Potential confounding mutations in Keio knockout strains: implications for research accuracy

The Keio library of single-gene knock-out mutants of Escherichia coli is useful for the research community. It has been used to analyze the role of various E. coli genes in alcohol tolerance, multi-drug resistance, and biofilm formation. The current study provides a general overview of potential single nucleotide polymorphisms (SNPs), insertion-deletion of bases (≤50 nucleotides, INDELs) in the genome of a set of 21 knock-out mutants of the Keio collection in comparison to the parent strain. A small number of SNPs and INDELs were predicted in the coding and intergenic regions of the knock-out mutants in comparison to the parental strain through sequencing and bioinformatic analysis. Mutations in the coding regions of genes (different from the actual gene knocked out in the mutants) led to different types of mutations in the affected genes, ranging from nonsense mutations to frameshift mutations, which could affect the functionality of the resulting gene products. These mutations in the intergenic and coding regions could lead to phenotypic differences in the single-gene knock-out mutant strains in comparison to the parent strain, independent of the desired gene deletion. This, in turn, could be misinterpreted by researchers using these strains as differences caused by the missing gene. While this is a preliminary study based on only 21 strains of the Keio collection, the deleted genes in the mutants used in this study were approximately evenly distributed across the entire genome. This study likely indicates the possibility of such mutations in other Keio strains, although a larger sample size of knock-out mutants would be required to understand the likelihood of such mutations across the library.IMPORTANCEThe Keio library of single-gene knock-out mutants of Escherichia coli has been widely used for a variety of studies. However, mutations might appear in the genome of these strains over time, leading to differences in the characteristics of the mutant and parent strains that are independent of the gene deletions of interest. This study predicts the presence of a few SNPs and INDELs in some of the knock-out mutants from the Keio collection, which could potentially alter the phenotypic attributes of the knock-out mutants with no role of the deleted gene towards this change. Therefore, this study highlights the possibility of the presence of such mutations in other strains of the library and the importance of conducting additional steps, such as complementation assays, to confirm the outcomes of studies comparing specific attributes of the knock-out mutants with the parental strain.