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# Complete genome sequences of two Bacillus thuringiensis serovar kurstaki strains isolated from Lebanon and Tunisia, highly toxic against lepidopteran larvae

Nancy Fayad, Rita Barssoum, Nathalie Marsaud, Rayan Nasseredine, Nouha Abdelmalek, Souad Rouis, Marie Ange Teste, Vincent Pailler, Veronique Gautier, Elodie Belmonte, et al.

► To cite this version:

Nancy Fayad, Rita Barssoum, Nathalie Marsaud, Rayan Nasseredine, Nouha Abdelmalek, et al. Complete genome sequences of two Bacillus thuringiensis serovar kurstaki strains isolated from Lebanon and Tunisia, highly toxic against lepidopteran larvae. Microbiology Resource Announcements, 2023, 12 (9), pp.00060-23. 10.1128/MRA.00060-23. hal-04287518

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# Complete genome sequences of two Bacillus thuringiensis serovar kurstaki strains isolated from Lebanon and Tunisia, highly toxic against lepidopteran larvae

Nancy Fayad, Rita Barssoum, Nathalie Marsaud, Rayan Nasseredine, Nouha Abdelmalek, Souad Rouis, Marie Ange Teste, Vincent Pailler, Veronique Gautier, Elodie Belmonte, et al.

► To cite this version:

Nancy Fayad, Rita Barssoum, Nathalie Marsaud, Rayan Nasseredine, Nouha Abdelmalek, et al. Complete genome sequences of two Bacillus thuringiensis serovar kurstaki strains isolated from Lebanon and Tunisia, highly toxic against lepidopteran larvae. Microbiology Resource Announcements, 2023, 12 (9), pp.00060-23. 10.1128/MRA.00060-23. hal-04287518

HAL Id: hal-04287518

https://hal.science/hal-04287518v1

Submitted on 17 May 2024

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entific research documents, whether they are pub-
lished or not. The documents may come from
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destinée au dépôt et à la diffusion de documents
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# Complete genome sequences of two *Bacillus thuringiensis* serovar *kurstaki* strains isolated from Lebanon and Tunisia, highly toxic against lepidopteran larvae

Nancy Fayad,¹,² Rita Barssoum,¹,³ Nathalie Marsaud,³,⁴ Rayan Nasseredine,¹ Nouha Abdelmalek,⁵ Souad Rouis,⁶ Marie Ange Teste,⁴ Vincent Pailler,⁷ Veronique Gautier,⁷ Elodie Belmonte,⁷ César Arturo Aceves Lara,³ Julien Cescut,⁸ Luc Fillaudeau,³ Mireille Kallassy Awad¹

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

**ABSTRACT** *Bacillus thuringiensis*-based products are key in the biopesticides market. *Bacillus thuringiensis kurstaki* strains Lip and BLB1 were isolated from Lebanese and Tunisian soils, respectively. These strains are highly toxic against lepidopteran larvae, *Ephestia kuehniella*. Here, we report Lip and BLB1 complete genomes, including their plasmid and toxin contents.

**KEYWORDS** *Bacillus thuringiensis*, genomes, whole-genome sequencing, PacBio

*Bacillus thuringiensis* serovar *kurstaki* (*Btk*) strains Lip and BLB1 were isolated from Lebanese and Tunisia soil samples, respectively (1–3). Both strains showed an increased toxicity against the lepidopteran *Ephestia kuehniella* larvae in comparison to the reference strain HD-1. Their entomopathogenic potential was evaluated by *in vivo* toxicity assays and showed a lethal concentration of 50% of the larvae (LC50) of 33.27 and 70 ng of toxin per milligram of flour for Lip and BLB1, respectively (1–3). In the context of IPM-4-Citrus (MSCA RISE, No. 734921, 2017–2023), a project aiming to optimize the culture of these strains on a wheat bran-based medium, a whole-genome sequencing (WGS) approach was adopted to elucidate all genomic aspects of *Btk* Lip and BLB1.

For WGS, strains were grown in liquid Luria-Bertani (LB) medium for 16 h at 30°C, after which DNA was extracted using the Monarch HMW DNA Extraction Kit as per manufacturer instructions. WGS was conducted using a PacBio Sequel II Sequencer (Pacific Biosciences, Menlo Park, CA, USA) (4, 5) at the Gentyane platform (Clermont-Ferrand, France). For PacBio sequencing, library was prepared using a SMRTbell prep kit3, following manufacturer instructions. Genomic DNA was sheared, cleaned of single-strand overhangs, repaired for damage and A A tailed. Then, barcoded overhang adapters were ligated to generate the SMRTBell templates. Fragments above 5 kb were then size selected with 35% AMPure PB Beads. A Fragment Analyzer (Agilent Technologies) and a Qubit fluorimeter (Life Technologies) allowed quantity and quality checks. A ready-to-sequence SMRTBell Polymerase Complex was created using a Binding Kit 3.2 (PacBio) and the Sequel II primer 3.2. A consensus sequencing (CCS) mode was adopted, and reads were later demultiplexed with Bam2fastx software (Bioconda) under default parameters.

