1. Introduction
Bacillus thuringiensis (Bt) is the most successful and widely used microbial control agent for the management of insect pests [
1]. This entomopathogenic bacterium produces parasporal inclusion bodies comprised of crystal (Cry) and cytolytic (Cyt) proteins upon the sporulation [
2,
3]. Due to the importance of Bt in the management of insect pests, many studies have focused on the development of Bt-based bio-insecticides as well as transferring Bt genes into the agricultural crops [
4,
5]. Indeed, several Bt strains have been characterized so far based on their genome sequencing and/or protein composition following mass-spectrometry based approaches to identify their gene content and proteome, as well to predict their biological activity [
6,
7,
8,
9,
10,
11,
12]. Molecular based characterization methods, through proteomics- and/or genomics-based analyses, have provided a rapid and comprehensive strategy for the identification of Bt toxins [
13,
14]. On the other hand, the extensive application of Bt products and Bt crops has led to the emergence of resistance to Bt toxins in insects [
15,
16]. Therefore, the identification of novel Bt strains and toxins may enhance the potency of Bt-based bioinsecticides and the efficiency of Bt crops. Thus, the discovery of new Bt genes or proteins with different biological activity, a wider spectrum of activity, and higher insecticidal potency can lead to better management of insect pests.
The whole genome of newly isolated Bt strains is being sequenced to speed up the detection of new putative insecticidal Bt genes [
9,
10]. Indeed, genome sequencing has been employed by many researchers to describe pesticidal and virulence factors genes [
11,
12,
17,
18,
19] as a fast and efficient means of Bt strain characterization.
On the other hand, it is believed that the insecticidal activity of Bt strains is mostly attributed to the type, amount, and relative abundance of the proteins produced [
20,
21]. Hence, proteomics analysis accelerates the screening studies and can reveal the protein composition of the Bt parasporal crystals, provided that the detected protein sequences exist in the protein databases. Different proteomic approaches enable the researchers to investigate the protein profile of Bt strains for different purposes such as the phylogenetic classification of
Bacillus spp. [
22], Bt protease profile [
23], bacteriocin characterization [
24], spore proteomes [
25], crystal protein composition [
26,
27], the protein composition of solubilized spore/crystal mixture [
6], proteome involved in carbon metabolism [
28], the metabolic regulation of sporulation and crystal formation [
29,
30], and the protein composition of whole Bt cell [
31]. Also, several studies have coupled whole-genome sequencing with the liquid chromatography and tandem mass spectrometry (LC-MS/MS) to investigate the gene content, the insecticidal proteins, and the bioactive compounds of newly isolated or highly potent Bt strains [
8,
18,
32].
The Bt strain KhF was originally isolated from a soil sample in Iran. We had observed that the spore/crystal mixture of KhF was toxic for second instar larvae of
Plodia interpunctella (Lep.: Pyralidae). Following the conventional solubilization processes, the crystal proteins of the KhF strain could not be completely solubilized. The PCR-based method was employed to screen the presence of Bt toxin genes belonging to the main family groups (
cry1,
cry1Aa,
cry1Ab,
cry1Ac,
cry1Ad,
cry1Ag,
cry1C,
cry1D,
cry1H,
cry1I,
cry1G,
cry2,
cyt1,
cyt2,
vip1,
vip2,
vip3,
ps1,
ps2,
ps3,
ps4,
sip1, and
sip2 genes). The results revealed only the possible presence of the
cry2 gene [
21]. These characteristics alongside the insecticidal activity of the KhF spore/crystal mixture against lepidopteran
P. interpunctella led us to conclude that most probably KhF harbored new Bt genes and proteins different from the known ones. Therefore, in this study, genomics and proteomics approaches were coupled and used to identify the possible toxic agents in the spores and crystals mixture. Due to the applicability of LC-MS/MS analysis incorporated with full genome sequencing in the identification of new Bt proteins, our results allowed us to identify two novel Bt proteins.
3. Discussion
Bt based insecticides are eco-friendly alternatives for pest control. The emergence of insect resistance to Bt toxins is one of the major constraints that threaten the application of Bt in the microbial control of pests. Therefore, the identification of novel Bt strains or toxins is of scientific and economic interest and is critical for efficient control of insect pests and the management of insect resistance.
