Synchronous replication initiation of the two Vibrio cholerae chromosomes

The genome of Vibrio cholerae, the causative agent of cholera, is divided between two circular replicons. Genomic analysis has revealed that several other bacterial species have more than one chromosome. However, to date, the dynamics of chromosome replication in bacteria with more than one chromosome have not been investigated. As in V. cholerae chromosome II (1.07 Mb) is smaller than chromosome I (2.96 Mb) and contains fewer essential genes, it was proposed that it originated as a plasmid [1]. The replication of chromosome II shows both plasmid and chromosome-like features [2]. Whereas prokaryotic and eukaryotic chromosomes replicate once per cell cycle [3], plasmids replicate autonomously and independently of the host chromosome [4]. Here we report that replication of both V. cholerae chromosomes is initiated synchronously, once per cell cycle. To decipher the replication kinetics of the two V. cholerae chromosomes, we used MeselsonStahl density shift experiments [5]. With this method, nonsynchronized cells are initially grown in medium containing heavy isotopes and then switched to medium with light isotopes. As the cells undergo semi-conservative DNA replication, heavy (HH) DNA gives rise to heavy-light (HL) DNA. Light-light (LL) DNA appears only after the initiation of HL DNA replication. Therefore, the time until appearance of LL DNA defines the minimal inter-replication period: the time interval between successive initiation events from the same origin [6]. There has been some debate in the literature regarding the degree of autonomy of the replication of certain low-copy plasmids, particularly the F-plasmid [7–9]. However, in density shift experiments, the inter-replication periods of the Escherichia coli chromosome and those of resident low copy plasmids clearly differ, which reflects the disparate factors controlling replication of the chromosome and plasmids [10,11]. We grew V. cholerae in ‘heavy’ medium until early exponential phase, and then shifted the cells to ‘light’ medium. At various times after this switch, DNA was extracted, sheared, and separated according to density by centrifugation in CsCl gradients. Gradient fractions were hybridized with radioactive DNA probes specific for the ori region of chromosome I (chrI) or chromosome II (chrII) to determine the relative amount of DNA in each fraction. As expected, with increasing time after the switch from heavy to light medium, the DNA density became lower, transitioning from HH to HL to LL (Figure 1). The persistence of some HH DNA until 60 min after the switch, which is longer than the doubling time of the culture (see Supplemental data), suggests that some cells were slow to recover from the density shift. Strikingly, the time at which LL DNA was first apparent (∼40 min) was the same for both chromosomes, indicating that they have the same inter-replication period. Thus, although the replicons differ in size, the time between successive initiation events is the same for both origins. Furthermore, this time was equal to the doubling time of the cells (see Supplemental data, Figure S1), indicating that each