# Complete genome sequences of Yersinia pestis 6/69 strain isolated from a bubonic plague patient in Madagascar and its isogenic strain cured of pPCP1

Emelyne Bougit, Guillem Mas Fiol, Pierre Lê-Bury, Charlotte Balière, Valérie Caro, Javier Pizarro-Cerdá, Olivier Dussurget

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## To cite this version:

Emelyne Bougit, Guillem Mas Fiol, Pierre Lê-Bury, Charlotte Balière, Valérie Caro, et al.. Complete genome sequences of Yersinia pestis 6/69 strain isolated from a bubonic plague patient in Madagascar and its isogenic strain cured of pPCP1. Microbiology Resource Announcements, 2025, 14 (3), pp.e0102124. 10.1128/mra.01021-24. pasteur-05086934

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Distributed under a Creative Commons Attribution 4.0 International License
# Complete genome sequences of *Yersinia pestis* 6/69 strain isolated from a bubonic plague patient in Madagascar and its isogenic strain cured of pPCP1

Emelyne Bougit,¹ Guillem Mas Fiol,¹,² Pierre Lê-Bury,¹,²,³ Charlotte Balière,⁴ Valérie Caro,⁴ Javier Pizarro-Cerdá,¹,²,⁵ Olivier Dussurget¹,²

**AUTHOR AFFILIATIONS** See affiliation list on p. 3.

**ABSTRACT** We report the complete genome sequences of two valuable strains to investigate plague pathogenesis: (i) *Yersinia pestis* strain 6/69, which was isolated from a bubonic plague patient in Madagascar and contains pCD1, pMT1, and pPCP1 virulence plasmids, and (ii) the 6/69 strain cured of pPCP1.

**KEYWORDS** *Yersinia pestis*, genome, pPCP1, *pla* gene, plague

Yersinia pestis causes plague (1), a life-threatening zoonosis still prevalent in Africa, Asia, and America (2), whose pathogenesis remains to be fully characterized. Here, we report the genome sequences of *Y. pestis* 6/69 and 6/69*pPCP1*, lacking pPCP1 virulence plasmid. The 6/69 strain was isolated in 1969 at the Institut Pasteur of Madagascar from a patient displaying a bubo in the Fandriana province of Madagascar (3). It belongs to Orientalis biovar, sublineage 1.0RI3 and harbors virulence plasmids pMT1, pCD1, and pPCP1, also known as pFra, pYV, and pPla, respectively (4, 5). Genomic characterization of the 6/69 strain is of interest as it has been used as a reference strain and, along with 6/69*pPCP1*, is useful for studying virulence genes, in particular, plasminogen activator gene *pla* harbored by pPCP1 (5–11).

To eliminate pPCP1, the 6/69 strain was subcultured 11 times at 4°C on trypticase soy agar with 0.002% hemin. For sequencing, 6/69 and 6/69*pPCP1* strains were grown for 16 hours in lysogeny broth at 28°C. DNA was extracted using PureLink Genomic DNA kit (Invitrogen) and quantified using Qubit 2.0 (ThermoFisherScientific). Default parameters were used except where otherwise noted. DNA was sequenced on a MinION-Mk1b using Oxford Nanopore Technologies (ONT) Rapid Barcoding Kit 24 V14 (SQK-RBK14.24), a R10.4.1 flowcell (FLO-MIN114) and MinKNOW/23.04.3 software, generating 80,000 and 33,000-bp length reads for 6/69 and 6/69*pPCP1*, respectively. Dorado/0.3.0 (https://github.com/nanoporetech/dorado) in sup mode was used for base calling. In parallel, a library was prepared using Illumina DNA Prep kit and sequenced on a MiSeq system (Illumina), generating 1,359 and 2,158 thousand 2 × 151 bp length reads for 6/69 and 6/69*pPCP1*, respectively. Hybrid genome assembly based on ONT and Illumina sequencing data was performed using the Easy pipeline (12). ONT reads were filtered with Filtlong/0.2.0 (https://github.com/rrwick/Filtlong) and assembled using Trycycler/0.5.0 (13), Flye/2.9 (14), Raven/1.6.0 (15), and NECAT/0.0.1 (16). Reads were polished using Medaka/1.11.3 (https://github.com/nanoporetech/medaka) and Polypolish/0.1.3 (17). Illumina reads were quality controlled using fastqc/0.12.1 and assembled using fq2dna/23.12 (gitlab.pasteur.fr/GIPhy/fq2dna), fqCleanER/23.12 (https://gitlab.pasteur.fr/GIPhy/fqCleanER), and SPAdes/3.15.5 (18). Annotation was performed with Bakta/1.5.0 (19). All reads were provided as FASTQ files.

