the deletion ofseqA leads to asynchronous replication. In our study, hemimethylated DNA was detected at both origins ofV. cholerae , suggesting that these origins are also subject to sequestration. The overproduction of SeqA led to a loss of viability, the condensation of DNA, and a filamentous morphology. Cells with abnormal DNA content arose in the population, and replication was inhibited as determined by a reduced ratio of origin to terminus DNA in SeqA-overexpressing cells. Thus, excessive SeqA negatively affects replication inV. cholerae and prevents correct progression to downstream cell cycle events such as segregation and cell division.