Av. Karlyshev et al., Application of high-density array-based signature-tagged mutagenesis to discover novel Yersinia virulence-associated genes, INFEC IMMUN, 69(12), 2001, pp. 7810-7819
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.