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Article

Isolation, Genomic, and Proteomic Characterization of a Novel Neotropical Strain of Bacillus thuringiensis with Mosquitocidal Activities

by
Giselly Batista Alves
1,
Marcelo Leite Dias
1,
Eugenio Eduardo de Oliveira
2,
Gil Rodrigues dos Santos
3,
Bergmann Morais Ribeiro
4 and
Raimundo Wagner de Souza Aguiar
1,*
1
Department of Biotechnology, Universidade Federal do Tocantins, Gurupi 77410-530, TO, Brazil
2
Department of Entomology, Universidade Federal de Viçosa, Viçosa 36570-900, MG, Brazil
3
Laboratory of Phytopathology, Universidade Federal do Tocantins, Gurupi 77410-530, TO, Brazil
4
Department of Cell Biology, Universidade de Brasília, Brasília 70910-900, DF, Brazil
*
Author to whom correspondence should be addressed.
Processes 2023, 11(5), 1455; https://doi.org/10.3390/pr11051455
Submission received: 24 February 2023 / Revised: 11 April 2023 / Accepted: 25 April 2023 / Published: 11 May 2023
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Abstract

:
The combination of genomic and proteomic analyses is a useful tool for the study of novel Bacillus thuringiensis (Bt) strains, as these approaches allow the accurate identification of pesticidal proteins and virulence factors produced. Here, we isolated and evaluated the potential of a novel Neotropical Bt strain (TOD651) for controlling larvae of Aedes aegypti and Culex quinquefasciatus mosquitoes. Aiming for the full comprehension of the TOD651 larvicidal potential, we further evaluated the whole TOD651 genome and conducted the proteomic analysis of the TOD651 spore–crystal mixtures. Our results showed that Bt TOD651 similarly killed both A. aegypti (0.011 µg/mL) and C. quinquefasciatus (0.023 µg/mL) larvae, exhibiting similar potency to the commercial Bt strain. The genome sequence revealed that Bt TOD651 harbors cry11Aa3, cry10Aa4, cry4Aa4, cry4Ba5, cyt1Aa5, cyt1Ca1, cyt2Ba13, mpp60Aa3, and mpp60Ba3. The proteomic analysis revealed no expression of Mpp60Aa3, while all the other pesticidal proteins were expressed (Cry4Ba5 was more abundant than Cyt1Aa5). The expression of the Mppe showed the major proportions between proteases. The virulent factor neutral protease B and spore coat proteins were also expressed. The expression of relevant pesticidal proteins (e.g., Cry, Cyt, Mpp, and other pathogenic factors), whose actions can occur in a synergic relation, indicates that the biocontrol using Bt TOD651 may contribute to delaying the selection of resistant individuals.

Graphical Abstract

1. Introduction

The control of insect vectors of different diseases is of great importance for public health. Mosquito species such as Aedes aegypti (Linnaeus, 1762) (Diptera: Culicidae) and Culex quinquefasciatus (Say, 1823) (Diptera: Culicidae) can transmit several diseases that affect human life. For instance, A. aegypti can transmit yellow fever virus, dengue virus, Chikungunya virus, and Zika virus, while C. quinquefasciatus is capable of transmitting arboviruses such as West Nile Virus (WNV) and the Wuchereria bancrofi nematode, responsible for lymphatic filariasis disease [1].
The use of chemical insecticides for the control of mosquitoes, which harm the environment, has been slowly substituted around the world by biological control strategies, such as Bacillus thuringiensis (Bt) [2]. Bt is a Gram-positive bacterium known for its toxicity and specificity towards insect hosts due to its ability to produce and release crystal proteins (Cry and Cyt) during the sporulation stage [3]. Bacillus thuringiensis serovar israelensis (Bti) is one of the subspecies’ most effective larvicides for mosquito control, being recommended by the World Health Organization (WHO) [4,5]. Bti produces Cry and Cyt crystals (Cry4Aa, Cry4Ba, Cry10Aa, Cry11Aa, Cyt1Aa, and Cyt2Ba) that exhibit toxicity against mosquito species from the genus Aedes, Anopheles, and Culex, and for the black fly (Simuliidae) [4,6,7,8]. These proteins synergistically interact and can decrease the incidence of resistance in insect populations [4]. Some Bti strains can also harbor Mpp60A and Mpp60B proteins, which are present in other subspecies such as jegathesan and malayensis [8].
Improvements in the control of mosquitoes using Bt have involved the constant identification and characterization of novel strains and pesticidal proteins [9,10,11,12]. In addition, next-generation sequencing (NGS) has allowed the whole-genome sequencing of novel Bt strains and their characterization. Genome information has been important in research and applications of Bt because pesticidal genes are easily detected [13,14,15]. Furthermore, other genes related to the pathogenicity of Bt can be explored through this technique, such as virulence factors and other secondary metabolites [16,17].
Despite the characterization of genes coding pesticidal proteins allowing strain classification, it is the expression of these genes that determines their spectrum of activity [18,19]. In the genome sequence, not all annotated coding regions are expressed, and the genomic approach may not suffice to fully explain the toxicity differences between Bt strains [20,21]. For instance, many pesticidal proteins are cryptic or have insignificant levels of expression [22]. Thus, proteomic analysis of the pesticidal proteins that make up the parasporal crystal is essential to understand the toxicity of novel and commercial Bt strains [19]. In this context, the combination of genomic and proteomic analyses is a powerful tool for the accurate identification of pesticidal proteins and virulence factors of Bt strains [23,24,25], and estimations of the abundance of such proteins can be achieved in purified parasporal crystals and spore–crystal mixtures [25,26].
Several studies have been conducted to investigate the genomics of mosquitocidal Bt strains [21,27,28,29]. Interestingly, the proteomic analysis for pesticidal proteins responsible for mosquitocidal activities in Bt strains remains scarce and underexploited. Therefore, we isolated a novel Bt strain (Bt TOD651) and evaluated its insecticidal activity against larvae of A. aegypti and C. quinquefasciatus. To explore and better understand its toxicity, we sequenced the whole genome of TOD651 and performed proteomic analysis of the spore–crystal mixture.

