Abstract
Yersinia pestis, the bacterium that causes the plague, has a dynamic genome with highly conserved fragments prone to rearrangement, influencing gene function and evolution. However, understanding these patterns is limited by few complete genomes and analytical methods. We developed a dual-validation strategy to analyze 242 complete genomes of Y. pestis natural isolates from diverse phylogroups. We detected 459 rearrangements, which enhanced phylogenetic resolution and resolved the third pandemic’s polytomy. Rearrangements are primarily mediated by four common insertion sequences, with IS1661 and IS100 showing the highest activity. These rearrangements are under strong positive selection, evidenced by 43 hotspots and convergent evolution in the rpsO-pnp operon, whose disruptions and reconnections altered gene expressions and temperature stress responses. We also identified unique structural alterations in human avirulent phylogroups, inactivating three genes and reordering 17 intergenic regions, some affecting virulence-related genes. This study provides a fresh perspective on Y. pestis evolution, revealing experimental targets and establishing a methodology for microbes with frequent rearrangements.
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Data availability
The complete genome assemblies and long-read sequencing data generated in this study have been deposited in GenBank and the Sequence Read Archive under BioProject no. PRJNA1190511 and in the NMDC under BioProject no. NMDC10018925. Accession numbers for each genome are listed in Supplementary Table 1. Publicly available complete genomes and the RNA-seq data used in this study are provided in Supplementary Tables 2 and 10, respectively. Insertion sequence types were identified using sequences from the ISfinder database. Functional annotations were assigned using the online eggNOG database. Source data are provided with this paper.
Code availability
The code relevant to this manuscript is publicly available via GitHub at github.com/WuYarong/YP_Rearrangement_Analysis and via Zenodo at https://doi.org/10.5281/zenodo.15152926 (ref. 54). It was implemented in Python v.3.7.10, using the packages SciPy (v.1.5.2), NumPy (v.1.18.1) and statsmodels (v.0.11.1). The insertion sequence copy number was calculated using a custom script based on the BLASTn v.2.13.0+ output format 6.
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Acknowledgements
This work was supported by the National Key Research and Development Program of China (no. 2022YFC2305304 to Y.C.), the National Natural Science Foundation of China (no. 31970002 to Y.C.) and the State Key Laboratory of Pathogen and Biosecurity (no. SKLPBS2215 to Y.W.).
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Y.C. designed the study. Y.G. conducted the DNA library construction and long-read sequencing. K.M. performed the complete genome assembly. Y.W. and C.Y. analyzed the genomic rearrangements. Y.W. and Y.C. drafted the manuscript. Y.S. provided valuable advice and insights. Y.C. and R.Y. supervised the project. All authors read, revised and approved the final manuscript.
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Extended data
Extended Data Fig. 1 Pipeline for identifying rearrangement events in Y. pestis phylogroups.
a, Synteny block construction without a reference genome using SibellaZ and maf2synteny tools. The adjacency marks synteny block neighbors, with ‘h’ for the head and ‘t’ for the tail of blocks. For instance, ‘2h-3t’ illustrates that the head of block no. 2 is adjacent to the tail of block no. 3. b, Pairwise rearrangement detection based on reference genomes with SyRI software. SYN denotes syntenic regions, INV for inversions, TRANS for translocations, INVTR for inverted translocations, and DUP for duplications. Our focus of genomic rearrangements was on INV, TRANS, and INVTR. c, Synteny block analysis combined with pairwise rearrangement identification. d, Determining rearrangement events in the last common ancestor (LCA) of phylogroups. e, Detecting rearrangement events within specific phylogroups.
Extended Data Fig. 2 Synteny blocks dynamics in Y. pestis.
a, Clustered heatmap of 113 accessory blocks in Y. pestis strains, based on their copy number variations. Color intensity reflects copy number, with dendrogram showing clustering. Names of 242 Y. pestis strains and Y. pseudotuberculosis outgroup are listed on the right. b, Close-up on the phylogroup-specific distribution of 43 synteny blocks, a subset derived from panel a, using the same heatmap color legend. Blocks are arranged by variations across phylogroups in the heatmap, highlighted with purple outlines. Parallelism scores from PareBrick analysis are shown above, while blue lines below mark the merging of adjacent blocks into larger segments, with prophage and ribosomal RNA (rRNA) related blocks labeled.
Extended Data Fig. 3 Principal component analysis (PCA) of phylogroup clustering in Y. pestis and comparison of synteny diversity with Y. pseudotuberculosis.
a, PCA cumulative variance plot of 178 synteny block adjacencies associated with rearrangements. The first six principal components (PCs) accounted for over 60.2% of the variance. b, PCA cumulative variance plot of 3,185 single nucleotide polymorphisms (SNPs) and clustering of Y. pestis phylogroups on PC1 and PC2. The first six PCs accounted for 69.8% of the variance. The shaded area represents the cluster of early-diverging 0.PE phylogroups, including 0.PE7, 0.PE2, 0.PE4A, 0.PE4B, and 0.PE4C. c, PCA cumulative variance plot of 2,023 indels and clustering of Y. pestis phylogroups on PC1 and PC2. The first six PCs accounted for 58.6% of the variance. The shaded area represents the cluster of early-diverging 0.PE phylogroups, including 0.PE7, 0.PE2, 0.PE4A, 0.PE4B, and 0.PE4C. d, Synteny diversity comparison between 242 Y. pestis and 25 Y. pseudotuberculosis strains. A sliding window analysis was performed using the IP32953 chromosome as the reference. Synteny diversity in 50-kb windows (10-kb steps) is visualized in purple for Y. pestis and orange for Y. pseudotuberculosis, with 5-kb windows (1-kb steps) in grey for both.
