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The genome ofVibrio choleraeconsists of two circular chromosomes of different sizes. Here, a comparative analysis of the replication origins of the large chromosomes (oriCI VC) of classical and El Tor biotypes of the pathogen is reported. Extensive nucleotide sequence analyses revealed that theoriCI VC region has six DnaA boxes instead of the five found inEscherichia coli oriC. The additional DnaA box, designated RV, was unique inV. choleraeas well as in other members of the familyVibrionaceae. However, RV was not found to be essential for the autonomous replication function of the 307-bporiCI VC minimal region. In contrast to El Tor and the recently evolvedV. choleraeO139 strains, theoriCI VC region of the classical biotype showed only a single base transition (T?G) in a highly conserved AT-rich 13-mer R repeat region. From the minichromosome copy number and its transformational efficiency analyses, it appears that the single base substitution in theoriCI VC of the classical biotype has a significant effect on its replication initiation. [ABSTRACT FROM AUTHOR]