Draft Genome Sequence of Bacillus thuringiensis INTA 103-23 Reveals Its Insecticidal Properties: Insights from the Genomic Sequence

Abstract

The genome of Bacillus thuringiensis strain INTA 103-23 was sequenced, revealing a high-quality draft assembly comprising 243 contigs with a total size of 6.30 Mb and a completeness of 99%. Phylogenetic analysis classified INTA 103-23 within the Bacillus cereus sensu stricto cluster. Genome annotation identified 6993 genes, including 2476 hypothetical proteins. Screening for pesticidal proteins unveiled 10 coding sequences with significant similarity to known pesticidal proteins, showcasing a potential efficacy against various insect orders. AntiSMASH analysis predicted 13 biosynthetic gene clusters (BGCs), including clusters with 100% similarity to petrobactin and anabaenopeptin NZ857/nostamide A. Notably, fengycin exhibited a 40% similarity within the identified clusters. Further exploration involved a comparative genomic analysis with ten phylogenetically closest genomes. The ANI values, calculated using fastANI, confirmed the closest relationships with strains classified under Bacillus cereus sensu stricto. This comprehensive genomic analysis of B. thuringiensis INTA 103-23 provides valuable insights into its genetic makeup, potential pesticidal activity, and biosynthetic capabilities. The identified BGCs and pesticidal proteins contribute to our understanding of the strain’s biocontrol potential against diverse agricultural pests.

Dataset: The raw genome sequencing data were submitted to the NCBI with BioSample number SAMN39459532, under BioProject PRJNA1065439. The assembled genome is available in the NCBI WGS project, under accession number JAYWUX000000000.

Dataset License: CC-BY license

1. Summary

Bacillus thuringiensis, an adept Gram-positive bacterium with spore-forming capabilities, has established itself as a predominant resident in diverse ecosystems, particularly thriving in soil environments [1]. Renowned for its remarkable protein synthesis machinery, B. thuringiensis synthesizes a diverse array of insecticidal proteins, including Cry, Cyt, Vip, Vpa/Vpb, and Sip, each exhibiting a high specificity against various insect orders [2]. The integration of genes encoding these insecticidal proteins into crops, known as Bt crops, imparts targeted resistance against insect pests [3].

Bacillus thuringiensis proteins exhibit toxicity upon ingestion, primarily targeting the host’s midgut epithelium [4]. In the form of crystalline inclusions, such as the bacterial parasporal crystal containing Cry and Cyt proteins, they are solubilized to release protoxins. These protoxins are activated by midgut proteases and then cross the peritrophic matrix and bind to specific receptors on the midgut epithelial membrane. The oligomerization of B. thuringiensis proteins forms pores, each likely consisting of four toxin monomers assembled into a pore-forming oligomer, observed in both Cry and Vip3 proteins [5,6]. Upon insertion into the epithelial membrane, these pores disrupt it, allowing gut bacteria invasion into the hemolymph, ultimately resulting in septicemia and insect mortality [7].

In addition to its well-established insecticidal prowess, select strains of B. thuringiensis showcase additional advantageous activities, manifesting nematicidal, bactericidal, fungicidal, and plant growth-promoting activities in specific contexts [8,9]. Notably, the environmentally friendly characteristics of B. thuringiensis have fueled their widespread adoption as integral components in global crop pest control initiatives [10].

The utility of these proteins has catalyzed ongoing efforts to explore novel B. thuringiensis isolates, with the goal of identifying and characterizing insecticidal proteins possessing unique specificities. Addressing the challenge of insect genetic resistance, particularly in transgenic crops expressing B. thuringiensis genes, necessitates the exploration of alternative strategies. The advent of revolutionary next-generation sequencing technology has facilitated the sequencing of numerous genomes from B. thuringiensis, aiming to unveil new invertebrate toxic proteins applicable to the biological control of agricultural pests and disease vectors (mosquitoes) [11,12,13].

This paper aims to bridge this knowledge gap by presenting the first sequenced and analyzed genome of the Argentine B. thuringiensis strain INTA 103-23, shedding light on its genetic makeup and potential applications in biocontrol strategies. Through elucidating the genomic underpinnings of this strain, we aim to contribute to the advancement of sustainable pest management practices and the development of environmentally friendly solutions for agricultural and public health challenges.

