Multipartite Regulation ofrctB, the Replication Initiator Gene ofVibrio choleraeChromosome II

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Abstract
Replication initiator proteins in bacteria not only allow DNA replication but also often regulate the rate of replication initiation as well. The regulation is mediated by limiting the synthesis or availability of initiator proteins. The applicability of this principle is demonstrated here for RctB, the replication initiator for the smaller of the two chromosomes ofVibrio cholerae. A strong promoter for therctBgene namedrctBpwas identified and found to be autoregulated inEscherichia coli. Promoter activity was lower inV. choleraethan inE. coli, and a part of this reduction is likely to be due to autorepression. Sequences upstream ofrctBp, implicated earlier in replication control, enhanced the repression. The action of the upstream sequences required that they be present incis, implying long-range interactions in the control of the promoter activity. A second gene specific for chromosome II replication,rctA, reducedrctBtranslation, most likely by antisense RNA control. Finally, optimalrctBpactivity was found to be dependent on Dam. Increasing RctB intransincreased the copy number of a miniplasmid carryingoriCIIVC, implying that RctB can be rate limiting for chromosome II replication. The multiple modes of control on RctB are expected to reduce fluctuations in the initiator concentration and thereby help maintain chromosome copy number homeostasis.