Predicted and Unsuspected Alterations of the Genomes Structure of Genetically DefinedBacillus subtilis168 Strains

Abstract

We scrutinized the genome structures of Bacillus subtilis 168 derivative strains the isolation procedure of which was well described. NotI and SfiI restriction fragment analyses indicated that strains constructed by transformation using non-168 Bacillus strains as donors had replacement of DNA segments in the predicted region. Interesting examples were found in a lineage including strains 168trpC2-YSl 1-RM125-MI112, the last two of which have a restriction-deficiency mutation (hsdM) introduced from a non-168 strain DNA. As expected, the loss of the hsdM gene was a result of replacement of the hsdM region by non-168 DNA fragments. Moreover, RM125 acquired a previously unnoticed NotI site that gave no obvious phenotypes. Among 168 derivative strains investigated, the most extreme case had changes at six different regions of the genome, probably introduced by repeated transformations. Unsuspected DNA alterations were found even in isogenic strains constructed by phage PBS1-mediated transduction. These acquired structural variations of the B. subtilis genome were distinct from the spontaneous DNA deletions previously characterized [M. Itaya, J. Bacteriol., 175, 741–749 (1993)]. Caution should therefore be taken for nominally isogenic strains that are thought to have identical genome structure except for the gene(s) of interest. We propose the term isogenomic strains for strains that shows the same macrorestriction fragment pattern.