A Vibrio cholerae deletion mutant lacking VS2773, a parA partitioning gene homolog located in a parAB operon on the large chromosome, displays altered positioning of the large chromosome origin. Deletion of a second parA homolog on the large chromosome (VC2061) does not affect its origin positioning. The origin position of the small chromosome is unchanged by either or both of these deletions, suggesting that VC2773 function is specific to the replicon on which it is carried. VC2773 and VC2772 form a parABS system with inverted repeats found near the large chromosome origin.In recent years, it has become possible to study the internal organization of the bacterial cell in great detail. DNA positioning and movement have been studied in various bacteria, using both live-and fixed-cell models. While it is clear that the chromosome is highly organized within the cell, the mechanisms underlying these processes are still not fully understood (18). Studies of bacteria with single chromosomes, such as Escherichia coli, Bacillus subtilis, and Caulobacter crescentus, have revealed variations in origin positioning and the timing of this placement (13,24,29). In the multireplicon-containing alpha-proteobacteria Agrobacterium tumefaciens and Sinorhizobium meliloti, origins localize predominantly to the cell poles, although they remain nonoverlapping (15). Fogel and Waldor have studied origin positioning in the bipartite genome of Vibrio cholerae, using a live-cell model and a fluorescent repressor-operator system (6). Their findings of distinct positioning patterns and cell cycle timing of movement for the two origins were independently confirmed by Fiebig et al. (5a) and suggest that a separate segregation mechanism may exist for each chromosome.Early studies of bacterial DNA partitioning focused on the maintenance of low-copy-number plasmids, such as F factor, P1, and R1. The genetic loci responsible for plasmid partitioning encode members of the ParA and ParB protein families. Homologs of these genes have been discovered on many bacterial chromosomes, with the exception of E. coli and related enteric organisms, and work in model systems such as B. subtilis, C. crescentus, Streptomyces coelicolor, and Pseudomonas putida has demonstrated a role for ParA and ParB in chromosome segregation. However, the segregation defects in strains with mutants of these genes have often been mild, with stronger phenotypes manifested under specific growth conditions or during developmental processes such as sporulation (9,12,16,19,23).The Vibrio species studied to date all have a divided genome consisting of two chromosomes; each chromosome carries a parAB locus (1,10,21,26). The locus on the large chromosome is related more closely to other bacterial chromosomal par loci than that on the small chromosome, which bears homology to plasmid-carried loci (10). Given the distinct localization patterns of the replication origins in V. cholerae, the parAB loci are factors that may confer specificity in positioning. Vibrio species also have a secon...

Although most bacteria contain a single circular chromosome, some have complex genomes, and all Vibrio species studied so far contain both a large and a small chromosome. In recent years, the divided genome of Vibrio cholerae has proven to be an interesting model system with both parallels to and novel features compared with the genome of Escherichia 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 in E. 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 of seqA leads to asynchronous replication. In our study, hemimethylated DNA was detected at both origins of V. 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 in V. cholerae and prevents correct progression to downstream cell cycle events such as segregation and cell division.DNA replication in bacteria is a highly complex process, requiring controlled initiation in a manner allowing progeny cells to inherit a normal genetic complement. Multiple factors influencing this process have been described for model bacteria with single chromosomes, such as Escherichia coli. A variety of bacteria from different subgroups have been found to have multipartite genomes, but it is unclear how correct replicon stoichiometry is maintained or may be modulated. All Vibrio species characterized thus far have divided genomes consisting of one large and one small chromosome, and in the last few years, Vibrio cholerae has become a primary model system for studying the replication of a complex bacterial genome.Although the timing of the replication of the V. cholerae chromosomes remains under investigation, a number of factors are known to be important for replication itself. DnaA and Dam methylation are both required for the replication of V. cholerae origin minichromosomes in an E. coli host (12). The novel factors RctA and RctB, an RNA and a protein, are involved in the replication of the small V. cholerae chromosome (12, 41). In the initial characterization of the V. cholerae replication origins, SeqA was also implicated as a required factor for the replication of its large chromosome origin in the E. coli host. Although the SeqA gene can be deleted in E. coli, a null muta...

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