Unexpected results from the application of signature-tagged mutagenesis to identify Yersinia pestis genes required for adherence and invasion

The signature-tagged mutagenesis (STM) method was applied in a protocol designed to identify genes required for Yersinia pestis invasion into epithelial cells. A library of 3060 mutants of Y. pestis CO99-3015 was made and assayed using an in vitro invasion assay with gentamicin protection. Initial results from the screen identified a set of 23 genes that might be required for invasion; however, screening of individual mutants for decreased invasion, even in a competition assay with the parent strain, failed to reveal obvious invasion defects. Altered colony character or size might have imposed a growth disadvantage for two of the mutants, which could possibly account for apparently decreased invasion. The sensitivity of the mutants to gentamicin was assayed to determine if the presence of the kanamycin-resistance cassette in the STM transposon changed the gentamicin resistance of the individual mutants. It was discovered that the mutants exhibited a variable range of resistance to killing by gentamicin, suggesting that the presence of the kanamycin-resistance cassette or the particular insertion mutation did in many cases affect the bactericidal potency of gentamicin. However, all mutants remained highly sensitive to growth inhibition in a disk assay on plates. These results may warrant precautions for use of kanamycin-resistance markers in studies with fully virulent Y. pestis, since gentamicin has been recommended for treatment of plague. Further, to use STM in the context of invasion assays, a selection other than gentamicin should be applied.