Independent control of replication initiation of the two Vibrio cholerae chromosomes by DnaA and RctB

Abstract

Although the two Vibrio cholerae chromosomes initiate replication in a coordinated fashion, we show here that each chromosome appears to have a specific replication initiator. DnaA overproduction promoted overinitiation of chromosome I and not chromosome II. In contrast, overproduction of RctB, a protein that binds to the origin of replication of chromosome II, promoted overinitiation of chromosome II and not chromosome I.

FIG. 1.

Distribution of the number of origins of the two V. cholerae chromosomes, established in four or more independent experiments. Only cells with one or more foci were analyzed. The number of cells used to determine each distribution is shown in the upper right in each panel. Fluorescence microscopy was carried out as described previously (7). Expression of TetR-YFP was induced with 0.04% arabinose, and expression of RctB and DnaA was induced with 100 μM IPTG.

FIG. 2.

Overproduction of DnaA stimulates chromosome I replication. IPTG (100 μM) was used to induce DnaA synthesis from pMR10 (12) in Bah-2 (22) grown at 37°C in minimal medium containing glycerol. In panel A, samples were taken every 5 min for Q-PCR with the following primers: 5′-CGCCAACCGAGTTTGGATTC-3′ and 5′-GAAAAAGCGCGTGAGCTTGG-3′ for oriCI_Vc, 5′-CTGAGGCGGATTTGGCACTC-3′ and 5′-GCTTGCGCCGCTTTTAACTG-3′ for terI_Vc, 5′-GCTCCACCTTCGGTGTTTCG-3′ and 5′-TGGTTTCGTGTGGCAGCAAT-3′ for oriCII_Vc, and 5′-TATCCGCACAGCCTCAGCAA-3′ and 5′-CACGCAAACAGACCGACACC-3′ for terII_Vc. Values were normalized to DNA extracted from a sample incubated with rifampin. Triangles, oriCI_Vc relative to terI_Vc; squares, oriCII_Vc relative to terI_Vc; circles, terII_Vc relative to terI_Vc. In panel B, IPTG was added at an OD₄₅₀ of 0.15, and at the indicated times samples were taken for a 4-h incubation with rifampin and cephalexin. Cells were fixed in ethanol and stained with mithramycin and ethidium bromide for flow cytometry as described previously (15).

FIG. 3.

Overproduction of RctB stimulates chromosome II replication. Bah-2 was grown as indicated in the legend to Fig. 2, except that RctB synthesis from pKGX was induced with 0.2% l-arabinose. At the indicated times, samples were incubated with rifampin and cephalexin prior to flow cytometric analysis as described in the legend to Fig. 2. Arrows indicate the additional DNA synthesized as a result of RctB overproduction.