Abstract
There is little knowledge of factors and mechanisms for coordinating bacterial chromosome replication and segregation. Previous studies have revealed that genes (and their products) that surround the origin of replication (oriCII) of Vibrio cholerae chromosome II (chrII) are critical for controlling the replication and segregation of this chromosome. rctB, which flanks one side of oriCII, encodes a protein that initiates chrII replication; rctA, which flanks the other side of oriCII, inhibits rctB activity. The chrII parAB2 operon, which is essential for chrII partitioning, is located immediately downstream of rctA. Here, we explored how rctA exerts negative control over chrII replication. Our observations suggest that RctB has at least two DNA binding domains—one for binding to oriCII and initiating replication and the other for binding to rctA and thereby inhibiting RctB's ability to initiate replication. Notably, the inhibitory effect of rctA could be alleviated by binding of ParB2 to a centromere-like parS site within rctA. Furthermore, by binding to rctA, ParB2 and RctB inversely regulate expression of the parAB2 genes. Together, our findings suggest that fluctuations in binding of the partitioning protein ParB2 and the chrII initiator RctB to rctA underlie a regulatory network controlling both oriCII firing and the production of the essential chrII partitioning proteins. Thus, by binding both RctB and ParB2, rctA serves as a nexus for regulatory cross-talk coordinating chrII replication and segregation.
Highlights
Efficient linkage of chromosome replication and chromosome segregation is necessary for all dividing cells
The primary agent governing replication initiation is RctB; initiation can be influenced by a previously characterized partitioning protein, ParB2, which we show counteracts rctA’s inhibitory effect upon chromosome II (chrII) replication
The autoregulatory parAB2 locus is the primary determinant of chrII segregation; this process can be influenced by RctB, which activates parAB2 expression by binding to rctA
Summary
Efficient linkage of chromosome replication and chromosome segregation is necessary for all dividing cells. It is important for maintaining balanced genetic content in organisms with more than a single chromosome, which includes a number of bacterial orders (e.g., Vibrionaceae, Photobacteriaceae [1]). For the gramnegative enteric pathogen Vibrio cholerae, whose genome is comprised of two circular chromosomes [2], distinct mechanisms that control the replication and segregation of each chromosome have been described, but no mechanisms for linking or coordinating these processes have been identified. The initiator of chromosome I (chrI) replication is DnaA, a conserved AAA+ ATPase protein found in most eubacteria [3,4,5,6]. It is likely that regulation of DnaA-mediated initiation of V. cholerae chrI parallels DnaA-dependent control of replication initiation in Escherichia coli [6,7]
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