Characterization of Chromosomal Regions Conserved in Yersinia pseudotuberculosis and Lost by Yersinia pestis

Abstract

The transformation of the enteropathogenic bacterium Yersinia pseudotuberculosis into the plague bacillus, Yersinia pestis, has been accompanied by extensive genetic loss. This study focused on chromosomal regions conserved in Y. pseudotuberculosis and lost during its transformation into Y. pestis. An extensive PCR screening of 78 strains of the two species identified five regions (R1 to R5) and four open reading frames (ORFs; orf1 to orf4) that were conserved in Y. pseudotuberculosis and absent from Y. pestis. Their conservation in Y. pseudotuberculosis suggests a positive selective pressure and a role during the life cycle of this species. Attempts to delete two ORFs (orf3 and orf4) from the chromosome of strain IP32953 were unsuccessful, indicating that they are essential for its viability. The seven remaining loci were individually deleted from the IP32953 chromosome, and the ability of each mutant to grow in vitro and to kill mice upon intragastric infection was evaluated. Four loci (orf1, R2, R4, and R5) were not required for optimal growth or virulence of Y. pseudotuberculosis. In contrast, orf2, encoding a putative pseudouridylate synthase involved in RNA stability, was necessary for the optimal growth of IP32953 at 37°C in a chemically defined medium (M63S). Deletion of R1, a region predicted to encode the methionine salvage pathway, altered the mutant pathogenicity, suggesting that the availability of free methionine is severely restricted in vivo. R3, a region composed mostly of genes of unknown functions, was necessary for both optimal growth of Y. pseudotuberculosis at 37°C in M63S and for virulence. Therefore, despite their loss in Y. pestis, five of the nine Y. pseudotuberculosis-specific chromosomal loci studied play a role in the survival, growth, or virulence of this species.

Delineation and Analysis of Chromosomal Regions Specifying Yersinia pestis

“…No growth defects were observed in the various mutants, compared to the level for the CO92p parental strain (data not shown). Similar results were previously obtained with the various Y. pseudotuberculosis strains deleted of each specific region (44). However, when these Y. pseudotuberculosis deletants were grown in the biochemically defined medium M63S, a growth defect could be observed for several mutants.…”

“…Remarkably, R1 appears to have been exchanged with a Y. pseudotuberculosisspecific region (YPTB0535 to -0537) coding also for a restriction/modification system. This region was shown to play a major role in Y. pseudotuberculosis pathogenicity (44).…”

“…Since the chromosome of Y. pestis is prone to frequent rearrangements (23), and since we found that the presence of pKOBEG-sacB had no significant impact on Y. pestis pathogenicity, we decided not to remove this plasmid in the deletion mutants, in order to limit as much as possible subcultures which would increase the occurrence of genomic reassortments. During our previous analysis of Y. pseudotuberculosis-specific regions, we found that two CDSs (YPTB1495 and YPTB3368) were not deletable, because they are essential for IP32953 survival (44). This was not the case for the Y. pestis-specific loci, as the eight specific regions were successfully deleted.…”

“…Surprisingly, it appears that the transformation of the enteropathogen Y. pseudotuberculosis into the plague bacillus has mostly been characterized by extensive loss of genetic material and functions (8). Ten chromosomal loci conserved in the species Y. pseudotuberculosis have been lost by Y. pestis during the course of its differentiation (43,44), and 208 genes present in the two species have been inactivated during the transformation process (8). The natural inactivation of one of these genes has recently been shown to have participated to the change in Y. pestis mode of transmission (19,57).…”

Molecular Darwinian Evolution of Virulence in Yersinia pestis

“…ORF3 and ORF4, with unknown function, are essential for the viability of Y. pseudotuberculosis, while ORF2 (a putative pseudouridylate synthase involved in RNA stability) and R3 (a genomic region composed mostly of genes of unknown functions) are necessary for its optimal growth in a chemically defined medium (51). Deletion of R1 (a genomic region responsible for the methionine salvage pathway) alters the mutant's virulence, suggesting that the availability of free methionine is severely restricted in vivo (51).…”