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

TL;DR

This paper presents the complete genome sequences of a bubonic plague-causing Yersinia pestis strain and a modified version without a specific virulence plasmid.

Contribution

The novel contribution is the sequencing of a clinical Y. pestis isolate and its isogenic derivative lacking the pPCP1 plasmid.

Findings

The genome of Yersinia pestis strain 6/69 was fully sequenced.

An isogenic strain of 6/69 cured of the pPCP1 plasmid was also sequenced.

The findings provide resources for studying plague pathogenesis.

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.

Figures

Genome maps of Yersinia pestis 6/69 (left) and 6/69pPCP1- (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.

Tables

TABLE 1. General features of Y. pestis 6/69 and 6/69pPCP1-

	6/69					6/69pPCP1-
Parameters	Chromosome	pMT1	pCD1	pPCP1	Whole genome	Chromosome	pMT1	pCD1	Whole genome
GenBank accession no.	CP166966.1	CP166967.1	CP166968.1	CP166969.1		CP166963.1	CP166964.1	CP166965.1
No. of circular contigs	1	1	1	1	4	1	1	1	3
Size (bp)	4,649,807	96,210	70,300	9,610	4,825,927	4,649,807	96,210	70,300	4,816,317
G + C content (%)	47.6	50.2	44.8	45.3	47.6	47.6	50.2	44.8	47.6
No. of annotated protein-coding genes	4,097	99	90	10	4,296	4,097	99	90	4,286
No. of pseudogenes	8	2	0	0	10	8	2	0	10
No. of rRNAs	19	0	0	0	19	19	0	0	19
No. of tRNAs	71	0	0	0	71	71	0	0	71
No. of ncRNAs	183	1	2	2	188	183	1	2	186
CRISPR array	4	0	0	0	4	4	0	0	4
Total no. of reads	80,656 (ONT)1,359,952 paired reads (Illumina)					33,370 (ONT)2,158,311 paired reads (Illumina)
ONT N50 length	14,027					14,960
Total no. of bases	624,569,231 (ONT)203,992,800 (Illumina)					249,181,216 (ONT)321,207,344 (Illumina)
Sequencing depth (×)	129 (ONT)42 (Illumina)					51 (ONT)66 (Illumina)

Full text

ANNOUNCEMENT

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.ORI3 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 basecalling. 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.

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_pPCP1-_ and 6/69 strains, highlighting their relevance as tools to study the role of pPCP1 in pathogenesis.

Genome maps of Yersinia pestis 6/69 (left) and 6/69pPCP1- (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.