3 IS-RFLP: a powerful tool for geographical clustering of global isolates of Yersinia pestis.

Multiple copies of several classes of insertion sequences (IS) are found in the genome of Yersinia pestis, the causative agent of bubonic and pneumonic plague. We used the genetic instability generated by these IS to develop a method (designated 3IS-RFLP) based on the restriction fragment length polymorphism of the IS100, IS285 and IS1541 elements for studying Y. pestis strains of worldwide origin. We show that 31S-RFLP is a powerful tool to group Y. pestis isolates according to their geographical origin, and therefore that this method may be valuable for investigating the origin of new or re-emerging plague foci or for addressing forensic issues.

The highly similar TMP kinases of Yersinia pestis and Escherichia coli differ markedly in their AZTMP phosphorylating activity.

Thymidine monophosphate (TMP) kinases are key enzymes in nucleotide synthesis for all living organisms. Although eukaryotic and viral TMP kinases have been studied extensively, little is known about their bacterial counterparts. To characterize the TMP kinase of Yersinia pestis, a chromosomal region encompassing its gene (tmk) was cloned and sequenced; a high degree of conservation with the corresponding region of Escherichia coli was found. The Y. pestis tmk gene was overexpressed in E. coli, where the enzyme represented over 20% of total soluble proteins. The CD spectrum of the purified TMP kinase from Y. pestis was characteristic for proteins rich in alpha-helical structures. Its thermodynamic stability was significantly lower than that of E. coli TMP kinase. However, the most striking difference between the two enzymes was related to their ability to phosphorylate 3'-deoxy-3'-azidothymidine monophosphate (AZTMP). Although the enzymes of both species had comparable Km values for this analogue, they differed significantly in their Vmax for AZTMP. Whereas E. coli used AZTMP as a relatively good substrate, the Y. pestis enzyme had a Vmax 100 times lower with AZTMP than with TMP. This fact explains why AZT, a potent bactericidal agent against E. coli, is only moderately active on Y. enterocolitica. Sequence comparisons between E. coli and Y. pestis TMP kinases along with the three-dimensional structure of the E. coli enzyme suggest that segments lying outside the main regions involved in nucleotide binding and catalysis are responsible for the different rates of AZTMP phosphorylation.

Insights into the evolution of Yersinia pestis through whole-genome comparison with Yersinia pseudotuberculosis.

Yersinia pestis, the causative agent of plague, is a highly uniform clone that diverged recently from the enteric pathogen Yersinia pseudotuberculosis. Despite their close genetic relationship, they differ radically in their pathogenicity and transmission. Here, we report the complete genomic sequence of Y. pseudotuberculosis IP32953 and its use for detailed genome comparisons with available Y. pestis sequences. Analyses of identified differences across a panel of Yersinia isolates from around the world reveal 32 Y. pestis chromosomal genes that, together with the two Y. pestis-specific plasmids, to our knowledge, represent the only new genetic material in Y. pestis acquired since the the divergence from Y. pseudotuberculosis. In contrast, 149 other pseudogenes (doubling the previous estimate) and 317 genes absent from Y. pestis were detected, indicating that as many as 13% of Y. pseudotuberculosis genes no longer function in Y. pestis. Extensive insertion sequence-mediated genome rearrangements and reductive evolution through massive gene loss, resulting in elimination and modification of preexisting gene expression pathways, appear to be more important than acquisition of genes in the evolution of Y. pestis. These results provide a sobering example of how a highly virulent epidemic clone can suddenly emerge from a less virulent, closely related progenitor.