The KhF was originally isolated from a soil sample in Iran. The strain produces spherical crystals and the spores/crystals mixture was found to be toxic for
P. interpunctella second instar larvae. Our previous studies have shown that this strain did not produce β-exotoxin (a Bt toxin analogous to ATP, which has a general toxic effect on all living organisms) and the PCR-screening showed that the strain probably carried
cry2 genes but not
cry1,
cry1Aa,
cry1Ab,
cry1Ac,
cry1Ad,
cry1Ag,
cry1C,
cry1D,
cry1H,
cry1I,
cry1G,
cry2,
cyt1,
cyt2,
vip1,
vip2,
vip3,
ps1,
ps2,
ps3,
ps4,
sip1, and
sip2 genes [
21]. Another characteristic of this strain is that only partial solubilization of crystals could be achieved. Due to the observation of the insecticidal activity against
P. interpunctella and incomplete solubility of KhF crystals, the discovery of novel Bt toxins in this strain was very promising. Therefore, the gene content and the protein composition of the spore/crystal mixture of this Bt strain have been investigated in this work.
The entire genome of KhF strain was sequenced through the Illumina NextSeq500 Sequencer. The genome assembly results and a subsequent search using two different databases revealed that the KhF strain comprises nine candidate insecticidal Bt proteins with the closest homology to Bt hypothetical protein, ricin-domain, and ETX/Mtx2 proteins based on the non-redundant protein database of NCBI and to Vip1Aa1, Vip1Ad1, Vip1Ba1, Vip2Ac1, Vip2Ad1, Vip4Aa1, and Cry49Aa1 toxins (with low similarity values ranging from 24% to 36%) based on the customized Bt toxins database (
Table 3). It is worth mentioning that following PCR-based screening of KhF strain gene content, the amplification of
vip1 and
vip2 genes was not observed [
21]. Therefore, the low similarity of
peg5936,
peg309,
peg2096,
peg3627,
peg5521, and
peg5522 genes to their corresponding
vip1 or
vip2 genes reinforce the novelty of the detected genes.
The candidate insecticidal protein genes detected in the genome of KhF were localized in six different scaffolds. Alongside the described candidate insecticidal Bt genes, the KhF strain contains 35 CDS encoding different classes of pathogenic factors (
Table S1). Among them, 10 coding sequences showed similarity to other known toxins present in
Bacillus spp., such as haemolytic enterotoxin (HE) and non-haemolytic enterotoxin (NHE). Due to the close phylogeny of
B. cereus sensu lato group, the haemolysin and non-haemolysin enterotoxin genes are conserved amongst the
Bacillus spp. and indeed, the presence of these genes has been reported in different Bt strains [
19,
39]. The genomic analysis of Bt strain HD-1 revealed the presence of the haemolysin Xh1A family genes and it has been proposed that these genes might play an important role in the toxicity process of HD-1 strain [
19].
The phylogenetic study of the KhF strain constructed based on the
gyrB gene showed that this Bt strain might have a close relationship with
B. thuringiensis strain MC28 and
B. toyonensis BV-17 (
Figure 3). According to the whole-genome sequencing, the
B. thuringiensis strain MC28 has three plasmids and harbours different
cry and
cyt genes, namely
cry4Cc1,
cry30Fa1,
cry53Ab1,
cry54Aa1,
cry54Ab1,
cry68Aa1,
cry69Aa1,
cry69Aa2,
cry70Ba1,
cyt1Da1, and
cyt2Aa3 [
40]. The other closely related
Bacillus strain to KhF is
B. toyonensis BV-17, as a non-pathogenic probiotic
Bacillus species [
41,
42]. The haemolysin BL secreted as three components by the
B. toyonensis BV-17 showed cytotoxicity against cancer cells [
42]. Interestingly,
peg3633,
peg3634, and
peg3632 genes from the KhF strain showed 100% similarity to haemolysin BL lytic component L1, component L2, and binding component, respectively.
The toxicity of the KhF strain has been assessed through bioassays, using mixtures of spores and crystals that are toxic for
P. interpunctella [
21] and
G. molesta (
Table 1). The protein composition of the spore/crystal mixture of the KhF strain was determined by LC-MS/MS analysis. It is worth mentioning that the Vip proteins are not present in crystals since they are produced and secreted to the media before the sporulation phase differently to the proteins that are present in the parasporal crystals. Under this point of view, seven out of the nine candidate insecticidal protein genes detected after genomic analysis in the KhF strain would not be detected in crystals.
