# Complete Genome Sequences of Bioluminescent Staphylococcus aureus Strains Xen31 and Xen36, Derived from Two Clinical Isolates

Océane Blaise, Clarisse Leseigneur, Thomas Cokelaer, Elena Capuzzo, Nadira

Frescaline, Olivier Dussurget

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Océane Blaise, Clarisse Leseigneur, Thomas Cokelaer, Elena Capuzzo, Nadira Frescaline, et al. Complete Genome Sequences of Bioluminescent Staphylococcus aureus Strains Xen31 and Xen36, Derived from Two Clinical Isolates. Microbiology Resource Announcements, 2023, 12 (3), pp.e0002423. 10.1128/mra.00024-23. pasteur-04120590

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# Complete Genome Sequences of Bioluminescent *Staphylococcus aureus* Strains Xen31 and Xen36, Derived from Two Clinical Isolates

Océane Blaise,ª,b,c Clarisse Leseigneur,ª Thomas Cokelaer,ª,e Elena Capuzzo,ª Nadira Frescaline,ª,b Olivier Dussurgetª

**ABSTRACT** Here, we report complete genome sequences of two clinical isolates of *Staphylococcus aureus*, namely, Xen31 and Xen36, which have been genetically modified to express an optimized *Photorhabdus luminescens* luciferase operon. Xen31 and Xen36 are bioluminescent strains used widely for investigation of bacterial pathogenesis, drug discovery, and development of novel therapies.

Staphylococcus aureus is a saprophytic bacterium that can infect multiple animal hosts, including humans (1–11). Methicillin-resistant S. aureus (MRSA) infections led to more than 100,000 deaths in 2019 worldwide (12). Thus, the development of innovative therapeutics is urgently needed. Bioluminescent microorganisms are powerful tools to use to investigate pathogenesis, screen antimicrobial drugs, and assess therapies (13–31). Here, we report genome sequences of bioluminescent S. aureus strains Xen31 and Xen36 (Perkin Elmer 119242 and 119243), derived from clinical isolates (16, 32). A modified *Photorhabdus luminescens luxABCDE* operon is integrated in the Xen31 chromosome and Xen36 plasmid. Genomic characterization of these strains is of interest as they are commonly used in infection models and preclinical studies (22, 27, 28, 31, 33–42).

Genomic DNA was isolated from bacteria grown in brain heart infusion broth (BD) using a PureLink DNeasy kit (Invitrogen), sheared in Covaris g-tubes at 5,500 rpm for 60 s twice, and size selected using Pacific Biosciences (PacBio) magnetic beads diluted 3.5-fold. Sequencing was performed using a PacBio Sequel I instrument. Libraries were prepared according to the PacBio protocol for multiplex SMRT sequencing with v3 chemistry using a SMRTbell express template prep kit 2.0 and barcoded adapters kit 8A/8B. Circular consensus sequences (CCSs) were constructed using SMRT Link software v6.2.0 (PacBio) with default settings except that minimum accuracy was set to 0.7. Bioconvert v0.6.2 (https://github.com/bioconvert/bioconvert) converted CCS files to FastQ files resulting in 40,653 and 29,054 CCS reads for Xen31 and Xen36 genomes, respectively. N₅₀ values were 9,122 and 9,890 bp, respectively. CCS data had high quality with a Phred score above 40 for 95% of reads. Taxonomic analysis using the Sequanamultitax pipeline (43) showed 99.9% *Staphylococcus aureus* reads. Assemblies were performed using the LORA pipeline from Sequana (43). Default parameters were used for all software. Using the Canu v1.6 assembler (44), four contigs were obtained for Xen31. Circularization was performed with Circlator v1.5.5 (45). The main circularized contig was 2,966,802 bp long with a G+C content of 32.95% (Table 1) and exhibited uniform coverage of 98×. The second circularized 32,188-bp contig had a uniform 105× coverage and was identified as plasmid CP041009.1 by BLAST. The 43,128-bp-long third contig had a uniform coverage

**Editor** Steven R. Gill, University of Rochester School of Medicine and Dentistry

**Copyright** © 2023 Blaise et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

Address correspondence to Olivier Dussurget, olivier.dussurget@pasteur.fr.
The authors declare no conflict of interest.

**Received** 13 January 2023

**Accepted** 4 February 2023

**Published** 22 February 2023

<footnote>ªInstitut Pasteur, Université Paris Cité, CNRS UMR 6047, Unité de Recherche Yersinia, Paris, France</footnote>

<footnote>ᵇInstitut de Recherche Biomédicale des Armées, INSERM UMRS-MD 1197, Centre de Transfusion Sanguine des Armées, Clamart, France</footnote>

<footnote>ᶜÉcole Polytechnique, Sorbonne Université, Laboratoire de Physique des Plasmas, CNRS, Palaiseau, France</footnote>

<footnote>ᵈInstitut Pasteur, Université Paris Cité, Plate-forme Technologique Biomics, Paris, France</footnote>

