Genetic variability of Yersinia pestis isolates as predicted by PCR-based IS100 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. In contrast, strains belonging to the glycerol-positive biovar antiqua showed a variety of fingerprinting profiles. Moreover, strains of the biovar medievalis (also glycerol positive) clustered together with the antiqua isolates originated from Southeast Asia, suggesting their close phylogenetic relationships. Interestingly, a Manchurian biovar antiqua strain Nicholisk 51 displayed a genotyping pattern typical of biovar orientalis isolates. Analysis of the glycerol pathway in Y. pestis suggested that a 93-bp deletion within the glpD gene encoding aerobic glycerol-3-phosphate dehydrogenase might account for the glycerol-negative phenotype of the orientalis biovar. The glpD gene of strain Nicholisk 51 did not possess this deletion, although it contained two nucleotide substitutions characteristic of the glpD version found exclusively in biovar orientalis strains. To account for this close relationship between biovar orientalis strains and the antiqua Nicholisk 51 isolate, we postulate that the latter represents a variant of this biovar with restored ability to ferment glycerol. The fact that such a genetic lesion might be repaired as part of the natural evolutionary process suggests the existence of genetic exchange between different Yersinia strains in nature. The relevance of this observation on the emergence of epidemic Y. pestis strains is discussed.

FIG. 1.

Digitized IS100-based fingerprints and dendrogram constructed by the UPGMA clustering method. Similarity analysis was performed using the Dice coefficient. The scale bar indicates dissimilarity proportions. The numbers 1 to 27 were assigned to each locus-specific primer vlm according to the size of the PCR fragment formed, using Y. pestis strain CO92 DNA as a template. The positions of the shifted fragments were followed in other strains. The IS100 genotypes of different groups shown are as follows: O1, O2a, O2b, and O2c are biovar orientalis; M1a, M1b, and M2 are biovar medievalis; A1a, A1b, A2, A3, and A4 are biovar antiqua; P1, and P2 are strains of the Pestoides group; and S1 and S2 are strains of Y. pseudotuberculosis.

FIG. 2.

Genes of glycerol metabolism that are defective in Y. pestis strain CO92 in comparison with strain KIM. The imperfect repeats that could account for a deletion are bold and underlined; lowercase letters represent mismatches within a repeat. (A) Cluster of genes, including the following: glpF, glycerol uptake facilitator; glpK, glycerol kinase; glpX, unknown function in glycerol metabolism. (B) glpD gene for glycerol-3-phosphate dehydrogenase.

FIG. 3.

Alignment of the partial sequences of the glpD gene of Y. pestis strains KIM, Nicholisk 51, and CO92. The consensus sequence is shown, and the asterisks represent differences. The imperfect repeats are designated in the same way as for Fig. 2.