The number of CCS reads was 524,791 for Lip and 374,689 for BLB1 (Table 1). Assembly was done with flye v2.5, under default parameters (6). Overlaps were manually removed. The final coverage was 417.3× and 746.6× for Lip and BLB1, respectively. Genome completeness was assessed by BUSCO v5.0, using default parameters (7), and was found to be at 99.78% for Lip and 96% for BLB1. Circular contigs were

Editor J. Cameron Thrash, University of Southern California, Los Angeles, California, USA

Address correspondence to Mireille Kallassy Awad, mireille.kallassy@usj.edu.lb.

The authors declare no conflict of interest.

See the funding table on p. 3.

Received 3 February 2023

Accepted 22 June 2023

Published 8 August 2023

Copyright © 2023 Fayad et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
# Complete genome sequences of two *Bacillus thuringiensis* serovar *kurstaki* strains isolated from Lebanon and Tunisia, highly toxic against lepidopteran larvae

Nancy Fayad,¹,² Rita Barssoum,¹,³ Nathalie Marsaud,³,⁴ Rayan Nasseredine,¹ Nouha Abdelmalek,⁵ Souad Rouis,⁶ Marie Ange Teste,⁴ Vincent Pailler,⁷ Veronique Gautier,⁷ Elodie Belmonte,⁷ César Arturo Aceves Lara,³ Julien Cescut,⁸ Luc Fillaureau,³ Mireille Kallassy Awad¹

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

**ABSTRACT** *Bacillus thuringiensis*-based products are key in the biopesticides market. *Bacillus thuringiensis kurstaki* strains Lip and BLB1 were isolated from Lebanese and Tunisian soils, respectively. These strains are highly toxic against lepidopteran larvae, *Ephestia kuehniella*. Here, we report Lip and BLB1 complete genomes, including their plasmid and toxin contents.

**KEYWORDS** *Bacillus thuringiensis*, genomes, whole-genome sequencing, PacBio

*Bacillus thuringiensis* serovar *kurstaki* (*Btk*) strains Lip and BLB1 were isolated from Lebanese and Tunisia soil samples, respectively (1–3). Both strains showed an increased toxicity against the lepidopteran *Ephestia kuehniella* larvae in comparison to the reference strain HD-1. Their entomopathogenic potential was evaluated by *in vivo* toxicity assays and showed a lethal concentration of 50% of the larvae (LC50) of 33.27 and 70 ng of toxin per milligram of flour for Lip and BLB1, respectively (1–3). In the context of IPM-4-Citrus (MSCA RISE, No. 734921, 2017–2023), a project aiming to optimize the culture of these strains on a wheat bran-based medium, a whole-genome sequencing (WGS) approach was adopted to elucidate all genomic aspects of *Btk* Lip and BLB1.

For WGS, strains were grown in liquid Luria-Bertani (LB) medium for 16 h at 30°C, after which DNA was extracted using the Monarch HMW DNA Extraction Kit as per manufacturer instructions. WGS was conducted using a PacBio Sequel II Sequencer (Pacific Biosciences, Menlo Park, CA, USA) (4, 5) at the Gentyane platform (Clermont-Ferrand, France). For PacBio sequencing, library was prepared using a SMRTbell prep kit3, following manufacturer instructions. Genomic DNA was sheared, cleaned of single-strand overhangs, repaired for damage and A A tailed. Then, barcoded overhang adapters were ligated to generate the SMRTBell templates. Fragments above 5 kb were then size selected with 35% AMPure PB Beads. A Fragment Analyzer (Agilent Technologies) and a Qubit fluorimeter (Life Technologies) allowed quantity and quality checks. A ready-to-sequence SMRTBell Polymerase Complex was created using a Binding Kit 3.2 (PacBio) and the Sequel II primer 3.2. A consensus sequencing (CCS) mode was adopted, and reads were later demultiplexed with Bam2fastx software (Bioconda) under default parameters.

