Incidental mutations occur frequently during transposon mutagenesis.

Transposon mutagenesis and subsequent phenotype-driven screening have been extensively used to annotate gene function and uncover the mechanisms of biological phenomena. During this process, the resulting phenotypic differences are specifically attributable to the disrupted genes. In this study, the complementation of the mini-Tn10 transposon-disrupted gene gntP did not restore the germination ability of the Bacillus thuringiensis MT1518-1 mutant to the wild-type level. Genome sequencing identified 186 unlinked mutations on the chromosome of the mutant MT1518-1, including one frameshift mutation in the germination-related gene exsA. We randomly selected 19 mini-Tn10 mutants for high throughput genome sequencing. The sequencing data revealed that incidental mutations occurred frequently on their chromosomes, including 2343 single-nucleotide polymorphisms, three insertions and one deletion. We also found that stressful conditions are the underlying cause for the appearance of incidental mutations. Caution is warranted when attributing the observed phenotypic changes to the transposon-disrupted genes.