2. Materials and Methods

2.1. Origin and Culture of Bt TOD651 Strain

The Bt TOD651 strain was isolated from a soil sample, collected in the state of Tocantins, Brazil (11°43′45″ S; 49°04′07″ W), according to Monnerat et al. [30]. This strain was cultured at 28 °C for 12 h in Luria–Bertani (LB) solid medium (10 gL−1 tryptone, 5 gL−1 yeast extract, 10 gL−1 NaCl, and 20 gL−1 agar). Posteriorly, a single colony of Bt TOD651 was transferred to an LB liquid medium and incubated (28 °C at 200 rpm for 16 h) for sporulation and DNA extraction steps. The Bti AM65-52 strain was isolated from a commercial sample (VectoBac®, Sumitomo, Tokyo, Japan) and used as a reference strain.

2.2. Crystal Protein Purification and SDS-PAGE Analysis

An aliquot of LB culture (3 mL) was transferred to CCY medium (30 mL) (13 mM KH2P04, 26 mM K2HP04, 0.002% (w/v) L-glutamine, 0.1% (w/v) casein hydrolysate, 0.1% (w/v) bacto casitone, 0.04% bacto yeast extract, 0.6% (w/v) glycerol, 0.05 M ZnCl2, 0.5 M MgCl2, 0.01 M MnCI2, 0.2 M CaCl2, 0.05 M FeCl3) and incubated for sporulation (28 °C at 200 rpm for 72 h). Then, the spore–crystal mixture was collected, and the crystal proteins were purified according to a previously described method [31]. Purified crystals were suspended in a small volume of phosphate-buffered saline (136 mM NaCl, 1.4 mM KH2PO4, 2.6 mM KCl, 8 mM Na2HPO4, and 4.2 mL H2O, pH 7.4), and fractionated by electrophoresis on a 10% SDS-PAGE gel [30].

2.3. Identification of Crystal Morphology

The morphological characterization of Cry protein crystals was performed by scanning electron microscopy. The spore–crystal mixture of Bt TOD651 was collected and diluted in sterile water. Then, a 100 µL aliquot of the diluted suspension was placed on metallic supports and dried for 24 h at 37 °C, covered with gold for 180 s using an Emitech apparatus (model K550; Quorum Technologies, Lewes, UK), and observed under a Zeiss scanning electron microscope (model DSM 962; Carl Zeiss AG, Oberkochen, Germany) at 10 or 20 Kv.

2.4. Larvae Rearing

The larvae of A. aegypti and C. quinquefasciatus were collected from fields without the application of insecticides, in regions of transition between urban and rural areas in the state of Tocantins, Brazil (11°40′55.7″ latitude S, 49°04′3.9″ longitude W). The insect colonies were established in the Entomology Laboratory of the Federal University of Tocantins, Gurupi Campus, according to Aguiar et al. [32]. The larvae were reared in plastic containers (40 cm × 25 cm × 8 cm) and fed a sterilized diet (an 80/20 mix of chick chow powder/yeast), and mosquitoes were provided with a 10% sucrose solution. We blood-fed the adult females five days after emergence with defibrinated sheep blood using a membrane feeder device [33].

2.5. Bioassays

Using the spore–crystal mixtures, bioassays were conducted on A. aegypti and C. quinquefasciatus third-instar larvae. The concentrations were determined according to Mclaughlin et al. [34]. Seven concentrations of the spore–crystal mixtures (0.05, 0.10, 0.15, 0.20, 0.25, 0.30, and 0.40 µg/mL) were tested, and sterile distilled water was used as a negative control. Bioassays were performed in 3 replicates with 25 larvae in 100 mL of distilled water. Treated larvae were kept at 26 ± 1 °C, 60.0 ± 5% RH, and a 12 h light–dark photoperiod. The number of dead and live larvae was counted after 24 h. The larvae that did not move when touched with a sterile stick were considered dead [35]. The spore–crystal mixture from the AM65-52 strain was used as a reference. Concentration–mortality curves were estimated by probity analysis using the PROBIT procedure in the SAS software [36].