Extended Data Fig. 4 Phylogroup LCA rearrangement events with varied reference genomes.
a, Rearrangements in the LCAs of branch 0 phylogroups, using the Y. pseudotuberculosis IP32953 chromosome as a reference. Synteny diversity (πsyn) for each phylogroup’s strains (5 kb sliding windows with a 1 kb step-size) is aligned with this reference genome. b, Rearrangements in the LCAs of phylogroups within branches 2-4, along with 1.ANT1 and 1.IN1 from branch 1, using the chromosome of strain 43005 (from 0.ANT3) as a reference. c, Rearrangements in 1.IN1-1.IN5 and 1.ORI1 LCAs, referenced against the chromosome of strain 15002 (from 1.IN2). d, Rearrangements in 1.ORI2 and 1.ORI3 LCAs, with the chromosome of strain El-Dorado (from 1.ORI1) as the reference. To be noted, 0.PE2, 0.PE4B, 0.PE4C, and 0.ANT3 (represented as 0.PE4-0.ANT1) have been presented in Fig. 3. In order to comprehensively depict the various populations of Y. pestis, they are listed here again.
Extended Data Fig. 5 Comparative synteny diversity in Y. pestis phylogroups.
a, Synteny diversity (πsyn) in IP32953 reference alignments. This panel illustrates a prominent inversion in the Y. pestis phylogenetic stem between 0.PE4 and 0.ANT1, along with a smaller-scale translocation, demarcated by shaded areas. b, Synteny diversity (πsyn) in 43005 reference alignments. This section highlights stepwise genomic rearrangements within branch 1, as indicated in color-coded shaded regions.
Extended Data Fig. 6 Heatmap of average genomic synteny proportion between pairwise strains across Y. pestis phylogroups.
Color gradient from blue to red represents increasing genomic collinearity from low to high.
Extended Data Fig. 7 Rearrangement patterns in branch 2 for the 2.ANT and 2.MED phylogroups.
Numerical labels represent rearrangement counts, with orange points highlighting events that delineate phylogenetic splits. Given the complexity of determining synteny block order for the common ancestor of the 2.ANT and 2.MED phylogroups, our analysis was limited to their rearrangements compared with strain 43005 from the 0.ANT3 phylogroup, as marked by asterisks. The same rearrangement variations occurring across different phylogroups were independently counted within each respective population.
Extended Data Fig. 8 Distribution of four common insertion sequences in various states across Y. pestis chromosomes.
Both single elements (for example, IS100 or IS1541) and combined elements (for example, ‘IS100-IS1541’, representing adjacent insertion sequences treated as a single unit) are shown (n = 242 chromosomes). Combined elements are counted separately and not included in single-element counts. Counts reflect the number of genomic fragments mapped to each insertion sequence type, irrespective of fragment length, which may result in slight differences from the copy number accumulation analyses described in the Supplementary Notes. In boxplots, the top, middle, and bottom lines indicate the 75th percentile, median, and 25th percentile, respectively. Whiskers extend to 1.5 times the interquartile range (IQR), and points beyond this range are shown as outliers.
Extended Data Fig. 9 Comparison of two methods for identifying rearrangement hotspots and reassessment using an alternative reference.
a, Comparison of the methods using strain 43005 as reference. Linear regression was performed between the -log10-transformed corrected p-values from the Poisson distribution method (y-axis) and the parallelism break scores from PareBrick (x-axis) in R, with hotspot 391t-42h (unfilled circle) excluded as an outlier. The solid line represents the fitted regression line (predicted mean response), with the shaded area indicating the 95% confidence interval (CI) of the fitted values. Adjusted r² and P value for the slope coefficient are shown. Horizontal and vertical dashed lines mark the detection thresholds of the Poisson method (corrected P = 0.05, -log10 scale) and PareBrick (score=1), respectively. Red diamonds indicate hotspots identified by the Poisson method only, while green highlights those identified by PareBrick but lacking statistical significance in the Poisson method. b, Profiling rearrangement hotspots using IP32953 chromosome as reference. See Fig. 5 for legend details. c, Comparison of the methods using strain IP32953 as reference. The figure legend is the same as in panel a.
Extended Data Fig. 10 Evolutionary changes of the rpsO-pnp operon within Y. pestis.
The left panel displays the phylogenetic tree of 242 Y. pestis strains, with Y. pseudotuberculosis IP32953 as the outgroup. The tree highlights five main branches of Y. pestis in distinct colors, with strain names positioned near the tree. The phylogenetic positions of the three global pandemics are annotated. Y. pestis strains from the first two, as ancient DNA without complete genomes, are not included in this study and marked by dashed arrows. The colored bar to the right of the tree represents designated phylogroups. The second bar denotes the rpsO-pnp operon’s state as disrupted or connected. The third bar, normalized to an average sequencing depth of 100, indicates the number of long reads supporting the operon’s connected state. The final bar shows whether there is an IS1661 insertion between the rpsO and pnp genes, with ‘IS1661-free’ marking the absence of such insertion.
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Wu, Y., Yang, C., Mu, K. et al. Insights into Yersinia pestis evolution through rearrangement analysis of 242 complete genomes. Nat Genet 57, 1994–2003 (2025). https://doi.org/10.1038/s41588-025-02264-5
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DOI: https://doi.org/10.1038/s41588-025-02264-5
