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The genome of the enteric pathogen Vibrio cholerae consists of two chromosomes. While the chromosome I replication origin and its cognate replication initiator protein resemble those of Escherichia coli, the factors responsible for chromosome II replication initiation display no similarity to any other known initiation systems. Here, to enhance our understanding of how this DNA sequence, oriCII, and its initiator protein, RctB, function, we used both targeted mutagenesis and a new random-mutagenesis approach (OriSeq) to finely map the oriCII structural features and sequences required for RctB-mediated DNA replication. Collectively, our findings reveal the extraordinary evolutionary honing of the architecture and motifs that constitute oriCII and reveal a new role for methylation in oriCII-based replication. Finally, our findings suggest that the OriSeq approach is likely to be widely applicable for defining critical bases in cis-acting sequences.

Molecular Dissection of the Essential Features of the Origin of Replication of the Second Vibrio cholerae Chromosome