Physical Mapping of Rhodobacter capsulatus: Cosmid Encyclopedia and High Resolution Genetic Map

Abstract

A combination of cosmid genome walking and pulse field gel electrophoresis was used to construct a high resolution physical and genetic map of the 3.8 Mb genome of Rhodobacter capsulatus SB1003. The mapping was done by grouping and further mapping of cosmids and bacteriophages from genomic libraries using PFGE-generated DNA fragments and SP6 and T7 specific transcripts corresponding to the ends of the cosmid inserts. Cosmid and phage clones formed two uninterrupted and ordered groups, one corresponding to the chromosome of Rb. capsulatus, the other to its 134 kb plasmid. Cos site end-labeling and partial EcoRV digestion of cosmids were used to construct a high resolution restriction map of the genome. Overlapping of the cosmids was confirmed by the resemblance of the cosmid restriction maps and by direct end-to-end hybridization. 34 genes or gene clusters were located in the ordered gene library and mapped with an accuracy of 1–10 kb. Three Rb. capsulatus strains; KB-1, St. Louis and 2.3.1., were chosen out of 14 others for a detailed comparison of their physical maps, which were partially constructed using the minimal cosmid set of Rb. capsulatus SB1003 as a source of ordering probes. Blots of the minimal set of 192 cosmids, covering the chromosome and the plasmid, with known map position of each cosmid, gives to Rb. capsulatus the same advantages that the Kohara phage panel gives to E. coli. Blots of this minimal cosmid set digested with EcoRV represent the entire genome split into gene-size pieces, and provide an opportunity for direct high resolution mapping of genes and transcripts.