2.6. Whole-Genome Sequencing, Assembly, and Annotation

Genomic DNA was extracted using the Wizard® Genomic DNA Purification Kit (Promega, Madison, WI, USA). Posteriorly, DNA concentration and purity were measured using the NanoDrop™ 8000 apparatus (Thermo Fisher Scientific, Waltham, MA, USA) and stored at −20 °C until further use. Sequencing was performed on Illumina Mi-Seq technologies, using a paired-end application, and reads with a mean length of 75.9 bp (Illumina, San Diego, CA, USA), generating a total of 15,425,426 reads with an average insert size of 200 bp and coverage of 426×. Sequence reads’ quality was assessed using FastQC software version 0.11.9 [37], and reads were trimmed using the Trim and Filter tool (error probability = 0.05) of Geneious version 10.2.6 [38]. The trimmed reads were used in de novo assembly with the SPAdes version 3.10.0 tool and default parameters [39], and contigs ≥ 1000 bp were discarded. The CDS of contigs were predicted using RASTtk (Domain: Bacteria; Taxonomy name: Bacillus thuringiensis; Genetic code: 11—Archaea and Bacteria). The chromosome was assembled using contigs and reference HD-789 (NCBI Accession No. CP003763) through reference-guided de novo assembly [40], using the Geneious map to reference tool to assess the virulence factors of related genes. Contigs unused in the chromosome assembly were filtered and used for predicting pesticidal protein-like genes. Related genes with virulence factors were predicted using the bacterial virulence factor database (VFDB) [41]. Putative pesticidal proteins were determined using Blastx through the Btoxin_Digger tool (scaffolds as a query) [42] and a customized database (CDS predicted as a query). The customized database was created from the Bt pesticidal protein list available at the Bt nomenclature website (http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/toxins2.html (accessed on 9 December 2022) through Geneious using the Add/Remove Database tool. CDS with homology to the Bt pesticidal proteins were filtered using parameters of an E-value of 0.001 and a word size of 6.

2.7. Phylogenetic Relationship

A phylogenetic tree was constructed using the gyrB gene (DNA gyrase subunit B), extracted from contigs sequences, and the gyrB genes of the Bacillus ssp. strains retrieved from GenBank. The alignment was performed using ClustalW, and the phylogenetic tree was created using MEGA 11 [43] from the neighbor-joining method, with 1000 replications.

2.8. LC-MS/MS Analysis

The liquid chromatography–tandem mass spectrometry (LC-MS/MS) method was used for protein detection in the spore–crystal mixture of the Bt TOD651 strain. The LC-MS/MS analysis was carried out at the Veritas/Life Sciences Department at the University of São Paulo (USP, Ribeirão Preto, SP, Brazil). Firstly, the spore–crystal sample was washed three times in 1× PBS (phosphate-buffered saline), resuspended in 750 µL of solubilization buffer (8 M urea, 0.5% octyl-glucopyranoside (OG), 0.05 M Tris-HCL, pH 8.8), and sonicated by 3 cycles (60 s, 30% amplitude, and shut off for 2 s) while maintained on ice. The quantification of solubilized protein was performed using the Bradford method (Protein Assay Dye Reagent Concentrate, Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions. In the sample preparation for advanced mass spectrometry, 50 µg of the sample was subjected to disulfide bridge reduction (50 µg of DTT (Dithiothreitol), 60 min of incubation at 37 °C), followed by alkylation (250 µg of I.A (iodoacetamide), 60 min at room temperature in the dark). Following this, the sample was diluted five times in Tris-hydrochloride (0.05 M Tris-HCl, pH 8.8), and incubated using 2 µg of trypsin (Promega, V511A) at 37 °C overnight. The cleanup and desalting of the sample were performed using C18 resin (Supleco, Bellefonte, PA, USA). The column was calibrated using 2% acetonitrile containing 0.1% formic acid, and the elution was performed with 50% acetonitrile. The sample was then dried in a speed vac and applied to a mass spectrometer (Thermo Fisher Orbitrap Eclipse) coupled to a nanoflow Nano LC-MS/MS chromatography system (Dionex Ultimate 3000 RLSCnano System, Thermofisher, Waltham, MA, USA). Peptides were separated for 90 min in a nanoEase MZ peptide BEH C18 column (130 A, 1.7 µm, 75 µm × 250 mm, Waters, Milford, MA, USA) at 300 nL/min, with a 4–50% acetonitrile gradient. The data were obtained on MS1 in the range of M/Z 375–1500 (120,000 resolution, AGC target 1 × 106, maximum time of injection of 100 ms). The most abundant ions were submitted to MS/MS (30% collision energy, 1.2 m/z, AGC target 1 × 105, 15,000 resolution).

