The Vibrio cholerae genome contains two unique circular chromosomes

Abstract

Vibrio cholerae, the etiologic agent of the diarrheal disease cholera, is a Gram-negative bacterium that belongs to the gamma subdivision of the family Proteobacteriaceae. The physical map of the genome has been reported, and the genome has been described as a single 3.2-Mb chromosome [Majumder, R., et al. (1996) J. Bacteriol. 178, 1105-1112]. By using pulsed-field gel electrophoresis of genomic DNA immobilized in agarose plugs and digested with the restriction enzymes I-CeuI, SfiI, and NotI, we have also constructed the physical map of V. cholerae. Our analysis estimates the size of the genome at 4.0 Mb, 25% larger than the physical map reported by others. Our most notable finding is, however, that the V. cholerae chromosome appears to be not the single chromosome reported but two unique and separate circular megareplicons.

Figure 1

PFGE of SfiI-digested V. cholerae DNA. λ concatamers (lane 1), O139 V. cholerae strain AI1837 (lane 2), El Tor V. cholerae strain E7946 (lane 3), and classical V. cholerae strain 395 (lane 4). The V. cholerae strain 395 fragments are identified to the right of lane 4. Pulse ramps were 13 to 28 sec for 60 hr at 180 V in 1.8% agarose.

Figure 2

PFGE of I-CeuI-digested V. cholerae DNA. Digested fragments were separated in different agarose concentrations to permit separation of both large and small fragments. (A) O139 V. cholerae strain AI1837 (lane 1), El Tor V. cholerae strain E7946 (lane 2), classical V. cholerae strain 395 (lane 3), and yeast-chromosome pulsed-field gel marker (lane 4). The first three bands are identified to the left of lane 1. Pulse ramps were 60 to 90 sec for 24 hr at 200 V in 1.0% agarose. Band 2 is circular, and so the molecular weight cannot be determined by comparison with linear molecular-weight markers. (B) O139 V. cholerae strain AI1837 (lane 1), El Tor V. cholerae strain E7946 (lane 2), and classical V. cholerae strain 395 (lane 3). Identification of the migration of λ concatamers is shown to the left of lane 1. Fragments 3 to 10 are identified on the right of lane 3. Pulse ramps were 13 to 28 sec for 60 hr at 180 V in 1.8% agarose.

Figure 3

PFGE of undigested versus I-CeuI-digested V. cholerae DNA. O139 V. cholerae strain AI1837 (lane 1) and classical V. cholerae strain 395 (lane 2). Lanes marked A are undigested and lanes marked B are I-CeuI-digested genomic DNA. Yeast-chromosome molecular-weight markers are in the far right lane. Pulse ramps were 60 to 90 sec for 60 hr at 200 V in 1.3% agarose.

Figure 4

The physical map of the two replicons of classical V. cholerae strain 395. The circles represent the I-CeuI (Inner) and SfiI (Outer) maps. The order of genes within each SfiI fragment are arbitrary. (A) Replicon I. (B) Replicon II.