**Editor** Vincent Michael Bruno, University of Maryland School of Medicine, Baltimore, Maryland, USA

Address correspondence to Olivier Dussurget, olivier.dussurget@pasteur.fr, or Javier Pizarro-Cerdá, pizarroj@pasteur.fr.

The authors declare no conflict of interest.

See the funding table on p. 3.

**Received** 17 September 2024

**Accepted** 20 January 2025

**Published** 20 February 2025

Copyright © 2025 Bougit et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
<table><caption>TABLE 1 General features of *Y. pestis* 6/69 and 6/69<sub>pPCP1</sub>-</caption><thead><tr><th rowspan="2">Parameters</th><th colspan="5">6/69</th><th colspan="4">6/69<sub>pPCP1</sub>-</th></tr><tr><th>Chromosome</th><th>pMT1</th><th>pCD1</th><th>pPCP1</th><th>Whole genome</th><th>Chromosome</th><th>pMT1</th><th>pCD1</th><th>Whole genome</th></tr></thead><tbody><tr><td>GenBank accession no.</td><td>CP166966.1</td><td>CP166967.1</td><td>CP166968.1</td><td>CP166969.1</td><td></td><td>CP166963.1</td><td>CP166964.1</td><td>CP166965.1</td><td></td></tr><tr><td>No. of circular contigs</td><td>1</td><td>1</td><td>1</td><td>1</td><td>4</td><td>1</td><td>1</td><td>1</td><td>3</td></tr><tr><td>Size (bp)</td><td>4,649,807</td><td>96,210</td><td>70,300</td><td>9,610</td><td>4,825,927</td><td>4,649,807</td><td>96,210</td><td>70,300</td><td>4,816,317</td></tr><tr><td>G + C content (%)</td><td>47.6</td><td>50.2</td><td>44.8</td><td>45.3</td><td>47.6</td><td>47.6</td><td>50.2</td><td>44.8</td><td>47.6</td></tr><tr><td>No. of annotated protein-coding genes</td><td>4,097</td><td>99</td><td>90</td><td>10</td><td>4,296</td><td>4,097</td><td>99</td><td>90</td><td>4,286</td></tr><tr><td>No. of pseudogenes</td><td>8</td><td>2</td><td>0</td><td>0</td><td>10</td><td>8</td><td>2</td><td>0</td><td>10</td></tr><tr><td>No. of rRNAs</td><td>19</td><td>0</td><td>0</td><td>0</td><td>19</td><td>19</td><td>0</td><td>0</td><td>19</td></tr><tr><td>No. of tRNAs</td><td>71</td><td>0</td><td>0</td><td>0</td><td>71</td><td>71</td><td>0</td><td>0</td><td>71</td></tr><tr><td>No. of ncRNAs</td><td>183</td><td>1</td><td>2</td><td>2</td><td>188</td><td>183</td><td>1</td><td>2</td><td>186</td></tr><tr><td>CRISPR array</td><td>4</td><td>0</td><td>0</td><td>0</td><td>4</td><td>4</td><td>0</td><td>0</td><td>4</td></tr><tr><td>Total no. of reads</td><td colspan="5">80,656 (ONT)<br/>1,359,952 paired reads (Illumina)</td><td colspan="4">33,370 (ONT)<br/>2,158,311 paired reads (Illumina)</td></tr><tr><td>ONT N<sub>50</sub> length</td><td colspan="5">14,027</td><td colspan="4">14,960</td></tr><tr><td>Total no. of bases</td><td colspan="5">624,569,231 (ONT)<br/>203,992,800 (Illumina)</td><td colspan="4">249,181,216 (ONT)<br/>321,207,344 (Illumina)</td></tr><tr><td>Sequencing depth (×)</td><td colspan="5">129 (ONT)<br/>42 (Illumina)</td><td colspan="4">51 (ONT)<br/>66 (Illumina)</td></tr></tbody></table>

The 6/69 strain possesses a 4.8 Mb circular chromosome and the three plasmids found in the CO92 reference strain (1) (Table 1; Fig. 1). The 6/69 and CO92 strains have an average nucleotide identity >99%. They differ by 52 chromosomal single nucleotide polymorphisms and 3 deletions of 1,510, 10,344, and 3,480 bp starting at positions 657344, 1310775, and 1534290 of CO92 chromosome (GCF_000009065.1). The absence of pPCP1 is the only difference between 6/69<sub>pPCP1</sub>- and 6/69 strains, highlighting their relevance as tools to study the role of pPCP1 in pathogenesis.