2.9. Proteomic Data Analysis

The proteomic data were processed using the PatternLabV [44]. Firstly, the customized database was created using translated CDS of the TOD651 genome through the Generate Search DB option, including a contaminant library (MS contaminant sequences, e.g., trypsin, keratins, and albumin). Then, proteomic data were analyzed against the customized database using the following parameters: The modifications selected in the search were carbamidomethyl (C), deamination (NQ), and oxidation (M). Enzyme trypsin (fully specific), two maximum missed cleavages, an initial precursor mass tolerance of 35 ppm, MS and MS/MS tolerance errors of 10 ppm, and acceptable FDR (false discovery rate) estimates of 3% at spectral, 2% at peptide, and 1% at protein levels were added as advanced parameters.
The functional annotation of the identified proteins was performed with the UniProtKB/Swiss-Prot database, and the summary graphic of functional classification was created using GO terms through the WEGO 2.0 tool (Web Gene Ontology Annotation Plot) [45].

2.10. Data Availability

The clean reads of the Bt TOD651 strain have been deposited at the Sequence Read Archive (SRA) under the accession number PRJNA907848.

3. Results

3.1. Protein Profile, Crystal Morphology, and Mosquitocidal Activity

The protein profile of Bt TOD651-purified crystals in an SDS-PAGE showed the main proteins with molecular weights of approximately 130, 70, and 27 kDa in size (Figure 1a). The ultra-structural analysis of the spore–crystal mixture indicated the presence of spherical crystals (Figure 1b).
The Bt TOD651 spore–crystal mixture showed mosquitocidal activity towards A. aegypti and C. quinquefasciatus, with 50% lethal concentration (LC50) values of 0.011 and 0.023 µg/mL, respectively (Table 1). High toxicity was observed, but the lethal concentration did not differ between the reference strain and Bt TOD651 for both species.

3.2. General Genomic Features

Most genes were classified into different functional classes using the RASTtk tool’s analysis. The amino acids and derivatives metabolism class (579 genes), carbohydrate metabolism (320 genes), cofactors, vitamins, prosthetic groups, pigment metabolism subsystems (215 genes), protein metabolism (155 genes), cell wall and capsule (163), nucleosides and nucleotides (159 genes), and protein metabolism were the most common (Figure 2). One hundred and eight (108) genes were grouped in the subsystem class “virulence, disease, and defense.” To assess the virulence factors, the chromosome was assembled at the draft level. This sequence consisted of a chromosome with ~5.4 Mb bp containing 35.9% GC, 5130 CDS, 1 rRNA, and 71 tRNA genes (Table 2). The sequences not used in the chromosome, and presumably belonging to plasmid sequences, were used for the cry/cyt gene screening.

3.3. Phylogenetic Analysis

Phylogenetic analysis using the gyrB gene showed that the Bt TOD651 strain formed a group closely related to three Bti strains (BGSC 4Q1, BGSC 4Q7rifR, and AM65-52), Bt MYBT18246, Bt ATCC 10792, and Bt serovar thuringiensis IS5056 (Figure 3).

3.4. Genes Associated with Bt TOD651 Pathogenicity

Different virulence-associated genes were detected in the genome sequence (Table S1, Figure 4). Among them, enzymes (inhA1 and sph), immune evasion (bpsC and polysaccharide capsule genes), iron acquisition genes (dhbA-C, dhbE, dhbF, hal, ilsA, and asbA-F), regulation genes (pagR-XO2, papR, plcR, and cheA), and toxins (hlyI-III, hblA, and nheA-C) were identified (Supplementary Table S1, Figure 4a).
A total of 10 CDS predicted in the Bt TOD651 genome were highly homologous to pesticidal proteins (Figure 4b, Table 3). Four cry genes (cry11Aa3, cry10Aa4, cry4Aa4, and cry4Ba5), three cyt (cyt1Aa5, cyt1Ca1, and cyt2Ba13), two mpp (mpp60Aa3 and mpp60Ba3), and one spp (spp1Aa1) genes were identified (Figure 4b, Table 3).

3.5. Proteomics of Spore–Crystal Mixture

The detected protein sequences were functionally classified into 10 GO terms related to cellular components, 8 GO terms related to molecular functions, and 14 terms related to biological processes (Figure 5). In the cellular component groups, most proteins were mainly related to the cell part and the cell, and the molecular function classification was represented by proteins with catalytic and binding activities. Moreover, in the biological process category, the major portion of proteins belonged to metabolic and cellular processes (Figure 5).
A comparison of genomic and proteomic data was performed to identify the predicted CDS that were expressed. A total of 43 CDS regions annotated in the genome were detected in the proteomic analysis with at least two peptides (Table 4). With respect to pesticidal proteins, only mpp60Aa3 did not show a unique peptide, and therefore its expression was not confirmed. Thus, the expression of Cry11Aa3, Cry10Aa4, Cry4Aa4, Cry4Ba5, Cyt1Aa5, Cyt1Ca1, Cyt2Ba13, and Mpp60Ba3 was confirmed. Cry4Ba5 was the most abundant peptide discovered (60), followed by Cry4Aa4 (47), Cry11Aa3 (46), Mpp60Aa3 (29), and Cry10Aa4 (29) (Table 4, Supplementary Table S2). Among the cytolytic proteins, Cyt1Aa was the most abundant, showing 13 unique peptides, while Cyt2Ba13 and Cyt1Ca1 showed 7 and 5 unique peptides, respectively (Table 4, Supplementary Table S2). Besides pesticidal proteins, other proteins were also identified. Metallophosphoesterase (Mppe) was the most abundant protease and showed 25 unique peptides (Table 4). Three unique peptides were from the virulence factor, extracellular neutral protease B. Proteins involved in sporulation (spore coat proteins and exosporium protein), protein biosynthesis (chaperone protein, heat-shock protein, translation elongation factor Tu, ribosomal proteins), and other functions (e.g., aminopeptidase, enolase, DNA-binding protein, RNA-binding proteins, and superoxide dismutase) were also identified (Table 4).

