The development of a partial physical map of Bacillus megaterium

Abstract

The techniques that would lead to a physical restriction map of B. megaterium were investigated. Methods of isolating whole chromosomal DNA were determined and optimized, and parameters for separation of large DNA molecules using Transverse Alternating Field Electrophoresis were established. Several rare cutting enzymes were tested on intact B. megaterium genomic DNA. Only two were found that could be used for physical mapping; the enzymes NotI and SfiI yielded 25 fragments and 7-8 fragments, respectively. Several new Tn917 and Tn917- 1acZ-cat insertional mutants were also isolated, including a unique tyr mutant. Using a Tn917 probe to hybridize with the DNA from the insertional mutants digested with NotI and SfiI, a partial map was formed. Six previously unmapped genetic loci were mapped within at least 0.1-5% of their actual physical distance on the chromosome. Additional mapping strategies were found to be unsuccessful, including partial digestions, double digestions, and hybridization of fragments from one enzyme digestion with probe made from another. An unsuccessful search for linker probes in a genomic library was also made. Nevertheless, significant progress has been made towards physically mapping B. megaterium.