2. Data Description

2.1. Isolation, Morphological Characterization, and Insecticidal Activity of Bacillus thuringiensis INTA 103-23

Bacillus thuringiensis INTA 103-23, isolated in 2005 from a waterlogged soil sample in Rafaela, Santa Fe, Argentina, exhibits distinctive characteristics when cultivated on a sporulation BM agar medium [14]. After 72 h at 29 °C, the strain forms round, elevated, opaque colonies with a matte texture, displaying a creamy white color (Figure 1A). The vegetative cells are rod-shaped and can form subterminal spores. Upon completion of the sporulation phase, ovoid parasporal crystals are released from the sporangia (Figure 1B).

INTA 103-23 was preliminary classified as B. thuringiensis based on its ability to produce parasporal crystals with insecticidal activity against second instar ladybird beetle (Epilachna paenulata, Coleoptera: Coccinellidae) [15] and neonate cotton boll weevil (Anthonomus grandis, Coleoptera: Curculionidae) larvae [14]. Furthermore, INTA 103-23 is characterized by the absence of thuringiensin (β-exotoxin) production [14].

2.2. Genome Assembly and Annotation

The genome of the INTA 103-23 strain was sequenced using the Illumina HiSeq X platform, generating high-quality paired-end reads (150 bp). Quality control with the FastQC v0.12.1 [16] program confirmed this, showing uniform quality scores, GC content, and acceptable N content across all bases for both raw and adapter-trimmed DNA libraries. SPAdes v3.15.4 [17] assembled the genome into 243 contigs with a total size of 6.30 Mb and an N50 of 0.92 Mb. Notably, it achieved 99% completeness and only 1% contamination, as assessed by CheckM v1.0.18 [18]. Additional details about the assembly are provided in

Table 1. Key characteristics of the draft genome assembly of INTA 103-23 obtained using QUAST v4.4 [19] and CheckM v1.0.18 [18].

Total contig count	243
Largest contig (nucleotide count)	304,352
Total length (nucleotide count)	6,302,053
GC-content (%)	34.94
N50 value	92,502
N75 value	48,719
L50 value	21
L75 value	43
Assembly completeness (%)	99.43
Suspected contamination (%)	0.57

.

When employing the BUSCO v5.5.0 program [20], our analysis revealed that the quantity of fully assembled single-copy orthologs reached a minimum of 99.8%, compared to the Bacillales_odb10 dataset (

Table 2. Assessment of BUSCO v5.5.0 marker [20] presence in protein-coding genes within the assembly. The table displays the count of orthologs identified in the assembly alongside their corresponding percentages.

Database	Bacillales_odb10
Single-copy orthologues assembled completely	449 (99.8%)
Orthologues present in one copy	444 (98.7%)
Multi-copy orthologues	5
Fragmented sequences	0
Orthologues missing from the assembly	1
Total count of singleton orthologs in the dataset	450

). These findings unequivocally attest to the high quality and comprehensiveness of the assembled genome.

A phylogenetic investigation performed with the Type Strain Genome Server (TYGS) [21] revealed that INTA 103-23 formed a distinct cluster alongside strain types B. thuringiensis ATCC 10792 and B. cereus ATCC 14579 (Figure 2). In accordance with the recently suggested classification, this cluster, referred to as Bacillus cereus sensu stricto, includes the majority of biovar Thuringiensis strains [22].

Subsequently, we selected ten bacterial genome assemblies from the Bacillaceae family available in the NCBI RefSeq database [23], focusing on those with the closest phylogenetic relationships. This selection process relied on the calculation of average nucleotide identity (ANI) values using the fastANI v0.1.3 tool [24], as detailed in

Table 3. Phylogenetically closest assemblies to the genome of INTA 103-23: ANI values calculated with the FastANI v0.1.3 software [24] and taxonomic assignment through the TYGS [21].

