Application of high-density array-based signature-tagged mutagenesis to discover novel Yersinia virulence-associated genes

Yersinia pestis, the causative agent of plague, and the enteropathogen Yers inia pseudotuberculosis have nearly identical nucleotide similarity yet cau se markedly different diseases. To investigate this conundrum and to study Yersinia pathogenicity, we developed a high-density oligonucleotide array-b ased modification of signature-tagged mutagenesis (STM). Y. pseudotuberculo sis YPIII mutants constructed with the tagged transposons were evaluated in the murine yersiniosis infection model. The DNA tags were amplified using biotinylated primers and hybridized to high-density oligonucleotide arrays containing DNA complementary to the tags. Comparison of the hybridization s ignals from input pools and output pools identified a mutant whose relative abundance was significantly reduced in the output pool. Sequence data from 31 transposon insertion regions was compared to the complete Y. pestis CO9 2 genome sequence. The 26 genes present in both species were found to be al most identical, but five Y. pseudotuberculosis genes identified through STM did not have counterparts in the Y. pestis genome and may contribute to th e different tropisms in these closely related pathogens. Potential virulenc e genes identified include those involved in lipopolysaccharide biosynthesi s, adhesion, phospholipase activity, iron assimilation, and gene regulation . The phospholipase A (PldA) mutant exhibited reduced phospholipase activit y compared to the wild-type strain and in vivo attenuation of the mutant wa s confirmed. The combination of optimized double tag sequences and high-den sity array hybridization technology offers improved performance, efficiency , and reliability over classical STM and permits quantitative analysis of d ata.