In the proteomics analyses, a total of 434 proteins were identified in the spore/crystal mixture of KhF. After eliminating the redundant proteins, 327 specific proteins with a confidence level of higher than 99% were detected. These proteins were compared to the translated proteins deduced from the KhF sequenced genome. To ensure the certainty of the existence of a detected protein in the strain, the proteins with the several discriminating peptides showing a confidence value higher than 99% that did not match with other detected proteins with a higher score were selected. According to such principles, among the insecticidal Bt genes detected in the genomics analysis, only two of them,
peg5936 and
peg5937, were expressed and identified in the spore/crystal mixture of the KhF strain and constituted a high percentage of the total protein content in the crystals (
Table 6). Moreover, two bands of 130 and 23 kDa coinciding with the predicted size of these two new genes, named KhFB (
peg5936) and KhFA (
peg5937), were visualized in the SDS-PAGE analysis of KhF crystals (
Figure 1, Panel B).
KhFA and KhFB proteins can be considered as new Bt-like proteins, different to the known Cry, Cyt, Vip, or Sip proteins because of their low similarity with the known Bt proteins. The phylogenetic analysis indicated that KhF proteins grouped with Mtx2 and Cry55Aa belong, respectively, to the ETX/Mtx2-like (now called Mpp) and the structurally unclassified Bt pesticidal proteins (now called Xpp) groups. None of the proteins of these groups are three domain Cry proteins, the most well-known pesticidal Bt toxins [
2].
Mtx2 family toxin, produced by some
Lysinibacillus sphaericus and Bt strains, exhibits mosquitocidal activity against different dipteran species [
43]. ETX/Mtx2 proteins have a β-sheet structure and are considered as pore forming pesticidal toxins [
44]. The toxic activity of Cry55 against nematodes and coleopterans has been reported [
45,
46], while neither the structural homolog nor the mode of action of this protein is known [
2,
44]. Due to the homology of Peg5937 to Cry55 protein and the existence of metalloproteinase as a toxic factor for nematodes [
47], the KhF strain may be considered as a nematicidal strain. Further experiments are required to investigate the insecticidal/nematicidal activity of the KhF strain.
Besides the Bt toxins, several virulence factor genes have been detected in the KhF strain. It has been demonstrated that in addition to the pesticidal toxins, Bt strains are capable of producing different pathogenic factors influencing the mechanism of action of a Bt strain [
19,
48]. The Immune Inhibitor A (InhA), bacillolysin, Spo0A and SpoVG proteins, enolase, and cold-shock proteins were found in the spore/crystal mixture of the KhF strain following the LC-MS/MS analysis. It has been demonstrated that the InhA and bacillolysin are metalloproteases produced during the expression of
cry genes at the early in sporulation phase in a Bt strain [
7,
31,
49,
50] and they may affect the insect immune responses [
51,
52,
53,
54]. Moreover, the proteomics results revealed the presence of enolase in the KhF strain and the possible function of this enzyme in the virulence of Bt strains has also been speculated [
25]. Besides the virulence factors, the Spo0A, SpoVG, and cold-shock proteins produced during the sporulation phase [
7] and involved in the sporulation process of
Bacillus spp. [
30,
55,
56,
57] were detected.
The bioassays revealed the toxicity of the spore/crystal mixture of KhF strain for
P. interpunctella and
G. molesta. The susceptibility of
P. interpunctella to different Bt proteins (Cry1A, Cry1B, Cry1C, Cry1D, Cry1E, Cry1F, Cry1I, Cry2A, Cry9, Cry39, and Cry40) has been demonstrated [
58,
59]. As well, the toxicity of Cry1A, Cry1Ca, Vip3Aa, and Vip3Af Bt toxins to
G. molesta has been shown [
60]. According to the PCR-based screening [
21] and the whole-genome sequencing and proteomics results presented in this work, these genes and their subsequent proteins are not present in the KhF strain. Nevertheless, the genomics study of the strain together with the proteomics analyses of spores and crystals have highlighted the presence of KhFA and KhFB, two novel putative Xpp and Mpp-like proteins, described for the first time in this study. These proteins, most probably together with some bioactive virulence factors found in the KhF strain, could cause the insecticidal activity of KhF spores and crystals mixtures. Further experiments will be required to assess the individual insecticidal potential of each one of these two new proteins.