4. Discussion

Here, we demonstrated that a novel Neotropical Bt strain (TOD651), isolated from Brazilian soil samples, exhibited significant larvicidal activities against larvae of A. aegypti and C. quinquefasciatus. Such larvicidal activities were similarly potent to those recorded in Bt strains that are already commercialized. In addition to the characterization of Bt TOD651 potential to be integrated into biorational programs of mosquito control, we further explored its genome and proteome, and discovered that the pathogenicity may be derived from the expression of pesticidal proteins (e.g., Cry11Aa3, Cry10Aa4, Cry4Aa4, Cry4Ba5, Cyt1Aa5, Cyt2Ba13, and Mpp60Ba3), virulence factors (e.g., Mppe and NprB), and spore coat proteins (e.g., CoatB, CoatE, CoatG, and CotY/CotZ family proteins).
The protein profiles and ultrastructure of the parasporal crystals of the Bt TOD651 strain were observed to be similar to those of other anti-dipteran Bt strains, including the reference strain [46,47]. The SDS-PAGE 130 kDa protein band represents the Cry4 protein, the 70 kDa band indicates the presence of Cry11/Cry10, and the 27 kDa band suggests the presence of the Cyt protein [4,8]. Further, the spherical crystals observed by scanning electron microscopy showed the usual the crystal morphology found in this type of Bt strain [48].
The phylogenetic analysis based on the gyrB gene showed that Bt TOD651 was closely related to Bti strains. In addition, a nematocidal strain, Bt MYBT18246 [49], a type strain, Bt ATCC 10792 (i.e., the nomenclatural type of the species Bt), and a strain toxic against lepidopteran insects, Bt thuringiensis IS5056 [50], were also closely related to Bt TOD651.
The Bt TOD651 strain’s whole-genome analysis revealed the following genes: cry11Aa3, cry10Aa4, cry4Aa4, cry4Ba5, cyt1Aa5, cyt1Ca1, cyt2Ba13, mpp60Aa3, and mpp60Ba3. Other genomic studies revealed a similar gene content in Bt strains with high mosquitocidal activity. For example, Bt AR23 has been described to harbor cry10Aa4, cry11Aa3, cry4Ba5, cry4Aa4, cyt2Ba, cyt1Aa, and cyt1Ca [28], while Bt LLP29 harbors cry4Aa4, cry10Aa4, cry11Aa4, cyt1Aa6, cyt2Ba1, and cry22Aa [29]. The Bt TOD651 genome also harbors different virulence factor genes, such as hemolysins, enterotoxins, proteases, and phospholipases. These virulence factor genes, conserved in the Bacillus cereus group, are responsible for the colonization and adaptation of Bt in insect hosts [51,52,53].
The proteomic analysis revealed the expression of Cry11Aa3, Cry10Aa4, Cry4Aa4, Cry4Ba5, Cyt1Aa5, Cyt2Ba13, and Mpp60Ba3 proteins. Thus, these proteins were responsible for the toxic activity of Bt TOD651 against A. aegypti and C. quinquefasciatus. Stein et al. [54] detected the transcripts of cyt1Ca but did not find Cyt1Ca protein. Here, Cyt1Ca1 protein was expressed. However, it may not be related to the toxicity of Bt TOD651, since neither mosquitocidal nor larvicidal activity function has been reported for Cyt1Ca [55].
Bt TOD651 showed the lower CL50 for A. aegypti. When tested individually, mosquitocidal Cry proteins have different toxicity levels between Aedes and Culex genera. The Cry4Ba protein is highly toxic against A. aegypti, while the Cry4Aa is highly toxic for Culex mosquitoes [56,57]. Cry11Aa has been linked to high toxicity against both the Aedes and Culex genera [56], whereas Cry10Aa is toxic to A. aegypti insects [8]. Mpp60 proteins show moderate insecticidal activity against C. quinquefasciatus larvae [58]. Cyt1Aa and Cyt2Ba exhibit toxicity towards both A. aegypti and C. quinquefasciatus [59,60]. However, these proteins act synergistically, which is mainly attributed to the Cyt1Aa toxin that can increase the activity of Cry4Aa, Cry4Ba, Cry11Aa, or Cry10Aa [61].
Cry10Aa has been reported as the minor protein component of Bti crystals [6]. In the proteomic analysis of Bti 4Q2-72, Cry10Aa presented a low abundance of peptides [62]. Cyt2Aa is also considered a minority pesticidal protein produced by Bti [63]. In the proteomic analysis of the commercial Bti AM65-52, Cry10Aa and Cyt2Ba were not expressed [19]. In line with the Cyt1Aa protein, the Cyt2Ba protein is also highly synergistic with the Cry proteins, and hence their combinations prevent the emergence of resistance in the target insects [4]. In addition, Cry10Aa seems to contribute to the overall toxicity of Bti [4]. Cry10Aa4 was one of the most abundant proteins in the spore–crystal mixture of Bt TOD651, and the peptide number of Cyt2Ba13 was sufficient to not consider it as a trace-level protein.
