title: Application of high-density array-based signature-tagged mutagenesis to discover novel Yersinia virulence-associated genes.
abstract: Yersinia pestis, the causative agent of plague, and the enteropathogen Yersinia pseudotuberculosis have nearly identical nucleotide similarity yet cause markedly different diseases. To investigate this conundrum and to study Yersinia pathogenicity, we developed a high-density oligonucleotide array-based modification of signature-tagged mutagenesis (STM). Y. pseudotuberculosis 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 signals 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 CO92 genome sequence. The 26 genes present in both species were found to be almost identical, but five Y. pseudotuberculosis genes identified through STM did not have counterparts in the Y. pestis genome and may contribute to the different tropisms in these closely related pathogens. Potential virulence genes identified include those involved in lipopolysaccharide biosynthesis, adhesion, phospholipase activity, iron assimilation, and gene regulation. The phospholipase A (PldA) mutant exhibited reduced phospholipase activity compared to the wild-type strain and in vivo attenuation of the mutant was confirmed. The combination of optimized double tag sequences and high-density array hybridization technology offers improved performance, efficiency, and reliability over classical STM and permits quantitative analysis of data.
authors.affiliation: Department of Infectious Diseases, London School of Hygiene and Tropical Medicine, University of London, London WC1E 7HT, United Kingdom.
authors.forename: A V
authors.initials: AV
authors.lastname: Karlyshev
authors.forename: P C
authors.initials: PC
authors.lastname: Oyston
authors.forename: K
authors.initials: K
authors.lastname: Williams
authors.forename: G C
authors.initials: GC
authors.lastname: Clark
authors.forename: R W
authors.initials: RW
authors.lastname: Titball
authors.forename: E A
authors.initials: EA
authors.lastname: Winzeler
authors.forename: B W
authors.initials: BW
authors.lastname: Wren
mesh_terms: D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D004251:DNA Transposable Elements; D005798:Genes, Bacterial; D007928:Lethal Dose 50; D008070:Lipopolysaccharides / Q000096:biosynthesis; D051379:Mice; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D010741:Phospholipases A / Q000235:genetics / Q000378:metabolism; D054466:Phospholipases A1; D014774:Virulence / Q000235:genetics; D015011:Yersinia pseudotuberculosis / Q000235:genetics* / Q000472:pathogenicity*; D015012:Yersinia pseudotuberculosis Infections
chemical_list: D001425:Bacterial Outer Membrane Proteins; D004251:DNA Transposable Elements; D008070:Lipopolysaccharides; D010741:Phospholipases A; D054466:Phospholipases A1; C412876:outer membrane phospholipase A