<footnote>ᵉInstitut Pasteur, Université Paris Cité, Hub de Bioinformatique et Biostatistique, Paris, France</footnote>
<table><caption>TABLE 1 General features of the *Staphylococcus aureus* strains Xen31 and Xen36<sup>a</sup></caption><thead><tr><th rowspan="2">Feature</th><th colspan="2">Data by strain</th></tr><tr><th>Xen31</th><th>Xen36</th></tr></thead><tbody><tr><td>Strain origin</td><td>Clinical isolate</td><td>Clinical isolate</td></tr><tr><td>Resistance type</td><td>MRSA</td><td>MSSA</td></tr><tr><td>Sequence type</td><td>ST247</td><td>ST8037</td></tr><tr><td>Genome size (bp)</td><td>2,966,802</td><td>2,749,617</td></tr><tr><td>G+C content (%)</td><td>32.95</td><td>32.91</td></tr><tr><td>No. of protein-coding genes</td><td>2,742</td><td>2,528</td></tr><tr><td>No. of rRNAs</td><td>19</td><td>19</td></tr><tr><td>No. of tRNAs</td><td>62</td><td>60</td></tr><tr><td>No. of tmRNAs</td><td>1</td><td>1</td></tr><tr><td>No. of CRISPR regions</td><td>5</td><td>10</td></tr><tr><td>Prophage (accession no.)</td><td>PT1028 (AY954948.1), φNM3 (NC_008617), φJB (NC_028669), AJ_2017 (NC_048644)</td><td>PT1028 (AY954948.1)</td></tr><tr><td>Plasmid accession no.</td><td>CP041009.1</td><td>CP082814 and CP029650.1</td></tr><tr><td>Antibiotic resistance genes</td><td>aac(6')-aph(2''), aadD, ant(9)-la, blaTEM-1A, bleO, cat(pC194), erm(A), erm(B), fosB, mecA, tet(M)</td><td>aph(3')-III and blaZ</td></tr><tr><td>Virulence determinant genes<sup>b</sup></td><td>adsA, agr, ahpCF, arlRS, aur, cap, chp, clf, coa, codY, crt, dlt, eap, ebp, efb, esa, ess, esx, flr, fnb, fur, geh, graRSX, hla, hld, hlgABC, hysA, ica, isd, katA, lukED*, lukGH, lytRS, mgrA, mprF, oatA, psm, RNAIII, rot, rsr, saeRS, sak, sar, sarT*, sarU*, saxG, sbi, scn, sdr, sea, see, selP*, set1*, set3*, set5*, set6*, set9*, set12*, set21*, set30-34*, set36-40*, set2, set4, set7, set8, sigB, sodA, sodM, spa, spI*, spn, srrAB, ssl, ssp</td><td>adsA, agr, ahpCF, arlRS, aur, cap, chp, clf, cna*, coa, codY, crt, dlt, eap, ebp, efb, esa, ess, esx, flr, fnb, fur, geh, graRSX, hla, hld, hlgABC, hysA, ica, isd, katA, lukGH, lytRS, mgrA, mprF, oatA, psm, RNAIII, rot, rsr, saeRS, sak, sar, saxG, sbi, scn, sdr, sea, seb*, sec*, see, seg*, sei*, selm*, seln*, selo*, selu*, set2, set4, set7, set8, set11*, set13*, set15*, set16*, set22*, sigB, sodA, sodM, spa, spn, srrAB, ssl, ssp, yent1,2*</td></tr></tbody></table>

of 500×. It was identified as phage ECel-2020n (CP062457.1) by BLAST and phage AJ_2017 (NC_048644) by the PHASTER Web server (46). The fourth contig of poor coverage was discarded. Analysis of the Xen36 sequence with Canu v1.6 and Flye v2.9 (47) resulted in a main contig of 2,749,617 bp with 80× coverage, 32.91% G+C content (Table 1), and 4 small contigs, identifying 28,816-bp plasmid CP082814 and 13,162-bp plasmid CP029650.1. Phages of the Xen31 and Xen36 main contigs were detected by PHASTER (Table 1).

A total of 2,742 and 2,528 protein-coding sequences were identified in Xen31 and Xen36, respectively (Table 1). Sequence types were defined using PubMLST. Putative clustered regularly interspaced short palindromic repeat sequences were identified by CRISPRCasFinder (48). Annotation was performed with Prokka v1.14.5 (49). Antimicrobial resistance genes were detected using ResFinder-4.1 (50) and GenBank. Virulence genes were identified using Virulence Factor of Pathogenic Bacteria Database (VFDB) and GenBank.

**Data availability.** Sequences were deposited in ENA under the accession number E-MTAB-12210. Raw data, assemblies, and annotations can be accessed online at https://doi.org/10.5281/zenodo.7517362.

## ACKNOWLEDGMENTS

We thank PerkinElmer for providing S. aureus strains Xen31 and Xen36 and Laurence Ma (Biomics Platform, Institut Pasteur) for strain sequencing.

We acknowledge funding from Institut Pasteur, Direction Générale de l'Armement, l'Agence Innovation Défense (AID), l'Agence Nationale de la Recherche (ANR-17-CE18-0011-02 and ANR-21-ASM1-0001-02), and Université Paris Cité and CNRS. O.B. was supported by an Ecole Polytechnique/AID doctoral fellowship. E.C. was supported by an Institut Pasteur/AID doctoral fellowship.

The Yersinia Research Unit is a member of the Laboratory of Excellence Integrative Biology of Emerging Infectious Diseases (ANR-LBX-62-IBEID).

<footnote><sup>a</sup> tmRNAs, transfer-messenger RNAs; MSSA, methicillin-susceptible *Staphylococcus aureus*.</footnote>

<footnote><sup>b</sup> *, Strain-specific genes.</footnote>
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