Cyt1Aa has been reported as the major component of Bti AM65-52 crystals [19], while Bti 4Q2-72 expressed higher levels of Cry11Aa [62]. In contrast, Bt TOD651 expressed mainly Cry4Ba5. Cyt1Aa plays an important role in the synergism of Bti strains and may also contribute to delaying the evolution of resistance to Cry proteins in low proportions [64].
With respect to Mpp proteins, only Mpp60Ba3 expression was detected in this study. Even though the mpp60Aa3 and mpp60Ba3 genes are part of the same operon, neither of them depends on the other to be expressed [58]. SDS-PAGE gel analysis revealed no detectable protein band for Mpp60Ba3. Sauka et al. [65] discovered an mpp homolog in a B. toyonensis strain’s genome but no protein bands by SDS-PAGE analysis, implying that these proteins are secreted and present in remnant fractions. The expression of Mpp60Aa and Mpp60Ba proteins has been detected in Bti AM65-52 [19] and Bt jegathesan [58] in low abundance. In contrast, Mpp60Ba3 represented a high proportion in Bt TOD651, suggesting that it plays an important role in the toxicity of this bacteria.
Metalloproteinases have been described as virulence factors involved in Bt pathogenesis, increasing the toxicity of pesticidal proteins [52]. The immune inhibitor A (InhA) has been identified as the main metalloproteinase associated with pesticidal proteins in spore–crystal mixtures of Bt strains [25,66]. Interestingly, in this study, the high protein homology to Metallophosphoesterase (Mppe) was the most abundant protease in Bt TOD651. A gene of the Mppe family has been identified in the B. cereus genome [67]. Mppe should play an important role in the stress resistance of bacteria to adapt to the environment/host [67]. Neutral protease B (NprB) (also named NprA and Npr99), also present in the spore–crystal mixture of Bt TOD651, has been associated with the virulence of Bacillus anthracis, degrading host tissues and increasing tissue permeability to the pathogen [68].
Other proteins that may contribute to Bt TOD651 toxicity were detected in the spore–crystal mixture, such as spore coat proteins. It has been demonstrated that spores in the spore–crystal mixture play a major role in Bt toxicity, not only due to septicemia from spore germination and outgrowth, but also due to a synergy between the spore coat protein and the crystal protein [69]. Heat-shock proteins and the elongation factor Tu were also detected, which are necessary for the formation of crystals in Bt strains [70,71].
The genomic and proteomic analysis of the Bt TOD651 and other Bt strains can provide insights into the genetic makeup and protein expression of the bacteria. This information can help identify the genes responsible for the bacterium’s insecticidal properties, which can be used for developing new, more effective insecticides. Additionally, genomic and proteomic analyses can provide information on the evolutionary history and diversity of different Bt strains, which can inform their classification to aid in the development of new strains with desired traits.
The Bt TOD651 strain can be used as an alternative for A. aegypti and C. quinquefasciatus control, and the combined genomic and proteomic analyses revealed the proteins directly related to their toxicity. In addition, Bt TOD651 exhibits a variety of protein content that can potentially delay the evolution of resistance. Furthermore, we detected other proteins that can also contribute to Bt TOD651’s pathogenicity.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/pr11051455/s1, Table S1: Virulence factors identified in the chromosome draft sequence of Bt TOD651 from the VFDB database. Table S2: Peptides identified for pesticidal proteins classified as unique (true) or non-unique (false).

Author Contributions

Conceptualization, all authors; methodology, G.B.A. and M.L.D.; writing—original draft preparation, G.B.A., M.L.D. and R.W.d.S.A.; writing—review and editing, B.M.R., G.R.d.S. and E.E.d.O.; supervision, R.W.d.S.A., G.R.d.S., E.E.d.O. and B.M.R.; project administration, R.W.d.S.A.; funding acquisition, R.W.d.S.A. and B.M.R. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Council of Scientific and Technological Development (CNPq—process numbers: 313455/2019-8, 427304/2018-0, 308576/2018-7), the Tocantins State Foundation for Research Aid (FAPT-SESAU/TO-DECIT/SCTIE/MS_CNPQ/No. 01/2017), and the Federal University of Tocantins (PROPESQ)—EDITAL No. 29/2020 PROPESQ, and PPGBIOTEC/UFT/GURUPI—Chamada pública para auxílio de tradução e/ou publicação de artigos cientificos—EDITAL No. 011/2020.

Data Availability Statement

All data and code generated appear in the submitted article.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

References

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Figure 1. SDS-PAGE analysis and scanning electron microscopic image of Bt TOD651-purified crystals and the spore–crystal mixture. (a) Protein profile of Bt TOD651-purified crystals: Lane 1—molecular mass marker; Lane 2—AM65-52-purified crystals; Lane 3—TOD651-purified crystals. (b) Scanning electron micrograph of spore–crystal mixture produced by Bt TOD651. Arrows indicate spore (yellow) and crystal (white).
Figure 1. SDS-PAGE analysis and scanning electron microscopic image of Bt TOD651-purified crystals and the spore–crystal mixture. (a) Protein profile of Bt TOD651-purified crystals: Lane 1—molecular mass marker; Lane 2—AM65-52-purified crystals; Lane 3—TOD651-purified crystals. (b) Scanning electron micrograph of spore–crystal mixture produced by Bt TOD651. Arrows indicate spore (yellow) and crystal (white).
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Figure 2. Subsystem category distributions in the genome of Bt TOD651-based functional classification.
Figure 2. Subsystem category distributions in the genome of Bt TOD651-based functional classification.
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Figure 3. Phylogenetic analysis of Bt TOD651 with other Bacillus spp. based on gyrB sequences. The phylogenetic tree was constructed using the neighbor-joining method with 1000 bootstrap replications. Bootstrap values < 50% were disregarded for branches.
Figure 3. Phylogenetic analysis of Bt TOD651 with other Bacillus spp. based on gyrB sequences. The phylogenetic tree was constructed using the neighbor-joining method with 1000 bootstrap replications. Bootstrap values < 50% were disregarded for branches.
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Figure 4. A graphical representation of the virulence factors and pesticidal protein-like genes found in the draft genome of Bt TOD651. (a) Distribution of virulence factor genes in the draft chromosome. (b) Position of pesticidal protein-like genes in contigs unused in chromosome assembly.
Figure 4. A graphical representation of the virulence factors and pesticidal protein-like genes found in the draft genome of Bt TOD651. (a) Distribution of virulence factor genes in the draft chromosome. (b) Position of pesticidal protein-like genes in contigs unused in chromosome assembly.
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Figure 5. Functional annotation and classification of proteins identified in the spore–crystal mixture of TOD651.
Figure 5. Functional annotation and classification of proteins identified in the spore–crystal mixture of TOD651.
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Table
Table 1. Lethal concentration estimations of Bt TOD651 to larvae of Aedes aegypti and Culex quinquefasciatus.
Table 1. Lethal concentration estimations of Bt TOD651 to larvae of Aedes aegypti and Culex quinquefasciatus.
StrainA. aegyptiC. quinquefasciatus
LC50
(µg/mL)		CL95
(µg/mL)		SLOPEχ2pLC50
(µg/mL)		LC95
(µg/mL)		SLOPEχ 2p
TOD6510.0110.0303.7265.620.050.0230.0554.3116.680.27
AM65-520.0130.0373.7254.330.360.0280.0694.4676.490.16
Table
Table 2. Draft chromosome features of the Bt TOD651 strain.
Table 2. Draft chromosome features of the Bt TOD651 strain.
General FeaturesValue
Mean depth coverage95.9×
Chromosome size (bp)5,409,948
Gapped sites (%)7.2
GC content (%)35.9
No. of CDS5130
No. of rRNA1
No. of tRNA71
Table
Table 3. Identification of genes coding pesticidal protein-like genes in the Bt TOD651.
Table 3. Identification of genes coding pesticidal protein-like genes in the Bt TOD651.
SequencePredicted CDS Length (aa)Homologous ProteinCoverage
(%)		Pairwise
Identity (%)		E-Value
Contig_1299peg.1190698Cry4Aa459.07 1/99.90 299.43 1,20.0
Contig_1369peg.1401645Cry11Aa3100.00 1,2100.00 1,20.0
Contig_305peg.58121161Cry4Ba5100.00 1,2100.00 1,20.0
Contig_370peg.6260674Cry10Aa497.19 1/100.00 2100.00 1,20.0
Contig_2018peg.338899Cyt2Ba1340.24 1/100.00 2100.00 1,20.0
Contig_3012peg.5774262Cyt1Aa5100.00 1100.00 10.0
Contig_551peg.7346291Cyt1Ca151.43 1/97.80 298.90 1,20.0
Contig_208peg.3554323Mpp60Aa3100.00 1,2100.00 1,20.0
Contig_208peg.3552319Mpp60Ba3100.00 1,2100.00 1,20.0
Contig_248peg.4529323Spp1Aa158.70 180.81 10.0
1 Btoxin_Digger. 2 Customized.
Table
Table 4. Pesticidal and other proteins identified in the spore–crystal mixture of the Bt TOD651 strain.
Table 4. Pesticidal and other proteins identified in the spore–crystal mixture of the Bt TOD651 strain.
CDS IDDescription 1Length
(bp)		Peptide
Sequence (No.)		Unique
Peptide (No.) 4		Coverage 5Protein
Score 6		NSAF 7
peg.5812Cry4Ba5 2113662600.5599211.3960.0492475
peg.1190Cry4Aa4 279151470.6587184.8080.0732234
peg.1401Cry11Aa3 264546460.6171161.1160.1816369
peg.3553Mpp60Ba3 230329290.811997.9150.0868883
peg.6260Cry10Aa4 270531290.4057104.1990.031742
peg.3039Metallophosphoesterase (Mppe) 347125250.711391.8430.0440183
peg.5774Cyt1Aa5 224913130.634550.490.1850302
peg.1543Heat-shock protein (GroEL)544990.139727.310.0060494
peg.5502L-alanyl-gamma-D-glutamyl-L-diamino acid endopeptidase325990.421533.370.0131636
peg.3700Spore coat protein (CotB)174880.54628.4160.0264785
peg.3439Aminopeptidase466880.283327.6350.0063558
peg.4239Elongation factor Tu320880.390631.320.0133693
peg.8774Enolase431770.287727.9990.0068719
peg.2515Spore coat protein (CotG)186770.182822.9460.0212316
peg.3388Cyt2Ba13 299770.676824.8950.0465379
peg.7343Cyt1Ca1 285550.258814.0950.0309731
peg.7029Dihydrolipoamide dehydrogenase of pyruvate dehydrogenase complex470440.072311.7440.003501
peg.8531Chaperone protein (DnaK)611440.091712.2560.0021544
peg.5180Spore coat protein (GerQ)139440.29512.9280.0165728
peg.5234LSU ribosomal protein L7p/L12p(P1/P2)119440.546213.2060.0110618
peg.7670NAD-dependent glyceraldehyde-3-phosphate dehydrogenase334440.239516.8490.0049265
peg.7030Dihydrolipoamide acetyltransferase component of pyruvate dehydrogenase complex227440.16748.9680.0072487
peg.7476Hypothetical protein250330.15610.9160.0052654
peg.2818Fructose-bisphosphate aldolase class II267330.17239.3140.0049302
peg.1753N-acetylmuramoyl-L-alanine amidase271330.11449.5470.0048574
peg.8176Uncharacterized protein YmfJ82330.46348.950.0120399
peg.4909N-acetylmuramoyl-L-alanine amidase327330.14378.2670.0030192
peg.3986Extracellular neutral protease B (NprB)426330.124411.3170.0038626
peg.9127SSU ribosomal protein S2p (SAe)233220.11595.1890.0028248
peg.7282Hypotetical protein143220.16784.5020.0046026
peg.4363Superoxide dismutase203220.18236.9770.0048634
peg.782Hypothetical protein129220.1865.0410.0051022
peg.4419Cell division trigger factor404220.09657.550.0016292
peg.3911DNA-binding protein (Hbsu)90220.36677.2820.0073131
peg.7358Spore coat protein (CotE)180220.21116.6410.0036565
peg.969Phage tail fiber protein431220.0514.8160.0015271
peg.3496Hypotetical protein108220.14814.3850.0060942
peg.9081Uncharacterized protein BA537368220.56.7170.0096791
peg.8583RNA-binding protein (Hfq)74220.32436.3230.0088943
peg.3589Spore coat protein of CotY/CotZ family155220.26.6490.0063695
peg.1544Heat-shock protein (GroES)94220.24475.2860.0070019
peg.5565Tricarboxylate transport sensor protein (TctE)92220.32613.9670.0071541
peg.8113Exosporium protein K118220.11025.9050.0083667
1 Annotation based on RASTtk. 2 Classification based on Btoxin_Digger and/or the customized Bt database. 3 Description based on Blastx from NCBI. 4 The number of peptide sequences that are unique to the protein. 5 The percentage of the protein sequence covered by identified peptides. 6 The sum of the ion scores of all peptides that were identified. 7 Normalized spectral abundance factor: calculated using the number of spectra divided by the protein length, and then normalized over the total of spectral counts/length for all the proteins in the sample.
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MDPI and ACS Style

Alves, G.B.; Dias, M.L.; Oliveira, E.E.d.; Santos, G.R.d.; Ribeiro, B.M.; Aguiar, R.W.d.S. Isolation, Genomic, and Proteomic Characterization of a Novel Neotropical Strain of Bacillus thuringiensis with Mosquitocidal Activities. Processes 2023, 11, 1455. https://doi.org/10.3390/pr11051455

AMA Style

Alves GB, Dias ML, Oliveira EEd, Santos GRd, Ribeiro BM, Aguiar RWdS. Isolation, Genomic, and Proteomic Characterization of a Novel Neotropical Strain of Bacillus thuringiensis with Mosquitocidal Activities. Processes. 2023; 11(5):1455. https://doi.org/10.3390/pr11051455

Chicago/Turabian Style

Alves, Giselly Batista, Marcelo Leite Dias, Eugenio Eduardo de Oliveira, Gil Rodrigues dos Santos, Bergmann Morais Ribeiro, and Raimundo Wagner de Souza Aguiar. 2023. "Isolation, Genomic, and Proteomic Characterization of a Novel Neotropical Strain of Bacillus thuringiensis with Mosquitocidal Activities" Processes 11, no. 5: 1455. https://doi.org/10.3390/pr11051455

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