<figure><img src="image_3.png" /><figcaption>FIG 1 Genome maps of *Yersinia pestis* 6/69 (left) and 6/69<sub>pPCP1</sub>- (right). Rings from the outside in: scale marks (1), protein-coding genes on the forward strand (2) or reverse strand (3), rRNA genes (4), tRNA genes (5), GC variation (6), and GC skew (7). Positions of the three identified deletions in 6/69 compared to CO92 are displayed (Del1, Del2, and Del3). Created using Circos.</figcaption></figure>
ACKNOWLEDGMENTS

We thank Cyril Savin for his advice on genome assembly and members of the Yersinia Research Unit and the French National Reference Center for Plague and Other Yersiniosis for insightful discussions.

The project received funding from Institut Pasteur and LabEX Integrative Biology of Emerging Infectious Diseases (ANR-10-LBX-62-IBEID). E.B. and P.L.-B. were supported by ANRS-MIE fellowships (ANRS0350b and ANRS0349b, respectively). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

AUTHOR AFFILIATIONS

¹Yersinia Research Unit, Institut Pasteur, Université Paris Cité, Paris, France

²WHO Collaborating Research and Reference Centre for Plague FRA-146, Institut Pasteur, Université Paris Cité, Paris, France

³Center for Immunology of Viral, Auto - immune, Hematological and Bacterial Diseases (IMVA - HB/IDMIT), Université Paris-Saclay, Inserm, CEA, Fontenay - aux - Roses, France

⁴Environment and Infectious Risk Unit, Laboratory for Urgent Response to Biological Threats, Institut Pasteur, Université Paris Cité, Paris, France

⁵Yersinia National Reference Laboratory, Institut Pasteur, Université Paris Cité, Paris, France

AUTHOR ORCIDs

Pierre Lê-Bury http://orcid.org/0000-0002-6010-2952

Valérie Caro http://orcid.org/0000-0001-8477-5708

Javier Pizarro-Cerdá http://orcid.org/0000-0002-4343-0508

Olivier Dussurget http://orcid.org/0000-0001-9924-0837

FUNDING

<table><thead><tr><th>Funder</th><th>Grant(s)</th><th>Author(s)</th></tr></thead><tbody><tr><td rowspan="8">Institut Pasteur</td><td></td><td>Emelyne Bougit</td></tr><tr><td></td><td>Guillem Mas Fiol</td></tr><tr><td></td><td>Pierre Lê-Bury</td></tr><tr><td></td><td>Charlotte Balière</td></tr><tr><td></td><td>Valérie Caro</td></tr><tr><td></td><td>Javier Pizarro-Cerdá</td></tr><tr><td></td><td>Olivier Dussurget</td></tr><tr><td rowspan="6">Agence Nationale de la Recherche (ANR)</td><td rowspan="6">ANR-10-LBX-62-IBEID</td><td>Emelyne Bougit</td></tr><tr><td>Guillem Mas Fiol</td></tr><tr><td>Pierre Lê-Bury</td></tr><tr><td>Charlotte Balière</td></tr><tr><td>Valérie Caro</td></tr><tr><td>Javier Pizarro-Cerdá</td></tr><tr><td></td><td>Olivier Dussurget</td></tr><tr><td>Agence Nationale de Recherches sur le Sida et les Hépatites Virales (ANRS)</td><td>ANRS0350b</td><td>Emelyne Bougit</td></tr><tr><td>Agence Nationale de Recherches sur le Sida et les Hépatites Virales (ANRS)</td><td>ANRS0349b</td><td>Pierre Lê-Bury</td></tr></tbody></table>
## AUTHOR CONTRIBUTIONS

Emelyne Bougit, Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review and editing | Guillem Mas Fiol, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – review and editing | Pierre Lè-Bury, Data curation, Formal analysis, Investigation, Methodology, Validation, Writing – review and editing | Charlotte Balière, Investigation, Methodology, Writing – review and editing | Valérie Caro, Investigation, Methodology, Writing – review and editing | Javier Pizarro-Cerdá, Conceptualization, Funding acquisition, Supervision, Writing – review and editing | Olivier Dussurget, Conceptualization, Funding acquisition, Project administration, Supervision, Writing – original draft, Writing – review and editing

## DATA AVAILABILITY

Genome sequences of *Y. pestis* 6/69 (IP304) and 6/69<sub>p</sub>PCP1- (IP343R) were submitted to NCBI under BioProject ID PRJNA1128573 and BioSample numbers SAMN42052713 and SAMN42053676. Assembled genomes are available in GenBank under numbers GCA_041410195.1 and GCA_041409745.1. Illumina and ONT data can be accessed on Sequencing Read Archive numbers SRR29554089, SRR29554088 and SRR29554145, SRR29554144, respectively. Furthermore, sequences are available on Yersiniomics (20), providing structural and functional properties of gene products.

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