The number of CCS reads was 524,791 for Lip and 374,689 for BLB1 (Table 1). Assembly was done with flye v2.5, under default parameters (6). Overlaps were manually removed. The final coverage was 417.3× and 746.6× for Lip and BLB1, respectively. Genome completeness was assessed by BUSCO v5.0, using default parameters (7), and was found to be at 99.78% for Lip and 96% for BLB1. Circular contigs were

Editor J. Cameron Thrash, University of Southern California, Los Angeles, California, USA

Address correspondence to Mireille Kallassy Awad, mireille.kallassy@usj.edu.lb.

The authors declare no conflict of interest.

See the funding table on p. 3.

Received 3 February 2023

Accepted 22 June 2023

Published 8 August 2023

Copyright © 2023 Fayad 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 Sequence features and accession numbers of replicons from Btk strains Lip and BLB1</caption><thead><tr><th></th><th>Replicon</th><th>Bioproject/biosample/NCBI SRA accession</th><th>Accession number</th><th>Length (bp)</th><th>CDS # (total)</th><th># Cry coding genes</th><th>GC content (%)</th><th>Number of PacBio CCS reads/reads N50</th></tr></thead><tbody><tr><td rowspan="12">Lip</td><td>Chromosome</td><td></td><td>CP116313</td><td>5,293,947</td><td>5,632</td><td></td><td>35.2</td><td rowspan="12">524,791/9,650</td></tr><tr><td>pLip2189</td><td></td><td>CP116316</td><td>2,189</td><td>4</td><td></td><td>35.3</td></tr><tr><td>pLip7635</td><td></td><td>CP116320</td><td>7,635</td><td>8</td><td></td><td>32.2</td></tr><tr><td>pLip7911</td><td></td><td>CP116321</td><td>7,911</td><td>9</td><td></td><td>32.3</td></tr><tr><td>pLip8513</td><td></td><td>CP116322</td><td>8,513</td><td>9</td><td></td><td>30.8</td></tr><tr><td>pLip12</td><td></td><td>CP116314</td><td>12,276</td><td>21</td><td></td><td>31.1</td></tr><tr><td>pLip15</td><td>PRJNA924104/</td><td>CP116315</td><td>15,008</td><td>28</td><td></td><td>34.9</td></tr><tr><td>pLip69</td><td>SAMN32746259/</td><td>CP116319</td><td>69,004</td><td>76</td><td></td><td>32.2</td></tr><tr><td>pLip91</td><td>SRX20261752</td><td>CP116323</td><td>91,357</td><td>102</td><td></td><td>31.2</td></tr><tr><td>pLip97</td><td></td><td>CP116324</td><td>97,437</td><td>85</td><td>2: Cry1Ab; Cry1Ac</td><td>34.5</td></tr><tr><td>pLip300</td><td></td><td>CP116317</td><td>300,451</td><td>267</td><td>5: Cry1Aa; Cry1Ac; Cry1la; Cry2Aa; Cry2Ab;</td><td>33.1</td></tr><tr><td>pLip457</td><td></td><td>CP116318</td><td>457,481</td><td>408</td><td></td><td>32.7</td></tr><tr><td rowspan="12">BLB1</td><td>Chromosome</td><td>PRJNA924104/</td><td>CP116325</td><td>5,677,911</td><td>6,060</td><td></td><td>35.3</td><td rowspan="12">374,689/8,967</td></tr><tr><td>pBLB1_7792</td><td>SAMN32746260/</td><td>CP116333</td><td>7,792</td><td>9</td><td></td><td>32.3</td></tr><tr><td>pBLB1_8398</td><td>SRX20261751</td><td>CP116335</td><td>8,398</td><td>10</td><td></td><td>29.8</td></tr><tr><td>pBLB1_8548</td><td></td><td>CP116336</td><td>8,548</td><td>8</td><td></td><td>30.8</td></tr><tr><td>pBLB1_12</td><td></td><td>CP116326</td><td>11,521</td><td>9</td><td>1: Cry1Ab</td><td>40.3</td></tr><tr><td>pBLB1_14</td><td></td><td>CP116327</td><td>14,174</td><td>21</td><td></td><td>32.5</td></tr><tr><td>pBLB1_15</td><td></td><td>CP116328</td><td>15,071</td><td>22</td><td></td><td>31.2</td></tr><tr><td>pBLB1_48</td><td></td><td>CP116330</td><td>47,643</td><td>73</td><td></td><td>35.4</td></tr><tr><td>pBLB1_56</td><td></td><td>CP116331</td><td>56,399</td><td>62</td><td></td><td>32.2</td></tr><tr><td>pBLB1_74</td><td></td><td>CP116332</td><td>73,558</td><td>88</td><td></td><td>30.6</td></tr><tr><td>pBLB1_81</td><td></td><td>CP116334</td><td>80,698</td><td>86</td><td></td><td>33.5</td></tr><tr><td>pBLB1_317</td><td></td><td>CP116329</td><td>317,321</td><td>282</td><td>5: Cry1Aa; Cry1Ac; Cry1la; Cry2Aa; Cry2Ab;</td><td>33.2</td></tr></tbody></table>

first highlighted by the Flye assembler repeat graphs and then further identified as plasmids via a database similarity search using a nucleotide BLAST+ (2.12.0+ [8]) executable *blastn* command. This allowed to search for identified plasmids against the complete *Bacillus thuringiensis* genomes from NCBI assembly (NCBI:txid1428). Additional checks of completeness, identity, and circularity of plasmids were done by multiple sequence alignment comparison with several reference Btk strains: HD-1 (ASM71753v1 [9]), YBT-1520 (ASM74754v1), and HD73 (ASM33875v1 [10]).

Each strain carried 11 different plasmids ranging between 2 and 457 kb. pLip15 was identified as a linear tectiviral prophage, quite similar to GIL16, a tectivirus also isolated from a *Bacillus thuringiensis* strain (11).

Genome annotation was done with NCBI's automated annotation pipeline Prokaryotic Genome Annotation Pipeline (PGAP) (12). Toxin content was mined using t.BLAST.n with the toxin protein sequences recovered from the Bacterial Pesticidal Protein Resource Center (https://www.bpprc-db.org [13]). Genes encoding toxins from the Cry1 and Cry2 families were detected in Lip on plasmids pLip300 and pLip97, and BLB1 on pBLB1_317 and pBLB1_12 (Table 1).
<table><caption>TABLE 1 Sequence features and accession numbers of replicons from *Btk* strains Lip and BLB1</caption><thead><tr><th></th><th>Replicon</th><th>Bioproject/biosample/NCBI SRA accession</th><th>Accession number</th><th>Length (bp)</th><th>CDS # (total)</th><th># Cry coding genes</th><th>GC content (%)</th><th>Number of PacBio CCS reads/reads N50</th></tr></thead><tbody><tr><td rowspan="13">Lip</td><td>Chromosome</td><td></td><td>CP116313</td><td>5,293,947</td><td>5,632</td><td></td><td>35.2</td><td rowspan="13">524,791/9,650</td></tr><tr><td>pLip2189</td><td></td><td>CP116316</td><td>2,189</td><td>4</td><td></td><td>35.3</td></tr><tr><td>pLip7635</td><td></td><td>CP116320</td><td>7,635</td><td>8</td><td></td><td>32.2</td></tr><tr><td>pLip7911</td><td></td><td>CP116321</td><td>7,911</td><td>9</td><td></td><td>32.3</td></tr><tr><td>pLip8513</td><td></td><td>CP116322</td><td>8,513</td><td>9</td><td></td><td>30.8</td></tr><tr><td>pLip12</td><td></td><td>CP116314</td><td>12,276</td><td>21</td><td></td><td>31.1</td></tr><tr><td>pLip15</td><td>PRJNA924104/</td><td>CP116315</td><td>15,008</td><td>28</td><td></td><td>34.9</td></tr><tr><td>pLip69</td><td>SAMN32746259/</td><td>CP116319</td><td>69,004</td><td>76</td><td></td><td>32.2</td></tr><tr><td>pLip91</td><td>SRX20261752</td><td>CP116323</td><td>91,357</td><td>102</td><td></td><td>31.2</td></tr><tr><td>pLip97</td><td></td><td>CP116324</td><td>97,437</td><td>85</td><td>2: Cry1Ab; Cry1Ac</td><td>34.5</td></tr><tr><td>pLip300</td><td></td><td>CP116317</td><td>300,451</td><td>267</td><td>5: Cry1Aa; Cry1Ac; Cry1la; Cry2Aa; Cry2Ab;</td><td>33.1</td></tr><tr><td>pLip457</td><td></td><td>CP116318</td><td>457,481</td><td>408</td><td></td><td>32.7</td></tr><tr><td rowspan="15">BLB1</td><td>Chromosome</td><td>PRJNA924104/</td><td>CP116325</td><td>5,677,911</td><td>6,060</td><td></td><td>35.3</td><td rowspan="15">374,689/8,967</td></tr><tr><td>pBLB1_7792</td><td>SAMN32746260/</td><td>CP116333</td><td>7,792</td><td>9</td><td></td><td>32.3</td></tr><tr><td>pBLB1_8398</td><td>SRX20261751</td><td>CP116335</td><td>8,398</td><td>10</td><td></td><td>29.8</td></tr><tr><td>pBLB1_8548</td><td></td><td>CP116336</td><td>8,548</td><td>8</td><td></td><td>30.8</td></tr><tr><td>pBLB1_12</td><td></td><td>CP116326</td><td>11,521</td><td>9</td><td>1: Cry1Ab</td><td>40.3</td></tr><tr><td>pBLB1_14</td><td></td><td>CP116327</td><td>14,174</td><td>21</td><td></td><td>32.5</td></tr><tr><td>pBLB1_15</td><td></td><td>CP116328</td><td>15,071</td><td>22</td><td></td><td>31.2</td></tr><tr><td>pBLB1_48</td><td></td><td>CP116330</td><td>47,643</td><td>73</td><td></td><td>35.4</td></tr><tr><td>pBLB1_56</td><td></td><td>CP116331</td><td>56,399</td><td>62</td><td></td><td>32.2</td></tr><tr><td>pBLB1_74</td><td></td><td>CP116332</td><td>73,558</td><td>88</td><td></td><td>30.6</td></tr><tr><td>pBLB1_81</td><td></td><td>CP116334</td><td>80,698</td><td>86</td><td></td><td>33.5</td></tr><tr><td>pBLB1_317</td><td></td><td>CP116329</td><td>317,321</td><td>282</td><td>5: Cry1Aa; Cry1Ac; Cry1la; Cry2Aa; Cry2Ab;</td><td>33.2</td></tr></tbody></table>

first highlighted by the Flye assembler repeat graphs and then further identified as plasmids via a database similarity search using a nucleotide BLAST+ (2.12.0+ [8]) executable *blastn* command. This allowed to search for identified plasmids against the complete *Bacillus thuringiensis* genomes from NCBI assembly (NCBI:txid1428). Additional checks of completeness, identity, and circularity of plasmids were done by multiple sequence alignment comparison with several reference *Btk* strains: HD-1 (ASM71753v1 [9]), YBT-1520 (ASM74754v1), and HD73 (ASM33875v1 [10]).

Each strain carried 11 different plasmids ranging between 2 and 457 kb. pLip15 was identified as a linear tectiviral prophage, quite similar to GIL16, a tectivirus also isolated from a *Bacillus thuringiensis* strain (11).

Genome annotation was done with NCBI's automated annotation pipeline Prokaryotic Genome Annotation Pipeline (PGAP) (12). Toxin content was mined using t.BLAST.n with the toxin protein sequences recovered from the Bacterial Pesticidal Protein Resource Center (https://www.bpprc-db.org [13]). Genes encoding toxins from the Cry1 and Cry2 families were detected in Lip on plasmids pLip300 and pLip97, and BLB1 on pBLB1_317 and pBLB1_12 (Table 1).
ACKNOWLEDGMENTS

This research was funded by the research council of Saint-Joseph University of Beirut CR-USJ under grant number FS65 and by the European Union's Horizon 2020 funds: H2020-MSCA-RISE-2016 #734921. M.K.A. mobility (Lebanon-France) was funded by Allocation de perfectionnement à la formation-recherche, November 2021, from the Agence Universitaire de la Francophonie.

AUTHOR AFFILIATIONS

¹Laboratory of Biodiversity and Functional Genomics, Université Saint-Joseph de Beyrouth, Beirut, Lebanon

²Multi-Omics Laboratory, School of Pharmacy, Lebanese American University, Byblos, Lebanon

³Toulouse Biotechnology Institute, Toulouse, France

⁴GenoToul GeT-BioPUCE, Toulouse, France

⁵Laboratoires Pharmaceutiques MédiS, Tunis, Tunisia

⁶Laboratory of Biopesticides, Centre of Biotechnology of Sfax, University of Sfax, Sfax, Tunisia

⁷Gentyane, GDEC, INRAE, Clermont-Ferrand, France

⁸Toulouse White Biotechnology, Toulouse, France

AUTHOR ORCIDs

Nancy Fayad http://orcid.org/0000-0002-8791-490X

Mireille Kallassy Awad http://orcid.org/0000-0003-1489-8686

FUNDING

<table><thead><tr><th>Funder</th><th>Grant(s)</th><th>Author(s)</th></tr></thead><tbody><tr><td>Saint Joseph University (USJ)</td><td>FS65</td><td>Mireille Kallassy Awad</td></tr><tr><td>EC | Horizon 2020 Framework Programme (H2020)</td><td>H2020-MSCA-RISE-2016 #734921</td><td>Rita Barssoum<br>Rayan Nasseredine<br>Nouha Abdelmalek<br>Souad Rouis<br>César Arturo Aceves Lara<br>Julien Cescut<br>Luc Fillauteur<br>Mireille Kallassy Awad</td></tr><tr><td>Agence Universitaire de la Francophonie (AUF)</td><td>Allocation de perfectionnement à la formation-recherche<br>November 2021</td><td>Mireille Kallassy Awad</td></tr></tbody></table>

AUTHOR CONTRIBUTIONS

Nancy Fayad, Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing – original draft, Writing – review and editing | Rita Barssoum, Formal analysis, Methodology, Writing – review and editing | Nathalie Marsaud, Formal analysis, Methodology, Writing – review and editing | Rayan Nasseredine, Methodology, Writing – review and editing, Resources | Nouha Abdelmalek, Investigation, Methodology, Writing – review and editing | Souad Rouis, Writing – review and editing, Resources, Funding acquisition | Marie Ange Teste, Investigation, Writing – review and editing | Vincent Pailler, Methodology, Software, Writing – review and editing, Resources | Veronique Gautier, Methodology, Writing – review and editing, Resources | Elodie Belmonte, Investigation, Writing – review and editing, Resources | César Arturo Aceves Lara, Investigation, Writing – review and editing, Funding acquisition | Julien
ACKNOWLEDGMENTS

This research was funded by the research council of Saint-Joseph University of Beirut CR-USJ under grant number FS65 and by the European Union's Horizon 2020 funds: H2020-MSCA-RISE-2016 #734921. M.K.A. mobility (Lebanon-France) was funded by Allocation de perfectionnement à la formation-recherche, November 2021, from the Agence Universitaire de la Francophonie.

AUTHOR AFFILIATIONS

¹Laboratory of Biodiversity and Functional Genomics, Université Saint-Joseph de Beyrouth, Beirut, Lebanon

²Multi-Omics Laboratory, School of Pharmacy, Lebanese American University, Byblos, Lebanon

³Toulouse Biotechnology Institute, Toulouse, France

⁴GenoToul GeT-BioPUCE, Toulouse, France

⁵Laboratoires Pharmaceutiques MédiS, Tunis, Tunisia

⁶Laboratory of Biopesticides, Centre of Biotechnology of Sfax, University of Sfax, Sfax, Tunisia

⁷Gentyane, GDEC, INRAE, Clermont-Ferrand, France

⁸Toulouse White Biotechnology, Toulouse, France

AUTHOR ORCIDs

Nancy Fayad http://orcid.org/0000-0002-8791-490X

Mireille Kallassy Awad http://orcid.org/0000-0003-1489-8686

FUNDING

<table><thead><tr><th>Funder</th><th>Grant(s)</th><th>Author(s)</th></tr></thead><tbody><tr><td>Saint Joseph University (USJ)</td><td>FS65</td><td>Mireille Kallassy Awad</td></tr><tr><td>EC | Horizon 2020 Framework Programme (H2020)</td><td>H2020-MSCA-RISE-2016 #734921</td><td>Rita Barssoum<br>Rayan Nasseredine<br>Nouha Abdelmalek<br>Souad Rouis<br>César Arturo Aceves Lara<br>Julien Cescut<br>Luc Fillauteur<br>Mireille Kallassy Awad</td></tr><tr><td>Agence Universitaire de la Francophonie (AUF)</td><td>Allocation de perfectionnement à la formation-recherche<br>November 2021</td><td>Mireille Kallassy Awad</td></tr></tbody></table>

AUTHOR CONTRIBUTIONS

Nancy Fayad, Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing – original draft, Writing – review and editing | Rita Barssoum, Formal analysis, Methodology, Writing – review and editing | Nathalie Marsaud, Formal analysis, Methodology, Writing – review and editing | Rayan Nasseredine, Methodology, Writing – review and editing, Resources | Nouha Abdelmalek, Investigation, Methodology, Writing – review and editing | Souad Rouis, Writing – review and editing, Resources, Funding acquisition | Marie Ange Teste, Investigation, Writing – review and editing | Vincent Pailler, Methodology, Software, Writing – review and editing, Resources | Veronique Gautier, Methodology, Writing – review and editing, Resources | Elodie Belmonte, Investigation, Writing – review and editing, Resources | César Arturo Aceves Lara, Investigation, Writing – review and editing, Funding acquisition | Julien
Cescut, Investigation, Writing – review and editing, Funding acquisition | Luc Fillaudeau, Writing – review and editing, Resources, Funding acquisition, Project administration | Mireille Kallassy Awad, Conceptualization, Investigation, Writing – review and editing, Resources, Funding acquisition, Project administration, Supervision

DATA AVAILABILITY

Whole-genome sequences for *Bacillus thuringiensis* serovar *kurstaki* strains Lip and BLB1 are available in NCBI's GenBank under the bioproject number PRJNA924104 for both strains and the accession numbers CP116313 to CP116324 for Lip and CP116325 to CP116336 for BLB1. Raw reads are also available on the NCBI SRA database under the bioproject number PRJNA924104.

REFERENCES

1. Saadaoui I, Rouis S, Jaoua S. 2009. A new Tunisian strain of *Bacillus thuringiensis kurstaki* having high Insecticidal activity and δ-endotoxin yield. Arch Microbiol 191:341-348. https://doi.org/10.1007/s00203-009-0458-y

2. El Khoury M, Azzouz H, Chavanieu A, Abdelmalak N, Chopineau J, Awad MK. 2014. Isolation and characterization of a new *Bacillus thuringiensis* strain lip harboring a new cry1Aa gene highly toxic to *Ephestia kuehniella* (Lepidoptera: Pyralidae) larvae. Arch Microbiol 196:435-444. https://doi.org/10.1007/s00203-014-0981-3

3. Fayad N, Abboud J, Driss F, Louka N, Kallassy Awad M. 2022. Optimization of culture conditions and wheat bran class selection in the production of *Bacillus thuringiensis*-based biopesticides. Fermentation 8:666. https://doi.org/10.3390/fermentation8120666

4. Rhoads A, Au KF. 2015. PacBio sequencing and its applications. Genomics Proteomics Bioinformatics 13:278-289. https://doi.org/10.1016/j.gpb.2015.08.002

5. Kanwar N, Blanco C, Chen IA, Seelig B. 2021. PacBio sequencing output increased through uniform and directional fivefold concatenation. Sci Rep 11:18065. https://doi.org/10.1038/s41598-021-96829-z

6. Kolmogorov M, Yuan J, Lin Y, Pevzner PA. 2019. Assembly of long, error-prone reads using repeat graphs. Nat Biotechnol 37:540-546. https://doi.org/10.1038/s41587-019-0072-8

7. Seppey M, Manni M, Zdobnov EM. 2019. BUSCO: assessing genome assembly and annotation completeness. Methods Mol Biol 1962:227-245. https://doi.org/10.1007/978-1-4939-9173-0_14

8. Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL. 2009. BLAST+: architecture and applications. BMC Bioinformatics 10:421. https://doi.org/10.1186/1471-2105-10-421

9. Day M, Ibrahim M, Dyer D, Bulla L. 2014. Genome sequence of *Bacillus thuringiensis* subsp. *kurstaki* strain HD-1. Genome Announc 2:e00613-14. https://doi.org/10.1128/genomeA.00613-14

10. Liu G, Song L, Shu C, Wang P, Deng C, Peng Q, Lereclus D, Wang X, Huang D, Zhang J, Song F. 2013. Complete genome sequence of *Bacillus thuringiensis* subsp. *kurstaki* strain HD73. Genome Announc 1:e0008013. https://doi.org/10.1128/genomeA.00080-13

11. Verheust C, Fornelos N, Mahillon J. 2005. GIL16, a new gram-positive tectiviral phage related to the *Bacillus thuringiensis* GIL01 and the *Bacillus cereus* pBClin15 elements. J Bacteriol 187:1966-1973. https://doi.org/10.1128/JB.187.6.1966-1973.2005

12. Li W, O'Neill KR, Haft DH, DiCuccio M, Chetvernin V, Badretdin A, Coulouris G, Chitsaz F, Derbyshire MK, Durkin AS, Gonzales NR, Gwadz M, Lanczycki CJ, Song JS, Thanki N, Wang J, Yamashita RA, Yang M, Zheng C, Marchler-Bauer A, Thibaud-Nissen F. 2021. RefSeq: expanding the prokaryotic genome annotation pipeline reach with protein family model curation. Nucleic Acids Res 49:D1020-D1028. https://doi.org/10.1093/nar/gkaa1105

13. Crickmore N, Berry C, Panneerselvam S, Mishra R, Connor TR, Bonning BC. 2021. A structure-based nomenclature for *Bacillus thuringiensis* and other bacteria-derived pesticidal proteins. J Invertebr Pathol 186:107438. https://doi.org/10.1016/j.jip.2020.107438
Cescut, Investigation, Writing – review and editing, Funding acquisition | Luc Fillaudeau, Writing – review and editing, Resources, Funding acquisition, Project administration | Mireille Kallassy Awad, Conceptualization, Investigation, Writing – review and editing, Resources, Funding acquisition, Project administration, Supervision

DATA AVAILABILITY

Whole-genome sequences for *Bacillus thuringiensis* serovar *kurstaki* strains Lip and BLB1 are available in NCBI's GenBank under the bioproject number PRJNA924104 for both strains and the accession numbers CP116313 to CP116324 for Lip and CP116325 to CP116336 for BLB1. Raw reads are also available on the NCBI SRA database under the bioproject number PRJNA924104.

REFERENCES

1. Saadaoui I, Rouis S, Jaoua S. 2009. A new Tunisian strain of *Bacillus thuringiensis kurstaki* having high Insecticidal activity and δ-endotoxin yield. Arch Microbiol 191:341-348. https://doi.org/10.1007/s00203-009-0458-y

2. El Khoury M, Azzouz H, Chavanieu A, Abdelmalak N, Chopineau J, Awad MK. 2014. Isolation and characterization of a new *Bacillus thuringiensis* strain lip harboring a new cry1Aa gene highly toxic to *Ephestia kuehniella* (Lepidoptera: Pyralidae) larvae. Arch Microbiol 196:435-444. https://doi.org/10.1007/s00203-014-0981-3

3. Fayad N, Abboud J, Driss F, Louka N, Kallassy Awad M. 2022. Optimization of culture conditions and wheat bran class selection in the production of *Bacillus thuringiensis*-based biopesticides. Fermentation 8:666. https://doi.org/10.3390/fermentation8120666

4. Rhoads A, Au KF. 2015. PacBio sequencing and its applications. Genomics Proteomics Bioinformatics 13:278-289. https://doi.org/10.1016/j.gpb.2015.08.002

5. Kanwar N, Blanco C, Chen IA, Seelig B. 2021. PacBio sequencing output increased through uniform and directional fivefold concatenation. Sci Rep 11:18065. https://doi.org/10.1038/s41598-021-96829-z

6. Kolmogorov M, Yuan J, Lin Y, Pevzner PA. 2019. Assembly of long, error-prone reads using repeat graphs. Nat Biotechnol 37:540-546. https://doi.org/10.1038/s41587-019-0072-8

7. Seppey M, Manni M, Zdobnov EM. 2019. BUSCO: assessing genome assembly and annotation completeness. Methods Mol Biol 1962:227-245. https://doi.org/10.1007/978-1-4939-9173-0_14

8. Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL. 2009. BLAST+: architecture and applications. BMC Bioinformatics 10:421. https://doi.org/10.1186/1471-2105-10-421

9. Day M, Ibrahim M, Dyer D, Bulla L. 2014. Genome sequence of *Bacillus thuringiensis* subsp. *kurstaki* strain HD-1. Genome Announc 2:e00613-14. https://doi.org/10.1128/genomeA.00613-14

10. Liu G, Song L, Shu C, Wang P, Deng C, Peng Q, Lereclus D, Wang X, Huang D, Zhang J, Song F. 2013. Complete genome sequence of *Bacillus thuringiensis* subsp. *kurstaki* strain HD73. Genome Announc 1:e0008013. https://doi.org/10.1128/genomeA.00080-13

11. Verheust C, Fornelos N, Mahillon J. 2005. GIL16, a new gram-positive tectiviral phage related to the *Bacillus thuringiensis* GIL01 and the *Bacillus cereus* pBClin15 elements. J Bacteriol 187:1966-1973. https://doi.org/10.1128/JB.187.6.1966-1973.2005

12. Li W, O'Neill KR, Haft DH, DiCuccio M, Chetvernin V, Badretdin A, Coulouris G, Chitsaz F, Derbyshire MK, Durkin AS, Gonzales NR, Gwadz M, Lanczycki CJ, Song JS, Thanki N, Wang J, Yamashita RA, Yang M, Zheng C, Marchler-Bauer A, Thibaud-Nissen F. 2021. RefSeq: expanding the prokaryotic genome annotation pipeline reach with protein family model curation. Nucleic Acids Res 49:D1020-D1028. https://doi.org/10.1093/nar/gkaa1105

13. Crickmore N, Berry C, Panneerselvam S, Mishra R, Connor TR, Bonning BC. 2021. A structure-based nomenclature for *Bacillus thuringiensis* and other bacteria-derived pesticidal proteins. J Invertebr Pathol 186:107438. https://doi.org/10.1016/j.jip.2020.107438