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

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 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).

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.