A High-Coverage Yersinia pestis Genome from a Sixth-Century Justinianic Plague Victim

Abstract

The Justinianic Plague, which started in the sixth century and lasted to the mid eighth century, is thought to be the first of three historically documented plague pandemics causing massive casualties. Historical accounts and molecular data suggest the bacterium Yersinia pestis as its etiological agent. Here we present a new high-coverage (17.9-fold) Y. pestis genome obtained from a sixth-century skeleton recovered from a southern German burial site close to Munich. The reconstructed genome enabled the detection of 30 unique substitutions as well as structural differences that have not been previously described. We report indels affecting a lacl family transcription regulator gene as well as nonsynonymous substitutions in the nrdE, fadJ, and pcp genes, that have been suggested as plague virulence determinants or have been shown to be upregulated in different models of plague infection. In addition, we identify 19 false positive substitutions in a previously published lower-coverage Y. pestis genome from another archaeological site of the same time period and geographical region that is otherwise genetically identical to the high-coverage genome sequence reported here, suggesting low-genetic diversity of the plague during the sixth century in rural southern Germany.

Keywords: Yersinia pestis; ancient DNA; justinianic plague; reconstruction; whole genome.

Fig. 1

The Altenerding plague burials. (A) Adult woman AE1175 (left) and adult man AE1176 (right) excavated at Altenerding and found positive for presence of Y. pestis. (B) The third molar sampled from individual AE1175 from which the Altenerding high-coverage genome was obtained. (C) Grave goods of individual AE1175 typical of the middle of the sixth century (© Archaeological State Collection Munich), Grave goods not shown true to scale (Detailed description in the historical and archaeological supplementary section). (D) Grave goods of individual AE1176 (© Archaeological State Collection Munich), Grave goods not shown true to scale (detailed description in the historical and archaeological supplementary section). (E) Geographical map specifying the location of the archaeological site “Altenerding”. The other site, where Y. pestis has been identified and its genome has been previously reconstructed (Wagner et al. 2014), is the cemetery of Aschheim, which is located approximately 20 km southwest of Altenerding.

Fig. 2

Maximum parsimony tree: maximum parsimony analysis of 1418 nucleotide positions from genomes of 133 Y. pestis strains. All positions containing missing data were eliminated. Bootstrap values are indicated next to the nodes and bootstrap values of 100% are indicated by an asterisk. The tree is rooted using the genome of Yersinia pseudotuberculosis (strain IP32953). Branches leading to isolates from the historical pandemics are colored red and purple representing the second and first pandemics, respectively. Monophyletic nodes have been collapsed and are represented by a triangle. The number of isolates in a collapsed node is indicated in brackets. Our analysis suggests the specifically derived SNPs represented by the Aschheim branch to be false positives, supporting that the Aschheim and the Altenerding genomes likely represent the same bacterial strain.

Fig. 3

Coverage of the false positive substitutions called for the Aschheim genome by Wagner et al., 2014 compared with the reanalyzed Aschheim genome and the Altenerding genome. Fold coverage was determined for each position where substitutions were erroneously reported in the Aschheim genome (Wagner et al. 2014). Values for the Altenerding genome, values published in Wagner et al. 2014 for the Aschheim genome and the values for the reanalyzed Aschheim genome are colored green, red, and blue, respectively. Most of the false positive positions in the Aschheim genome contain abnormal coverage peaks, significantly higher than the average coverage of the corresponding genome.

Fig. 4

Comparison of coverage and heterozygous positions between ancient Y. pestis genomes. The average coverage calculations and the calling of heterozygous positions were done against the CO92 chromosome (NC_003143.1) as a reference. The percentage of heterozygous positions was calculated out of the total number of SNPs called for each genome, using a mapping quality threshold of 30 and a coverage threshold of 5 reads. The raw data published in Wagner et al. 2014, Bos et al. 2011, and Bos et al. 2016 were reanalyzed and used for calculating the coverage and the percentage of heterozygous positions for the Aschheim Justinianic genome and the genomes from the second pandemic, respectively. The Black Death genome 8124 was excluded from this analysis since the coverage was not sufficient for calling of heterozygous positions. The Aschheim Justinianic genome contains a high percentage of heterozygous positions compared with the other ancient Y. pestis genomes. Heterozygous positions called for a genome of the haploid Y. pestis indicate nonendogenous reads.