Genome sequence of the deep-rooted Yersinia pestis strain Angola reveals new insights into the evolution and pangenome of the plague bacterium

Abstract

To gain insights into the origin and genome evolution of the plague bacterium Yersinia pestis, we have sequenced the deep-rooted strain Angola, a virulent Pestoides isolate. Its ancient nature makes this atypical isolate of particular importance in understanding the evolution of plague pathogenicity. Its chromosome features a unique genetic make-up intermediate between modern Y. pestis isolates and its evolutionary ancestor, Y. pseudotuberculosis. Our genotypic and phenotypic analyses led us to conclude that Angola belongs to one of the most ancient Y. pestis lineages thus far sequenced. The mobilome carries the first reported chimeric plasmid combining the two species-specific virulence plasmids. Genomic findings were validated in virulence assays demonstrating that its pathogenic potential is distinct from modern Y. pestis isolates. Human infection with this particular isolate would not be diagnosed by the standard clinical tests, as Angola lacks the plasmid-borne capsule, and a possible emergence of this genotype raises major public health concerns. To assess the genomic plasticity in Y. pestis, we investigated the global gene reservoir and estimated the pangenome at 4,844 unique protein-coding genes. As shown by the genomic analysis of this evolutionary key isolate, we found that the genomic plasticity within Y. pestis clearly was not as limited as previously thought, which is strengthened by the detection of the largest number of isolate-specific single-nucleotide polymorphisms (SNPs) currently reported in the species. This study identified numerous novel genetic signatures, some of which seem to be intimately associated with plague virulence. These markers are valuable in the development of a robust typing system critical for forensic, diagnostic, and epidemiological studies.

FIG. 1.

(A) Circular representation of the Y. pestis Angola chromosome. Circles, from outer to inner: predicted open reading frames encoded on the plus (1) and minus strands (2), colored according to the respective MANATEE roles; (3) G+C skew; (4) sSNP; (5) nsSNP; (6 and 7) genome-wide distribution of SNPs; (8) chromosomal regions of interest; (9) mobile elements; (10 to 18) comparative analysis of the Angola proteome to strains CO92 (10), KIM (11), Antiqua (12), Nepal516 (13), 91001 (14), and Pestoides F (15); interspecies comparison to Y. pseudotuberculosis strains IP32953 (16) and IP31758 (17) and Y. enterocolitica 8081 (18); (19) chi-square values. (B) Chimeric plasmid pMT-PCP. Circles, from outer to inner: (1 and 2) predicted open reading frames; (3) G+C skew; (4) mobile elements. Circle fragments for comparison to the pPCP plasmid are on the outside and show Y. pestis strains CO92 (1), KIM (2), 91001 (3), Antiqua (4), and Nepal516 (5); circle fragments for comparison to the pMT plasmids are on the inside and show strains CO92 (6), KIM (7), 91001 (8), Antiqua (9), Nepal516 (10), Pestoides F (11), and G8787 (12); circles for interspecies comparison to S. enterica CT18 pHCM2 plasmid (13); (14) chi-square values; (15) chimeric plasmid composition. Plasmid regions secondarily lost in the Y. pestis evolution, such as ribonucleases nrdAB and the Nepal516-specific 1,220-bp deletion of four hypothetical proteins with no assigned function (brown). (C) Architecture of pMT-PCP. The plasmid features a pPCP dimer integrated into the pMT plasmid in a head-to-tail arrangement.

FIG. 2.

Ancestral genome features of Y. pestis Angola. (A) Prevalence of the methionine salvage locus. Strain Angola and the deep-branching Pestoides group isolate F are the only known Y. pestis isolates that carry the methionine salvage pathway on their chromosomes. Its presence in the phylogenetic ancestors Y. pseudotuberculosis and Y. enterocolitica argues for a secondary loss of this metabolic property in descending Y. pestis isolates. The scale, in base pairs, indicates the genomic location of the methionine salvage locus composed of the seven genes mtnKADCEBU. Genes shared between these highly conserved and syntenically arranged loci are colored accordingly. (B) Prevalence of the nrdAB locus. Strain Angola encodes another locus on its chimeric plasmid, the ribonucleotide diphosphate reductase nrdAB operon, which most likely was lost secondarily in descending Y. pestis isolates. Corresponding loci are found only in other deep-branching Y. pestis strains: the Pestoides group isolates F and G8786 and the 0.PE4 microtus strain 91001. Besides Y. pestis, this locus also is present in the phylogenetically related pHCM2 plasmid of S. enterica CT18. In Y. pestis strain 91001, one of the two conserved hypothetical proteins appears to be degenerated (YP_pMT44, YP_pMT45). The scale, in base pairs, indicates the genomic location of the nrdAB operon. Genes shared between these loci are colored accordingly.

FIG. 3.

Genomic architecture of the F1 capsular antigen. The F1 capsule is present and syntenically organized in all previously sequenced Y. pestis genomes, while strain Angola is the only currently known Y. pestis isolate that lacks the complete operon. Genes shared between these loci are colored accordingly.

FIG. 4.

Phylogenetic position of Y. pestis Angola. The phylogenetic tree is based on 424 sSNPs and 1,006 nsSNPs and reveals a deep branching of the 0.PE3 strain Angola within Y. pestis. Branch designations were assigned according to the nomenclature introduced by Achtman et al. (1). Strains biochemically classified into the same biovar are colored accordingly.

FIG. 5.

Core genes and gene discovery in human pathogenic Yersinia. (A) Core genes. For each number of genomes n, circles are the permutations for Y. pestis values obtained for a sampling size of 1,000. Diamonds and triangles give median and mean values for each distribution. The curve represents the exponential regression of the least-squares fit of F_core(n) = κ_c exp[−n/τ_c] + tg_c(θ). The extrapolated core genome size is shown as a horizontal dashed red line. (B) Gene discovery. The numbers of new genes found are plotted for increasing values of n. The curve is the least-squares fit of the exponential decay F_new(n) = κ_n exp[−n/τ_n] + tg_n(θ) based on the means of the distribution. The value of tg_n(θ) shown represents the number of new genes asymptotically predicted for further genome sequencing. Core genes (C, E) and gene discovery (D, F) when Y. pseudotuberculosis strains IP31758 and IP32953 and Y. enterocolitica 8081 are included.

FIG. 6.

Pangenome of human pathogenic Yersinia. (A) Pangenome of Y. pestis. The total number of genes found according to the pangenome analyses is shown for increasing values of the number n of Yersinia genomes sequenced using medians and an exponential fit. Red diamonds indicate the means of the distributions. The dashed line represents the total number of genes that comprise the respective pangenome asymptotically predicted for further genome sequencing. (B) The two-species pangenome computed for Y. pestis and the closest evolutionary ancestor Y. pseudotuberculosis. (C) The three-species Yersinia pangenome computed for Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica.