Genetic Variability of Yersinia pestis Isolates as Predicted by PCR-Based IS 100 Genotyping and Analysis of Structural Genes Encoding Glycerol-3-Phosphate Dehydrogenase ( glpD )

Abstract

A PCR-based genotyping system that detects divergence of IS100 locations within the Yersinia pestis genome was used to characterize a large collection of isolates of different biovars and geographical origins. Using sequences derived from the glycerol-negative biovar orientalis strain CO92, a set of 27 locus-specific primers was designed to amplify fragments between the end of IS100 and its neighboring gene. Geographically diverse members of the orientalis biovar formed a homogeneous group with identical genotype with the exception of strains isolated in Indochina. Yersinia pestis, the causative agent of bubonic plague, is a recently evolved clone of Yersinia pseudotuberculosis serotype O:1b (1, 29). Strains of Y. pestis are divided into three biovars on the basis of their abilities to ferment glycerol and to reduce nitrate. These phenotypic differences have proven useful in distinguishing strains thought to be responsible for the three plague pandemics (7). Isolates of the biovar antiqua (able to ferment glycerol and reduce nitrate) are believed to remain as holdovers from the first pandemic that started with the Justinian plague of the 6th century. Strains of the biovar medievalis (glycerol positive and nitrate negative) evidently caused the second pandemic of Europe (Black Death), which was initiated during the 14th century, and strains of the biovar orientalis (glycerol negative and nitrate positive) are responsible for the third plague pandemic of modern times (15,22).Several molecular methods that generate fingerprinting patterns of Y. pestis DNA have been successfully used for genotyping strains of this microorganism, such as pulse-field gel electrophoresis and ribotyping (15,16,18,25) as well as newly described variable-number tandem repeat analysis (2). Genotyping of Y. pestis was also accomplished by restriction fragment length polymorphism (RFLP) detected on Southern blots using probes originating from IS100, which is found at least singly on each of the three plasmids of the species and as multiple copies within the chromosome (24). This insertion sequence (IS) element was sequenced (11, 23) and then used by Filippov et al. (9) in conjunction with the novel IS285 to obtain fingerprint patterns establishing phylogenetic relationships. Further IS100-and IS285-based RFLP analysis became the method of choice used by many researchers for extensive analysis of Y. pestis collections (1,5,14,20). Finally, a third insertion sequence (IS1541) was discovered that disrupts inv, which encodes invasin (28). This element is now known to be present in many copies in the Y. pestis genome and has also been used to obtain RFLP fingerprinting profiles (21, 28).Many insertions of IS1541 in the genome of Y. pestis strain 6/69 M biovar orientalis were characterized and then tested to see if the insert flanked the same genes in other strains of Y. pestis (21). The authors employed five unrelated strains (four of biovar orientalis and one of medievalis) of different ribotypes and IS1541 hybridization patterns that were isol...

Genomic Signature Tags (GSTs): A System for Profiling Genomic DNA

“…However, one tag that is predicted to be present four times in the genome seems to be under represented in our database. This tag is associated with an IS100 element that is known to be a source for genetic variability in different Y. pestis isolates (Motin et al 2002), which may in part explain our results. The two plasmids, pMT1 and pPCP1, thought to be present in the EV766 genome, each contain a single BamHI site, and each should have contributed two unique tags to our library.…”

Genome plasticity in Yersinia pestis The GenBank accession numbers for the sequences reported in this paper can be found in Table 1 T1 ; the GenBank accession number for DFR4 is AF426171.

“…DFR4 is also deleted from Nicholisk 51, an antiqua strain, which has recently been shown to have an IS100-based fingerprint more typical of the orientalis biovar (Motin et al, 2002). This observation led to the hypothesis that this antiqua strain has restored ability to ferment glycerol.…”

“…DFR5 is found in only one strain of the antiqua biovar : Y. pestis Nicholisk 51 (strain 65, Table 2). Interestingly, this strain represents a variant of the antiqua biovar with restored ability to ferment glycerol (Motin et al, 2002). The PCR data from the Y. pestis collection were correlated as profiles designated A-M (Table 2).…”

Application of DNA Microarrays to Study the Evolutionary Genomics of Yersinia pestis and Yersinia pseudotuberculosis

“…The initial ribotyping studies, in conjunction with data on the geographical sources of the strains, were used to correlate the different biovars with the three plague pandemics (Guiyoule et al 1994). This has been further validated by restriction fragment length polymorphism (RFLP) analysis of IS100 element insertions from strains of the three biovars (Achtman et al 1999) and PCR-based IS100 genotyping by use of the Y. pestis CO-92 genome sequence as a reference (Motin et al 2002). Y. pseudotuberculosis strains are grouped in 21 serotypes, on the basis of differences in their lipopolysaccharide (LPS) content.…”