Physical map of the genome of Vibrio cholerae 569B and localization of genetic markers

Abstract

A combined physical and genetic map of the genome of the classical O1 hypertoxinogenic strain 569B of Vibrio cholerae has been constructed. The enzymes NotI, SfiI and CeuI generated DNA fragments of suitable size distribution that could be resolved by pulsed-field gel electrophoresis. The digests produced 37, 22, and 7 fragments, respectively. The CeuI maps of the genomes of strains 569B and O395, constructed by partial restriction digestion, were identical, and the data are consistent with the concept of circular chromosomes. The genome size of each of the strains was estimated to be about 3.2 Mb. The NotI and SfiI digestion profiles of the genomic DNAs of strains 569B and O395 exhibited distinct restriction fragment length polymorphism. The linkages between the 37 NotI fragments of the genome of strain 569B were determined by combining three approaches: isolation of linking clones, analysis of partial digestion fragments, and identification of NotI fragments in isolated CeuI and SfiI fragments. To align linked fragments precisely, NotI-digested genomic DNA was end labeled and separated in the same gel with the NotI-digested DNA to be probed with linking clones. This also allowed the identification of smaller restriction fragments that are not visible in ethidium bromide- stained gels. The presence of repetitive DNA sequences in the V. cholerae 569B genome has been demonstrated. Twenty cloned homologous and heterologous genes and seven rrn operons have been positioned on the physical map. The two copies of the Ctx genetic element in the genome of strain 569B are located about 1,000 kb apart.