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Infection and Immunity, December 2001, p. 7810-7819, Vol. 69, No. 12
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.12.7810-7819.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Application of High-Density Array-Based Signature-Tagged Mutagenesis To Discover Novel Yersinia Virulence-Associated Genes

A. V. Karlyshev,1 P. C. F. Oyston,2 K. Williams,1 G. C. Clark,2 R. W. Titball,2 E. A. Winzeler,3,dagger and B. W. Wren1,*

Department of Infectious Diseases, London School of Hygiene and Tropical Medicine, University of London, London WC1E 7HT,1 and Defense Science and Technology Laboratory, CBS Porton Down, Salisbury, Wiltshire SP4 OJQ,2 United Kingdom, and Department of Biochemistry, Stanford University School of Medicine, Stanford, California 943305-53073

Received 14 June 2001/Returned for modification 24 July 2001/Accepted 8 August 2001

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.

* Corresponding author. Mailing address: Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, University of London, Keppel Street, London WC1E 7HT, United Kingdom. Phone: 44 (0) 207 927 2288. Fax: 44 207 637 4316. E-mail: brendan.wren{at}lshtm.ac.uk.

dagger Present address: Novartis Institute for Functional Genomics, San Diego, CA 92121-1125.

Infection and Immunity, December 2001, p. 7810-7819, Vol. 69, No. 12
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.12.7810-7819.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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