After writing my review of the Proceedings of the National Academy of Sciences USA article “Targeted enrichment of ancient pathogens yielding the pPCP1 plasmid of Yersinia pestis from victims of the Black Death”, I vaguely wondered if the authors could have sequenced more than a single 10kb plasmid. If the single-copy chromosomal DNA was too scarce, maybe one of the other Yersina pestis plasmids that may exist at a higher copy number (e.g., pMT1) might be sequenced. Well, that question was answered on Wednesday, October 12, when Nature posted a Letter online titled “A draft genome of Yersinia pestis from victims of the Black Death”. It turns out the Nature Letter, published six weeks after the PNAS article, was authored by the same researchers, and they had sequenced the entire genome of the ancient strain of Y. pestis that caused the Black Death in Europe during 1347–1351. In addition to the 10kb Y. pestis plasmid pPCP1 sequenced in the PNAS article, two other plasmids and the chromosomal DNA were fished out of the soup of ancient human DNA, cloned, sequenced and assembled to reconstruct the genetic details of the Y. pestis strain that so devastated the population in Europe.
What makes this draft genome of Y. pestis so newsworthy? This is the first ancient organism that had its entire genome sequenced. The researchers had to overcome three main problems in dealing with ancient DNA from the bacterial pathogen:
- How to prevent contamination with modern DNA.
- How to isolate only the relevant bacterial DNA from a milieu of ancient human DNA.
- How to accurately assemble an intact genome from the tiny fragments of ancient DNA.
The authors were able to address each of these points, culminating in the technique described in the PNAS paper and used in the Nature Letter: using immobilized modern Y. pestis sequences to fish out sequences that bind to it—basically other Y. pestis sequences. The blood-borne bacterial pathogen may be better preserved in dental pulp compared to other skeletal remains because teeth are not as porous as bone, but there is still a large quantity of human DNA that will interfere with analysis. Enriching for the species-specific DNA was one way to increase the amount of Y. pestis DNA for sequencing and reduce the human DNA, which may interfere with a genome sequence analysis.
So after the painstaking process of extracting DNA from teeth of Black Plague victims, enriching for Y. pestis DNA and finally, using high-throughput sequencing to decode the genetic sequence from four victims, the researchers were able to publish the full sequence of Y. pestis, including its chromosome and the two remaining plasmids: pMT1 and pCD1.
The researchers now had deciphered the sequence of this ancient and deadly strain of bacteria. How does it differ from modern Y. pestis? Answer: Not much. The modern reference genome used, CO92, had only 103 single-nucleotide differences compared to the ancient Y. pestis chromosomal DNA and its two plasmids. However, ancient Y. pestis had some sections of the genomes rearranged in comparison to most modern strains, but the rearranged segments closely matched similar sections of genome in the more ancient strains (e.g., Mictrotus 91001). Based on phylogenetic analysis, the authors place the Black Plague as the ancestor to all modern strains of Y. pestis. As a result of this association between modern and 660-year-old Y. pestis, the authors question the etiology of the Justinian plague outbreak in the sixth to eighth century, widely thought to be caused by Y. pestis. Because that plague was substantially earlier than the Black Death Y. pestis, and the results of phylogenetic analysis place the Black Death near the root of all modern pathogenic Y. pestis strains, the authors infer the Plague of Justinian is unlikely to share genetic similarity with the modern strains, and suggest it is a completely unique strain that died out or even a different pathogenic agent altogether. However, a genetic comparison between the Black Death and the Plague of Justinian would better resolve this issue.
Even with the remarkably few changes between modern and Black Death Y. pestis strains, there are still differences to be explored. Substituting sequences from the Black Death strain in a modern strain can answer questions about infectivity or growth. However, the authors acknowledge that there may be an array of factors that contributed to the high mortality of the Black Death, including a cold, rainy climate, malnutrition and other diseases that immunocompromised the humans in 1347–1351.
Learning how scientists uncover mysteries like what was the cause of the Black Death and how the Black Death differs from modern Y. pestis fascinates me as evidenced by the blog posts I have written on the subject. In addition, I like putting a face to the scientists whose publications I read so I avidly watched a PBS Newshour interview of one of the authors, Henrick Poinar, and link to the video for your viewing pleasure.
Bos KI, Schuenemann VJ, Golding GB, Burbano HA, Waglechner N, Coombes BK, McPhee JB, Dewitte SN, Meyer M, Schmedes S, Wood J, Earn DJ, Herring DA, Bauer P, Poinar HN, & Krause J (2011). A draft genome of Yersinia pestis from victims of the Black Death. Nature PMID: 21993626