NCBI RefSeq Assembly	Strain	ANI	TYGS Taxonomy
GCF_900114335.1	5MFCol3.1	98.2317	B. cereus sensu stricto
GCF_001583685.1	FSL K6-0043	98.2235	B. cereus sensu stricto
GCF_000412975.1	BAG1O-3	98.2222	B. cereus sensu stricto
GCA_000293725.1	HuB1-1	98.2017	B. cereus sensu stricto
GCF_000633295.1	H1m	98.1528	B. cereus sensu stricto
GCF_000291035.1	BAG1X1-2	98.1277	B. cereus sensu stricto
GCF_001883875.1	B-2012	98.0455	B. cereus sensu stricto
GCF_900142585.1	BC15	98.0288	B. cereus sensu stricto
GCF_002146345.1	BGSC 4AD1	97.9884	B. cereus sensu stricto
GCF_002146395.1	BGSC 4AG1	97.9505	B. cereus sensu stricto

. The strains displaying the highest degree of similarity in nucleotide sequences among all the genomes were identified as members of Bacillus cereus sensu stricto, thus affirming the accurate taxonomic classification of the INTA 103-23 strain (Table 3).

The annotation using RASTtk—v1.073 revealed that the genome of INTA 103-23 contains 6993 genes, with 6736 being coding sequences and 2476 of them marked as hypothetical proteins (Supplementary Data S1). The draft genome sequence of INTA 103-23 harbors potential pesticidal proteins and virulence factors associated with insect pathogenesis. Eleven coding sequences (CDS) showed significant BlastX [25] similarity to known pesticidal proteins such as Mpp46, Spp1, Xpp22, Cry73, Cry54, Cry4, Tpp49, and Tpp36, which have demonstrated efficacy against a broad spectrum of insects, spanning five different orders (

Table 4. Pesticidal proteins repertoire identified in the INTA103-23 genome. Experimentally derived data from the Bacterial Pesticidal Protein Resource Center (BPPRC) specificity database for the target species [27].

Contig	Insecticidal Proteins	Percent of Identity	Target Order	Target Species	Assay Method
28	Mpp46Ab1	34	Diptera	Culex pipens	Addition to water
35	Spp1Aa1	80	Blattodea	Blattella germanica	Injection
			Lepidoptera	Spodoptera litura	Injection
87	Xpp22Ab1	34	Coleoptera	Cylas brunneus	Diet incorporation
			Coleoptera	Cylas puncticollis	Diet incorporation
			Coleoptera	Anthonomus grandis	Diet incorporation
			Coleoptera	Diabrotica virgifera virgifera	Surface contamination
			Lepidoptera	Plutella xylostella	Surface contamination
			Lepidoptera	Trichoplusia ni	Surface contamination
104	Cry73Aa1	39	ND a	ND	ND
152	Cry54Ba2	34	ND	ND	ND
153	Cry4Ba2	31	Diptera	Aedes aegypti	Addition to water
168	Cry4Ba4	47	Diptera	Anopheles albimanus	Addition to water
			Diptera	Anopheles gambiae	Addition to water
			Diptera	Anopheles stephensi	Addition to water
			Diptera	Culex pipens	Addition to water
			Diptera	Culex quinquefasciatus	Addition to water
			Diptera	Simulium spp.	Addition to water
206	Tpp49Aa4	52	Diptera	Culex quinquefasciatus	Addition to water
212	Tpp36Aa1	38	Coleoptera	Diabrotica virgifera virgifera	Surface contamination
215	Tpp36Aa1	31	Coleoptera	Diabrotica virgifera virgifera	Surface contamination

^a ND, not determined.

). Additionally, INTA 103-23 contains other coding sequences encoding ten putative chitinases and six chitin-binding proteins. As expected, genes associated with thuringiensin synthesis, such as thuE, were absent [26]. The demonstrated insecticidal efficacy of INTA 103-23 against E. paenulata [15] and A. grandis [16] may be attributed to coleoptericidal proteins encoded by genes identified within its genome (Table 4). Furthermore, the presence of genes encoding pesticidal toxins known to target mosquito and lepidopteran larvae in the genome of INTA 103-23 suggests a potential, broader insecticidal activity (Table 4). However, further validation through bioassays is warranted to confirm and characterize this association.

The application of the antiSMASH v6.1.1 tool [28] identified a total of 13 biosynthetic gene clusters (BGCs), as outlined in

Table 5. Predicted biosynthetic gene clusters in the INTA 103-23 genome with the antiSMASH v6.1.1 [28] program.

Contig	Type/Activity	Location (Relative Coordinate, b.p. a)	Most Similar Known Cluster	Percent of Similarity
4	Betalactone	1–18,969 (18,969)	Fengycin	40
4	NRPS	92,175–158,083 (65,909)	-	-
27	LAP, RiPP-like	9582–33,088 (23,507)	-	-
28	Ladderane	1–36,711 (36,711)	S-layer glycan	26
34	Siderophore	5069–18,776 (13,708)	Petrobactin	100
37	NRPS-like	4398–47,979 (43,582)	-	-
45	NRPS	4439–44,738 (30,300)	-	-
46	Terpene	7890–29,743 (21,854)	Molybdenum cofactor	17
61	RiPP-like	6156–16,377 (10,222)	-	-
72	NRPS	1–18,492 (18,492)	Anabaenopeptin NZ857/nostamide A	100
75	Ranthipeptide	1594–16,138 (14,545)	-	-
90	NRPS	1–10,777 (10,777)	Bacillibactin	23
97	RiPP-like	1–8503 (8503)	-	-

^a The total length of the regions is provided in brackets following the genomic coordinates relative to each contig.

. Notably, among the identified BGCs, the ones bearing the highest similarity were associated with well-known entities such as petrobactin and anabaenopeptin NZ857/nostamide A, exhibiting 100% similarity. Additionally, it is noteworthy that fengycin showed a 40% similarity within the identified clusters.

The present study unveils the genetic makeup and potential applications of the Argentine B. thuringiensis INTA 103-23 strain. By leveraging next-generation sequencing technology, we sequenced and analyzed the genome of INTA 103-23, shedding light on its genomic characteristics and insecticidal potential. Our findings contribute to the broader understanding of B. thuringiensis and its role in pest management strategies.

In conclusion, the sequencing and analysis of the INTA 103-23 genome offers valuable insights into its genetic composition and potential applications. The presence of genes encoding pesticidal proteins and other virulence factors underscores its potential as a biocontrol agent against agricultural pests and disease vectors. Moreover, the absence of genes associated with thuringiensin synthesis suggests alternative mechanisms for insecticidal activity. The elucidation of the genomic underpinnings of INTA 103-23 paves the way for the further exploration and utilization of this strain in sustainable pest management practices. This research contributes to the advancement of knowledge regarding B. thuringiensis and its applications in agriculture and public health.

3. Methods

3.1. DNA Extraction, Library Construction, and Massive Genome Sequencing

DNA, comprising both chromosome and plasmids, was extracted utilizing the Wizard genomic DNA purification kit (Promega, Madison, WI, USA). Subsequently, the extracted DNA underwent electrophoresis, was stained with SYBR Safe, and quantified through a Multiskan SkyHigh µDrop Plate spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). A comprehensive Illumina library was then constructed using the purified DNA and subjected to high-throughput Illumina sequencing at the Unidad Operativa Centro Nacional de Genómica y Bioinformática (ANLIS Malbrán, Santiago del Estero, Argentina) in the paired-end mode, producing reads with a length of 2 × 150 bp. Finally, the short nucleotide reads underwent quality control using FastQC v0.12.1 [16].

3.2. Genome Assembly, Sequence Analysis, and Annotation

Genome assembly was performed de novo using the SPAdes v3.15.4 genome assembler [13], and the resulting assembly underwent a rigorous quality control assessment with CheckM v1.0.18 [18] and QUAST v4.4 [19]. To evaluate the taxonomy-wise completeness, the percentage of one-copy orthologs from the “Bacillales_odb10” database was determined using BUSCO v5.5.0 [20]. Species delimitation was performed using the TYGS [21].

In the subsequent phase, we employed fastANI v1.33 [24] to identify the ten phylogenetically closest genomes downloaded from the NCBI RefSeq database [23], selecting those with the highest ANI values when compared to our assembly. Initial genome annotation was carried out using the NCBI Prokaryotic Genome Annotation Pipeline (2023 release) and RASTtk—v1.073 [29].

Following annotation, a targeted BLAST analysis was conducted to identify genes encoding pesticidal proteins, utilizing a customized non-redundant insecticidal protein database. The putative susceptible insect species affected by the identified virulence factors were obtained from the Bacterial Pesticidal Protein Resource Center (BPPRC) specificity database [27]. The prediction and characterization of biosynthetic gene clusters were accomplished using antiSMASH v6.1.1 [29].