Integrated Physical and Genetic Mapping of Bacillus cereus and Other Gram-Positive Bacteria Based on IS 231 A Transposition Vectors

The genome structure of Bacillus cereus is relatively complex, its DNA being modulated between a size-varying chromosome and large plasmids. To study the genetic organization of the B. cereus type strain ATCC 14579, thermosensitive transposition vectors were designed on the basis of IS 231 A-derived cassettes containing uncommon restriction sites. A highly preferred insertion site for IS 231 A was detected in the chromosome by Southern blotting and pulsed-field gel electrophoresis (PFGE) analyses of independent insertion mutants. However, once this insertional hot spot was occupied, secondary IS 231 A insertions occurred randomly, as demonstrated by isolation of independent B. cereus auxotrophs at a frequency of approximately 0.6%. The hot-spot site, as well as several auxotrophic mutations, were mapped by using Not I, Sfi I, and Asc I PFGE restriction profiles. It was confirmed by sequencing that one of the insertions, generating an Ade − phenotype, had disrupted a gene of the purine synthesis pathway. These results showed that combined PFGE and sequencing analyses of mini-IS 231 A insertions enable the construction of integrated physical and genetic maps of B. cereus type strain. Moreover, the presence of the ultrarare I- Sce I restriction site in the mini-IS 231 A allowed the isolation, in double-insertion mutants, of contiguous and nonoverlapping large chromosomal fragments, convenient for direct sequencing. The system detailed in this report is therefore a powerful tool for comparative genetic studies among members of the B. cereus group (i.e., B. cereus , B. thuringiensis , B. mycoides , and B. anthracis ) and could also be applied to more distantly related gram-positive bacteria.